Elementary Study On Immortalization Of Rabbit Chondrocytecells By Transfection With Human Telomerase Reverse Transcriptase

Objective:The purpose is to explore if the hTERT gene can activate the telomerase activity of the rabbit articular cartilage cells and to extend the life-span in vitro by transfecting into the second generation of New Zealand white big articular cartilage cells with exogenous gene hTERT.It is the foundation to make the immortalization of rabbit chondrocyte cells in the future.Methods:1.Human telomerase reverse transcriptase(hTERT) gene was respectively transfected into PT67 packing cells with steady and transient method by lipofectamine method.After collecting virus supernatant, viral titers were estimated by NIH3T3 cells.2. The rabbit second-generation cartilage cells were respectively infected by two kinds of the virus supernatant, and to be selected by G418 and to obtain the G418-resistant colonise which were subcultucted.It is to establish preliminarily an immortalized rabbit articular cartilage cells.3.Expression of hTERT in establishment of an immortalized rabbit chondrocyte cells were detected by reverse transcription polymerase chain reaction (RT-PCR),which.were compared with the normal second generation of the rabbit articular cartilage cells.Results:1.Plasmid steady transfect packing cells were selected by G418,and to obtain a stable virus producing cell line.The expression hTERT-mRNA of resistance PT67 and normal PT67 cell were detected by RT-PCR.the result indicated that they all express hTERT-mRNA band, but the express of hTERT-mRNA band in resistance PT67 was obviously stronger than normal PT67(p<0.05).then viral titers of steady transfect packing cells was 2.0×105CFU/ml. transient transfer packaging cells produce a virus supernatant,whose viral titers was 1.0×106CFU/ml.2.The second rabbit articular cartilage cells were infected by steady transfect packing cells with the flow cytometric detection whose infection rates is 28.55±6.99%; The rabbit second-generation cartilage cells were infected by transient transfer packaging cells whose infection rates is 81.42±11.45%.Compared with the second generation of the rabbit uninfected control articular cartilage cells,the control infection rates is 0.23±0.05%. By the analysis of variance, the difference is significant(P<0.05).3.The expression of hTERT-mRNA of an immortalized rabbit chondrocyte cells were detected by reverse transcription polymerase chain reaction (RT-PCR). It show that an immortalized rabbit chondrocyte cells expressed a specificity band in 145bp;Whereas,the expression of hTERT-mRNA of the rabbit normoal second generation chondrocyte cells were not detected the band. The results show that the hTERT gene was transferred into rabbit chondrocyte cells and can be expressed.Conclusions:1.A stable virus encoding hTERT producing cell line was successfully constructed.2.Human telomerase reverse transcriptase(hTERT) gene was respec-tively transfected into PT67 packing cells with steady and transient method by lipofectamine method.Infection rate of transient method is higher by the flow cytometric detection.3.Exogenous hTERT could activate the telomerase activity of the rabbit articular cartilage cells by the retrovirus mediated...