CN111534483B - 一种胰岛素样生长因子结合蛋白7激活剂在人脐带间充质干细胞成软骨分化中的应用 - Google Patents
一种胰岛素样生长因子结合蛋白7激活剂在人脐带间充质干细胞成软骨分化中的应用 Download PDFInfo
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Abstract
本发明公开了一种胰岛素样生长因子结合蛋白7激活剂在人脐带间充质干细胞成软骨分化中的应用。蛋白聚糖和Ⅱ型胶原蛋白为软骨特异性基质产物，对照诱导组hUC‑MSCs在基础诱导培养基的诱导下出现了明显的成软骨分化，异茜草素诱导组比对照诱导组中蛋白聚糖和Ⅱ型胶原蛋白含量更高，说明异茜草素诱导组中的异茜草素可以进一步促进hUC‑MSCs成软骨分化，且具有剂量效应。异茜草素可能是通过激活IGFBP7发挥作用，异茜草素为有效的IGFBP7激活剂。可见，胰岛素样生长因子结合蛋白7激活剂异茜草素具有在体外促进脐带间充质干细胞成软骨分化的作用，可用于制备促进脐带间充质干细胞成软骨分化的培养基。
Description
技术领域
本发明属于干细胞领域，涉及脐带间充质干细胞诱导分化，具体涉及一种胰岛素样生长因子结合蛋白7激活剂在人脐带间充质干细胞成软骨分化中的应用。
背景技术
软骨组织工程是治疗软骨损伤疾病的重要生物工程手段。在软骨组织工程中，种子细胞的选择主要考虑以下几点：细胞易获取并且来源充足，对取材对象损伤小；较低的免疫排斥；具有诱导分化潜力并且增殖分化能力较强等；可以通过体外扩增技术获取大量传代细胞。
软骨修复和再生以往集中在软骨细胞方面，尽管这些细胞取材方便和免疫原型较低，但其获取细胞量很少并且具有去分化等缺点，这限制了软骨修复再生的发展。在诱导具有分化潜力的细胞向软骨向分化的研究中，各种来源的细胞被报道，其中间充质干细胞具有不可替代的成软骨诱导分化能力被广泛关注。虽然人骨髓间充质干细胞等作为干细胞源是软骨组织工程的主要对象，但受年龄限制的骨髓移植过程不可避免地伴随着疼痛和损伤，而且骨髓基质干细胞的利用率很低，增殖分化能力随年龄的增长而下降。相较于其它干细胞，脐带间充质干细胞具有低免疫原型，同时具备成软骨潜力，可以作为软骨组织工程的细胞来源之一。
人脐带间充质干细胞(hUC-MSCs)是一类具有多向分化能力和自我更新能力的间充质干细胞。因脐带具有取材容易，不容易被污染，不涉及道德、伦理和法律方面的问题等诸多优点，被广泛用于hUC-MSCs的提取。而且，研究发现hUC-MSCs的体外扩增和多向分化能力比其他组织来源的间充质干细胞更强。
冯学涛等发现，血小板裂解液在体外具有定向诱导人脐带间充质干细胞分化成软骨细胞中的作用(血小板裂解液对人脐带间充质干细胞体外成软骨分化的影响，中国修复重建外科杂志，2011年10期)。
罗二梅等发现，还原型谷胱甘肽对人脐带间充质干细胞的成软骨诱导具有促进作用(还原型谷胱甘肽对人脐带间充质干细胞成软骨诱导的影响，中国生物工程杂志，2013年03期)
胰岛素样生长因子结合蛋白7(IGFBP7)是一种分泌型糖蛋白，在机体内广泛表达，可以与胰岛素样生长因子结合，正向或负向调节IGF信号通路，也可独立发挥功能，参与调控细胞的生长分化、增殖和凋亡以及组织重塑等重要生理过程。研究发现，IGFBP7具有促进人脐带间充质干细胞的软骨分化的作用(IGFBP7促进人脐带间充质干细胞的软骨分化，南京医科大学学报自然科学版，2019年8月第39卷第8期)。
发明内容
本发明是为了克服现有技术中存在的不足，提供一种胰岛素样生长因子结合蛋白7激活剂在人脐带间充质干细胞成软骨分化中的应用。
本发明技术方案如下：
一种胰岛素样生长因子结合蛋白7激活剂在体外促进脐带间充质干细胞成软骨分化中的应用，所述胰岛素样生长因子结合蛋白7激活剂为异茜草素。
一种胰岛素样生长因子结合蛋白7激活剂用于制备促进脐带间充质干细胞成软骨分化培养基的用途，所述胰岛素样生长因子结合蛋白7激活剂为异茜草素。
一种用脐带间充质干细胞制备软骨细胞的培养基，含有一种胰岛素样生长因子结合蛋白 7激活剂，所述胰岛素样生长因子结合蛋白7激活剂为异茜草素。
有益技术效果：
本发明发现，一种胰岛素样生长因子结合蛋白7激活剂异茜草素具有在体外促进脐带间充质干细胞成软骨分化的作用，可以用于制备促进脐带间充质干细胞成软骨分化的培养基。
附图说明
图1为hUC-MSCs的表型流式鉴定图；
图2为各组蛋白聚糖的含量比较；
图3为各组Ⅱ型胶原蛋白的含量比较；
图4为胰岛素样生长因子结合蛋白7(IGFBP7)表达量的Western blot图。
具体实施方式
下面通过具体的实施例详细介绍本发明实质性内容，但不能以此限定本发明的保护范围。
一、试验材料
DMEM/F12培养基和胎牛血清购自Gibco公司。
谷胺酰胺和双抗购自南京森贝伽生物科技有限公司。
PBS缓冲液按照配方配制后于4℃保存，24h内使用完。
Ⅳ型胶原酶和胰酶购自上海懋康生物科技有限公司，按照说明书使用。
异茜草素纯度不低于98％。
二、试验方法
1、hUC-MSCs的提取培养和鉴定
使用的hUC-MSCs与专利2020104333269、2020104333714中相同，制备和鉴定方法为：
取正常足月剖腹产新生儿脐带约12cm(脐带采集后4℃保存于含1％双抗的PBS缓冲液中，6h内提取干细胞培养)，用含1％双抗的PBS缓冲液冲洗去除脐动静脉及脐带外膜，剪成约1mm3大小的组织块，置于37℃恒温震荡仪内，先后加入Ⅳ型胶原酶、胰酶分别消化60min 和30min提取细胞，再用含20％FBS、25mmol/L谷胺酰胺和1％双抗的DMEM/F12培养基重悬细胞，以1.0×106/mL接种于细胞培养瓶内，培养4d后更换培养基，之后2～3d换液1次，待细胞80％融合时传代。取第5代细胞进行实验。
取第5代hUC-MSCs，胰酶消化，充分吹打均匀，制备成单细胞悬液，加入CD34-PE、CD45-PE、CD73-PE、CD90-PE和CD105-FITC。室温避光孵育30min，多聚甲醛固定后，流式细胞仪进行检测。
2、hUC-MSCs向软骨细胞的诱导分化
2.1分组
低浓度异茜草素诱导组(A组)：使用含终浓度为6.25mg/L胰岛素、6.25mg/L转铁蛋白、 10μg/L转化生长因子β1、0.1μmol/L地塞米松、50mg/L维生素C、5μM异茜草素(DMSO助溶)、5％FBS和1％双抗的DMEM/F12培养基诱导培养；
高浓度异茜草素诱导组(B组)：使用含终浓度为6.25mg/L胰岛素、6.25mg/L转铁蛋白、 10μg/L转化生长因子β1、0.1μmol/L地塞米松、50mg/L维生素C、10μM异茜草素(DMSO 助溶)、5％FBS和1％双抗的DMEM/F12培养基诱导培养；
对照诱导组(C组)：使用含终浓度为6.25mg/L胰岛素、6.25mg/L转铁蛋白、10μg/L转化生长因子β1、0.1μmol/L地塞米松、50mg/L维生素C、5％FBS和1％双抗的DMEM/F12 培养基(基础诱导培养基)诱导培养，培养基中添加与A、B组等体积的DMSO溶媒。
2.2诱导培养
取第5代生长良好的细胞hUC-MSCs，胰酶消化，充分吹打均匀，制备成单细胞悬液，以2×105/孔接种于6孔板中，待细胞85％融合时，按照上述分组更换培养基，诱导培养18d，每3d更换一次对应的培养基。每组设3个复孔。
3、蛋白聚糖的含量测定
诱导18d后，取各组细胞上清200μL，加入40μL木瓜蛋白酶消化24h；加入100μL8mol/L 盐酸胍和750μL 0.25％阿利新蓝溶液，4℃反应1h；12000r/min离心15min，弃上清，加入150μL 异丙醇溶解沉淀，600nm波长处测定吸光度值。以一系列浓度梯度的硫酸软骨素标准液制作标准曲线，将吸光度值代入标准曲线求得蛋白聚糖含量。
4、Ⅱ型胶原蛋白的含量测定
诱导18d后，取各组细胞上清250μL，加入羟脯氨酸消化液50μL，加入羟脯氨酸反应液 2mL，60℃水浴15min，冷却后3500r/min离心10min，取150μL上清，560nm波长处测定吸光度值。以一系列浓度梯度的羟脯氨酸标准液制作标准曲线，将吸光度值代入标准曲线求得Ⅱ型胶原蛋白含量。
5、Western blot测定IGFBP7表达量
诱导18d后，收集各组细胞，PBS洗涤3遍以去除细胞表面的培养基及培养基中的成分，裂解液裂解，使用BCA试剂盒测定裂解液中的蛋白浓度，取50μg蛋白进行SDS-PAGE电泳，并转移至PVDF膜上，经5％脱脂奶粉封闭，分别加入IGFBP7、GAPDH(内参)一抗4℃孵育过夜，次日用TBS-T洗涤液洗膜，加入二抗室温孵育1h，ECL法显色、曝光，化学发光成像系统拍照，用图像分析软件Image J分析灰度。
6、数据分析
在SPSS 17.0中，数据以均值±SD表示，并进行t检验，P＜0.05说明差异有统计意义。
三、试验结果
1、hUC-MSCs的提取培养和鉴定结果
hUC-MSCs的表型鉴定流式结果如表1和图1所示，CD34-PE和CD45-PE阴性表达，CD73-PE、CD90-PE和CD105-FITC阳性表达，符合hUC-MSCs的表型特征。
表1 hUC-MSCs的表型鉴定结果
| 表达率 | |
| CD34-PE | 0.35％ |
| CD45-PE | 0.47％ |
| CD73-PE | 95.2％ |
| CD90-PE | 96.8％ |
| CD105-FITC | 98.5％ |
2、蛋白聚糖的含量测定结果
各组蛋白聚糖的含量如表2和图2所示，与对照诱导组(C组)相比，低浓度异茜草素诱导组(A组)和高浓度异茜草素诱导组(B组)蛋白聚糖的含量显著升高，且存在明显的剂量依赖性，B组高于A组。
表2各组蛋白聚糖的含量
| 组别 | 含量(μg/mL) |
| 对照诱导组(C组) | 20.9±3.1 |
| 低浓度异茜草素诱导组(A组) | 35.2±3.4 |
| 高浓度异茜草素诱导组(B组) | 58.5±3.2 |
3、Ⅱ型胶原蛋白的含量测定
各组Ⅱ型胶原蛋白的含量如表3和图3所示，与对照诱导组(C组)相比，低浓度异茜草素诱导组(A组)和高浓度异茜草素诱导组(B组)Ⅱ型胶原蛋白的含量显著升高，且存在明显的剂量依赖性，B组高于A组。
表3各组Ⅱ型胶原蛋白的含量
| 组别 | 含量(μg/mL) |
| 对照诱导组(C组) | 197.5±18.2 |
| 低浓度异茜草素诱导组(A组) | 320.6±21.3 |
| 高浓度异茜草素诱导组(B组) | 572.8±23.5 |
4、IGFBP7表达量测定结果
Western blot结果如图4所示，与对照诱导组(C组)相比，低浓度异茜草素诱导组(A 组)和高浓度异茜草素诱导组(B组)IGFBP7蛋白的含量显著升高，且存在明显的剂量依赖性，B组高于A组。
本领域技术人员知道，蛋白聚糖和Ⅱ型胶原蛋白为软骨特异性基质产物，对照诱导组hUC-MSCs在基础诱导培养基的诱导下出现了明显的成软骨分化，异茜草素诱导组比对照诱导组中蛋白聚糖和Ⅱ型胶原蛋白含量更高，说明异茜草素诱导组中的异茜草素可以进一步促进 hUC-MSCs成软骨分化，且具有剂量效应。异茜草素可能是通过激活IGFBP7发挥作用，异茜草素为有效的IGFBP7激活剂。由此可见，胰岛素样生长因子结合蛋白7激活剂异茜草素具有在体外促进脐带间充质干细胞成软骨分化的作用，可以用于制备促进脐带间充质干细胞成软骨分化的培养基。
Claims (3)
1.一种胰岛素样生长因子结合蛋白7激活剂在体外促进脐带间充质干细胞成软骨分化中的应用，所述胰岛素样生长因子结合蛋白7激活剂为异茜草素。
2.一种胰岛素样生长因子结合蛋白7激活剂用于制备促进脐带间充质干细胞成软骨分化培养基的用途，所述胰岛素样生长因子结合蛋白7激活剂为异茜草素。
3.一种用脐带间充质干细胞制备软骨细胞的培养基，含有一种胰岛素样生长因子结合蛋白7激活剂，其特征在于：所述胰岛素样生长因子结合蛋白7激活剂为异茜草素。
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