JOURNAL ARTICLE

[Chondrogenesis of bone marrow mesenchymal stem cells induced by transforming growth factor beta3 gene in Diannan small-ear pigs]

Huijian Wang, Yanlin Li, Jianming Chen, Xin Wang, Fengkai Zhao, Shuhai Cao
Chinese Journal of Reparative and Reconstructive Surgery 2014, 28 (2): 149-54
24796184

OBJECTIVE: To observe transforming growth factor beta3 (TGF-beta3) gene expression and the chondrogenesis of bone marrow mesenchymal stem cells (BMSCs) after TGF-beta3 gene is transfected into BMSCs of Diannan small-ear pig.

METHODS: Recombinant adenovirus 5 (rAd5) was extracted as gene vector and packed into recombinant adenovirus rAd5-TGF-beta3, double enzyme digestion and PCR identification were performed. BMSCs were isolated and cultured from bone marrow of 2-month-old Diannan small-ear pigs (weighing, 12-15 kg), and the 2nd generation of BMSCs were harvested for experiments. The experiments were divided into 3 groups. BMSCs were transfected with rAd5-TGF-beta3 as experimental group and with empty vector as control group, and non-transfected BMSCs were used as blank control group. The transfection efficiency of exogenous gene was identified by flow cytometry, TGF-beta3 protein expression by immunofluorescence and Western blot. The cell morphology of experimental group was observed by inverted phase contrast microscope, and the expression of collagen type II in each group was detected by Western blot.

RESULTS: The rAd5-TGF-beta3 recombinant adenovirus was successfully constructed and transfected into BMSCs. Green fluorescence was observed by immunofluorescence microscope. Flow cytometry test showed the best transfection at 72 hours (transfection efficiency of 84.86%). Immunofluorescence staining showed that the expression of TGF-beta3 protein was obvious at 72 hours; Western blot showed that there was a TGF-beta3 positive band with a relative molecular mass of 30 x 10(3), while the control group and blank control group had no positive band. Obvious chondrogenic differentiation was observed in the experimental group after transfection in vitro, while the control group and blank control group had no obvious chondrogenic differentiation. Western blot showed that there was collagen type II positive band with a relative molecular mass of 130 x 10(3) at 21 days after culture, while the control group and blank control group had no positive band.

CONCLUSION: rAd5-TGF-beta3 gene can be successfully transfected into BMSCs via adenovirus vectors, and stable expression of TGF-beta3 protein can be observed, enhancing BMSCs differentiation into chondrocytes, which may provide an experimental basis for gene therapy of joint cartilage defects.

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