| 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,BMScs 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. |
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