Studies On The Culture System And Immortalization Of Mammary Epithelial Cells In Swamp Buffaloes

The purpose of the study was to establish an in vitro culture system of immortalization mammary epithelial cells in swamp buffaloes, so as to provide more evidences for further studies on development of mammary gland and its milking mechanism. The mid-laction mammary gland tissue in swamp buffaloes was used as the research material in the study as follows:The first part of the experiment was to isolate, purify,characterize and culture the swamp buffalo mammary epithelial cells .Primary buffalo mammary epithelial cells were cultivated from the mid-laction mammary gland tissue by either gland explant or separation by trypsin-collagenase. The method of the different adhesive and the sensitivity of trypsin-collagenase can be used to isolate and purify swamp buffalo mammary epithelial cells. After purification, Morphological observation indicates that the major morphology of epithelial cells are cobblestone, and the cell growth curve assumes "S" shape. Analysis showed that the normal chromosome number(2n=48) and the type also possed in the cultured cells; The results for immunochemical identification of cytokeratin 8 and amplification of a-casein gene in mammary epithelial cells indicated that the cells were swamp buffalo mammary epithelial cells.The method of swamp buffalo mammary epithelial cells immortalization was also studied. SV40 was transfected into the fourth generation of purification buffalo Mammary Epithelial cells by the DNA- calcium phosphate precipitation method. After process screening, the cells were continuously cultured . By using Telomerase TRAP Detection Kit with silver staining to detect the cells telomerase activity, it showed that the telomerase activity of the transfected cells was higher than the non-transfected, and the transfected cells enlarged. However, there was no significant difference in growth rate between transfected and non-transfected cells.Two primers were designed based on the Bos Taurus ER and PRLR cDNA released at the GeneBank. Estrogen receptor a and the prolactin receptor were amplified by RT-PCR technology from the immortalized mammary epithelial cells in swamp buffaloes. The partial sequences of ER and PRLR with length of 171bp and 253bp were obtained respectively. The cloned ER and PRLR sequences shared 100% and 99.2%, respectively, homologue to the response sequence in cows. The result confirmed that ER and PRLR also could be expressed in the immortalized mammary epithelial cells compared to those in the primary mammary epithelial cells. Cell proliferation dealed with different concentrations of estradion was measured by MTT detection. The result showed the concentration of 5mg/L estradion had the most significant stimulating effect on cells proliferation.In conclusions: (1)The primary mammary epithelial cell could be easily obtained from the gland explant; (2) Mammary epithelial cells in swamp buffaloes were identified by PCR amplification of a-casein gene and immunochemical identification of cytokeratin8; and the cells had the normal function of secreting protein casein; (3) SV40 was expressed in mammary epithelial cells in swamp buffaloes by the DNA-calcium phosphate precipitation method. The telomerase activity of transfected cells was higher than non-transfected, and the epithelial cells was sucessfully immortalizated. (4) Estrogen receptor a and prolactin receptor espression still worked in the transferred cells; (5) The proliferation of mammary epithelial cells in swamp buffaloes were promoted by appropriate concentration of estrogen. However, high concentrations of estradion could suppress the cells proliferation. The concentration of 5mg/L estradion had the most significant stimulating effect on cells proliferation.