本文關(guān)鍵詞： 慢病毒 角膜上皮細(xì)胞 有效性 安全性 細(xì)胞實驗　出處：《重慶醫(yī)科大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的觀察慢病毒(lentivirus)載體用于角膜上皮細(xì)胞基因轉(zhuǎn)染的有效性及安全性。 方法角膜上皮細(xì)胞的原代及傳代培養(yǎng)并應(yīng)用免疫熒光技術(shù)做細(xì)胞鑒定。慢病毒載體介導(dǎo)的增強型綠色熒光蛋白(lenti-EGFP)以不同的感染復(fù)數(shù)(MOI=0,1,10,50,100,500)轉(zhuǎn)染實驗細(xì)胞,于轉(zhuǎn)染后24、48、72、96h運用倒置熒光顯微鏡觀察不同感染復(fù)數(shù)(MOI)下增強型綠色熒光蛋白(EGFP)的表達(dá)并計算細(xì)胞轉(zhuǎn)染率,測定最佳轉(zhuǎn)染劑量,即最適感染復(fù)數(shù);RT-PCR方法檢測EGFP基因的表達(dá)情況。lenti-EGFP以最適感染復(fù)數(shù)轉(zhuǎn)染角膜上皮細(xì)胞,通過組織化學(xué)技術(shù)(HE染色、透射電子顯微鏡)觀察正常角膜上皮細(xì)胞及轉(zhuǎn)染細(xì)胞的形態(tài)及超微結(jié)構(gòu)變化;流式細(xì)胞技術(shù)(FCM)檢測慢病毒載體對角膜上皮細(xì)胞凋亡的影響。 結(jié)果EGFP于轉(zhuǎn)染48h即開始有表達(dá),隨著轉(zhuǎn)染時間的延長其表達(dá)增強。MOI=1、10、50、100時,角膜上皮細(xì)胞轉(zhuǎn)染率隨著感染復(fù)數(shù)的增加而增加,各感染復(fù)數(shù)下的細(xì)胞轉(zhuǎn)染率差異有統(tǒng)計學(xué)意義(P0.05),MOI在100與500時,轉(zhuǎn)染率差異無統(tǒng)計學(xué)意義(P0.05),即最適感染復(fù)數(shù)為100。RT-PCR結(jié)果提示轉(zhuǎn)染組細(xì)胞內(nèi)EGFP基因有明確表達(dá)。當(dāng)MOI=100時,角膜上皮細(xì)胞HE染色提示轉(zhuǎn)染細(xì)胞形態(tài)規(guī)則,與正常角膜細(xì)胞形態(tài)一致;透射電子顯微鏡觀察轉(zhuǎn)染細(xì)胞超微結(jié)構(gòu)與正常細(xì)胞無明顯差別。流式細(xì)胞術(shù)檢測轉(zhuǎn)染組細(xì)胞凋亡率與正常組細(xì)胞無明顯差別(P0.05)。 結(jié)論lenti-EGFP能夠有效、安全地轉(zhuǎn)染離體兔角膜上皮細(xì)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(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R774.1

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