本文選題：PDX-1　切入點：cDNA　出處：《安徽醫(yī)科大學》2012年碩士論文　論文類型：學位論文

【摘要】：目的：構建PDX-1基因的克隆載體，為真核表達載體的構建及研究PDX-1誘導干細胞分化的作用機制提供基礎。 方法：采用Trizol方法從大鼠胰島細胞瘤細胞株中提取RNA，通過RT-PCR方法在體外擴增大鼠PDX-1cDNA，瓊脂糖凝膠電泳鑒定，割膠純化后回收目的基因，與pMD-18T克隆載體連接構建，，經限制性內切酶雙酶切及DNA序列分析鑒定pMD-18T-PDX-1的正確構建。 結果：正確構建了含有PDX-1cDNA的克隆載體pMD-18T-PDX-1，其中PDX-1cDNA全長為908bp。 結論：在體外成功克隆了PDX-1基因，并正確構建了PDX-1基因克隆載體，對進一步進行PDX-1在真核細胞中的表達及研究PDX-1在干細胞定向分化中的作用具有重要的意義。 目的：體外培養(yǎng)人臍帶間充質干細胞，將間充質干細胞誘導分化為胰島素分泌細胞，探討誘導分化過程中干細胞向胰島素分泌細胞分化的潛能及功能變化趨勢。 方法：體外培養(yǎng)人臍帶間充質干細胞，培養(yǎng)至貼壁80%，將間充質干細胞分兩組，A組為誘導組，B組為對照組。A組先后予以1mmol/L2-巰基乙醇培養(yǎng)2天，10ng/ml表皮生長因子、10ng/ml堿性成纖維細胞生長因子及2%B27培養(yǎng)7天，之后添加20mmol/L尼克酰胺及艾塞那肽培養(yǎng)7天。B組不添加試劑，繼續(xù)換液培養(yǎng)。通過觀察細胞形態(tài)變化、RT-PCR體外擴增兩組細胞的PDX-1基因的cDNA、放射免疫法測定兩組細胞胰島素分泌量三種方法來鑒定胰島素分泌細胞。 結果：（1）通過形態(tài)學觀察，可見未分化的臍帶間充質干細胞呈長梭形，分界清楚，傳代后細胞呈纖維樣生長，而A組細胞逐漸變小，呈圓形，折光性變強，細胞聚集成團，類似胰島樣細胞，B組細胞仍呈長梭形，分界清楚，纖維樣生長；（2）RT-PCR： A組細胞可擴增出PDX-1基因的cDNA，經瓊脂糖凝膠電泳鑒定，PDX-1目的片段為912bp，與pubmed公布基因片段符合；B組細胞不能擴增出人PDX-1基因的cDNA。（3）通過放射免疫法測定兩組細胞胰島素分泌量，A組可檢測出胰島素分泌，B組未檢出胰島素分泌。 結論：成功在體外微環(huán)境下將人臍帶間充質干細胞誘導分化為胰島素分泌細胞，為干細胞在糖尿病治療領域的應用提供基礎。
[Abstract]:Aim: to construct the clone vector of PDX-1 gene for the construction of eukaryotic expression vector and to study the mechanism of differentiation of stem cells induced by PDX-1. Methods: Trizol method was used to extract RNAs from rat islet cell tumor cell lines. Rat PDX-1cDNAwas amplified by RT-PCR method in vitro, and identified by agarose gel electrophoresis. The target gene was purified by tapping and constructed by ligation with pMD-18T clone vector. The correct construction of pMD-18T-PDX-1 was identified by restriction endonuclease digestion and DNA sequence analysis. Results: the clone vector pMD-18T-PDX-1 containing PDX-1cDNA was constructed correctly, in which the total length of PDX-1cDNA was 908bp. Conclusion: PDX-1 gene was successfully cloned in vitro and PDX-1 gene clone vector was constructed correctly, which is of great significance for further expression of PDX-1 in eukaryotic cells and study of the role of PDX-1 in stem cell differentiation. Aim: to culture human umbilical cord mesenchymal stem cells and differentiate them into insulin secreting cells. Methods: human umbilical cord mesenchymal stem cells were cultured in vitro. The mesenchymal stem cells were divided into two groups: group A: induction group: group B: control group. Group A was treated with 1 mmol / L 2-mercaptoethanol for 2 days and then cultured for 2 days with 10 ng / ml epidermal growth factor 10 ng / ml basic fibroblast growth factor and 27 days for 7 days. Group B was treated with 20mmol/L nicotinamide and Isenapeptide for 7 days. The PDX-1 gene cDNAs of the two groups of cells were amplified by RT-PCR in vitro and the insulin secretion of the two groups was determined by radioimmunoassay to identify the insulin-secreting cells. Results from morphological observation, the undifferentiated umbilical cord mesenchymal stem cells were shown to be fusiform, with clear boundary and fibroid growth after passage, while the cells in group A gradually became smaller, round, refractive change, and the cells gathered into clusters. The cells in group B of islet like cells were still fusiform, and the boundaries were clear. The cDNAs of PDX-1 gene were amplified by agarose gel electrophoresis (agarose gel electrophoresis). The target fragment of PDX-1 was 912bp.The cDNA of PDX-1 gene could not be amplified from pubmed group B cells by radioimmunoassay (RIA). Insulin secretion was detected in group A and no insulin secretion was detected in group B. Conclusion: human umbilical cord mesenchymal stem cells were successfully induced to differentiate into insulin secreting cells in vitro, which provided the basis for the application of stem cells in the field of diabetes treatment.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R329;R587.1

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