Expression And Regulation Of Human Telomerase Reverse Transcriptase Gene On Epidermal Stem Cells Derived From Human Skin

As the skin tissue-specific stem cells, epidermal stem cells have strong proliferative potential and self-renewal capacity. They are also the main source for generation, reparation and rebuilding of skin, which make them seed cells in tissue engineering. Proliferation and differentiation of epidermal stem cells to understand regulatory mechanisms is an important prerequisite for their application, and theoretical basis. The research on epidermal stem cells has focus on stem cell niches, integrin family, Wnt signaling pathway, Notch signaling pathway, c-Myc and p63, p16 gene expression and other pathways, which have made progress. Recent study found that after epidermal stem cells in vitro and in vivo the proliferation of performance are very different, that is very easy to lose their stem cell properties, in vitro replication more difficult, limiting further research.. The loss of telomerase activity are known and proliferation related gene expression changes are caused by a variety of adult stem cells in vitro' replication and the main reason for limited expansion. However, epidermal stem cells caused by the reasons for this difference is also true, is not yet clear.Telomerase is a telomere end can extend and maintain telomere length ribosomal protein, it to its own RNA synthesis of telomeric DNA as a template and added to the chromosome ends, add the missing chromosome telomere DNA replication in order to extend telomeres, thus extending life and even to immortalized cells. Human telomerase includes a telomere DNA complementary to the RNA subunit (hTR) and a reverse transcriptase activity of telomerase reverse transcriptase (hTERT), and other associated protein (TPI). Of the three subunits of telomerase, only human telomerase reverse transcriptase (hTERT) expression is coincide with telomerase activation.In humans, telomerase activity present in stem cells, germ cells, some capacity for regeneration of body cells and the vast majority of malignant tumors. Its expression is highly active malignant proliferation of tumor cells is an important condition, but the amount of expression but also has extended the role of cell life. With the conduct of stem cell differentiation, telomerase activity may also decrease to terminally differentiated cells can no longer measured telomerase. Up and maintenance of telomerase activity in stem cells, stem cells to improve the replication and amplification, and reveals the stem cell aging mechanisms will be important. Number of applications has been reported in the literature hTERT gene into human bone marrow mesenchymal stem cells, fibroblasts, osteogenic cells, umbilical cord blood mesenchymal cells, retinal pigment cells, endothelial cells and other cells to extend the life of the success of cell proliferation try. Preliminary results from both significantly prolong the cell life cycle, and maintain normal phenotype and no tendency to malignant transformation. Developmental biology of skin current study found that epidermal stem cells in the epidermis is rich in the basal layer and hair follicle growth phase in both the expression of telomerase activity, suggesting that telomerase activity may be related mechanism of proliferation and differentiation of epidermal stem cells also exist between important internal links. Present in vitro telomerase activity of human epidermal stem cells and its expression characteristics of epidermal stem cell proliferation and differentiation is still a lack of understanding of the relationship.In this topic, we observed the hTERT gene and its protein and telomerase activity in cultured epidermal stem cells in vitro. To investigate its effects on cultured human epidermal stem cell biological characteristics of the role and significance. To construct the recombinant plasmid pIRES2-EGFP-hTERT encoding human telomerase reverse transcriptase and enhanced green fluorescent protein and transfect into cultured human fetal epidermal stem cells by liposome-mediated transfection. The expression of hTERT mRNA transcription and protein, telomerase activity and biological characteristic were observed, which may offer new ideas and theories in skin tissue engineering.PartⅠIsolation, cultivation and expression of human telomerase reverse transcriptase in human epidermal stem cellsObjectives:To observe the hTERT gene and protein expression of telomerase activity characteristics of cultured human skin stem cells. To explore the biological characteristics of epidermal stem cell function and significance.Method:The skin samples of fetus were taken from the accidental abortion (24-26 weeks gestational age) which provided by the Department of Maternity, the First Affiliated Hospital of Nanchang University. The use of these samples were obtained with consent from parturients and patients and approved by Medical Ethics Committee of the Hospital. The sample was disposed with Trypsin-EDTA respectively and got the epidermis, and then we digested the epidermis and harvested the epidermal cells. The type IV collagen was used to isolate and purify the human epidermal stem cells, and the epidermal growth factor and the keratinocyte serum free medium were used to culture the cells. To observe the human epidermal stem cells under inverted phase contrast microscope morphology. Theβ1 integrin, keratin 19 and p63 transcription factor were identified by the immunocytochemical stain. The colony forming efficiency were calculated. The proliferation and cycle of human epidermal stem cells were detected with flow cytometry. The expression of hTERT mRNA and hTERT protein levels were respectively measured by reserve transcriptase-polymerase chain reaction (RT-PCR) and Western blot. The telomerase activity was detected by TRAP-ELISA.Results:The cultured cells revealed colony growth. The cloning efficiency was significantly higher than the control group keratinocytes, the difference was statistically significant. (P< 0.05). The cells instage G0/G1 accounted for 82.64% by cell cycle analysis. The (31 integrin, k19 and p63 had positive expression in the epidermal stem cells but not the keratinocyte cells. The hTERT was expressed weakly at mRNA level and protein levels in the human fetal epidermal stem cells. The telomerase activity was weakly positive.Conclusion:1. In the study, the cultured cells isolated by trypsin-EDTA digestion andⅣrapid adhesion revealed colony growth. Most cells were in the G0/G1 phase, which shows that the cultured human fetal epidermal stem cells have the stem cell biological characteristics.2. The hTERT was expressed weakly at mRNA level and protein levels in the human fetal epidermal stem cells. The telomerase activity was weakly positive. The expression and regulation of telomerase reverse transcriptase in human epidermal stem cells may be of great significance to maintain of their self-renewal capacity.PartⅡTransfection of human telomerase reverse transcriptase gene into human epidermal stem cellsObjectives:To transfect the recombinant plasmid pIRES2-EGFP-hTERT encoding human telomerase reverse transcriptase and enhanced green fluorescent protein into cultured human fetal epidermal stem cells, and to observe the hTERT gene and protein expression and telomerase activity of cultured human skin stem cells. To explore the biological characteristics of epidermal stem cell function and significance, which it offer new ideas and theories for the skin tissue engineering. Method:To construct the recombinant plasmid pIRES2-EGFP-hTERT encoding human telomerase reverse transcriptase and enhanced green fluorescent protein and transfect into cultured human fetal epidermal stem cells by liposome-mediated transfection. The positive cells were selected with G418. The plasmid pIRES2-EGFP and plasmid pIRES2-EGFP-hTERT encoding hTERT was transfected into cultured human fetal epidermal stem cells by liposome-mediated transfection. The positive cells were selected with G418. The expression of hTERT mRNA and hTERT protein levels were respectively measured by reserve transcriptase-polymerase chain reaction (RT-PCR) and Western blot. The telomerase activity was detected by TRAP-ELISA. The colony forming efficiency was calculated. The cell cycle of human epidermal stem cells was detected with flow cytometry. The growth curve was detected with MTT. The karyotype analysis in parallel was detected.Results:The plasmid pIRES2-EGFP-hTERT was successfully amplified and constructed recombinant eukaryotic expression. The restriction fragment length consistent with the theoretical expectations. The recombinant plasmid was transfected into cultured human fetal epidermal stem cells. The expression of EGFP-positive cells was observed at 488nm wavelength fluorescence microscope after 24h. The whole cells showed green. The cells selected with G418. The expression of hTERT mRNA and protein and telomerase activity was positive. The expression was higher than the non-transfected group and empty vector group (P<0.05). The difference was statistically significant between non-transfected group and empty vector group. The cells in transfected group did not change shape, revealed colony growth. The cloning efficiency was significantly higher than non-transfected group and empty vector group (P<0.05). Compared with non-transfected group and empty vector group, transfected group cells grew rapidly by growth curve. Cell cycle analysis showed that the cells instage G0/G1 accounted for 84.60%. The chromosomal morphology of cells was normal, diploid karyotypea, without distortion.Conclusion:1. The recombinant plasmid pIRES2-EGFP-hTERT can be successfully transfected into cultured human fetal epidermal stem cells by liposome-mediated transfection method. The expression of hTERT mRNA and protein and telomerase activity was significantly increased.2. The human epidermal stem cells transfed hTERT gene revealed colony growth, enhanced proliferation and life cycle extension. And most of the epidermal stem cells remains relatively quiescent (G0/G1), maintaining the characteristics of stem cells. The karyotype analysis of cells has no distortion. The human epidermal stem cells of hTERT gene regulation can be stably cultured, which may become ideal seed cells of tissue engineering.