- 學位論文
利用Pax-4基因轉殖方法增進鼠胚胎幹細胞體外分化為胰島素製造細胞
Enhancement of Insulin-Producing Cell Differentiation from Embryonic Stem Cell Using Pax-4-Nucleofection Method
摘要
如何能夠有效成功的應用幹細胞，其方法包括：誘導分化及基因轉殖等等。在胰臟發育過程所必需的轉錄因子Pax4已經被證實是一個誘導分化為胰島素製造細胞（insulin producing cells）的重要基因，以前有關研究發現含Pax4的穩定細胞株（stable clone）確實可以增加胰島素製造細胞的形成。雖然我們的實驗並無直接將基因組嵌入細胞株內，造成細胞無法穩定且持久的保有此基因，但是我們目前的實驗結果仍然可以促使胚胎幹細胞誘導分化成為胰島素製造細胞。 首先使胚胎幹細胞形成embryonic body (EB)，四天後將EB打成單顆細胞。之後，用一個有效的基因轉殖法將基因送到細胞內，一組是Pax4，而另一組是送入空的質體當控制組，合併G418選殖，挑出帶有Pax4的胚胎幹細胞，而Pax4的表現會隨著實驗一直到實驗結束，再將有表現Pax4的胚胎幹細胞培養在事先用fibronectin-coated 的培養皿上，驅使細胞朝向內胚層的細胞分化，並且用quantitative PCR（Q-PCR）來偵測內胚層或胰臟相關基因的表現，也分別利用流式細胞儀、免疫染色、酵素免疫分析等方法來偵測胰島素的分泌量，並準備事先打STZ破壞胰臟功能的裸鼠，再將帶有Pax4的胚胎幹細胞打入老鼠體內，並且評估血糖的恢復情形。 利用轉殖基因技術（nucleofection）合併G418 來篩選細胞發現有高達70%左右的效率。這種高效率的eGFP表現能一直維持到研究的結束。胰臟的發展也因pax4 nucleofection 而加速其分化過程。在Pax4 nucleofection 以後的第8天，藉由Q-PCR偵測到 foxa2 、mixl1 、pdx1 、胰島素和somatostatin等基因的表現。接受Pax4基因轉殖的胚胎幹細胞，在第18天時高達35%的分化細胞表現胰島素，而以空質體基因轉殖的細胞有10%的細胞會表現胰島素，而Pax4 RNAi載體轉入的細胞組僅有8%會表現胰島素。另外，將具有較高表現胰島素的細胞打入經由STZ處理過的裸鼠身上進行胚胎幹細胞的移植實驗，結果發現實驗組的裸鼠的血糖恢復正常值。 本研究中在細胞內和細胞外的實驗都顯示，經由Pax4 non-permanent nucleofection 的結果都可增加胰島素製造細胞的形成。藉由Pax4短暫的表現及G418 的篩選，並且引導胚胎幹細胞的分化意向，如此，不但增加胰島素製造細胞的形成，而且可降低畸形瘤（teratoma）形成的危險性，利用pax4 nucleofection 轉殖細胞之潛力也為未來醫學治療上帶來更多的希望。
並列摘要
For therapeutic considerations, the criteria for successful stem cell (SC) based applications should include the useful strategy that induces the stem cells commitment more effectively. The manipulation should compromise safety considerations. Pax4, a transcription factor, has been demonstrated to be essential in the insulin producing cells (IPC) formation. The stable clone of embryonic stem cells (ES) with constitutive external pax4 gene expression has been established in previous studies, and the IPC’s higher efficiency has been revealed. However, permanent/irreversible change of gene in cells is still unacceptable for clinical usage due to genome integrity consideration. In the current study, we demonstrate that transfection with non-permanent pax4 gene is still effective to force ES to become IPC. The method of embryonic body (EB) formation was used for endoderm/mesoderm commitment before the following pax4 gene transfection. The use of nucleofection provided high efficiency of gene delivery, and when combined with G418 selection, a high level of pax4 gene expressed cells was observed till the end of the experiment. The ES at day 4 after embryoid body formation was dissociated and nucleofected with pax4 or mock plasmid (control) and fibronectin was employed as the coating material to maintain the endoderm lineage differentiation. The expression of endoderm or pancreatic differentiation related gene was monitored by quantitative PCR. The population of IPC at day 18 of the experiment was investigated by FACS, and the content/secretion of insulin was estimated by immunofluorescent staining and the ELISA assay. STZ pretreated nude mice were used to eliminate the normoglucemia restoration of pax4+ ES implantation. A high efficiency (approximately 70%) was demonstrated when using nucleofection to deliver eukaryotic expression plasmid; approximately 70% of efficiency was shown. By G418 selection, the high percentage of cells express eGFP could be maintained till the end of the study. The pancreatic differentiation seems to be accelerated by the pax4 nucleofection. By Q-PCR, foxa2, mixl1, pdx1, insulin and somatostatin were expressed at day 8 after the nucleofection. Higher percentage (35%) of differentiated cells express insulin at day 18 of the experiment in the group of pax-4 transfected ES as compared to 10% in group of cells with mock plasmid and 8% in the group with pax-4 RNAi vector transfection. A higher IPC in the population also reflected in the insulin content by ELISA assay, and the glucose dependency was demonstrated in insulin secretion level. In the transplantation study of STZ pretreated nude mice, normoglucemia restoration was observed. The IPCs enhancement from embryoid body dissociated cells through pax-4 non-permanent nucleofection was demonstrated in the studies both in vitro and in vivo. By pax4 transient expression and G418 selection, the tendency of ES differentiation was guided. Not only the IPC formation becomes more effective, also less risk of teratoma formation was also shown. The potential of pax-4-nucleofection cells for medical treatment is promising.