Establishment Of Swine Intestinal Epithelial Cells (SIECs) Strain Transcripted By HTERT And The Influence Of TGEV On Activities Of SIECs

The intestinal epithelial cells are well characted model and commonly used for studying the function and the pathogenesis of intestine. However, the primary isolated cells of swine small intestinal have a finite proliferative lifespan, known as replicative senescence. In the culture of 10 passages, cells entered into the senescence phase, finally the population appears growth retardation, and cells can not subculture again. Many studies show the temolerase activity in cells is transcription control of TERT that acts as a reverse transcriptase. Introduction of hTERT could enhance the activity of temolerase in normal cells, elongate the lifespan of various cells. In this study, we isolated swine small intestinal epithelial cells (SIECs) in vitro, and the plasmid pCI-neo-hTERT was transfected into SIECs by lipofection. We also studied the influence of the Transmissible Gastroenteritis Virus (TGEV) on activities of SIECs. The experiment results obtained as follows:(1)The intestinal epithelial cells were isolated by mechanical separation. The results showed the cells looked like cobblestones and were stained positively cytokeratine 18, which also showed the cells we isolated and cultured were epithelial cells. The epithelial cells had good capacity of proliferation. In culture of about 10 passages, cells entered into the senescence phase, finally the population appears growth retardation, and cells could not subculture again.( 2 ) The intestinal epithelial cells were transfected with pCI-neo-hTERT by lipofectamine. 48 h after transfection, cells were screened with 400μg/mL G418 for 15 days, drug resistant cells were selected and matained in 200μg/mL to ensure a stably positive cell population. Positive cell population was expanded for further culture. The transfected cells were detected the expression of hTERT mRNA by RT-PCR. The results showed that the hTERT mRNA was expressed in transferred cells, the ectogenic gene hTERT had integrated into genome of transferred cells stably. The cells were cultured up to 80 passages after introduction of the hTERT. We named it as intestinal epithelial cells transfected hTERT(hTERT-SIECs)。(3)The 60th genenration SIECs were inoculated by Transmissible gastroenteritis virus (TGEV) M12F2 strain setting as normal control grouop as parallel groups. We measured the activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase of inoculated cells and control cells at 24,36 and 72 h respectively, and detected the cell cycle of the 2rd and 5th genenration cells which had been inoculated. The results showed that SIECs inoculated by M12F2 strain of TGEV had no obvious changes. Compared with control group, activities of Na+-K+-ATPase and Ca2+-Mg22+-ATPase of cells were lower, cells in S phase were decreased and cell apoptosis was increased .On the basis of isolation and culture of swine small intestinal epithelial cells successfully, the hTERT was transfected into SIECs by lipofection. After transfection of hTERT, the SIECs had been passaged to 80 generation, and had no obvious changes inoculated with TGEV. The TGEV depressed the activities of SIECs. It may provide a new strategy of pathogenesis of TGEV.