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Effects of Theaflavin and Transforming Growth Factor Beta 1 on the Proliferation and Differentiation of Rabbit MSCs into Chondrocytes

Author: WuXianFeng
Tutor: ShangXiFu
School: Anhui Medical University,
Course: Surgery
Keywords: Bone marrow stromal cells Chondrocytes Theaflavins Transforming growth factor-β1
CLC: R329
Type: Master's thesis
Year: 2009
Downloads: 39
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Abstract


Objective: Theaflavins is the main ingredient in black tea, regulate blood lipids, a variety of pharmacological activities of anti-oxidation, anti-tumor, anti-heart and cerebrovascular disease. The previous studies Theaflavins bone marrow stromal stem cells into less investigated the effects of theaflavins and transforming growth factor-β1 (TGF-β1) proliferation and induced differentiation of chondrocytes in vitro cultured rabbit bone marrow stromal cells (BMSCs). Method: experimental 2008-05/8 in Anhui Medical University Affiliated Provincial Hospital Central Laboratory. (1) in experimental animals: clean grade 8-week-old rabbits 12, Anhui Medical University animal testing center, met the animal ethical standards for the disposal of animal experiment. ② experimental methods: rabbit systemic heparin were sacrificed under anesthesia, bilateral femur and humerus, the removal of soft tissue resection on both sides, including the epiphyseal plate epiphyseal end, whole bone marrow method isolated and cultured bone marrow stromal stem cells at 5 × 10 4 / L of the cell density inoculated digested when the cells were grown to 80% ~ 90% confluence and passaged. Taken spread to the third generation cells at 1 × 10 4 / ml were seeded added containing recombinant human insulin 10 mg / L dexamethasone 10 -8 mmol / L, TGF-β 1 5 ng / L of DMBM chondrogenic induction medium cultured for 2 weeks. The 3rd generation BMSC divided into three groups: control group, B group: the complete medium plus dexamethasone (10 -8 mmol / L), TGF-β1 (5 ng / L), vitamin C (10mmol / L). Group C: complete medium plus dexamethasone (10 -8 mmol / L) of TGF-β1 (5 ng / L), vitamin C (10mmol / L), theaflavin (30 mg / L ). (3) experimental evaluation: cell growth observed under an inverted microscope, stained with toluidine blue, alcian blue colorimetric determination of GAG content in each hole of each board, and collagen type II immunohistochemistry to identify. Results: ① The cell growth curve: Bone marrow stromal cells have strong proliferative capacity, culture 1d cell adaptation period, 3d logarithmic growth phase, 8d into the platform of cell proliferation after rapid slow down the decline in the number of cells. The ② observed cell growth: bone marrow isolated BMSCs proliferation in vitro cell growth has slowed down significantly after the induction of TGF-β1 and theaflavins. Compared with the control group, after the two weeks after the induction, the induction group BMSCs with the control group, a significant difference (P <0.05), also exists between the difference (P <0.05) in the induction group. ③ toluidine blue staining and immunohistochemistry identified: two weeks after the induction of TGF-β1 and theaflavins cells with toluidine blue metachromatic positive staining of collagen type II, showed the biological characteristics of the chondrocytes. Conclusion: theaflavins (30 mg / L) in the TGF-β1-existing conditions can effectively promote BMSCs into chondrocytes in vitro.

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