The feasibility of establishing immortal caprine mammary epithelial cell line by transfection of telomerase
| Autor: | Meng-Wei Ke, 柯孟韡 |
|---|---|
| Rok vydání: | 2005 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 93 Establishment of a stable telomerase-expressed caprine mammary epithelial cell line Abstract Production of recombinant biopharmaceuticals from the mammary gland of domestic animals through transgenic nuclear transfer technology is a dominant system in scientific agriculture. Low efficiency, high costs and time-consuming, however, are common problems in producing transgenic domestic animals. Furthermore, improper plasmid constructs and gene source from different species or tissues are more serious problems making protein lose its biological activity. In order to solve the above problems, the culture of primary mammary epithelial cell for pioneer testing the expressed recombinant protein biological activity and characteristics is recommended, but the finite life span of cultured primary mammary epithelial cell is more concerned. SV40 large T-antigen transformed cells are also immortal but they accompany with carcinogenesis and lose of normal cellular functions. Stable expression of telomerase reverse transcriptase (TERT) in primary culture cells can induce cells immortalized and maintain cells with both normal cellular morphology and functions. The aim in this study is to establish a telomerase-immortalized caprine mammary epithelial cell line (CMEC) as a pioneer testing platform to examine the biological activity of interesting gene expressed in transgenic animal. In the beginning, the characteristics of the cultured CMEC were examined and verified it specifically expressed cytokeratin 18 and formed alveolar structure on MATRIGEL matrix, showing the CMEC still possess the normal function of mammary epithelial cell. Further, we transfected CMEC with hTERT and selected with proper antibiotics for establishing a stable hTERT-expressed caprine mammary epithelial cell line. As the immortal CMEC cell line is established, it can not only be used for mammary gland development study but also for cellular senescence and breast cancer research. Using mRNA subtractive hybridization and differential display techniques, caveolin-1 was seem to be lost or down-regulated in human mammary adenocarcinoma-derived cells comparing to normal mammary epithelial cell. For further researches, a rabbit polyclonal peptide antibody specially recognizing different species’ caveolin-1 protein was developed. This antibody could against 22-24 kDa caveolin-1 protein extracted from human epithelial cell line A431, CMEC, mice fibroblast cell line NIH3T3 and recombinant caveolin-1 transiently-expressed rat pituitary epithelial cell line GH3 by western blotting and immunostain techniques. This result had proved this antibody can be used for examining caveolin-1 protein expressed in mouse, rat, goat and human species. To explore whether this antibody can be applied for breast cancer diagnosis, we performed immunohistochemistry on breast tumor biopsy and showed significant difference in caveolin-1 expression between normal with tumor was observed. To the conclusion, once the immortal CMEC being established, we can study the development of mammary gland and the production of recombinant protein biopharmaceuticals. Further, combining with caveolin-1 antibody, the study of cellular senescence and signal transduction through caveolin-1 can also be achieved. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
| Externí odkaz: |
|