CN115389499B - A hair follicle detection kit, method and application thereof - Google Patents
A hair follicle detection kit, method and application thereof Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/84—Systems specially adapted for particular applications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
本发明涉及医学检测技术领域，尤其涉及一种毛囊检测试剂盒、方法及其应用。本发明毛囊检测试剂盒包含毛囊处理液和染色液，毛囊处理液包括曲拉通X‑100和溶剂，或包括曲拉通X‑100、Ⅰ型胶原酶和溶剂；其中，曲拉通X‑100的体积百分浓度为0.1～0.5％，Ⅰ型胶原酶的质量百分浓度为0.01～0.5％。本发明将曲拉通X‑100和Ⅰ型胶原酶按照特定比例组合用于处理离体毛囊，增加毛囊通透性和透光度，更利于染色液渗透到组织内部。利用本发明处理后的检测方法不仅将检测时间显著缩短，还能够减少毛囊或细胞在切片过程中损坏的风险，并能够提供毛囊组织结构和毛囊血管的完整信息，本发明的方法仅需要半小时就能完成毛囊的检测和出具检测报告。
The present invention relates to the field of medical detection technology, and in particular to a hair follicle detection kit, method and application thereof. The hair follicle detection kit of the present invention comprises a hair follicle treatment solution and a dyeing solution, and the hair follicle treatment solution comprises Triton X-100 and a solvent, or comprises Triton X-100, type I collagenase and a solvent; wherein, the volume percentage concentration of Triton X-100 is 0.1-0.5%, and the mass percentage concentration of type I collagenase is 0.01-0.5%. The present invention combines Triton X-100 and type I collagenase in a specific ratio for treating in vitro hair follicles, increasing the permeability and transmittance of the hair follicles, and being more conducive to the penetration of the dyeing solution into the tissue. The detection method after treatment by the present invention not only significantly shortens the detection time, but also reduces the risk of damage to hair follicles or cells during the slicing process, and can provide complete information on the hair follicle tissue structure and hair follicle blood vessels. The method of the present invention only takes half an hour to complete the detection of hair follicles and issue a test report.
Description
Technical Field
The invention relates to the technical field of detection, in particular to a hair follicle detection kit, a hair follicle detection method and application thereof.
Background
Alopecia and baldness have become the biggest problem for the current generation, and more young people have added the army of alopecia. Only hair has been of interest in treating hair loss over the past few decades. In order to obtain a healthy and dense hair, many people select various advanced hair washing and caring products, but only simply nourish the hair, do not treat the cause of alopecia, and cannot improve and treat alopecia at all.
Common factors that cause hair loss include genetic factors, diseases, hormonal changes, chemotherapeutics, stress, malnutrition, excessive hair dressing, aging, and the like. Although there are many causes of alopecia, the root is the hair follicle regardless of the cause.
Hair follicles are biological factories of hair processing, consisting of hair shafts, inner root sheaths, accompanying layers, outer root sheaths, hair matrix, long Tubu, and dermal interstitial parts (dermal sheaths and dermal papilla). The function of hair follicles depends on more than 20 cells in the hair follicle, of which the most critical 5 types of cells are hair follicle stem cells and melanocytes located at Long Tubu, dermal papilla cells located at dermal papilla, and hair matrix cells and melanocytes located at the hair matrix portion, respectively. Wherein:
Hair follicle stem cells, located at Long Tubu, are the raw material for most of the functional cells of hair follicle, and provide new cells for growing hair follicle continuously. Hair follicle stem cells, which are one of adult stem cells, have the potential to self-replicate and differentiate. The hair cycle circulation is maintained by hair follicle stem cells. Hair follicle stem cells responsible for hair growth are typically dormant, but rapidly activate and divide during a new cycle of hair growth. Due to the activation or resting state of the hair follicle stem cells, the normal shedding and regeneration of the hair is ensured. Hair loss can result if hair follicle stem cells are not normally activated, if damaged or reduced in number.
Dermal papilla cells dermal microenvironment formed by dermal papilla cells has been considered as a key signaling center for regulating hair follicle growth and rest, and dermal papilla cells are critical for activating hair follicle regeneration. During the growth induction phase of hair follicles, dermal papilla cells can control proliferation and differentiation of hair follicle stem cells by secretion control signals. Prepare for the next hair cycle. Therefore, dermal papilla is essential for hair growth, and if dermal papilla is missing, hair follicles cannot be activated in telogen. In addition, the number of dermal papilla cells affects the size of hair follicles, and the size of dermal papilla is related to the thickness of hair.
Hair matrix cells-in the growth of hair, hair matrix cells located in the hair matrix portion are continually dividing to form the "raw material" of the hair. With the continuous supply of "stock", the hair shaft is pushed out of the scalp and becomes macroscopic hair, and the number of hair matrix progenitor cells and hair bulb size affect hair density and thickness.
The states and the numbers of the two types of cells play a decisive role in the color of hair, the melanocytes are differentiated into melanocytes, the melanocytes synchronously generate melanin in hair matrix, and the melanin is transferred to Mao Ganzu cells and hair marrow cells, fur cytoplasm cells and Mao Xiaopi cells of filial generation of the cells to color the hair, so that the colored finished hair is finally formed. Hair whitening can be caused if melanocytes fail to differentiate normally or if the number of melanocytes is reduced.
The normal growth of hair also requires the feeding of nutrients to the hair follicle by the perifollicular microvasculature, which is responsible for the delivery of large amounts of nutrients to the dermis where the follicle grows. The early stages of hair follicle growth are when its nutritional needs are most vigorous, and thus vessel density increases significantly. In the growing period, blood vessels around hair follicles are actively remodelled, and if there is a problem in the angiogenesis at this time, the supply of nutrients and oxygen for hair formation becomes insufficient, and the number of micro-blood vessels around hair follicles is small or blood flow is insufficient, which causes hair loss.
In summary, hair follicle circulation and hair regeneration require cooperation and response between various cells in the follicle, require that the cells that synthesize the hair remain active, require sufficient supplementation after cell depletion, and require vascular delivery of large amounts of nutrients to sustain hair follicle growth. The hair follicle atrophy can be caused by the occurrence of any link, and the newly grown hair is thin, soft, withered and white, and the complete disappearance of the hair follicle can be seriously caused, so that the new hair can not grow out, and the sparse and top-withering of the hair can be caused.
However, currently, a dermatoscope is generally used for hair follicle detection. The dermatoscope is a skin magnifier with polarized light source, which can only detect scalp and hair which has grown outside the skin, but can not detect whether the structure of hair follicle is defective, whether the hair follicle component cells are complete and whether the blood supply around the hair follicle is sufficient.
Although the histopathological detection method (frozen section or paraffin section) can detect the hair follicle structure and cells, the 3-10 μm thin sheet obtained by cutting the hair follicle needs to be dyed, only the local plane structure is displayed, the three-dimensional structure of the hair follicle tissue and the reticular structure of the hair follicle blood vessel cannot be reflected, and the paraffin section needs to be subjected to the treatments of tissue fixation, embedding, section and the like, so that the time consumption is long, and the waiting time is long. While frozen sections are quick to slice, frozen microtomes are expensive and have high requirements on slicing technology of detection personnel.
Therefore, a rapid and efficient hair follicle detection kit and a detection method are urgently needed to acquire complete information of hair follicle tissue structures, hair follicle cells and peripheral capillary vessels of hair follicles, so as to judge the integrity degree of hair follicle tissues and cells and the blood supply condition of hair follicles, and evaluate the growth state and regeneration capacity of hair follicles.
Disclosure of Invention
In view of the above, the invention provides a hair follicle detection kit, a method and application thereof. The hair follicle treatment liquid in the hair follicle detection kit can effectively increase the permeability and the transmittance of hair follicles, and is more beneficial to dyeing and detection of hair follicle tissue structures, hair follicle cells and hair follicle peripheral micro-blood vessels after dyeing.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a hair follicle detection kit, which comprises hair follicle treatment liquid and staining liquid, wherein the hair follicle treatment liquid comprises triton X-100 and a solvent, or comprises triton X-100, type I collagenase and a solvent, the staining liquid is NBT/BCIP staining liquid or a combination of NBT/BCIP staining liquid and hematoxylin staining liquid, the volume percentage concentration of the triton X-100 is 0.1-0.5%, and the mass percentage concentration of the type I collagenase is 0.01-0.5%.
The hair follicle treatment liquid in the kit is used for treating hair follicles, so that proper porosity and openings can be formed in cell membranes, the permeability and the transmittance of the hair follicles are increased, the penetration of staining liquid into tissues is facilitated, and the hair follicle treatment liquid can be used for microscopic detection without slicing.
In some embodiments, the hair follicle treatment liquid comprises triton X-100, type I collagenase and a solvent, wherein the staining liquid is NBT/BCIP staining liquid or a combination of NBT/BCIP staining liquid and hematoxylin staining liquid, the volume percentage concentration of the triton X-100 is 0.1-0.5%, and the mass percentage concentration of the type I collagenase is 0.01-0.5%.
In some preferred embodiments, the volume percentage concentration of the triton X-100 in the hair follicle treating liquid is 0.1-0.3%, specifically 0.1%, 0.2% or 0.3%, and the mass percentage concentration of the type I collagenase is 0.01-0.1%, specifically 0.01%, 0.05% or 0.1%.
When the mass percentage concentration of the type I collagenase is too high (more than or equal to 1 percent), the tissue structure of the hair follicle can be damaged, excessive dye is resident in the tissue of the hair follicle, the background color is darkened, the tissue transmittance is poor, the contrast between the hair follicle structure and blood vessels is not obvious, the blood vessel distribution is not clear, the score of the picture definition and the score of the blood vessel dyeing effect are lower, the cell membrane and the nuclear membrane can be degraded, the dyeing is too deep, the contrast between the cell nucleus and the cell plasma is not obvious, and the hair follicle cells cannot be clearly observed. And when the concentration of the triton X-100 is too low (less than or equal to 0.05 percent), less dye enters the blood vessel, the blood vessel is incompletely dyed, the lumen of the hair follicle blood vessel is unclear, and the definition of the dyed picture and the scoring of the blood vessel dyeing effect are low.
In some embodiments, the volume percent concentration of triton X-100 is 0.3% and the mass percent concentration of type I collagenase is 0.1%.
In some embodiments, the volume percent concentration of triton X-100 is 0.1% and the mass percent concentration of collagenase type I is 0.1%.
In some embodiments, the solvent is PBS buffer or double distilled water.
In some embodiments, the staining solution is an NBT/BCIP staining solution.
In some embodiments, the staining solution is a combination of NBT/BCIP staining solution and hematoxylin staining solution, and in such embodiments, the hair follicle detection kit of the present invention further comprises a differentiation solution and a bluing solution.
In the invention, in the NBT/BCIP solution, the concentration of NBT is 0.1-0.6 mg/ml, the concentration of BCIP is 0.05-0.3 mg/ml, and the solvent is PBS buffer solution.
In the invention, the volume percentage of the hematoxylin stock solution in the hematoxylin solution is 50-100%.
The hair follicle detection kit provided by the invention is used for detection, no section is needed, after hair follicle is cleaned, the hair follicle is treated by the hair follicle treatment liquid, and then the hair follicle is dyed, or the hair follicle treatment liquid and the dyeing liquid are used simultaneously.
The invention provides a detection method of an in-vitro hair follicle, which comprises the following steps of treating with hair follicle treatment liquid and then dyeing:
a1, cleaning hair follicles to be tested, treating the hair follicles with a hair follicle treatment liquid for 8-20 min, cleaning, and then dyeing the hair follicles with an NBT/BCIP dyeing liquid for 3-15 min;
b1, cleaning the dyed hair follicle, placing the hair follicle on a detection slide, dripping 80-95% of glycerol, sealing the slide, and observing under a microscope.
In some embodiments, in step a1, the treatment time of the hair follicle treatment liquid is 8-15 min.
In some embodiments, in step a1, the follicular treatment fluid is treated for a period of 10 minutes.
In some embodiments, the NBT/BCIP solution is stained for 10 minutes.
Or alternatively
A2, cleaning hair follicles to be tested, treating the hair follicles with a hair follicle treatment liquid for 8-20 min, cleaning, then dyeing the hair follicles with an NBT/BCIP solution for 3-15 min, and then dyeing the hair follicles with a hematoxylin solution for 2-10 min;
And b2, sequentially treating the dyed hair follicle by a differentiation liquid and a blue-returning liquid, placing the hair follicle on a detection slide, dripping 80-95% of glycerol, sealing the slide, and observing under a microscope.
In some embodiments, in step a2, the treatment time of the hair follicle treatment liquid is preferably 8-15 min. In some embodiments specifically 10 minutes.
In some embodiments, the NBT/BCIP solution staining time is 10 minutes.
In some embodiments, the hematoxylin solution staining time is 3 minutes.
The differentiation liquid used in the differentiation is hydrochloric acid alcohol, wherein the volume percentage concentration of the concentrated hydrochloric acid is 0.2-1.5%, the differentiation time is 0.5-5 minutes, and the preparation method of the differentiation liquid comprises the steps of respectively dissolving 0.2ml, 0.5ml, 1ml or 1.5ml of concentrated hydrochloric acid (37%) into 200ml of 75% ethanol solution, and uniformly stirring to obtain the differentiation liquid with the volume percentage concentration of the concentrated hydrochloric acid of 0.2%, 0.5%, 1% or 1.5%.
The blue-returning liquid is an ammonia water solution, the mass percentage concentration of ammonia is 0.1-0.5%, and the blue-returning time is 0.5-3 minutes. The preparation method of the ammonia water bluing liquid comprises the steps of respectively dissolving 0.5ml, 1ml or 2.2ml of 28% ammonia water in 400ml of double distilled water, and uniformly stirring to obtain the bluing liquid with the ammonia mass percentage concentration of 0.11%, 0.22% or 0.5%.
The invention also provides a detection method of the in-vitro hair follicle, which comprises the following steps when the hair follicle treatment liquid and the staining liquid are used for treatment simultaneously:
a3, after the hair follicle to be detected is cleaned, adding hair follicle treatment liquid and NBT/BCIP staining liquid for treatment for 8-20 min;
And b3, cleaning the dyed hair follicle, placing the hair follicle on a detection slide, dripping 80-95% of glycerol, sealing the slide, and observing under a microscope.
Or alternatively
A4, after the hair follicle to be detected is cleaned, adding hair follicle treatment liquid and NBT/BCIP staining liquid to treat for 8-20 min;
b4, dyeing the hair follicle after NBT/BCIP dyeing by adopting a hematoxylin solution for 2-10 min;
And c4, sequentially treating the dyed hair follicle by a differentiation liquid and a blue-returning liquid, placing the hair follicle on a detection slide, dripping 80-95% of glycerol, sealing the slide, and observing under a microscope.
After the hair follicle is treated and dyed by adopting the detection method, the length, the diameter and the melanin content of the hair follicle can be measured under a microscope, and the number of blood vessels around the hair follicle can be counted. The detection of hair follicles and the detection report can be completed only in half an hour, so that the method is suitable for rapid detection before hair follicle transplantation, the traditional detection time is greatly shortened, and the labor and time cost and the expensive antibody reagent cost are saved. And meanwhile, the integrity of hair follicles and cells is not damaged, and the complete information of the tissue structure of the hair follicles and the blood vessels of the hair follicles can be provided.
In some embodiments, the washing solution is PBS buffer solution, and the washing time is 0.5-1 min. The PBS buffer solution consists of double distilled water and the following components with the concentration of 8.18g/L of sodium chloride, 0.20g/L of potassium chloride, 1.42g/L of disodium hydrogen phosphate, 0.245g/L of potassium dihydrogen phosphate and the pH value of 7.3.
According to the detection method provided by the invention, NBT/BCIP staining solution is used for detecting blood supply around hair follicle, hematoxylin staining is used for detecting hair follicle structure, hair follicle state and hair follicle cell number, the hair follicle structure and the hair follicle state comprise at least one of hair follicle length, hair follicle thickness, dermal papilla size, long Tubu thickness, hair bulb size and hair shaft melanin content, and the hair follicle cells comprise one or more of hair follicle Long Tubu stem cells, dermal papilla cells or hair matrix cells.
Compared with the prior art, the invention has the following beneficial effects:
1) The hair follicle detection kit provided by the invention detects the form of hair follicle, peripheral blood supply of hair follicle and hair follicle cell composition, analyzes the health state of hair follicle from multiple dimensions, and provides support for the subsequent evaluation of the effect after hair follicle transplantation and targeted treatment.
2) After the hair follicle detection kit is processed, the hair follicle structure and the cell morphology can be directly observed under a microscope, the detection time is obviously shortened, the integrity of hair follicles and cells is not damaged, the complete information of the hair follicle tissue structure and the hair follicle blood vessels can be provided, the hair follicle detection kit can be used for completing the detection of the hair follicles and the detection report only by half an hour, is suitable for the rapid detection before hair follicle transplantation, greatly shortens the traditional detection time, saves the labor and time cost and the expensive antibody reagent cost, and provides a convenient and rapid detection method for hair follicle detection.
3) Compared with the traditional blood vessel detection method, the hair follicle blood supply detection kit provided by the invention has the advantages that the hair follicle permeability and the transmittance can be increased by adopting the tropull X-100 or the combination of the tropull X-100 and the type I collagenase, the penetration of the dyeing liquid into tissues is facilitated, the effect of the hair follicle blood supply after dyeing is better, the distribution condition of blood vessels and the vascular lumen structure can be clearly seen, and thus, whether the hair follicle blood supply is sufficient or not can be known.
4) The hair follicle detection kit of the invention adopts tissue treatment liquid to treat, then sequentially performs vascular staining and hematoxylin staining, or combines the steps of combined treatment and vascular staining to treat, then performs hematoxylin staining, has clear staining effect, and can shorten the staining time by adopting the latter, thereby achieving the purpose of faster detection. Meanwhile, by adopting pictures at different layers, the hair bulb size, dermal papilla and blood supply of hair bulb parts of hair follicles and the contents of stem cells and melanin of the hair bulb parts of the hair bulbs can be obtained, so that whether the whole hair follicle is in a healthy state or not can be obtained through analysis.
Drawings
FIG. 1 shows the results of rapid staining of C57 mouse hair follicles using the method of example 4, FIG. 1A is the results of staining of hair bulb, FIG. 1B is the results of staining of blood vessels around hair follicles, FIG. 1C is the results of staining Long Tubu and hair shafts, and FIG. 1D is the results of staining Long Tubu and hair follicle stem cells;
FIG. 2 is a photograph showing the staining of hair follicle stem cells, FIG. 2A is a photograph showing the immunohistochemical staining of hair follicle stem cells, and FIG. 2B is a photograph showing the rapid staining of hair follicle stem cells;
FIG. 3 shows the results of rapid staining of C57 mouse hair follicles using the method of example 5, FIG. 3A is the results of staining of hair bulb, FIG. 3B is the results of staining of blood vessels around hair follicles, FIG. 3C is the results of staining Long Tubu and hair shafts, and FIG. 3D is the results of staining Long Tubu and hair follicle stem cells;
FIG. 4 is a front view of the hair follicle detecting device, wherein the 1-label area, the 2-first bracket, the 3-second bracket, the 4-sample detecting area, and the 5-clamp area;
FIG. 5 is a perspective view of a device for hair follicle detection, wherein the 1-tag region, 2-first scaffold, 3-second scaffold, 5-clamping region, 6-cavity, 7-slot;
FIG. 6 shows a side view of a hair follicle testing device, 1-marker area, 5-clamp area, 6-cavity, 7-slot, 8-test slide;
Fig. 7 shows a front view of a detection slide, wherein an 8-detection slide,
FIG. 8 shows a side view of a test slide, wherein 801-on slide, 802-off slide;
FIG. 9 shows the NBT/BCIP staining of C57 mouse hair follicles, FIGS. 9A-D are the NBT/BCIP staining for treatment for 5, 10, 15 and 30min in order;
FIG. 10 shows the NBT/BCIP staining results of C57 mouse hair follicles, FIG. 10A shows the staining results of triton X-1000.05% (v/v) +type I collagenase 0.1% (w/w), FIG. 10B shows the staining results of triton X-1000.1% (v/v) +type I collagenase 0.1% (w/w), FIG. 10C shows the staining results of triton X-1000.3% (v/v) +type I collagenase 0.1% (w/w), FIG. 10D shows the staining results of triton X-1000.5% (v/v) +type I collagenase 0.1% (w/w), FIG. 10E shows the staining results of triton X-1001% (v/v) +type I collagenase 0.1% (w/w), and FIG. 10F shows the staining results of triton X-1000.3% (v/v).
FIG. 11 shows the results of NBT/BCIP staining and hair follicle cell staining of C57 mice hair follicles, FIG. 11A shows the results of staining with triton X-1000.1% (v/v) +type I collagenase 0.01% (w/w), FIG. 11B shows the results of staining with triton X-1000.1% (v/v) +type I collagenase 0.1% (w/w), FIG. 11C shows the results of staining with triton X-1000.1% (v/v) +type I collagenase 0.5% (w/w), FIG. 11D shows the results of staining with triton X-1000.1% (v/v) +type I collagenase 1% (w/w), FIG. 11E shows the results of staining with triton X-1000.3% (v/v) +type I collagenase 0.01% (w/w), FIG. 11F shows the results of staining with triton X-1000.3% (v/v) +type I collagenase 0.1% (w/w), FIG. 11G shows the results of staining with triton X-1000.3% (v/v) +type I type I collagenase 0.1% (w/w), and FIG. 11E shows the results of staining with triton X-3565% (v/v) +type I type I collagenase 0.1% (w/w).
FIG. 12 illustrates the detection of hair follicles using the hair follicle detection device of the present invention;
FIG. 13 shows the results obtained with the HE staining method for hair follicles in C57 mice;
FIG. 14 shows the results obtained with immunohistochemical staining of the hair follicle bulb of C57 mice obtained with CD 31.
Detailed Description
The invention provides a hair follicle detection kit, a hair follicle detection method and application thereof. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
Reagents, instruments, materials, equipment used in the present invention are commercially available.
The stock hematoxylin solution is a commercially available hematoxylin dye solution, and all of the currently commercially available hematoxylin dye solutions can be used for the staining of the present invention, for example, harris hematoxylin dye solution (bead halyard corporation, etc.), cole's hematoxylin dye solution (gold cloning biotechnology, etc.), mayer hematoxylin dye solution (Nanjing aerospace, etc.), modified Lillie-Mayer hematoxylin dye solution (Solarbio, etc.), ehrlich hematoxylin dye solution (Shanghai, ji, etc.), carazzi hematoxylin dye solution (Nanjsen Bei Ga, etc.), gill modified hematoxylin dye solution (Beijing Lei Gen, etc.). 100% of hematoxylin is the stock solution of hematoxylin.
The preparation method of the triton X-100 solution in the hair follicle treatment liquid comprises the steps of respectively dissolving 10 mu L, 20 mu L, 30 mu L, 40 mu L or 50 mu L of triton X-100 in 10ml of 0.01mol/LPBS buffer solution to obtain PBS solution with volume concentration of 0.1%, 0.2%, 0.3%, 0.4% or 0.5% of triton X-100.
The preparation method of the type I collagenase solution in the hair follicle treating liquid comprises the steps of respectively dissolving 5mg, 10mg, 20mg, 30mg, 40mg or 50mg of type I collagenase in 10ml of 0.01mol/L PBS buffer solution to obtain PBS solutions with mass concentrations of 0.05%, 0.1%, 0.2%, 0.3%, 0.4% and 0.5% of type I collagenase.
The PBS buffer solution and PBS cleaning solution of the invention are aqueous solutions with the pH of 7.3, wherein the formulation of the PBS buffer solution and the PBS cleaning solution is 8.18g/L of sodium chloride, 0.20g/L of potassium chloride, 1.42g/L of disodium hydrogen phosphate and 0.245g/L of monopotassium phosphate;
The vascular staining solution adopts NBT/BCIP staining solution, wherein NBT/BCIP is English abbreviation of (nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate).
The invention is further illustrated by the following examples:
EXAMPLE 1 follicular treatment fluid of the present invention
The hair follicle treating liquid comprises the following components:
0.3% of triton X-100 (v/v) and PBS buffer as solvent.
EXAMPLE 2 follicular treatment fluid of the present invention
The hair follicle treating liquid comprises the following components:
0.3% of triton X-100 (v/v) and 0.1% of collagenase I (w/w), and the solvent is PBS buffer solution.
EXAMPLE 3 follicular treatment fluid of the present invention
The hair follicle treating liquid comprises the following components:
0.1% of triton X-100 (v/v) and 0.1% of collagenase type I (w/w), and the solvent is PBS buffer.
Example 4 Hair follicle detection kit and detection method of the present invention
The hair follicle detection kit comprises 0.3% (v/v) of triton X-100+0.1% (w/w) of a PBS buffer solution (hair follicle treating liquid) of type I collagenase, a PBS solution (0.4 mg/ml NBT, 0.19mg/ml BCIP) of NBT/BCIP, a hematoxylin solution (with the concentration of 100 percent), a hydrochloric acid alcohol differentiation solution (with the concentration of 1 percent by volume of concentrated hydrochloric acid in hydrochloric acid alcohol), an ammonia water bluing solution (with the concentration of 0.22 percent by mass of ammonia) and a PBS cleaning solution (with the concentration of 0.01 mol/L).
1. Method for rapidly detecting isolated hair follicle
1) Taking 6 centrifuge tubes, and adding one of the hair follicle treatment liquid, NBT/BCIP staining liquid, hematoxylin solution, hydrochloric acid alcohol differentiation liquid, bluing liquid or PBS cleaning liquid into each centrifuge tube, wherein the adding volume of each solution is 1ml.
2) The hair follicle to be detected is taken out from the physiological saline and is washed for 1 minute by a washing liquid;
3) Treating hair follicle by adopting hair follicle treating liquid for 10 minutes, and cleaning the hair follicle by using cleaning liquid for 1 minute;
4) The treated hair follicles were stained with NBT/BCIP for 10 minutes;
5) The hair follicle after NBT/BCIP staining is stained with hematoxylin solution for 3 minutes, and is washed with a cleaning solution for 0.5 minute;
6) Placing hair follicle into differentiation solution, differentiating for 3 min, and cleaning with cleaning solution for 0.5 min;
7) The hair follicle is placed in the bluing liquid for 2 minutes and is cleaned by the cleaning liquid for 1 minute;
8) 2 drops of glycerin are dripped on the detection slide, and the hair follicle is placed in the glycerin and sealed by the detection slide.
9) The capped hair follicle was placed on a hair follicle detection device for observation and detection, and photographed with a positive optical microscope (80 i, nikon) and an image acquisition system (SPOT FLEX).
Firstly, treating with triton X-100+I collagenase for 10min, and then sequentially carrying out NBT/BCIP staining and hematoxylin staining, wherein after staining, not only the hair bulb, dermal papilla, long Tubu and Mao Ganjie structures of the hair follicle are obvious, but also the nucleus staining of the hair follicle stem cells is clear, and the vascular lumen and the vascular three-dimensional structure of the hair follicle are also clear (figures 1A-1D).
2. Hair follicle detection index and measuring method
2.1 Detection of the Length and thickness of Hair follicle
Hair follicle length and thickness, namely placing the washed hair follicle to be detected on a scale in parallel, and measuring by using analysis software Image J1.52 k (NIH);
2.2 follicular structure and State detection
Long Tubu thickness, namely collecting the clearest layer of the hair shaft at the bulge under the 200-time visual field of an optical microscope, and measuring the linear distance between the hair shaft at one side and the edge of the hair follicle to represent the thickness of the bulge.
The size of the hair bulb is that the hair bulb is collected under the 200 times visual field of the optical microscope, and the longest length of the hair bulb is measured by using a self-contained measuring scale.
Melanin content the most clear aspect of the hair shaft at the bulge area was collected under 200 x field of light microscope, the black hair shaft area and the optical density value in that area were measured with Image J software, the average optical density value = the optical density value in that area/black hair shaft area, and the melanin content was expressed as the average optical density value.
Dermal papilla the most clear level of the hair bulb melanin was collected under an optical microscope at 200 x field, and the longest length of the transparent portion (similar to an oval shape) of the melanin concave region was measured, indicating the dermal papilla size.
2.3 Perifollicular blood supply detection
The hair follicle to be detected which is dyed by the hair follicle rapid dyeing method is collected under the 200 times visual field of an optical microscope and approaches to the hair bulb blood vessels, and the number of the blood vessels which are transversely distributed around the hair follicle is counted.
2.4 Follicular cell detection
Marking hair follicle stem cells, dermal papilla cells and hair matrix cells by an immunohistochemical staining method, obtaining the positions of the hair follicle stem cells, the dermal papilla cells and the hair matrix cells in the hair follicle according to the immunohistochemical staining result, and counting the quantity of the hair follicle stem cells, the dermal papilla cells and the hair matrix cells of the hair follicle to be detected in the rapid staining of the hematoxylin of the hair follicle according to the obtained cell position information.
The immunohistochemical staining method comprises the following steps:
1) The tissue block is directly put into 4% paraformaldehyde for fixation for 24 hours after the tissue block is obtained, and then the tissue block is washed overnight with flowing water.
2) Slicing, namely putting the tissue block into 30% sucrose solution for dehydration for 16 hours, taking out the wiped water, and performing OCT embedding slicing.
3) Fixing: frozen sections were removed from the freezer and left to air dry at room temperature for a few moments.
4) Washing, namely washing the substrate for 5min multiplied by 3 by a PBS shaking table at 50 r/min.
5) Circling, namely, sucking up the water around the tissues and drying the slide. Carefully stitch the outline with the PAP pen windings.
6) Well-punching, namely dropwise adding 0.1% TritonX-100 to each slice and incubating for 10 minutes at room temperature.
7) Washing, namely washing the substrate for 5min multiplied by 3 by a PBS shaking table at 50 r/min.
8) Blocking the sections were blocked with a solution containing 2% fetal bovine serum (PBS) at 37℃for 1 hour, the amount of BSA being based on complete immersion of the tissue.
9) Primary antibody, blotted dry blocking buffer, sections were placed in wet boxes and incubated for 1 hour at 37 ℃ with primary antibody diluted with 2% fetal bovine serum solution (the antibodies were diluted according to recommended concentrations and pre-experimental results).
10 Washing, throwing away the primary antibody and washing with PBS buffer 5 times.
11 Secondary antibody was incubated at 37 ℃ with 2% BSA solution drop-wise (1:1000) on sections for 1 hour.
12 Washing, namely, throwing away the secondary antibody, and washing the secondary antibody by using PBS buffer solution for 5min multiplied by 3.
13 Staining nuclei, incubating with 1ug/ml DAPI working solution for 5min at room temperature.
14 Washing with PBS for 5min×3.
15 Cover plate, namely wiping off moisture on the surface of the slice, dropwise adding a drop of anti-fluorescence quenching buffer glycerol into each slice, and covering a cover glass.
16 Observation under a laser confocal microscope or preservation in a dark place at-20 ℃.
The immunohistochemical staining method serves to mark the positional information of hair follicle stem cells, dermal papilla cells and hair matrix cells in the hair follicle. The conventional immunohistochemical staining method of the hair follicle stem cells comprises the steps of preparing paraffin sections after material selection, preparing paraffin sections, carrying out dehydration, wax dipping, embedding and the like on the paraffin sections, then slicing, carrying out rehydration, marking by using a hair follicle stem cell marker antibody SOX9 through immunohistochemical, and obtaining results as shown in figure 2A only after at least four days, wherein only one enzyme substrate reaction staining is needed, and only the rapid staining method is needed for detecting the hair follicle cells after subsequent detection, so that the quantity and the state of all the hair follicle stem cells of the whole hair follicle can be seen only in ten minutes (figure 2B).
2.5 Evaluation of health status of hair follicle to be measured
And evaluating the detection result of the hair follicle to be detected by taking the index range of the normal hair follicle as a reference standard, thereby obtaining whether the state of the hair follicle is normal.
Example 5 Hair follicle detection kit and detection method
The kit for detecting the hair follicle in vitro comprises a PBS solution (NBT of 0.4mg/ml and BCIP of 0.19 mg/ml) of 0.3% triton X-100 (v/v) +0.1% type I collagenase (w/w) +NBT/BCIP, a hematoxylin solution (the concentration of hematoxylin is 100%), a hydrochloric acid alcohol differentiated solution (the volume percentage concentration of concentrated hydrochloric acid in the hydrochloric acid alcohol is 1%), an ammonia water bluing solution (the mass percentage concentration of ammonia is 0.22%) and a PBS cleaning solution (the concentration of PBS is 0.01 mol/L).
1. Method for rapidly detecting isolated hair follicle
1) 5 Centrifuge tubes were taken, and one of PBS solution, hematoxylin solution, alcohol hydrochloride differentiation solution, bluing solution or PBS washing solution of 0.3% triton X-100 (v/v) +0.1% type I collagenase (w/w) +NBT/BCIP was added to each centrifuge tube, and the addition volume of each solution was 1ml.
2) The hair follicle to be detected is taken out from the physiological saline and is washed for 1 minute by a washing liquid;
3) Hair follicles were treated with 0.3% triton X-100 (v/v) +0.1% collagenase type i (w/w) +nbt/BCIP in PBS for 10min and washed with wash solution for 1 min;
4) The hair follicle after NBT/BCIP staining is stained with hematoxylin solution for 3 minutes, and is washed with a cleaning solution for 0.5 minute;
5) Placing hair follicle into differentiation solution, differentiating for 3 min, and cleaning with cleaning solution for 0.5 min;
6) The hair follicle is placed in the bluing liquid for 2 minutes and is cleaned by the cleaning liquid for 1 minute;
7) 2 drops of glycerin are dripped on the detection slide, and the hair follicle is placed in the glycerin and sealed by the detection slide.
8) The capped hair follicle was placed on a hair follicle detection device for observation and detection, and photographed with a positive optical microscope (80 i, nikon) and an image acquisition system (SPOT FLEX).
The hair follicle is dyed by adopting a mixed solution of triton X-100+0.1% type I collagenase (w/w) +NBT/BCIP, then adopting hematoxylin for dyeing, the background after dyeing is shallower than that of example 4, the hair bulb, dermal papilla, long Tubu and Mao Ganjie structures of the hair follicle after dyeing are more obvious, the nucleus of the hair follicle stem cells is dyed clearly, the vascular lumen and the vascular stereo structure of the hair follicle are clearer (figures 3A-3D), and the hair follicle is processed by adopting the triton X-100+type I collagenase and NBT/BCIP in a mixed mode, so that the dyeing time is further shortened.
2. Hair follicle detection index and measuring method
The "hair follicle detection index and measurement method" of example 5 are the same as those of example 4.
Example 6 Hair follicle detection device
The hair follicle detection device comprises a fixing device and a detection unit, wherein the fixing device consists of a marking area 1, a clamping area 5 and a fixing area, the fixing area is positioned between the marking area and the clamping area, one side, close to the fixing area, of the marking area 1 and one side, close to the fixing area, of the clamping area 5 are respectively provided with a slot 7, the fixing area consists of a first support 2 and a second support 3, the first support 2 and the second support 3 are respectively provided with a cavity 6 for accommodating the detection unit, a rectangle enclosed by the first support 2, the second support 3, the marking area and the clamping area is a sample detection area 4, and the detection unit is a detection slide 8. See fig. 4-6 for front, perspective and side views of the hair follicle detection device.
In one or more embodiments, the test slide is divided into an upper slide 801 or a lower slide 802, hair follicles are placed between the upper slide 801 and the lower slide 802, and a test slide 8 with hair follicles is placed in the cavity of the first bracket 2 and the second bracket 3 (the test slide is shown in fig. 7-8).
In one or more embodiments, the marking area 1, the clamping area 5 and the fixing area are integrally formed, and the first bracket 2 or the second bracket 3 of the fixing area is composed of a left bracket and a right bracket, which are detachably connected.
The device can enable the slide to be easily fixed and can be detected at multiple angles under a light microscope or a microscope, and the hair follicle detection device comprises a fixing device and a detection unit.
Example 7 vascular staining test
(One) experiment of influence of different staining time on the staining result of the perifollicular blood vessel NBT/BCIP
To examine the effect of different staining times on NBT/BCIP staining results, the vessels of the beard hair follicle of C57 mice were examined at 5min, 10min, 15min and 30min, with NBT concentrations of 0.4mg/ml and BCIP concentrations of 0.19mg/ml.
The results are shown in fig. 9A-9D and table 1, when the NBT/BCIP is dyed for 5 minutes, the dyeing is insufficient, the blood vessel is light, the lumen of the hair follicle blood vessel is not clear, when the NBT/BCIP is dyed for 10-30 minutes, the dyeing effect is good, the dyeing is sufficient, the lumen of the hair follicle blood vessel is clear, but after the dyeing time is 10 minutes, the darker the dyeing background is along with the time, the contrast between the blood vessel and the background is not obvious, the observation of the blood vessel is affected, the time is consumed, and the time cost is increased.
TABLE 1 results of NBT/BCIP staining of perifollicular vessels at different staining times
(II) experiment of influence of different follicular treatment fluids on the result of NBT/BCIP staining of perifollicular vessels
The effect of different hair follicle treatment fluids on the staining results was examined, and the effect of the combination of type I collagenase and triton X-100 (0.05%, 0.1%, 0.3%, 0.5% and 1%) at different volume concentrations on the staining effects was examined, and the hair follicles of C57 mice were examined using the staining kit of test group 5-10, the composition of the staining kit of test group 5-10 being shown in Table 2, and the detection method was as follows:
1) Taking out the hair follicle to be detected from the physiological saline, and cleaning the hair follicle with a cleaning solution for 1 minute;
2) Treating hair follicle by adopting hair follicle treating liquid for 10 minutes, and cleaning the hair follicle by using cleaning liquid for 1 minute;
3) The hair follicle is dyed for 10 minutes by using NBT/BCIP dyeing liquid, and is washed for 0.5 minutes by using a washing liquid;
4) 2 drops of glycerin are dripped on the detection slide, and the hair follicle is placed in the glycerin and sealed by the detection slide.
5) The capped hair follicle was placed on a hair follicle detection device for observation and detection, and photographed with a positive optical microscope (80 i, nikon) and an image acquisition system (SPOT FLEX).
6) And (3) evaluating the definition of the dyed hair of the experimental group 5-10 by adopting image definition evaluation green version 0.4 software, and grading the blood vessel dyeing effect according to the following criteria, namely, whether the color depth of blood vessel dyeing outside hair follicle and inside dermis sheath is proper, whether the overall blood vessel lumen and blood vessel distribution of the dyed hair follicle are clear, and evaluating the four grades of A, B, C and T. The scoring standard of the dyeing result is that grade A tablets are more than or equal to 90 (excellent), grade B tablets are 75-89 (good), grade C tablets are 60-74 (basically qualified), and grade D tablets are less than or equal to 59 (unqualified).
As can be seen from fig. 10A to 10F and table 2, the treatment with fralaton X-100 alone for 10min or with fralaton X-100+i type collagenase for 10min, the clarity of the picture after staining and the vascular staining effect were superior to those of the experimental group 2 which had not been treated with the hair follicle treating liquid.
Meanwhile, NBT/BCIP staining definition (more than 95%) and vascular staining effect score (grade A) after treatment of 0.1% (w/w) type I collagenase plus 0.1-0.3% (v/v) of triton X-100 (experimental group 6-7) are superior to NBT/BCIP staining definition (88.2%) and vascular staining effect score (grade B) after independent treatment of triton X-100 (experimental group 10). In addition, the collagenase type I is treated for 10min by 0.1% (w/w) +triton X-1000.3% (v/v), and is dyed for 10min by NBT/BCIP, so that the vascular lumen of the hair follicle is clearer, the vascular distribution condition can be clearly seen, and the dyeing effect is optimal and is obviously superior to the combination of other concentrations. The combination of 0.3% triton X-100 (v/v) +0.1% type I collagenase (w/w) is shown to be better able to form a gel with proper porosity and openings on the vessel wall of the hair follicle and the dermis sheath of the hair follicle, which is beneficial for the uniform penetration of NBT/BCIP dye into the vessel lumen.
TABLE 2 Effect of different follicular treatment fluids on vascular staining results
Example 8 Effect of perifollicular vascular staining and follicular cell staining experiment results
In order to simultaneously improve the dyeing effect of peripheral blood vessels and hair follicle cells of the C57 mice, the effects of different ratios of the triton X-100 and the type I collagenase on the dyeing effect of the hair follicle blood vessels and the hair follicle cells of the C57 mice are examined by combining the triton X-100 solution and the type I collagenase. The composition of the hair follicle detection kit of experimental groups 11-18 is shown in Table 3, and the detection method of experimental groups 11-18 is as follows:
1) Taking out the hair follicle to be detected from the physiological saline, and cleaning the hair follicle with a cleaning solution for 1 minute;
2) Treating hair follicle by adopting hair follicle treating liquid for 10 minutes, and cleaning the hair follicle by using cleaning liquid for 1 minute;
3) The hair follicle is dyed for 5 or 10 minutes by using NBT/BCIP dyeing liquid, and is washed for 0.5 minutes by using a washing liquid;
4) The hair follicle after NBT/BCIP staining is stained with hematoxylin solution for 3 minutes, and is washed with a cleaning solution for 0.5 minute;
5) Placing hair follicle into differentiation solution, differentiating for 3 min, and cleaning with cleaning solution for 0.5 min;
6) The hair follicle is placed in the bluing liquid for 2 minutes and is cleaned by the cleaning liquid for 1 minute;
7) 2 drops of glycerin are dripped on the detection slide, and the hair follicle is placed in the glycerin and sealed by the detection slide.
8) The capped hair follicle was placed on a hair follicle detection device for observation and detection, and photographed with a positive optical microscope (80 i, nikon) and an image acquisition system (SPOT FLEX).
9) The evaluation method is to evaluate the tissue transparency of the 7 th item, the nucleus and the cytoplasm contrast of the 8 th item and whether the blood vessel staining is clear according to the HE staining standard of the clinical technical operation Specification and pathological division book under the condition that other items are unchanged. The scoring standard of the dyeing result is that grade A tablets are more than or equal to 90 (excellent), grade B tablets are 75-89 (good), grade C tablets are 60-74 (basically qualified), and grade D tablets are less than or equal to 59 (unqualified).
TABLE 3 staining results of perifollicular blood vessels and follicular cells with different follicular treatment fluids
As can be seen from FIGS. 11A-11H and Table 3, when the mass concentration of the triton X-100 is 0.1% or 0.3%, the mass concentration of the class I collagenase is 0.01% -0.1% (experiments 11, 12, 15 and 16) of hair follicle cells and hair follicle blood vessel staining effects are better (class A or class B), the staining effects are better than those of experiments 13, 14, 17 and 18, and when the mass percentage concentration of the class I collagenase is more than or equal to 1% (v/v%), the tissue transmittance after staining is poor, the staining is too deep, the cell nucleus and cell plasma are relatively poor, and the blood vessel staining is unclear (class D).
Example 9 comparative experiment of the detection method of the present invention with the conventional staining method
The detection method and detection result (comprising hair follicle cells and blood vessels around hair follicle) of the invention are shown in example 4, the physiological saline after hair follicle of a mouse is washed, the tissue sample after the dyeing is placed in the dyeing reagent of example 4 for dyeing treatment, the dyed tissue sample is placed on a detection slide, 90% glycerol is dripped to cover the tissue, the detection slide is used for covering the tissue, a detection unit comprising the tissue sample is inserted and fixed on a fixing device from a cavity at the side of the fixing device, and the tissue form can be observed by reversing the front and back sides of the detection unit under a microscope (see fig. 12).
(1) The hair follicle cell detection method in the prior art adopts a conventional HE staining method, the experimental result is shown in fig. 12, which is an image shot by the HE staining of a conventional section, and the section of the HE method is thinner, so that only cells and structures of one plane can be displayed, and the method is not stereoscopic.
Compared with the conventional HE method, the method and the device can clearly display cells and structures at different layers of hair follicles without slicing images shot by the hair follicles of the C57 mice, and have a stereoscopic impression (see FIG. 13).
(2) Existing follicular vascular detection
The detection of tissue, namely mouse hair follicle, immunohistochemical CD31 hair follicle vascular staining, comprises the steps of firstly cleaning, embedding and slicing the hair follicle, then punching by using triton, blocking by using 2% BSA, incubating the primary antibody overnight, incubating the secondary antibody, and finally observing by using a microscope, wherein the detection result is shown in figure 14. The immunohistochemical CD31 staining process takes two days, and needs to pay attention to that the hair follicle must be parallel to the bottom of the embedding box when embedding, bubbles are easy to generate around the hair follicle, a complete hair follicle longitudinal section is cut when slicing, a plurality of hair follicles need to be cut first and constantly adjusted and checked by a microscope in time so as to determine whether the hair follicle longitudinal section is cut, and the immunofluorescence staining process is difficult to fall off part of tissues due to the fact that the hair follicle structure is complex, especially the dermal papilla part of the hair bulb part can cause partial vascular loss, the detected vascular number can be influenced, and the number of blood vessels of a certain section is only needed, so that whether the whole blood supply of the hair follicle is normal can not be judged.
Compared with immunohistochemical CD31 hair follicle vascular staining, the invention does not need to carry out steps such as slicing when carrying out vascular staining, and adds the combination of triton or triton and type I collagenase to treat the hair follicle, and the treated hair follicle has better vascular staining effect, can clearly and completely display the vascular distribution conditions of different layers of the hair follicle, the number of blood vessels and the three-dimensional structure of the vascular lumen, thereby knowing whether the blood supply of the hair follicle is sufficient.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
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