Study On Culture And Immortalization Of Renal Tubular Epithelial Cells In Newborn Goat

Recently,the researchers focused their attention on cells physiology, cell injury and repair studies with renal tubular epithelial cell of newborn goat and avoided the complexity of the whole animal or organ. However, primary culture of renal tubular epithelial cell have a finite proliferative lifespan before they undergo permant growth arrest and these facts make studies difficult. In this study, kidney was collected for primary renal tubular epithelial cell culture with collagenaseⅣin vitro. Human Telomerase Reverse Transcriptase (hTERT) gene was transfected into the purified primary cell in order to obtain stable renal tubular epithelial cell line, working out a reliable cell system for further studies. The results of this research obtained as follows:(1)Isolated renal tubular epithelial cells were obtained by collagenaseⅣ. The results showed that a number of pure and highly active cell aggregates were obtained by inverted phase contrast microscope. After 3 days, the endothelial cells merged and looked like cobblestones. The cells was covered the bottom pan by 4 days and cultured up every 3 days. Cells by us could be successively propageted 12 times and in the culture of 13 passages, cells entered into senescence phase and can not subculture again.(2)The results showed the purified primary cells were stained positively for cytokeratin 18 antigen and Alkaline phosphatase assay. Cells were with characteristic of microvilli and desmosome by TEM.(3)The renal tubular epithelial cells transfected with pCI-neo-hTERT were screened with G418 for 14～15 days, the negative cells died and durg resistant cells were matained to ensure a stably positive population. The cells clones were transmitted using small filter papper and expanded for further culture.The renal tubular epithelial cells transfected with pCI-neo-hTERT have a characteristic of polygonal cobblestone. Growth curve, was typical"S"type, showed that transfected cells had more powerful proliferation activities.(4)We have detected the expression of hTERT in the cells by RT-PCR and Western blotting. The transfected cells were stained positively for cytokeratin 18 antigen and Alkaline phosphatase assay, it showed phenotype antigen of transfected cells had no loss. The cell cycles and cell apoptosis of cells were detected by FCM, we found that transfected cells had higher proliferation activity than primary cells and had lower apoptosis than cells of 10 passages. The results displaied that the hTERT was transfected into renal tubular epithelial cells successfully and the cell life was prolonged and transfected cells maintained properties of primary cells.Cells has been maintained in culture, up to 20 passages till now, and can be used in future immortaliaed research.