骨髓间充质干细胞和肌腱干细胞构建组织工程化肌腱的对比研究

发布时间：2018-03-20 09:34

本文选题：骨髓间充质干细胞　切入点：肌腱干细胞　出处：《中国人民解放军医学院》2017年硕士论文　论文类型：学位论文

【摘要】：背景:肌腱组织由于其独特的解剖和组织结构,因此缺乏自身修复能力,愈合后损伤部位以常以瘢痕组织代替。为解决此问题,组织工程化肌腱成为研究热点,但目前使用的人工生物材料载体存在老化、排异等缺点。骨髓间充质干细胞(BMSCs)作为老牌种子细胞在组织再生领域应用广泛,但有报道称其应用后出现了异位钙化、干细胞形成肿瘤等情况。肌腱干细胞(TDSCs)由于其来源,用于肌腱修复研究理论上更具优势。结缔组织生长因子(CTGF)可诱导两种细胞向肌腱细胞方向分化。因此,课题组使用CTGF和这两种细胞进行体外实验,构建出一种不含其它材料的组织工程化肌腱用于肌腱损伤的修复,并通过对比两种细胞选取更适合肌腱损伤修复的种子细胞。目的:1、验证结缔组织生长因子(CTGF)诱导骨髓间充质干细胞(BMSCs)和肌腱干细胞(TDSCs)成肌腱分化能力,并对比两种干细胞诱导前后的成肌腱潜能。2、利用结缔组织生长因子(CTGF)诱导成肌腱分化能力,利用这两种干细胞体外构建组织工程化肌腱。3、初步探索组织工程化肌腱在肌腱损伤修复中的作用,并对比两种组织工程化肌腱的效果。方法:1、分离培养绿荧光大鼠的骨髓间充质干细胞(BMSCs)和肌腱干细胞(TDSCs),并进行鉴定。2、取第3代的骨髓间充质干细胞(BMSCs)和肌腱干细胞(TDSCs), 25ng/ml的CTGF进行诱导分化培养2周,选取scleraxis和tenomodulin这两个成肌腱相关基因,利用RT-qPCR进行mRNA表达检测。验证CTGF诱导BMSCs和TDSCs成肌腱分化能力,并对比两种干细胞诱导前后的成肌腱潜能。3、取第3代的BMSCs和TDSCs,体外诱导成肌腱分化2周,并构建出干细胞组织工程化肌腱。4、将BMSCs和TDSCs构建的组织工程化肌腱分别植入裸鼠皮下,8周、12周后取材,进行组织形态学检测、免疫组化染色。结果:1、RT-qPCR结果显示,CTGF能显著增加BMSCs和TDSCs中tenomodulin和scleraxis的mRNA表达(p0.05),对比发现,诱导分化前后TDSCs中tenomodulin 和 scleraxis 的 mRNA 表达均明显高于 BMSCs (p0.05)。2、利用CTGF诱导BMSCs和TDSCs体外构建两种组织工程化肌腱,组织形态学分析结果显示两种组织工程化肌腱均具有不成熟肌腱样结构,TDSCs组结构更加理想。3、组织形态学和免疫组织化学分析结果显示,两种组织工程化肌腱均可在裸鼠体内形成肌腱样组织,其中TDSCs组种植后结构成熟更快,细胞外基质成分更加接近正常肌腱。结论:1、CTGF配合抗坏血酸可有效诱导BMSCs和TDSCs成肌腱分化,TDSCs比BMSCs有着更大的成肌腱分化潜能。2、仅利用二维条件进行细胞培养,利用CTGF诱导BMSCs和TDSCs的成肌腱分化作用,两种细胞均可在体外构建出一种不含任何合成人工材料的组织工程化肌腱。3、体外构建出两种组织工程化肌腱在裸鼠体内种植均可形成类肌腱样组织。TDSCs构建的组织工程化肌腱优于BMSCs。 TDSCs可能比BMSCs更适合在组织工程学领域用于肌腱损伤修复。
[Abstract]:Background: because of its unique anatomy and tissue structure, tendon tissue lacks the ability to repair itself. Scar tissue is often used to replace the injured site after healing. In order to solve this problem, tissue engineered tendon has become a hot research topic. However, the artificial biomaterial carriers used at present have some disadvantages, such as aging and rejection. Bone marrow mesenchymal stem cells (BMSCs) are widely used in the field of tissue regeneration as old seed cells, but there are reports of ectopic calcification after their application. Because of its origin, TDSCs have a theoretical advantage in tendon repair. Connective tissue growth factor (CTGFs) can induce two kinds of cells to differentiate into tendon cells. CTGF and these two kinds of cells were used to construct a kind of tissue engineered tendon without other materials to repair tendon injury. By comparing the two kinds of cells, seed cells were selected to repair tendon injury. Objective: 1 to test the ability of bone marrow mesenchymal stem cells (BMSCs) and tendon stem cells (TDSCs) induced by connective tissue growth factor (CTGF) to differentiate tendon. The ability of tendon differentiation induced by connective tissue growth factor (CTGF) was compared between the two kinds of stem cells before and after induction. The two kinds of stem cells were used to construct tissue engineered tendon in vitro to explore the role of tissue engineered tendon in the repair of tendon injury. Methods the bone marrow mesenchymal stem cells (BMSCs) and the tendon stem cells (TDSCs1) of green fluorescent rats were isolated and cultured. TDS cells were cultured for 2 weeks with 25 ng / ml CTGF. Scleraxis and tenomodulin were selected to detect the expression of mRNA by RT-qPCR. The ability of CTGF to induce the differentiation of BMSCs and TDSCs tendons was verified. The tendon potential of the two stem cells before and after induction was compared. The third generation of BMSCs and TDSCs were taken and induced into tendon differentiation for 2 weeks in vitro. The tissue engineered tendon of stem cells was constructed. The tissue engineered tendons constructed by BMSCs and TDSCs were implanted into nude mice for 8 weeks and 12 weeks, respectively. Results the expression of mRNA in tenomodulin and scleraxis in BMSCs and TDSCs was significantly increased by RT qPCR. The results showed that CTGF could significantly increase the mRNA expression of tenomodulin and scleraxis in BMSCs and TDSCs. The mRNA expression of tenomodulin and scleraxis in TDSCs was significantly higher than that in BMSCs p0.05. 2before and after differentiation. CTGF was used to induce BMSCs and TDSCs to construct two kinds of tissue engineered tendons in vitro. The results of histomorphology analysis showed that both of them had immature tendonoid structures. The structure of TDSCs was more ideal. The results of histomorphology and immunohistochemical analysis showed that the two kinds of tissue engineered tendons had immature tendon-like structures. Both kinds of tissue engineered tendons could form tendon-like tissues in nude mice. In TDSCs group, the structure matured more quickly after implantation. Conclusion BMSCs and TDSCs can induce tendon differentiation of BMSCs and TDSCs more effectively than that of BMSCs. TSCs can only be cultured under two-dimensional conditions, and the differentiation potential of TSCs is higher than that of BMSCs, and the extracellular matrix components are closer to normal tendons. Conclusion: TSCs can induce tendon differentiation of BMSCs and TDSCs effectively, and the differentiation potential of TSCs is higher than that of BMSCs, and only two dimensional conditions can be used for cell culture. The tendon differentiation of BMSCs and TDSCs was induced by CTGF. Both kinds of cells could construct a kind of tissue engineered tendon without any synthetic material in vitro. In vitro, two kinds of tissue engineered tendons could be implanted in nude mice to form tendonoid tissue. TDSCs could be constructed. Engineering tendons are superior to BMSCs. TDSCs may be more suitable for tendon injury repair than BMSCs.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R686

【参考文献】