CN110090300A - A kind of method and its application for the Regeneration and Repair promoting histoorgan - Google Patents

Abstract

The present invention relates to a kind of method and its application of Regeneration and Repair for promoting histoorgan.The inventors discovered that Myosin inhibitor can destroy cell machinery stress system stable state, cytogenetics repair ability is greatly improved using stress reaction similar to stress reaction and regenerative response during regeneration in excitation.The Myosin inhibitor is preferably (-)-Blebbistatin.Method of the invention only needs single small molecule or single factor test to handle, easy to operate, reproducible, can be used as excitation stress reaction, excitation regenerative response, a kind of new method for improving cytogenetics repair ability and/or promoting the Regeneration and Repair of histoorgan.

Description

A method for promoting the regeneration and repair of tissues and organs and its application

technical field

The invention relates to the field of biotechnology, in particular to a method for promoting the regeneration and repair of tissues and organs and related applications thereof.

Background technique

Tissues and organs of multicellular organisms are composed of parenchymal cells, mesenchymal cells and their secreted extracellular matrix. Parenchymal cells refer to the main structural and functional cells of tissues and organs (for example, the parenchymal cells of the brain are neurons, and the parenchymal cells of the liver are hepatocytes), and the interstitial cells and extracellular matrix constitute the interstitial part of tissues and organs (mainly interstitial cells, collagen, laminin, fibronectin, elastin, proteoglycans, glycoproteins, glycosaminoglycans), which mainly play the role of mechanical support and connection, and the extracellular matrix constitutes and maintains the physiological activities of cells The microenvironment is a bridge for signal transduction between cells, participates in and regulates various physiological and pathological processes, and plays an important role in tissue wound repair, regeneration and fibrosis.

In the regeneration process of lower organisms, injury leads to a rapid stress response, and a series of stress proteins such as the heat shock protein family are up-regulated, and the stress response occurs within 24 hours of injury; it is triggered on the 2-2.5 day after injury During regeneration, some regeneration initiation-related genes and tissue-specific genes are up-regulated; specific differentiated tissue cells appear in the final stage of regeneration, and new tissues are regenerated.

For evolutionary reasons, mammals have a very limited ability to regenerate. Injury to tissue cells in higher mammals will cause tissue cell degeneration, necrosis, and inflammatory response. If the damage is small, the normal parenchymal cells surrounding the damaged tissue cells will proliferate and repair, thereby completely restoring the normal tissue structure and function. However, if the large injury or repeated injury exceeds the regenerative repair ability of the surrounding parenchymal cells, in order to prevent excessive bleeding and reduce the risk of infection, the body will produce a severe stress protection mechanism to produce coagulation, immune and inflammatory reactions, and Promote the fibrous connective tissue (extracellular matrix) of the interstitial part to proliferate in large quantities to repair the defective tissue, accompanied by pathological changes of fibrosis. Although the proliferating fibrous connective tissue repairs the defect and protects the relative integrity of the tissue and organ to the greatest extent, this repair not only inhibits the normal regenerative repair, but makes the repair unable to have the structure and function of the original organ. It may even lead to organ fibrosis, hardening and loss of function due to this excessive, strong and uncontrolled repair response.

Worldwide, tissue and organ fibrosis is the main cause of disability and death in many diseases, and tissue and organ fibrosis plays an important role in the occurrence and development of diseases related to major organs of the human body. According to relevant statistics, nearly 45% of the patients who died of various diseases in the United States can be attributed to tissue and organ fibroproliferative diseases. Therefore, controlling the fibrosis process in mammals is an important idea for the treatment of various diseases.

Stress fibers are microfilament bundle structures widely present in eukaryotic cells, which are composed of a large number of parallel microfilaments, and are closely related to the adhesion between cells or cells and the surface of the substrate. has an important role. Its components are actin, myosin, tropomyosin and α-actinin. The main mechanical stress system of the cell is composed of stress fibers, extracellular matrix, cell membrane receptors (such as integrins), nuclear skeleton and linker complex of cytoskeleton, nuclear lamina, and chromosome skeleton. The mechanical stress system endows cells with the regulation of mechanical stiffness, participates in the induction, transmission and generation of mechanical forces inside and outside cells, and regulates the assembly, distribution and expression of genetic material. Mechanical stress System homeostasis is one of the indicators characteristic of a particular cell. The homeostasis of the cell mechanical stress system helps to maintain and establish specific cell properties, maintain and stabilize cell-specific genetic regulation and expression characteristics, and break cell mechanical homeostasis as a kind of cell damage that stimulates cell stress and stimulates strong cell damage repair ability.

The present invention uses a method without loss and bleeding to stimulate the stress response and regeneration response similar to the regeneration process of lower organisms by destroying the steady state of the cell mechanical stress system, and the stress response can greatly improve the genetic repair of cells. ability. Because the invention is easy to handle, it can be used as a new method for mobilizing somatic cells in mammals to participate in cell renewal, inhibiting tissue fibrosis, and improving tissue repair and organ regeneration in the process of pathological damage. It can also be used as a new method to improve the homologous recombination ability of the cell background and the homologous recombination ability mediated by double-strand breaks.

Contents of the invention

The present invention is based on the following findings of the inventors: Myosin inhibitors can destroy the homeostasis of the cellular mechanical stress system, stimulate stress responses and regenerative responses similar to those in the regeneration process of lower organisms, and use stress responses to greatly improve cell Genetic repair ability, and then completed the present invention.

Thus, in one embodiment, the present invention relates to the use of Myosin inhibitors for eliciting a stress response.

In one embodiment, the present invention relates to the use of Myosin inhibitors to elicit a stress response by disrupting the homeostasis of cellular mechanical stress systems.

In one embodiment, the present invention also relates to the use of Myosin inhibitors to elicit regenerative responses.

In one embodiment, the present invention relates to the use of Myosin inhibitors to stimulate regenerative responses by disrupting the homeostasis of cellular mechanical stress systems.

In one embodiment, the present invention also relates to the application of Myosin inhibitors in improving the genetic repair ability of cells.

In one embodiment, the present invention relates to the use of Myosin inhibitors in enhancing the genetic repair capacity of cells by disrupting the homeostasis of cellular mechanical stress systems.

In one embodiment, the stimulating stress leads to the activation of a high genetic damage repair response, thereby increasing the up-regulation of the body homologous recombination repair gene and improving the recombination ability.

In one embodiment, the present invention also relates to the application of Myosin inhibitors in promoting the regeneration and repair of tissues and organs.

In one embodiment, the present invention relates to the use of Myosin inhibitors in promoting the regenerative repair of tissues and organs by disrupting the homeostasis of the cellular mechanical stress system.

In one embodiment, the present invention also relates to the application of Myosin inhibitors in the preparation of drugs or reagents for stimulating stress response, stimulating regeneration response, improving cell genetic repair ability and/or promoting regeneration and repair of tissues and organs.

In one embodiment, the present invention also relates to the preparation of Myosin inhibitors for stimulating stress response, stimulating regeneration response, improving cell genetic repair ability and/or promoting regenerative repair of tissues and organs by destroying the homeostasis of cell mechanical stress system. Application in medicine or reagent.

In one embodiment, the present invention also relates to the application of Myosin inhibitors in the preparation of drugs or reagents for treating diseases related to stress response, regenerative response, cell genetic repair ability and/or regenerative repair of tissues and organs.

In one embodiment, the disease is scleroderma.

In one embodiment, the Myosin inhibitor is (-)-Blebbistatin.

In one embodiment, the organ is the liver.

The method of the present invention only needs single small molecule or single factor treatment, simple operation and good repeatability, and can be used as a new method for stimulating stress response, stimulating regenerative response, improving cell genetic repair ability and/or promoting regeneration and repair of tissues and organs method.

The terms used in this document have the following meanings:

High-sugar DMEM: a high-sugar DMEM medium (dulbecco's modified eagle medium, DMEM), a commercial medium containing various glucose and amino acids, developed on the basis of MEM medium.

N2B27: A 1:1 mixture of DMEM/F12 basal medium and neurobasal basal medium, a well-defined cell culture medium containing N2 supplement and B27 supplement, which is reported to be beneficial to the differentiation of mouse embryonic stem cells towards nerves.

DMEM/F12: A commercial basal culture medium mixed with 1:1 of DMEM medium and F12 medium, which is suitable for the cultivation of colony density.

Neurobasal: A commercially available basal medium that facilitates the culture of neuronal cells.

GlutaMAX: A cell culture supplement that directly replaces L-glutamine in cell culture media.

Double Antibody: Penicillin and streptomycin are two antibiotics commonly used in cell culture to prevent bacterial contamination during cell culture.

N2 Supplement: A commercial serum-free cell culture supplement.

B27 Supplement: A commercial serum-free cell culture supplement.

Description of drawings

Figure 1 shows a diagram of Myosin inhibitors disrupting cellular mechanical system homeostasis and inducing cellular softening.

Figure 2A shows that the DMSO-treated mice in the bile duct ligation liver fibrosis model control group were lethargic and their activity was weakened. Compared with the Myosin inhibitor (-)-Blebbistatin significantly inhibited treatment group, the mice were more active and full of energy.

Figure 2B shows that the Myosin inhibitor (-)-Blebbistatin significantly inhibited the treatment group to have better fibrous macula compared to the control group.

Figure 2C shows that the Myosin inhibitor (-)-Blebbistatin significantly inhibits the treatment group and improves the survival rate of fibrotic mice.

Figure 2D shows that the Myosin inhibitor (-)-Blebbistatin significantly inhibits fibrotic deposits.

Figure 2E shows the quantification results of statistical fibrosis degree of at least 5 mice in each group.

Fig. 3A shows the data analysis results of transcriptome sequencing of (-)-Blebbistatin-treated human fibroblast samples.

Figure 3B and Figure 3C show that the mRNA expression of these genes started to be down-regulated at day 1 and day 2. After 22 days of induction in the neural induction system, regeneration initiation-related genes such as FST and neurogenesis-related genes were significantly up-regulated.

Figure 4A shows the experimental flow chart.

Figures 4B-I show the effect of (-)-Blebbistatin against experiments in the treatment of scleroderma.

Figure 5 shows the experimental procedure and the graph that (-)-Blebbistatin significantly inhibits fibrosis.

Figure 6 shows a graph showing that disruption of the homeostasis of the cellular machinery system induces regeneration-related responses to promote regeneration and repair of liver tissue.

Figure 7A shows 5-50 micromolar (-)-Blebbistatin treated human fibroblasts, 6-hour transcriptome analysis, DNA replication-related, homologous recombination-related genes, mismatch repair-related genes, nucleotide excision repair and base Excision repair genes were significantly upregulated.

Figure 7B shows that further analysis found that almost all important genes involved in homologous recombination were significantly up-regulated.

Figure 8 shows a graph showing that a high genetic damage repair event accompanied by an elicited stress response should increase the background homologous recombination gene repair process.

Detailed ways

The specific examples are set forth below and illustrate the implementation of the present invention in detail, but the following content should not be construed as limiting the present invention in any way.

Example 1: Myosin inhibitors disrupt cell mechanical system homeostasis and induce cell softening

Figure 1A is a schematic diagram of laser confocal scanning, Figure 1B shows DMSO (control), (-)-Blebbistatin (also referred to as Blebbistatin or Bleb or Ble) (20 micromolar) treated human fibroblasts, phalloidin exchange staining , cells in the control group (upper row) have abundant stress fibers arranged in parallel, and the edges of the nuclei are smooth and round or oval. In the experimental group, after Ble treatment, the stress fibers disintegrate, and the nuclei appear wrinkled and irregular. Figure 1C shows that lamin A/C has a strong uniform distribution at the DNA periphery, and there is a clear polarity in the basal-tip direction, with lamin A/C being significantly higher in the apical portion than in the basal portion. Heterochromatin HP1 protein showed that the control group was significantly stronger than the experimental group. The results in Figure 1D show that the nuclear localization signal of the stress effect protein YAP1/TAZ in more than 90% of the cells in the experimental group was significantly stronger than the cytoplasmic localization, while only 35% of the cells treated with drugs in the experimental group had a more obvious and localized signal. Figure 1E Quantitative PCR determined that the proteins related to the regulation of cell mechanical stiffness were significantly down-regulated after small molecule treatment. Using fluorouridine-labeled ribosomes to transcribe RNA, Figure 1F shows that the transcriptional active area of the control group is mainly concentrated near the center of the nucleus, while the experimental group shows that fluorouridine-labeled active transcription areas are also distributed near the periphery of the nucleus, Three biological replicates. The effect of immunofluorescence staining on epigenetics of Ble, as shown in Figure 1G, shows that Ble significantly reduced the modification of H3K27 trimethylation on day 4, further verified the modification complex component EZH2 of H3K27 trimethylation, and found that EZH2 in the control group had obvious nuclei Localization, Ble treatment EZH2 protein level was down-regulated and showed cytoplasmic localization, as shown in Figure 1H.

Example 2: Disruption of the homeostasis of the cell mechanical system improves the survival rate of fibrotic mice

The ICR mice used in the experiment were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd., the curved tweezers, finger tweezers, scissors, needle holders, suture needles, and sutures were purchased from Azowon Trading Co., Ltd., and the antibiotics were purchased from Gibco ( 15240-062).

8-week-old female mice were selected, fasted for 24 hours before the experiment, anesthetized the mice with 5% chloral hydrate, injected intraperitoneally at 8 ml/kg, fixed the mice after anesthesia, made an incision in the abdomen, exposed the organs, and found the closest For the duodenum at the stomach end, gently pull the duodenum to find the bile duct, carefully peel off the bile duct with forceps, ligate the bile duct with suture, drip antibiotics, and then suture to close the abdominal cavity. Fasting for 12 hours after surgery.

14-20 days after the operation, it can be observed that the skin of the mouse is yellow-green, and the drug treatment is performed. Adopt implanted capsule osmotic pressure pump, 0.25 microliters/hour, sustained release for two weeks (ALZEN, 1002), drug concentration. The mice were anesthetized with 5% chloral hydrate, injected intraperitoneally at 8 ml/kg, fixed the mice after anesthesia, made an incision in the abdomen to expose the organs, carefully pushed away the liver and intestines, implanted the osmotic pump into the abdominal cavity, and added antibiotics dropwise , after closing the abdominal cavity with sutures.

Results Figure 2A shows that the DMSO-treated mice in the bile duct ligation liver fibrosis model control group were lethargic and their activity was weakened. Compared with the Myosin inhibitor (-)-Blebbistatin significantly inhibited the treated mice, they were more active and full of energy. Figure 2B shows that the Myosin inhibitor (-)-Blebbistatin significantly inhibited the treatment group to have better fibrous macula compared with the control group, and the Myosin inhibitor (-)-Blebbistatin significantly inhibited the treatment group to improve the survival of fibrotic mice rate, as shown in Figure 2C, the results are from 5 mice. The results of Sirius red staining showed that Myosin inhibitor (-)-Blebbistatin significantly inhibited the accumulation of fibers caused by tube ligation, as shown in Figure 2D-E.

Example 3: Stresses related to regeneration induced by disruption of cell mechanical system homeostasis and responses related to regeneration initiation and tissue generation

(-)-Blebbistatin treated human fibroblasts, and samples were collected for 0 hour, 6 hours, 1 day, and 2 days for transcriptome sequencing. The data analysis is shown in Figure 3A. A large number of HSP70 families and HSP90 Genes involved in the general stress response are upregulated. On day 1 and day 2 the mRNA expression of these genes started to be down-regulated. After 22 days of induction in the neural induction system, regeneration initiation-related genes such as FST and neurogenesis-related genes were significantly up-regulated, as shown in Figure 3B and Figure 3C.

Example 4: The application of regeneration-related responses induced by disruption of the homeostasis of the cell mechanical system in the treatment of scleroderma

Scleroderma is a connective tissue disorder characterized by localized or diffuse fibrosis of the skin and internal organs, followed by sclerosis and atrophy. The disease can cause multi-system damage, and its exact etiology and pathogenesis are still unclear, and there is no effective treatment. In the present invention, local injection of bleomycin (BLM) on the back of ICR mice successfully induces skin sclerosis in mice, and intraperitoneal administration of Myosin inhibitor (-)-Blebbistatin for treatment. The experimental process is shown in Figure 4A.

One week after the injection of bleomycin, the skin at the injection site on the back of the mice appeared thickened and hardened, with poor elasticity and no change in hair growth. Table ulcers. In the normal saline control group, the hair in the shaved injection area on the back of the mice continued to grow, and no obvious skin hardening or thickening was seen, as shown in Figure 4B. Compared with the control group administered DMSO intraperitoneally, the Myosin inhibitor (-)-Blebbistatin (1-3 mg/kg/day, dissolved in 2% DMSO+25% PEG400+2% Tween 80+ddH ₂ O)3 was administered intraperitoneally Zhou can significantly reduce the degree of induration and scab, accompanied by a smaller degree of superficial ulcer, as shown in Figure 4B. Further experiments verified that Myosin inhibitor (-)-Blebbistatin promotes hair growth in the induration area as shown in Figure 4C. Significantly reduced epidermal and dermal thickness, and promoted the number of hair follicles and glands, as shown in Figure 4D-F. Fiber staining results showed that (-)-Blebbistatin significantly reduced the accumulation of interstitial fibers, as indicated by the arrow in Figure 4G. The results of Ki-67 staining showed that (-)-Blebbistatin promoted the proliferation of hair follicle cells, thereby promoting the formation of hair follicles as shown in Figure 4H. Surprisingly, (-)-Blebbistatin can promote the emergence of neural fate in the induration area, and some cells express nuclear-localized mature neuron Marker NeuN as shown in Figure 4I, suggesting whether the induction of neural fate contributes to damage repair and tissue Regeneration awaits further study.

Example 5: Application of regeneration-related responses induced by homeostasis disruption of cell mechanical system in promoting liver tissue regeneration and repair

Induction of CCl ₄ liver injury model

The ICR mice used in the experiment were purchased from Speiford (Beijing) Biotechnology Co., Ltd., carbon tetrachloride was purchased from Aladdin Reagent (Shanghai) Co., Ltd. (C131583-1L), and corn oil was purchased from Sigma (C8267). The experimental procedure is shown in Figure 5A. Eight-week-old female mice were selected and raised in the barrier for one week. They were randomly divided into two groups and injected with CCl ₄ /corn oil (preparation volume 2:5) and corn oil at a dose of 2 ml/kg. , twice a week for 8 consecutive weeks. After 8 weeks, mice were anesthetized with 5% chloral hydrate, part of liver pages were taken out, fixed with 4% paraformaldehyde, dehydrated and soaked in wax, and stained for fibrosis using Sirius red (YY-R20384-100ML) kit, in which collagen fibers Red, as shown in Figure 5B, C. After the identification of successful modeling, drug treatment was carried out. Intraperitoneal injection and tail vein injection were used. Dissolve small molecules in dimethyl sulfoxide, dilute with normal saline, the concentration of small molecules is 1 mg/kg per day, or add sequentially (>1 mg/kg, sequentially add 5% DMSO+30% propylene glycol+3% Tween80+ddH ₂ O or 2% DMSO+25% PEG400+2% Tween 80+ddH ₂ O), 1 ml syringe to draw the drug, puncture the skin and abdominal wall muscle with a hypodermic syringe, and inject the liquid into the abdominal cavity, taking care not to injure the diaphragm and For other organs, stay for a while before pulling out the needle to avoid fluid leakage. When the tail vein was injected, the mouse was fixed in the holder, the tail was straightened and taut, and the bleeding was stopped with cotton after the injection. Continuous injection for 7 days, stop the injection. Sirius red staining was carried out again to identify the results, as shown in Figure 5D and E, the Myosin inhibitor (-)-Blebbistatin significantly inhibited fibrosis.

Liver function regeneration and repair identification

Further use Alb-cre×mTmG hybrid mice, the mature liver cells express GFP, and CCl4 is used for fibrosis modeling. After drug treatment, most of the cells expressing GFP express Alb, while DMSO—a part of the cells expressing GFP do not express Alb , indicating that the Myosin inhibitor (-)-Blebbistatin can prevent the loss of liver function during liver injury, as shown in Figure 6A. Blood biochemical analysis (fasting for 12-16 hours before sampling) further confirmed that the Myosin inhibitor (-)-Blebbistatin can reduce the levels of liver damage indicators such as ALT, AST and GGT, as shown in Figure 6B. Further staining and identification found that (-)-Blebbistatin significantly promoted cell proliferation after liver injury, as shown in Figure 6C-D, and reduced cell apoptosis during liver injury, as shown in Figure 6E-F. In conclusion, the Myosin inhibitor (-)-Blebbistatin promotes liver regeneration by inhibiting liver injury fibrosis, promoting liver cell proliferation and reducing apoptosis, thereby maintaining liver function.

Embodiment 6: (-)-Blebbistatin induces a high genetic damage repair response activated by stress, and improves the gene upregulation of body homologous recombination repair

As shown in Figure 7A, human fibroblasts were treated with 5-50 micromolar (-)-Blebbistatin, and the transcriptome was analyzed for 6 hours, DNA replication-related, homologous recombination-related genes, mismatch repair-related genes, nucleotide excision repair and base excision repair genes were significantly upregulated. As shown in Figure 7B, further analysis found that almost all important genes involved in homologous recombination were significantly up-regulated.

Example 7: (-)-Blebbistatin stimulates high genetic damage repair response activated by stress response, and its application in improving the gene process of body homologous recombination repair

The experimental process is shown in Figure 8A. First, a reporter plasmid system for homologous recombination repair was constructed. Only when homologous recombination occurs between exogenous homologous sequences and cell homologous fragments, the cells emit green fluorescence. At the same time, mRuby red fluorescent protein was transferred to mark the efficiency of transfected cells, and the ratio of green fluorescent protein to mRuby was analyzed by flow cytometry to determine the efficiency of homologous recombination. The control group was treated with DMSO for 4 hours 18 hours after the transfection, and the experimental group was treated with (-)-Blebbistatin for 4 hours at 18 hours, 24 hours, and 36 hours after the transfection. The HDR efficiency was analyzed by flow cytometry 72 hours after the transfection. The results showed that (-)-Blebbistatin significantly improved the background homologous recombination efficiency, as shown in 8B-C. Since Nrf2 is a significantly up-regulated gene in oxidative stress response and is an effector protein of oxidative stress, it is further verified that stress response can improve homologous recombination ability by simulating oxidative stress with a small molecule Nrf activator, as shown in Figure 8D-E. The experimental procedure is as follows, the control group was treated with DMSO after the transfection medium change, the experimental group was treated with 250nM RTA408 after the transfection medium change, and the HDR efficiency was analyzed by flow cytometry 72 hours after transfection.

The foregoing merely illustrates the principles of the invention, and it should be understood that the scope of the invention is not intended to be limited to the exemplary aspects described herein, but rather includes all currently known and future developed equivalents. In addition, it should be pointed out that several improvements and modifications can be made without departing from the technical principle of the present invention, and these improvements and modifications should also be regarded as the scope of the present invention.

Claims (12)

1.Myosin inhibitor is in excitation stress reaction, excitation regenerative response and/or improves answering in cytogenetics repair ability With.

2.Myosin inhibitor is in preparation for exciting stress reaction, excitation regenerative response and/or improving cytogenetics reparation energy The drug of power or the application in reagent.

3. such as application of any of claims 1-2, which is characterized in that the excitation stress reaction, excitation regeneration are anti- Answering and/or improve cytogenetics repair ability is realized by destroying cell machinery stress system stable state.

4. application as claimed in any one of claims 1-3, which is characterized in that the excitation stress send out should cause to activate it is high Genetic damage reparation hair is answered, and then improves the up-regulation of ontology homologous recombination repair gene, improves recombination ability.

Application of the 5.Myosin inhibitor in the Regeneration and Repair for promoting histoorgan.

6.Myosin inhibitor is preparing the application in drug or reagent for promoting the Regeneration and Repair of histoorgan.

7. the application as described in any one of claim 5-6, which is characterized in that it is described promote histoorgan Regeneration and Repair be It is realized by destroying cell machinery stress system stable state.

8.Myosin inhibitor is related to stress reaction, regenerative response and/or cytogenetics repair ability for treating in preparation Disease drug or the application in reagent.

9.Myosin inhibitor is in the drug or reagent that preparation is used to treat disease relevant to the Regeneration and Repair of histoorgan Application.

10. the application as described in any one of claim 8-9, which is characterized in that the disease is chorionitis.

11. such as application of any of claims 1-10, which is characterized in that the Myosin inhibitor is (-)- Blebbistatin。

12. the application as described in any one of claim 5-7,9-11, which is characterized in that the organ is liver.