| Background and PurposeCartilage tissue engineering and gene therapy have become the focus of osteoarthritis cartilage repairment to date. How to acquire a large functional cartilage seed cells have restricted cartilage tissue engineering construction. This experiment infected hBMP - 2 gene to rabbit articular chondrocytes in vitro ,and cervus and cucumis polypeptide was added into the cultivation system. The proliferation of chondrocytes and secretion function of collagen typeⅡof chondrocytes was observated. We try to find out a new direction for acquiring a large of functional cartilage seed cells and to treat osteoarthritis and osteoclasia using hBMP - 2 genes and cervus and cucumis polypeptide in clinical practice.Methods1.Experimental group: non-transfected group, Ad-GFP group, Ad - hBMP2 - GFP group, cervus and cucumis polypeptide combining with Ad - hBMP2 - GFP group transfection.2. hBMP2 - GFP viral vectors was packed by human embryo kidney293 cell , then viral vectors was amplificated, and the virus drops of degrees was further determined.3. Primeval generation rabbit chondrocytes were separated, cultured, and identified, , the third generation cells were used as model. , transfect hBMP-2 was transfed into rabbit chondrocytes via gene transfecting technology. After 48h, RT - PCR was used to detect hBMP - 2 mRNA expression, hBMP - 2 protein and collagen typeⅡexpression in chondrocytes were detected using cellular immune fluorescence and the Western Blot. Non-transfected group and empty virus group for controls.4. MTT method screened the best drug concentration and time of cervus and cucumis polypeptide after adding to chondrocytes 24h,48h and 72 h.5. Growth cycle of chondrocytes were detected after culturing 48h by flow cytometry. MTT method determined articular chondrocytes proliferation following cultivate 48h, 72h,, Western blot was used to test the expressions of hBMP - 2 protein and collagen typeⅡ.6. Statistical methods: All of statistical treatments were performed by SPSS16.0 statistical software. All data were analyzed with ( (x|-)±s), the single factor analysis of variance was used, interblock difference was performed with q test. P < 0.05 was considered as statistical significance.Results1. The titre of Ad - hBMP2 - GFP was 2 x 10~6PFU/ml after infected 293 cells.2. Infecting hBMP - 2 after 48h, infecting group had hBMP -2 mRNA expression, visible red fluorescent expression of hBMP -2 protein located in cell cytoplasm,but not in nuclei, non-infecting group and empty virus group were negative. After 48h and 72h, compared with expression quantity, gray-scale value of hBMP - 2 protein and collagen typeⅡin hBMP - 2 carried group signnificntly increased more than those in empty virus group .3. 4~32ug/ml drug concentration of cervus and cucumis polypeptide for chondrocytes accelerated proliferation for 48h to 72h.The best concentration and time are 8ug/ml and 48h.4. hBMP-2 combined Cervus and cucumis polypeptide shorten the growth cycle of chondrocytes, promoted the proliferation of chondrocytes, and promoted the expression of the hBMP-2 and collagen typeⅡprotein.ConclusionsAdenovirus-mediated hBMP-2 gene can efficiently transfect chondrocytes of rabbits in vitro, and 8ug/ml concentrations of cervus and cucumis polypeptide was added into the cultivation system. the both can promote the proliferation of chondrocytes and secretion function of collagen typeⅡof chondrocytes, that chondrocytes can be proliferated considerably and maintein their good biological characteristics during they are being cultured. This reserch provide a new direction for getting a large number of cartilage seed cells in cartilage tissue engineering...
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