关键词: 人端粒酶逆转录酶；转染；软骨细胞；细胞增殖

Abstract:

Abstract:Objective To explore the way and technique to gain adequate seed cells by introducing human telomerase reverse transcriptase(hTERT) gene into rabbit chondrocytes.  Methods The chondrocytes of rabbit were isolated and cultivated,the expression vector pcDNA3.1/ zeo(+)-hTERT was transfected into chondrocytes, in control group it was zero load pcDNA3.1/ zeo(+),the positive clones were selected and amplified,the reverse transcription-polymerase chain reaction(RT-PCR) was used to detect the expression of hTERT mRNA, toluidine blue staining was used to analyze whether or not the chondrocytes transfected with hTERT gene possessed bionomics of chondrocytes.Flow cytometry examination was used to analyze cell cycle and karyotype.The condition of tumor formation of nude mouse was observed by tumorigenesis experiment.Results pcDNA3.1/ zeo(+)-hTERT was transfected into chondrocytes detected by RT-PCR on the RNA level.The morphology of chondrocytes infected with pcDNA3.1/ Zeo(+)-hTERT still kept polygon, which was same as the normal chondrocytes cultivated in vitro.After toluidine staining, polysaccharide was stained amethyst, small amethyst particles of proteoglycan were well-distributed.It suggested that the transformed cells still possessed the function of secreting cartilage correlated matrix.The multiplicate ability of transformed cells could be analyzed by measuring the cycle of cells.The multiplicate ability increased remarkably, the way of cell division was based on diploidy keeping the stabilization of karyotype.No tumorigenesis was found in nude mouse.Conclusion The proliferative ability of chondrocytes transfected with hTERT are enhanced obviously and the malignant transformation does not take place, they can serve as an excellent tool to study the biochemical and physiological aspect of chondrocytes and be used as seeding cells in tissue engineering.

Key words: human telomerase reverse transcriptase；transfection；chondrocytes；cell proliferation