本文選題：多潛能干細胞(iPSCs) + 基因治療�。� 參考：《中南大學(xué)》2011年碩士論文

【摘要】：目的：通過異位表達某些轉(zhuǎn)錄因子,可以將體細胞重編程成誘導(dǎo)性多潛能干細胞(iPSCs),它能避開ES細胞帶來的倫理問題,減少免疫排斥,給自體化干細胞基因治療的臨床應(yīng)用提供了可能性,為遺傳性疾病體外研究提供了一個很好的平臺。 方法：目前有多種方法可誘導(dǎo)獲得iPS細胞,包括利用各種病毒載體、純化的重組蛋白質(zhì)、修飾的RNA分子等。同時研究發(fā)現(xiàn)多種小分子化合物也可促進iPS的產(chǎn)生,如Vc能加速基因表達水平的改變,促進前體iPS細胞轉(zhuǎn)變成完全重編程的iPS細胞；VPA在重編程過程中,可上調(diào)ES特異性基因的表達,下調(diào)MEF特異性基因的表達。 在本研究中,我們利用逆轉(zhuǎn)錄病毒介導(dǎo)的轉(zhuǎn)染系統(tǒng)將Oct4,Sox2,c-Myc和Klf4這4個轉(zhuǎn)錄因子導(dǎo)入人皮膚成纖維細胞(HDF),同時在誘導(dǎo)過程中添加Vc和VPA,最終獲得iPS細胞。然后,通過TRA-1-60/TRA-1-81活細胞染色,機械法挑取陽性克隆傳代培養(yǎng)。采取形態(tài)學(xué)、堿性磷酸酶染色以及干細胞表面多潛能標(biāo)志物染色的方法對傳代后的克隆進行初步鑒定。 結(jié)果：(1)用脂質(zhì)體Lipofectamine2000將逆轉(zhuǎn)錄病毒載體及包裝質(zhì)粒共同轉(zhuǎn)染293T細胞,可獲得較高質(zhì)量的逆轉(zhuǎn)錄病毒；(2)誘導(dǎo)出的克隆在形態(tài)上與hES細胞相似,外源基因GFP不表達,經(jīng)過活細胞染色發(fā)現(xiàn)其TRA-1-60/TRA-1-81有表達,挑取上述克隆傳代后,AP染色結(jié)果呈陽性,ES細胞表面標(biāo)志免疫熒光檢測呈陽性。傳至P7代,仍然具有ES樣克隆形態(tài)。 結(jié)論：利用逆轉(zhuǎn)錄病毒系統(tǒng)將人皮膚成纖維細胞重編程為iPS細胞,經(jīng)初步鑒定可表達多潛能性表面標(biāo)志物。
[Abstract]:Objective: by ectopic expression of some transcription factors, somatic cells can be reprogrammed into inducible multipotential stem cells, which can avoid the ethical problems caused by es cells and reduce immune rejection. It provides the possibility for the clinical application of autologous stem cell gene therapy and provides a good platform for the in vitro study of genetic diseases. Methods: there are many methods to induce iPS cells, including the use of various virus vectors, purified recombinant proteins, modified RNA molecules and so on. At the same time, it was found that many kinds of small molecular compounds can also promote the production of iPS, such as VC can accelerate the change of gene expression level and promote the transformation of precursor iPS cells into fully reprogrammed iPS cells during the reprogramming process. It can up-regulate the expression of es specific gene and down-regulate the expression of MEF specific gene. In this study, we used a retrovirus-mediated transfection system to transfer the four transcription factors Oct4nSox2Oc-Myc and Klf4 into human skin fibroblasts, and then added VC and VPA during the induction process to obtain iPS cells. Then, the positive clones were subcultured by TRA-1-60/TRA-1-81 staining, and the positive clones were subcultured by mechanical method. The clones were identified by morphology, alkaline phosphatase staining and multipotential marker staining on stem cell surface. Results using liposome Lipofectamine2000 to co-transfect retrovirus vector and packaging plasmid into 293T cells, a high quality retroviral vector was obtained. The clone induced by the recombinant plasmid was similar to that of hES cells in morphology, and the foreign gene GFP was not expressed. The expression of TRA-1-60/TRA-1-81 was found by living cell staining, and the AP staining was positive after the above clones were selected. The surface markers of es cells were detected positive by immunofluorescence. After transmission to P7 generation, it still had es like clone morphology. Conclusion: human skin fibroblasts were reprogrammed into iPS cells by retrovirus system, and multipotential surface markers could be expressed.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329

【參考文獻】

相關(guān)博士學(xué)位論文 前1條

1 余樹民;小鼠胚胎干細胞建系的研究[D];西北農(nóng)林科技大學(xué);2007年

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