人发角蛋白诱导人脐带间充质干细胞向雪旺细胞分化的实验研究

发布时间：2018-01-19 22:39

本文关键词： 脐带间充质干细胞角蛋白雪旺细胞诱导分化　出处：《暨南大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的：研究人脐带间充质干细胞（hUC-MSCs）的基本生物学特性。摸索人发角蛋白（HHK）萃取的最佳条件，同时观察其与hUC-MSCs的生物相容性。采用不同的方法诱导hUC-MSCs向雪旺细胞(SCs)分化，探讨HHK在其中的作用，并寻找最优的诱导分化方案，为hUC-MSCs用于周围神经和脊髓损伤修复提供理论依据。方法：用酶消法分离hUC-MSCs，通过普通光学显微镜观察其生长状态，原子力显微镜观察其形态学特征，采用MTT法观察P3代生长曲线及P1-P8的细胞增殖活力，流式细胞术测定P1-P6的细胞生长周期和鉴定细胞表型。还原法提取HHK，观察其对hUC-MSCs的增殖及趋化作用。用不同浓度的HHK溶液包被培养瓶，观察其对hUC-MSCs贴壁和粘附的影响。随机将hUC-MSCs分为A、B、C、D、E、F共6组，分别采用复合诱导因子+HHK、复合诱导因子、共培养+HHK、共培养、HHK、普通培养基培养，9天后采用HE染色和原子力显微镜观察其观察其形态学及超微结构变化。免疫细胞荧光观察分化后细胞S-100和GFAP特异性蛋白表达，，流式细胞仪定量检测分化后S-100蛋白表达阳性率及细胞凋亡率。结果：采用酶消法成功分离得到了纯度较高的hUC-MSCs，P1-P6代细胞周期及P1-P8代细胞的生长活力无明显差异。流式细胞术鉴定细胞表达CD105、CD90、CD73、CD54、CD44、CD13、CD29，不表达CD34、CD14、CD45、HLA-DR。采用还原法成功萃取了HHK，适当浓度的HHK有提高细胞增殖活力、促进细胞迁移、加速细胞贴壁和增加粘附力的作用。经SCs定向诱导分化后，细胞逐渐变成长梭形，两极有细长突起，原子力观察细胞核表面突起增多。细胞免疫荧光法观测到诱导后hUC-MSCs表达S-100蛋白和GFAP蛋白明显增高，流式细胞仪定量检测到hUC-MSCs表达S-100蛋白增高、细胞凋亡率降低。6组中前四组发生不同程度的诱导分化，后两组未见细胞分化，其中分化效率以细胞因子组高于共培养组，加HHK组高于未加HHK组；细胞凋亡率以共培养组低于细胞因子组，加HHK组低于未加HHK组。分化效率与细胞凋亡率成正相关，但HHK可以降低细胞凋亡率提高诱导分化率。结论：本实验成功在体外分离、培养及鉴定了hUC-MSCs。采用还原法萃取的HHK具有较好的生物相容性，可提高hUC-MSCs增殖活力、促进细胞迁移和粘附。HHK联合复合因子为最佳诱导方案，可以显著提高hUC-MSCs向SCs分化效率并降低细胞凋亡率。
[Abstract]:Objective: to study the basic biological characteristics of human umbilical cord mesenchymal stem cells (hUC-MSCs) and to explore the optimum conditions for extraction of human hair keratin (HHK). At the same time, the biocompatibility of hUC-MSCs with hUC-MSCs was observed. Different methods were used to induce the differentiation of hUC-MSCs into Schwann cells, and to explore the role of HHK in the differentiation. The optimal differentiation induction scheme was found to provide theoretical basis for the application of hUC-MSCs in the repair of peripheral nerve and spinal cord injury. Methods: the growth state of hUC-MSCs was observed by ordinary optical microscope and morphological characteristics were observed by atomic force microscope (AFM). The growth curve of P3 generation and the proliferative activity of P1-P8 were observed by MTT. The cell cycle and phenotype of P1-P6 were determined by flow cytometry. HHK was extracted by reduction method. The proliferation and chemotaxis of hUC-MSCs were observed. The culture bottle was coated with different concentrations of HHK solution. To observe the effect of hUC-MSCs on adhesion and adhesion of hUC-MSCs, hUC-MSCs was randomly divided into 6 groups, which were treated with HHK. Compound induction factor, co-culture HHK, co-culture HHK, common culture medium. After 9 days, the morphological and ultrastructural changes were observed by HE staining and atomic force microscopy, and the expression of S-100 and GFAP specific proteins were observed by immunofluorescence. The positive rate of S-100 protein expression and apoptosis rate after differentiation were quantitatively detected by flow cytometry. Results: high purity hUC-MSCs was obtained by enzyme digestion. There was no significant difference in cell cycle of P1-P6 passage and growth activity of P1-P8 passage cells. Flow cytometry was used to identify the expression of CD105, CD90, CD73, CD54 and CD44. CD13, CD29, CD34, CD14, CD45, HLA-DR.HHK was successfully extracted by reductive method, and the appropriate concentration of HHK could improve the proliferation of HHKcells. Promote cell migration, accelerate cell adhesion and increase adhesion. After SCs induced differentiation, the cells gradually became fusiform, with slender processes at the two poles. The expression of S-100 protein and GFAP protein in hUC-MSCs was significantly increased after induction by atomic force. Flow cytometry showed that the expression of S-100 protein in hUC-MSCs was increased, and the apoptosis rate was decreased in the first four groups, but no differentiation was found in the latter two groups. The differentiation efficiency of cytokine group was higher than that of co-culture group, and that of HHK group was higher than that of no HHK group. The rate of apoptosis in co-culture group was lower than that in co-culture group, and that in HHK group was lower than that in untreated HHK group. The differentiation efficiency was positively correlated with the apoptosis rate, but HHK could decrease the apoptosis rate and increase the induced differentiation rate. Conclusion: HHK extracted by reductive method has good biocompatibility and can improve the proliferative activity of hUC-MSCs. Promoting cell migration and adhesion of HHK combined with compound factor could significantly increase the differentiation efficiency of hUC-MSCs into SCs and decrease the rate of apoptosis.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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