Effects of Theaflavin and Transforming Growth Factor Beta 3 on the Proliferation and Differentiation of Rabbit MSCs into Chondrocytes
|School||Anhui Medical University,|
|Keywords||Chondrocyte Bone marrow stromal cells Theaflavin Transforming growth factorβ3(TGF-β3)|
Objective:It’s hard to repair the chondrogenic. MSCs is an ideal seedage.Theaflavins and transforming growth factor beta 3 both have the effects on the proliferation and chondrogenic differentiation of MSCs.Our experiment to abserve the effects of theaflavin and transforming growth factor beta 3 on the proliferation and chondrogenic differentiation of rabbit marrow-derived mesenchymal stem cells in vitro.Methods:Our experiment completed in center laboratory of Anhui provincial hospital affiliated Anhui medical university.⑴Exprimntal animal:15 rabbits of cleaning grade and 67 weeks old are provided by animal experiment center of Anhui medieal university.The disposal of ammal in process of experiment refer to ethical standard of animals.⑵exprical 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 6×104 /L,and when the cells grew to the fusion of 90%, digesting and going down to the future generation.We inoculate cells of the third generation according to the density of 1×104 /L,and the DMEM chondrocytes-induced liquid contained rh-insulin 10mg/L,dexamethasone 10-8mmol/L,and TGF-β3 10ng/L to be cultured for 2 weeks.MSCs from the third passage were grown in complete medium,inductive medium with 10μg/L TGF-β3,and inductive medium with 30mg/l theaflavin and with 10μg/L TGF-β3 respectively.⑶Evaluation:inverted phase contrast microscope,MTT assay toluidine blue staining,alcian blue staining and cartilage Specific collageull were used to investigate cell biological characteristics.Resuets: MSCs have vigorous reproduetive activity,24 hours to be cultured is in the cell adaptive phase,after 72 hours is in the increased logarithmic phase,one week in the platform pharse,cell proliferation quickly steps down.MSCs isolated from rabbit bone marrow proliferated actively in vitro.MSCs in monolayer cultures treated with TGF-β3 and theaflavin showed growth deceleration.In comparing with the control group , MTT assay demonstrated that the cell protiferation of all three groups,showed a significant difference（P<0.05） after two weeks.The same result was showed between groups.Identification result of toluidine blue staining and collagen type immunohistocyto staining indicating that cartilage specific matrix was produced in vitro.Conelusion: The theaflavin combination with TGF-β3 can facilitate the proliferation of BMSCs and induce the chondrogenic differentiation actively.