Effect Of Human Telomerase Reverse Transcriptase On Biological Behaviour Of Human Embryonic Fibroblast Cell

Background: Telomerase is associated with malignant proliferation and aging of cells. On one hand, over expression of telomerase plays a vital role in the infinite proliferation of tumor cells. On the other hand, the activation of telomerase is an important way to extend the life-span of somatic cells. Of the three subunits of telomerase, only human telomerase reverse transcriptase (hTERT/hTRT/hEST2) expression is coincide with telomerase activation. Studies have showed that ectopic expression of hTERT in somatic cells which didn't have telomerase expression could largely extend the cell life-span. So there are researches about using somatic cells transfected with hTERT as seed cells in tissue engineering. As a chief component of dermis, the fibroblast also encounters the problem of senescence and the difficulty of amplification. Therefore, in this paper, we preliminary study the effect of exogenous hTERT on the biological behaviours of human embryonic fibroblasts(hEF) and futher explore possibility of hEF cells transfected with hTERT gene to be seeds of dermis.Aim: Sense hTERT fluorescent eukaryotic expressing vector was constructed and then transfected into hEFs to explore the effects of exogenous hTERT on biological behaviours of hEF. Methods: Construct the whole length cDNA of hTERT gene into fluorescent eukaryotic expressing vector pIRES2-EGFP with DNA recombinant technology. After identification by enzyme digestion, the sense recombinant plasmid of hTERT was transfected into hEF by Lipofectin reagent. We then use PCR,RT-PCR and Western blot to identify the correction oftransfection from DNA,mRNA and protein levels. Subsequently, telomerase activity by TRAP-ELISA, telomeric restriction fragment (TRF) by Southern blot, cell growth curve by MTT, cell cycle distribution by flow cytometry, expression of PCNA, ID1,Ⅰand Ⅲ collagen by Western blot, cell morphology under light telescope and transmissive electronic telescope, tumorigenesis in nude mice were examined and evaluated in transfected and untransfected cells.Results: (1) Successfully construct the hTERT-pIRES2-EGFP fluorescent eukaryotic expressing vector. (2) Sense hTERT-pIRES2-EGFP fluorescent eukaryotic expressing vector and pIRES2-EGFP vector were transfected into hEFs with Lipofectin reagent and gained one clone through G418 selection respectively, which were named as hEF-hTERT and hEF-EGFP. The cells were examined from DNA, mRNA and protein levels to determine the correction of transfection. It showed that exogenous hTERT gene could be transcripted and translated in hEF-hTERT cells. (3) The telomerase activity was significantly higher in hEF-hTERT than that in hEF and hEF-EGFP(0.290±0.040 vs 0.110±0.012, 0.093±0.032,P<0.01). Terminal restriction fragment (TRF) length in hEF-hTERT, hEF-EGFP, hEF was 6.0kb,5.3kb,5.4kb respectively. After cultured in vitro, the hTERT expression in hEF and hEF-EGFP cells was decreased gradually, the cell growth became slow, whereas the hTERT expression in hEF-hTERT cells maintained at a high level and cell doubled through 2-3 days after 70 passages. (4) Exogenous hTERT transfection has no effect on the mRNA transcription of hTR and TP1 in both transfected and untransfected cells. (5) Compared with hEF and hEF-EGFP cells, proliferation in hEF-hTERT cells was increased obviously. Expression level of PCNA,Id1,Ⅰ and Ⅲ collagen in hEF-hTERT was also higher than that in hEF or hEF-EGFP cells. Flow cytometric analysis displayed an accumulation of transfected hEFs in S and G2M phase of cell cycle and proliferation index (PI)and a decreased percentage of G0/G1 phase in hEF-hTERT. Ultrastructure under transmission electronic microscope revealed that organelles such as mitochondria or endoplasmic reticulum in hEF-hTERT cells were more abundant compared with hEF or hEF-EGFP cells. (6) After transfection, the number of chromosomes in hEF-hTERT was still 46, which was the same as that of normal primary hEF cells. Flow cytometry displayed no abnormal DNA ploidy. No tumorigenicity in nude mice wa...