茶黄素与TGF-β3对兔骨髓间充质干细胞向软骨细胞增殖和诱导分化地影响

发布时间：2018-01-09 03:08

本文关键词：茶黄素与TGF-β3对兔骨髓间充质干细胞向软骨细胞增殖和诱导分化地影响　出处：《安徽医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的:软骨损伤在临床上非常多见,但修复非常困难,组织工程的发展使软骨修复的研究得到长足的发展,间充质干细胞被认为是一种理想的种子细胞。转化生长因子β3可以诱导软骨的代谢和增殖,而茶黄素也有促进软骨代谢和增殖的作用,本实验探讨在茶黄素和转化生长因子β3的联合作用下对骨髓间充质干细胞向软骨细胞诱导分化和增殖的影响。方法:本实验于2009-09/12在安徽医科大学附属省立医院中心实验室完成。⑴实验动物:健康6~7周龄清洁级新西兰大白兔15只,雌雄不拘,由安徽医科大学动物试验中心提供,实验过程中对家兔处置符合动物伦理学标准。⑵实验方法:全身肝素麻醉状态下处死动物,取双侧肱骨和股骨,剔去附丽软组织,切除两侧骺端(包括骺板),全骨髓法分离及培养骨髓间充质干细胞,按6×10~4 /L的细胞密度接种,当细胞生长至90%左右融合时消化传代。取第3代细胞按1×10~4 /ml地密度接种,置入含地塞米松10~(-8)mmol/L、转化生长因子β3 10μg/L和重组人胰岛素15mg/L的DMBM成软骨诱导液培养二周,取第三代骨髓间充质干细胞分为3组:对照组:完全培养基加地塞米松10~(-8) mmol/L、维生素C 10mmol/L。转化生长因子组:完全培养基加地塞米松10~(-8) mmol/L、维生素C 10mmol/L、转化生长因子β3 10μg/L。茶黄素组:完全培养基加地塞米松10~(-8) mmol/L、维生素C 10mmol/L、转化生长因子β3 10ng/mL、茶黄素30mg/L。⑶评估:在倒置显微镜下观察细胞的生长状况和形态变化,用甲苯胺蓝染色和阿利新蓝比色法测定各板每孔中葡萄糖氨基聚糖含量,免疫检测仪测定3组的吸光度值鉴别II型胶原。结果:骨髓基质干细胞增值能力旺盛,培养24小时为细胞适应期,72小时为对数增长期,一周后进入平台期,以后速度减慢,细胞数减少;骨髓中分离获得地骨髓间充质干细胞在体外增殖旺盛,细胞经茶黄素和转化生长因子β3诱导后生长明显减缓。加入诱导液诱导二周后,诱导组与对照组均有显著的差异性(P0.05),诱导组之间同样具有差异性(P0.05)。经甲苯胺蓝染色和免疫组化测定结果,经诱导后透明软骨特征性标志II型胶原化学染色阳性,甲苯胺蓝异染明显。结论:茶黄素与转化生长因子β3联合作用能有效促进骨髓间充质干细胞在体外向软骨细胞分化。
[Abstract]:Objective: cartilage injury is very common in clinic, but it is very difficult to repair. The development of tissue engineering has made great progress in the research of cartilage repair. Mesenchymal stem cells are considered to be ideal seed cells. Transforming growth factor 尾 3 can induce the metabolism and proliferation of cartilage, while theaflavin can promote the metabolism and proliferation of cartilage. The effects of theaflavine and transforming growth factor 尾 3 on the differentiation and proliferation of bone marrow mesenchymal stem cells into chondrocytes were investigated. Methods: this experiment was completed in the Central Laboratory of Provincial Hospital affiliated to Anhui Medical University on 2009-09 / 12. 1 Experimental Animals: 15 clean New Zealand white rabbits of 6 ~ 7 weeks of age, male and female. The animal test center of Anhui Medical University provided 2 experimental methods for the treatment of rabbits in accordance with animal ethics standard .2: the animals were killed under general heparin anesthesia and bilateral humerus and femur were taken. The bone marrow mesenchymal stem cells were isolated and cultured by removing the soft tissue of epiphysis (including epiphyseal plate) and inoculating with 6 脳 10 ~ (4 / L) / L of cell density. The third passage cells were inoculated at 1 脳 10 ~ (4) / ml density and implanted with dexamethasone 10 ~ 10 ~ (-1) -8 mmol / L. Transforming growth factor 尾 3 10 渭 g / L and recombinant human insulin 15 mg / L DMBM chondrocytes were cultured for 2 weeks. The third generation of bone marrow mesenchymal stem cells were divided into three groups: control group: complete culture medium plus dexamethasone 10 + -8) mmol/L. Vitamin C 10 mmol / L. Transforming growth factor group: complete medium plus dexamethasone 10% -8) mmol / L, vitamin C 10 mmol / L. Transforming growth factor 尾 3 10 渭 g / L. Theafin group: complete medium supplemented with dexamethasone 10% -8) mmol / L, vitamin C 10 mmol / L. Transforming growth factor 尾 3 10 ng / mL, theaflavin 30 mg / L.3 evaluation: cell growth and morphological changes were observed under inverted microscope. The content of glucosaminoglycan in each hole of each plate was determined by toluidine blue staining and alimine blue colorimetry. The absorbance of type II collagen was identified by immunoassay. Results: the proliferation of bone marrow stromal cells (BMSCs) was exuberant. The cells were cultured for 24 hours in adaptation phase and 72 hours in logarithmic growth period. One week later they entered the plateau phase and then the speed slowed down and the number of cells decreased. The proliferation of mesenchymal stem cells isolated from bone marrow was strong in vitro, and the growth of cells induced by theaflavin and transforming growth factor 尾 3 was slowed down obviously. There was significant difference between the induction group and the control group (P 0.05), and there was also difference between the induction group and the control group (P 0.05). The results were determined by toluidine blue staining and immunohistochemistry. The characteristic marker of hyaline cartilage, type II collagen, was positive after induction, and toluidine blue was positive. Conclusion: the combination of theaflavins and transforming growth factor 尾 3 can effectively promote the differentiation of bone marrow mesenchymal stem cells into chondrocytes in vitro.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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