| Objective: According to the gene transfection technology, use the adenovirus tomediate TGF-beta1gene transfecting Epiphyseal plate cartilage cells (EpiphysealChondrocytes, ECs), and observe the expression of TGF-beta1and ECs synthesisand secretion of cartilage matrix, to provide a experimental basis for building a newtype of Epiphyseal plate cartilage tissue engineering.Methods:1. ECs were isolated from3-week-old New Zealand white rabbits’femoraldistal epiphyseal plate cartilage. Then ECs were cultured and amplified in vitro. ECs atpassage3were used for the transfect ion.2. Build a TGF-beta1gene adenovirusvector.3. The experiment was divided into three groups, respectively named TGF-beta1vector group, empty vector group, and control group. After transfection, the cellmorphology was observed under fluorescence microscope. Meanwhile, the number andfluorescence intensity of fluorescent cells were observed under fluorescence microscope.The transfection efficiency was estimated by counting fluorescent cells in each field ofvision.And the cell Proliferation activity was assayed by using MTT. The TGF-beta1conceniration in the culture media was detected by ELISA method.Use Collagen Ⅱimmunofluorescence to evaluate cartilage capability of transfect cells. Use RT-PCR todetect TGF-beta1mRNA expression in each cell, and Western-blot to detect eachgroup of cells Ⅱ collagen type and TGF-beta1protein expression.Results:1. ECs was successfully isolated from rabbit epiphyseal plate cartilage andsubcultured and identified.2. Each group of transfected cells was observed under afluorescence microscope, TGF-β1carrier group and empty vector group have morefluorescent cells, while the control group did not have fluorescent cells. It has beenproved that the proliferative capacity of adenovirus-mediated TGF-β1gene transfection of ECs has no effect in the short term by MTT assay.3.14days after transfection,compared with the control group the observed collagen type II significantly increasedthrough TGF-β1carrier group immunofluorescence.4.RT-PCR detected that in thetransfection group specimens TGF-β1mRNA expression significantly increasedcompared with the control group5.Western-blot results showed that the transfectedgroup Collagen Ⅱ and TGF-β1expression also significantly enhanced.Conclusion:1. ECs was successfully isolated from rabbit epiphyseal plate cartilageand subcultured and identified, rabbits in vitro epiphyseal plate3th represents the typeof stable cells, the expression of biology and functional characteristics of a goodschool.2. The recombinant adenovirus carrying hTGF-β1gene had been constructedsuccessfully,use the recombinant adenovirus vector to transfect ECs successfully, andthe transfection rate is very high, meanwhile, small toxicity to cells.3. Using the genetransfection technology to make TGF-beta1gene transfect ECs, and strengthen theability of ECs synthesis and secretion of cartilage matrix. |
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