The Establishment Of Caprine Ovarian Interstitial Cells By Transfecting HTERT Gene

The culture in vitro of ovarian interstitial cells(OICs) is an important method to study the ovarian functions and action mechanism, there are many advantages to use the cell lines in the experiments, such as the higy purity, the stable characters and the user-friendly control. The establishment of caprine OIC line will offer excellent experimental materials for the further study about ovary. In this study, the aim plasmid namely pCI-neo-hTERT was transfected to primary caprine ovarian interstitial cells (COICs) to be immortalized. Experimental results obtained as follows:1. Caprine ovaries were collected on the aseptic condition and then cutted, incubated for 3 hours with proper amount collagenase II ( the concentration is 1.5mg/mL), purified with differential velocity adherent technique , cultured at 37℃and 5% CO2. The result showed that the culture cells adhered to the wall, presented long fusiform or mutiple shape, a confluent monolayer was former with the cells being interlaced network. In the immunocytochemical experiment, the isolated cells were positive vimentin and negative keratin.2. The second generation caprine ovarian interstitial cells (COICs) were transfected with pCI-neo-hTERT by lipofectamine. Cells were screened with the optimum screening concentration, which was 400μg/ml G418, for 14 days. positive clones was acquired and then digested to enlarged passage culture. The transfected COICs remain consistent in the progress of passage culture, namely fibroblast-like as same as primary cells. Cells grow well until the 53rd generation now. In the immunocytochemical staining, the 50th generation cells transfected by hTERT showed positive of hTERT while the untransfected cells were negative, which proved that hTERT gene has integrated in the cellular genome and at the same time expressed stably. The transfected cells still showed positive vimentin and negative keratin with the normal surface marker. Cells transfected by hTERT has contact inhibition like the primary cells according to the cell curves, which shows the ordinary biology characteristics of the immortalized cells.3. The cell cycle analysis of the untransfected second generation cells and transfected 50th generation cells demonstrated that the immortalized cells has the normal cell cycle and the occupation probabilityof s-phase is obvious large than untransfected cells. The apoptosis results show that apoptosis rate of transfected cells is distinct higher and the living cells dominated over 90%. The DNA ploidy analysis results illustrate that the transfected 50th generation cells remain normal control diploid. In the soft agar cloneing test of untransfected and transfected cells as well as a positive control namely SP20, The COICs including untransfected and transfected cells could not grow in the soft agar, which shows there were no neoplastic transformation in thest immortalized cells.In summary, hTERT gene was transfected into the caprine ovarian interstitial cells with the basic of the primary cells successfully isolated and cultured in this study. Telomerase was activated in the cells transfected by hTERT gene, which extended the cellular lifespan enormously, so the immortalized caprine ovarian interstitial cell line was established. The transfected cell line shows no neoplastic transformation and at the same time maintains normal cells'biological property, which is a foundation for the further studies.