Immortalization Of Goat Endometrial Stromal And Epithelial Cells

Endometrium is a exceptive immunology location. In gestational period, It ensure the fetus with half antigen not to be rejection and the need of nutrition, meanwhile, it guarantee the mother not be impaired by fetal antigen and uterus not be infected by pathogenic bacteria. Endometrium educes very important effect in the whole theriogenologic process. Therefore it is important to establish culture system in vitro. It is time-consuming and need great effort to prepare primary cells. Because of common variations in the primary cell's physiology, it is difficult to compare results from experiments with different cell preparations or passages. Immortalization of these cells which have so many good qualities including homogeneity, stability and easy handling would greatly facilitate studies. Many studies showed temolerase activity in cells is transcription control of TERT that act as a reverse transcriptase. Introduction of hTERT could enhance the activity of temolerase in normal cells, elongate the life span of various cells and even result in immortalisation. In this study, we isolated goat ESCs and EECs in vitro, the plasmid pCI-neo-hTERT was transfected into ESCs and EECs by Sunma-sofast. Experimental results obtained as follows:1. Goat endometrium were digested by 1.5 g/mL collagenasesⅡ, then the single cells or the glandule mass cells were isolated by brachytely centrifugalization-nature settled, cultured in DF12 + 10% new calf serum at 37℃and 5% CO2 condition. The single cells became adherent after 0.5 h of culture. Two shapes cells were found, spindle and polygonal. Immunocytochemical staining method showed that this 2 shapes of stromal cells were positive for vimentin, with 95% of positive rate, and negative for cytokeratin. It shows cells cultured are ESCs. The glandule mass became adherent after culture 12 h, the cells looked like cobblestones and were stained positively for cytokeratin relative antigen , whicht shows cells cultured are EECs and the purity was exceed 95%.2. ESCs were transfected with pCI-neo-hTERT by summa-sofast. 24 h after transfection, cells were screened with 600μg/mL G418 for 14 days. Drug resistant cells were selected and matained in 300μg/mL to ensure a stably positive population, positive clones were expanded for further culture. The vimentin which is important marker of ESCs were detected. The result showed hTERT-ESCs were stained positively for vimentin and negetively for cytokeratin. Immunocytochemical staining method shows that positive for hTERT in transfected ESCs. One of clones, was cultured up to 60 passages after introduction of the hTERT. The results showed that activity of telomerase in goat ESCs can be active through ectogenic gene hTERT. The cells after transfected was named hTERT-ESCs.3. Through map of growth curve and observation of cell culture, it was found that the transfected cells have the normal cell generation cycle and contact inhibition. hTERT-ESCs were injected into nude mice, after 2 months, no tumors developed in nude mice. 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 these two cells failed to form any colonies after 2 weeks. The proliferative ability of hTERT-ESCs cultured in medium containing different concentration serum was detected by MTT assay. The result showed that the proliferative ability of hTERT-ESCs cultured in medium containing high concentration of serum is higher than it in low concentration of serum. The serum requirement of the immortalised cells was not lose. 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.4. EECs were transfected with pCI-neo-hTERT. 24 h after transfection, cells were screened with 500μg/mL G418 for 14 days. Drug resistant cells were selected and matained in 250μg/mL to ensure a stably positive population, positive clones were expanded for further culture. The confluent monolayer of transfected EECs looked like cobblestones, it were positive for cytokeratin. Immunocytochemical staining method shows that positive for hTERT in transfected EECs while not in untransfected EECs. The transfected EECs was cultured up to 55 passages. The results showed that activity of telomerase in goat EECs can be active through ectogenic gene hTERT. The transfected EECs was named hTERT-EECs.5. Through map of growth curve and observation of cell culture, it was found that the hTERT-EECs have the normal cell generation cycle and contact inhibition. hTERT-EECs were injected into nude mice, after 2 months, no tumors developed in nude mice. The ability of hTERT-EECs and untransfected EECs to grow in anchorage-independent manner by using a clonogenic soft agar assay. The result showed that these two cells failed to form any colonies after 2 weeks. The serum requirement of hTERT-EECs was not lose. 100 nmol/L E2 can stimulate the proliferation of transfected hTERT-EECs;50 nmol/L,100 nmol/L P4 can inhibit the proliferation of hTERT-EECs.Overall results suggested that the hTERT was transfected into goat ESCs and EECs successfully, the cell proliferation life-span was elongated in vitro, cells immortalised by hTERT retain their original characteristic. Therefore we concluded that the goat immortalised ESCs and EECs were established. It was first reported in domestic and foreign.