Isolation And Culture Of Goat Endometrial Stromal Cell And Study On Its Immortalization

Obtaining primary cells in vitro studies is limited by the time and cost involved in preparation, and frequent variations in the cells'physiology. These facts make it difficult to compare results from experiments using different cell preparations or passages. Immortalized cell lines possess advantages over primary cells, such as better uniformity of cultures, easier availability and easier genetic manipulation. So, in order to overcome this problem and provide the homogeneous seed cells for futher studies, the aim of this research is to establishment the immortalized goat endomentrail cell lines by transfected the plasmid pCI-neo-hTERT. Experimental results obtained as follows:1. Goat endometrium were digested by collagenasesⅡ, then were isolated by brachytely centrifugalization-nature settled, cultured in DF12 + 10 % new calf serum at 37℃and 5 % CO2 condition. The purity is about 95 %. When seeding at 1×106 cell/mL, most ESCs became adherent after 24 hours and monolayer were formed after culturing 5 days. ESCs show that the long spindle-shaped or the triangle and arrange parallel. Detected the cultural characterization of ESCs. The result show that the positive for vimentin and negative for cytokeratin.2. 600μg/mL of G418 is the most suitable concentration of transfection by concentration gradient sieving. The eukaryotic expression plasmid pCI-neo-hTERT was transfected into Goat ESCs. After sequential screening by of G418. Five monoclonal ESCs were gained finally and designated as A5, B5, B6, C4, and C6 respectively. One of the clones(A5) was cultured for up to 50 passages and stilly cultured. 3. Immunocytochemical staining method shows that positive for hTERT in transfected ESCs while not in untransfected ESCs. The transferred cells were detected the activity of telomerase by PCR-ELISA in 20th generation and 50th generation. The results showed the high activity of telomerase was positive in transferred cells. The activity of telomerase in ESCs can be active through ectogenic gene hTERT.4. The vimentin which is important marker of ESCs were detected. The result showed the transfected cells were stained positively for vimentin and negetively for cytokeratin. Through map of growth curve and observation of cell culture, we find that the transfected cells have the normal cell generation cycle and contact inhibition. The ability of transfected ESCs and untransfected ESCs to grow in anchorage-independent manner by using a clonogenic soft agar assay. The result showed that this two cells failed to form any colonies after 2 weeks. Above results indicated that the transfected ESCs maintained the propertied of normal cells, and almostly had no neoplastic transformation.5. Detect the effection of proliferation with different concentration and group of hormonal sex by MTT. The results indicated that the combination of 100 nmol/L E2 and 10 nmol/L P4 in media can stimulate the proliferation of transfected stromal cells.Results suggested that the hTERT was transfected into goat ESCs successfully, the cell proliferation life-span was elongated in vitro, cells immortalised by hTERT retain their original characteristic. This cell line will be a powerful and consistent resource for in vitro studies.