The Study Of Recombinant Adeno-associated Virus-mediated TGF-β₁ Gene Modifying Adipose-derived Stem Cells Into Cartilage Cells

Objective:To investigate the chondrocyte differentiation of ADSCs modified by recombinant adeno-associated virus-mediated TGF-β1 gene. It provids an new method of induction for cartilage tissue engineering. Methods:ADSCs were isolated from the subcutaneous adipose tissue which was from 6-month-old New Zealand white rabbits' back and neck by mechanical digestion and enzyme digestion, then ADSCs were cultured and amplified in vitro. ADSCs at passage 3 were used for the transfection. The experiment was divided into three groups, respectively named TGF-β1 vector group, empty vector group, and control group. In the TGF-β1 vector group and empty vector group, ADSCs at passage 3 were respectively infected with rAAV2-TGFβ1-IRES-EGFP and rAAV2-EGFP, and the multiplicity of infection was 5×105, the time of transfection was 90min. After transfection, the sodium butyrate was used for promoting the expression of target gene. Semi-volume media was changed every other day, and the culture medium swapped out every time was collected in EP tube.24 hours after transfection, the cell morphology was observed under reverse microscope. Meanwhile, the number and fluorescence intensity of fluorescent cells were observed under fluorescence microscope every other day. The transfection efficiency was estimated by counting fluorescent cells in each field of vision. And the cell proliferation activity was assayed by using MTT. The TGF-β1 concentration in the culture media was detected by ELISA method.14 days and 21 days after transfection, ADSCs were detected by collagen typeⅡimmunohistochemistry staining and toluidine blue staining of proteoglycan. Results:ADSCs isolated and cultured in vitro were flat, long-spindle and amplified stably, the cell morphology was uniformity. There were many green fluorescent cells in the TGF-β1 vector group and empty vector group, not in the Control group. The proliferation ability of ADSCs transfected with recombinant adeno-associated virus was similar in 3 groups.14 days and 21 days after transfection, ADSCs were detected by collagen typeⅡimmunohistochemistry staining and toluidine blue staining of proteoglycan, both of them presented positive. Conclusion:Recombinant adeno-associated virus-mediated TGF-β1 gene could be successfully transfected into ADSCs, and the transfection efficiency is high, the cell toxicity is low, but the expression of target gene need sodium butyrate help; ADSCs successfully differentiate into cartilage cells, induced by TGF-β1, the inducible factor is more steady, longer and more reliable, and the concentration of TGF-β1 in the media is low.