本文選題：載脂蛋白E　切入點：基因治療　出處：《山西醫(yī)科大學》2017年碩士論文

【摘要】：目的:1.將ApoE(載脂蛋白E)與陽離子脂質體Lipofectamine 2000偶聯(lián),構建能特異轉染肝(癌)細胞的基因運載體。2.檢測在體外細胞實驗中,ApoE-Lipofectamine 2000的轉染特異性及轉染效率,并檢測其對細胞的毒性作用。3.通過ApoE-Lipofectamine 2000介導的pcDNA3.1-pSurvivin-TK聯(lián)合更昔洛韋(GCV)系統(tǒng)的體內抑制試驗,觀察該系統(tǒng)對裸鼠肝癌模型的靶向殺傷作用。方法:1.將ApoE與Lipofectamine 2000偶聯(lián),制備ApoE修飾的Lipofectamine2000。2.在體外細胞實驗中,質粒包裹實驗分析脂質體與質粒的結合比;脂質體攜帶pGenesil-1質粒轉染肝細胞HL-7702、肝癌細胞HepG2、人胚腎細胞HEK293T、大腸癌細胞SW480,并通過熒光顯微鏡及流式細胞儀檢測轉染效率;MTT法分析脂質體對細胞的毒性作用。3.在體內試驗中,將HepG2細胞接種于裸鼠右腋皮下,建立肝癌裸鼠移植瘤模型;待腫瘤長至約為200 mm3時,將荷瘤裸鼠隨機分為三組;A組:腫瘤空白對照組;B組:Lipofectamine 2000+pcDNA3.1-pSurvivin-TK+GCV治療組;C組:ApoE-Lipofectamine 2000+pcDNA3.1-pSurvivin-TK+GCV治療組。觀察各組治療后瘤體積及瘤重的變化,計算抑瘤率;RT-PCR法與Western blotting法觀察脂質體介導的HSV-tk質粒在瘤組織中的表達情況;HE染色與TUNEL法觀察治療后移植瘤的凋亡壞死情況。結果:1.制備ApoE修飾的Lipofectamine 2000運載體,質粒包裹實驗顯示質粒與Lipofectamine 2000和ApoE修飾的Lipofectamine 2000的質量體積比分別為1:2,1:2.5時,可完全包裹質粒。2.流式結果表明,ApoE-Lipofectamine 2000對HL-7702和HepG2細胞的轉染效率明顯高于Lipofectamine 2000,轉染效率有顯著性差異,具有統(tǒng)計學意義(P0.05,P=0.03,P=0.001)。在HEK293T、SW480細胞中,兩種脂質體轉染效率無明顯差異,無統(tǒng)計學意義(P0.05,P=0.93,P=0.55)。3.MTT實驗表明ApoE修飾后的Lipofectamine 2000沒有增加Lipofectamine2000對HepG2細胞的毒性作用(P0.05)。4.成功建立裸鼠肝癌移植瘤模型;治療后,與對照組相比,瘤體積和瘤重均減小,B、C組抑瘤率分別為(29.46±2.51)%,(46.18±1.79)%,二者相比差異有統(tǒng)計學意義(P0.05,P=0.007)。5.Western blotting與RT-PCR結果顯示,在A組未見明顯條帶,B、C兩組均檢測到HSV-tk質粒成功表達。6.HE染色與TUNEL法顯示,兩種脂質體介導的HSV-tk質粒聯(lián)合GCV治療對肝癌移植瘤均具有一定的殺傷作用,但ApoE修飾后的Lipofectamine 2000介導的抑制作用更明顯。結論:1.成功構建了一種肝癌靶向基因遞送載體,ApoE-Lipofectamine 2000。2.ApoE修飾的Lipofectamine 2000對肝(癌)細胞具有較高的親和力,能夠靶向轉染肝(癌)細胞。3.ApoE-Lipofectamine 2000介導的pcDNA3.1-pSurvivin-TK聯(lián)合更昔洛韋(GCV)系統(tǒng)對裸鼠肝癌模型具有靶向殺傷作用。
[Abstract]:Objective 1. To couple apolipoprotein E (ApoE) with cationic liposome Lipofectamine 2000 to construct gene carrier .2. to detect the transfection specificity and transfection efficiency of ApoE-Lipofectamine 2000 in vitro. In vivo inhibition test of ApoE-Lipofectamine 2000 mediated pcDNA3.1-pSurvivin-TK combined with ganciclovir (GCV) system was used to observe the targeted killing effect of the system on nude mice liver cancer model. Methods: 1. ApoE was coupled with Lipofectamine 2000. ApoE modified Lipofectamine 2000.2 was prepared. In vitro cell experiment, plasmid encapsulation assay was used to analyze the binding ratio of liposomes to plasmids. Liposomes carrying pGenesil-1 plasmid were transfected into hepatocytes HL-7702, HepG2, HEK293T, SW480. the transfection efficiency was detected by fluorescence microscope and flow cytometry. HepG2 cells were inoculated subcutaneously in the right axilla of nude mice to establish the transplanted tumor model of hepatoma in nude mice, and when the tumor reached about 200 mm3, The nude mice were randomly divided into three groups: control group B (control group) and control group (group B) treated with 2000 pcDNA3.1-pSurvivin-TK GCV. The changes of tumor volume and tumor weight after treatment were observed in group C (group C) and group C (group C) treated with ApoE-Lipofectamine 2000 pcDNA3.1-pSurvivin-TK GCV. The expression of HSV-tk plasmid mediated by liposome in tumor tissue was observed by RT-PCR and Western blotting. The apoptosis and necrosis of transplanted tumor were observed by HE staining and TUNEL method. Results 1. The ApoE modified Lipofectamine 2000 carrier was prepared. Plasmid encapsulation assay showed that when the mass / volume ratio of the plasmid to Lipofectamine 2000 modified by Lipofectamine 2000 and ApoE was 1: 2 / 1: 2.5, the plasmid could be completely encapsulated. Flow cytometry showed that the transfection efficiency of ApoE-Lipofectamine 2000 on HL-7702 and HepG2 cells was significantly higher than that of Lipofectamine 2000, and there was significant difference in transfection efficiency between ApoE-Lipofectamine 2000 and Lipofectamine 2000. There was no significant difference in transfection efficiency between the two liposomes in HEK293TnSW480 cells. The results showed that Lipofectamine 2000 modified with ApoE did not increase the toxicity of Lipofectamine2000 to HepG2 cells. Compared with the control group, the tumor volume and tumor weight of the control group were significantly lower than that of the control group. The tumor inhibition rates in the group C were 29.46 鹵2.51g and 46.18 鹵1.79g, respectively. The difference between the two groups was statistically significant. The results of Western blotting and RT-PCR showed that the tumor volume and weight of the two groups were significantly lower than those in the control group. The successful expression of HSV-tk plasmid was detected in group A and group C. 6. He staining and TUNEL staining showed that both liposome-mediated HSV-tk plasmids combined with GCV therapy had a certain killing effect on liver cancer transplanted tumor. But the inhibitory effect of ApoE modified Lipofectamine 2000 was more obvious. Conclusion: 1. A hepatoma targeting gene delivery vector, Lipofectamine 2000 modified by 2000.2.ApoE, was successfully constructed, and Lipofectamine 2000 had high affinity to liver (cancer) cells. ApoE-Lipofectamine 2000 mediated pcDNA3.1-pSurvivin-TK combined with ganciclovir has targeted killing effect on nude mice liver cancer model. 3.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.7

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