【作者】 吴险峰；

【导师】 尚希福；

【作者基本信息】 安徽医科大学 ， 外科学， 2009， 硕士

【摘要】 目的:茶黄素是红茶中的主要成分,具有调节血脂、抗氧化、抗肿瘤、抗心脑血管疾病等多种药理活性。以往研究茶黄素对骨髓基质干细胞转化较少,本实验探讨茶黄素和转化生长因子β1（TGF-β1）对体外培养兔骨髓基质干细胞（BMSCs）增殖及向软骨细胞诱导分化的影响。方法:实验于2008-05/8在安徽医科大学附属省立医院中心实验室完成。①实验动物:清洁级8周龄家兔12只,由安徽医科大学动物试验中心提供,实验过程中对动物的处置符合动物伦理学标准。②实验方法:兔全身肝素化后麻醉状态下处死,取双侧股骨和肱骨,去除软组织,切除包括骺板在内的两侧骺端,采用全骨髓法分离培养骨髓基质干细胞,按5×10⁴/L的细胞密度接种,当细胞生长至80%～90%融合时消化传代。取传至第三代的细胞按1×10⁴/ml的密度接种,加入含重组人胰岛素10mg/L、地塞米松10^-8mmol/L、TGF-β₁5ng/L的DMBM成软骨诱导液培养2周。取第3代BMSC分为三组:A对照组,B组:完全培养基加地塞米松(10^-8mmol/L)、TGF-β1（5ng/L）、维生素C（10mmol/L）。C组:完全培养基加地塞米松(10^-8mmol/L)、TGF-β1（5ng/L）、维生素C（10mmol/L）、茶黄素（30 mg/L）。③实验评估:倒置显微镜下观察细胞生长状况,甲苯胺蓝染色、阿利新蓝比色法测定各板每孔中GAG含量、以及Ⅱ型胶原免疫组化鉴别。结果:①细胞生长曲线:骨髓基质干细胞具有旺盛的增殖能力,培养1d为细胞的适应期,3d为对数增长期,8d时进入平台期,之后细胞增殖迅速减慢,细胞数下降。②观察细胞生长:骨髓中分离获得的BMSCs在体外增殖旺盛,TGF-β1和茶黄素诱导后细胞生长明显减缓。与对照组相比,经过诱导2周后,诱导组BMSCs均与对照组有显著的差异性（P＜0.05）,诱导组之间也存在差异性（P＜0.05）。③甲苯胺蓝染色和免疫组化鉴定结果:经TGF-β1和茶黄素诱导2周后,细胞甲苯胺蓝异染明显,Ⅱ型胶原化学染色阳性,表现为软骨细胞生物学特性。结论:茶黄素（30 mg/L）在TGF-β1存在的条件下能有效促进BMSCs在体外向软骨细胞分化。更多还原

【Abstract】 Objective:Theaflavins are the major constituent of black tea,and havea lot of pharmacological activity,for example,regulating blood fat,antioxygen, anti-tumor,resisting cerebrovascular disease,and so on.To go forward,the study of theaflavins’ effect on mankind MSCs transformingWere relatively scarce,but our expriment to abserve the effects of theaflavin and transforming growth factor beta 1 on the proliferation and chondrogenic differentiation of rabbit marrow-derived mesenchymal stem cells in vitro.Methods:Our expriment completed in center laboratory of Anhui provincial hospitol affiliated Anhui medical university.①Exprimntal animal:12 rabbits of cleaning grade and 8 weeks old are provided by animal expriment center of Anhui medical university. The disposal of animal in process of expriment refer to ethical standard of animals.②exprical method:The rabbits are heparinized and put to death in drugged state,we obtain its sideway femur,tibia and humerus,remove its soft tissue,cut epiphysis of two sides including epiphyseal plate,isolate and culture the MSCs with whole bone marrow culture method technology,inoculate it according to the density of 5×10⁴/L,and when the cells grew to the fusion of 80%-90%,digesting and going down to the future generation.We inoculate cells of the third generation according to the density of 1×10⁴/ml,and add the DMEM chondrocytes-induced liquid contained rh-insulin 10mg/L,dexamethasone 10^-8mmol/L,and TGF-β₁5ng/L to be cultured for 2 weeks.MSCs from the third passage were grown in complete medium（Group A）,inductive medium with 5mg/ml FGF-β₁（Group B）,and inductive medium with 30mg/l theaflavin and with 5mg/ml TGF-β₁（Group C） respectively.③Exprical evaluation:inverted phase contrast microscope,MTT assay toluidine blue staining,alcian blue staining,and cartilage specific collagenⅡwere used to investigate cell biological characteristics.Resucts:①Cell gowth curve:MSCs have vigorous reproductive activity,1 days to be cultured is in the cell adaptive phase,after 3 days is in the increased logarithmic phase, 8 days in the platform pharse,and 8 days.cell proliferation quickly steps down.②Observing cell growth:MSCs isolated from rabbit bone marrow proliferated actively in vitro.MSCS in monolayer cultures treated with TGF-β₁ and theaflavin showed growth deceleration.In comparing with the control group,MTT assay demonstrated that the cell protiferation of goup A.B and C,showed a significant difference（p＜0.05） after two weeks.③Identification result of toluidine blue staining and collagen type immunohistocyto staining:after 2 weeks,BMS_cs treated with 5mg/ml TGF-β₁ and 30mg/l theaflavin were assessed by toluidine blue staining and collagen type immunohistocyto staining,indicating that cartilage spacific matrix was produced in vitro.Conclusion:On the condition of TGF-β1,theaflavin combination of TGF-β1 and theaflavin in concentration（30mg/L） can not only facilitate the proliferation of BMSCs,but also induce the chondrogenic differentiation actively.更多还原