Establishment of an immortalized porcine preadipocyte clone and adipocyte differentiation

動物細胞培養是研究細胞的生理、增生、分化與探討基因功能、表現調控機制等之重要模式。早期，科學家使用初代細胞培養來進行研究，但是初代細胞在數次的繼代後，即出現細胞生長停滯，甚或死亡的現象。所以每次的實驗都要重新採取一批細胞。但是每次由組織取得之細胞除了在試驗花費不貲外，所取得的細胞族群也雜而不齊，造成許多研究結果的不一致，所以一株穩定的不朽化細胞株對於做研究是非常重要的。 本論文的研究目的是藉著恢復端粒酶活性與表現人類乳突病毒第16型的E7 (HPV 16-E7)蛋白建立不朽化豬的脂肪前身細胞株。實驗所需的初代細胞由二週齡三品種雜交豬(Landrace, Yorkshire, and Duroc)的背脂分離出來，試驗所需要之反轉錄病毒由GP2-293細胞產出，在細胞第一代時利用反轉錄病毒轉染human telomerase reverse transcriptase (hTERT)後，以puromycin篩選一週，存活之細胞再進行HPV 16-E7的轉染，以histidinol作篩選。在histidinol篩選後，利用細胞選殖得到許多細胞殖系，並且利用RT-PCR確定其hTERT與HPV 16-E7的表現，其中一株細胞植系生長快速並且在以分化液(含有insulin, dexamethasone, rosiglitazone等)誘導分化後，能夠分化成脂肪細胞，將其命名為LYD1。試驗進一步測試了pref-1、PPARγ、aP2與LPL的基因表現，推測LYD1為脂肪前身細胞，並且具有分化能力。 因為LYD1細胞能夠表現hTERT與HPV 16-E7基因，我們推測LYD1細胞能夠無限制的分裂，此外，LYD1細胞能夠以分化液誘導分化成脂肪細胞。因此，若能確定與脂肪細胞分化相關的基因表現，即能確定LYD1細胞是一株不朽化的豬脂肪前身細胞株。Culture of animal cells is a basic and important technique for research and development of biotechnological products. It is used to investigate cell physiology, proliferation, differentiation, and function of genes and regulation of their expression. Originally, scientists employ primary cell culture for research but primary cultured cells will be senescent or die after multiple passages. For this reason, they have to isolate new cells from tissues for each experiment. Not only greater cost of time and money is needed but also the result of experiment will vary due to the differences among batches of cells. As a result, a stable clone is essential for better research. The aim of our study was to establish an immortalized pig preadipocyte by introduction of a combination of telomerase and human papillomavirus type 16 E7 (HPV 16-E7). The primary porcine stromal vascular cells were isolated from back fat of a two-week hybrid pig (Landrace, Yorkshire, and Duroc). Retrovirus with human telomere reverse transcriptase (hTERT) or HPV 16-E7 were produced by GP2-293 packaging cell line. Retrovirus with hTERT infected primary cells at passage one and then, cells were selected by puromycin for two weeks. The cells that were alive after treatment of puromycin, indicating a successful expression of telomerase. We also confirmed that the telomerase mRNA was present in the selected cells. They were infected with HPV 16-E7 containing retrovirus, and selected by histidinol treatments. After histidinol selection, pure cell strains were obtained by cell cloning. The expression of hTERT and HPV 16-E7 mRNA in several clones of porcine preadipocyte were confirmed by RT-PCR. One of the clones grew very fast and could be induced to differentiate into adipocyte when treated with adipocyte differentiation medium (insulin, dexamethasone, rosiglitazone, containing DMEM/F12). We named this cell clone LYD1. Expression of preadipocyte factor 1 (pref-1), peroxisome proliferator activated-receptor gamma (PPARγ）, lipoprotein lipase（LPL） and adipocyte-specific fatty acid binding protein（aP2） were determined. Because LYD1 cells can express hTERT and HPV 16-E7 genes, we conjecture that LYD1 cells can proliferate indefinitely. These LYD1 cells could be induced to differentiate into adipocyte by differentiation medium. Therefore, if we can confirm the expression of adipocyte specific gene, it will become a suitable porcine preadipocyte clonal cell line.致謝 Ⅰ 中文摘要 Ⅱ 英文摘要 Ⅲ 圖目錄 V 表目錄 VII 文獻檢討 1 材料方法 11 結果 19 討論 23 結論 38 參考文獻 39 附錄 4