TGF-ALFA 因子對豬早期胚體外發育及胚內源性基因表現之影響

本研究計畫旨在探討，豬發情配種後不同生理階段之輸卵管上皮細胞，其基因轉譯物對於豬胚早期發育之調控機制。供試豬隻於注射人類絨毛激性腺素 (hCG) 後50~60、96 及 120 小時，應用外科手術截取其輸卵管，分別抽取其 Total RNA，並經進行 反轉錄-聚合酵素連鎖反應 (DDRT-PCR)測試，比較各不同生理時段之豬輸卵管上皮細胞之基因表現差異性;試驗結果證實以隨機引子4 (arbitrary primer 4) 及錨定引子3(anchor primer 3)，可明確分辨豬輸卵管上皮細胞中(96/120-1-R4A3) 基因表現有生理時間特異性;進一步經選殖、定序及 GCG 軟體比對後，確認該(96/120-1-R4A3) 基因片段為豬轉型生長因子-α (TGF-α)。後續試驗針對豬胚體外培養系統中添加該轉型生長因子-α並探討其對胚源性基因表現之調控情形;結果發現添加20ng/ml 的轉型生長因子-α 條件下對於 4-細胞期之豬胚在進行共培養3 小時後，再抽取其 Total RNA 進行基因 差異表現之分析，證實豬胚添加有轉型生長因子-α 被培養者，表現有一基因 TIG-I，係有別豬胚之在未添加生長因子被培養者。而該TIG-I 基因進一步選殖、定序及 GCG 軟體比對後，證實其序列類似於小鼠早期胚之mo25 基因 (89%) 者然， 鑑於在小鼠之mo25 基因序列中具有鈣離子結合區，顯示其功能涉及細胞訊息之傳導;處於 4-細胞期豬胚表現之TIG-I，其作用是否亦類似於小鼠之mo25 者然，誠值得進一步試驗詳加探討。In the present study, timely expression of gene(s) in porcine oviduct epithelia was identified and the consequent effect of the specified ligant on expression of embryonic gene at early stages of development was elucidated. Total RNA extracted from the oviduct epithelia of gilts, fallotomized at 50-60, 96 and 120 h post the injection of human chorionic gonadotrophin (hCG), were subjected to analyses by mRNA differential display and by reverse transcription-polymerase chain reaction (DDRT-PCR), respectively. Results from sequencing analysis confirmed that the sequence of gene fragment of (96/120)-1-R4A3, expressed by porcine oviduct epithelia at 96 and 120 h post the hCG injection, was identical to that of gene encoding the porcine transforming growth factor-α(TGF-α). Moreover, by using an in vitro culture system, it was found that the expression of TIG-I gene in early 4-cell of porcine embryos does appear to be TGF-α dependent. The cDNA sequence of TIG-I gene was further confirmed to be homologous to that of mo25 cDNA found in mouse early embryos and the sequence also appeared to be having a Ca-binding domain located within the coding region. These results imply that TGF-α gene timely expressed in the epithelia of porcine oviducts is essentially required for porcine early embryos to activate their signal transduction pathway