本文關鍵詞： 慢病毒 角膜上皮細胞 有效性 安全性 細胞實驗　出處：《重慶醫(yī)科大學》2010年碩士論文　論文類型：學位論文

【摘要】： 目的觀察慢病毒(lentivirus)載體用于角膜上皮細胞基因轉染的有效性及安全性。 方法角膜上皮細胞的原代及傳代培養(yǎng)并應用免疫熒光技術做細胞鑒定。慢病毒載體介導的增強型綠色熒光蛋白(lenti-EGFP)以不同的感染復數(MOI=0,1,10,50,100,500)轉染實驗細胞,于轉染后24、48、72、96h運用倒置熒光顯微鏡觀察不同感染復數(MOI)下增強型綠色熒光蛋白(EGFP)的表達并計算細胞轉染率,測定最佳轉染劑量,即最適感染復數;RT-PCR方法檢測EGFP基因的表達情況。lenti-EGFP以最適感染復數轉染角膜上皮細胞,通過組織化學技術(HE染色、透射電子顯微鏡)觀察正常角膜上皮細胞及轉染細胞的形態(tài)及超微結構變化;流式細胞技術(FCM)檢測慢病毒載體對角膜上皮細胞凋亡的影響。 結果EGFP于轉染48h即開始有表達,隨著轉染時間的延長其表達增強。MOI=1、10、50、100時,角膜上皮細胞轉染率隨著感染復數的增加而增加,各感染復數下的細胞轉染率差異有統計學意義(P0.05),MOI在100與500時,轉染率差異無統計學意義(P0.05),即最適感染復數為100。RT-PCR結果提示轉染組細胞內EGFP基因有明確表達。當MOI=100時,角膜上皮細胞HE染色提示轉染細胞形態(tài)規(guī)則,與正常角膜細胞形態(tài)一致;透射電子顯微鏡觀察轉染細胞超微結構與正常細胞無明顯差別。流式細胞術檢測轉染組細胞凋亡率與正常組細胞無明顯差別(P0.05)。 結論lenti-EGFP能夠有效、安全地轉染離體兔角膜上皮細胞。
[Abstract]:Objective to observe the efficacy and safety of lentivirus vector in gene transfection of corneal epithelial cells. Methods the primary and passage culture of corneal epithelial cells was performed and identified by immunofluorescence. Lenti-EGFP mediated by lentivirus vector was expressed in different infective plural numbers (. MOI=0. After transfection, the experimental cells were transfected with 244872. At 96 h, the expression of enhanced green fluorescent protein (EGFP) under different infected complex moi was observed by inverted fluorescence microscope, and the transfection efficiency was calculated. The optimal transfection dose was determined, that is, the optimal complex number of infection. RT-PCR method was used to detect the expression of EGFP gene. Lenti-EGFP was transfected into corneal epithelial cells with the most suitable number of infections. The corneal epithelial cells were stained with HE by histochemical technique. The morphology and ultrastructure of normal corneal epithelial cells and transfected cells were observed by transmission electron microscope (TEM). Flow cytometry (FCM) was used to detect the effect of lentivirus vector on corneal epithelial cell apoptosis. Results the expression of EGFP began at 48h after transfection and increased with the extension of transfection time. The transfection efficiency of corneal epithelial cells increased with the increase of the complex number of infection. There was no significant difference in transfection efficiency (P 0.05), that is, the optimal complex number of infection was 100. RT-PCR results showed that the EGFP gene was expressed clearly in the transfected cells, when MOI = 100. The HE staining of corneal epithelial cells showed that the morphology of transfected cells was consistent with that of normal corneal cells. The ultrastructure of transfected cells was not different from that of normal cells by transmission electron microscope, but the apoptosis rate of transfected cells was not significantly different from that of normal cells by flow cytometry. Conclusion lenti-EGFP can effectively and safely transfect rabbit corneal epithelial cells in vitro.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R774.1

【參考文獻】

相關期刊論文 前1條

1 李寧;朱寶長;朱宛宛;王淑艷;任萍;關云謙;張愚;;慢病毒介導綠色熒光蛋白轉染人胚胎干細胞及其培養(yǎng)[J];基礎醫(yī)學與臨床;2008年10期

，

本文編號：1469004