ApoE修飾的Lipofectamine 2000介導(dǎo)HSV-tk基因?qū)Ω伟饔玫难芯?/H1>
發(fā)布時(shí)間：2018-03-28 22:03
本文選題：載脂蛋白E 切入點(diǎn)：基因治療 出處：《山西醫(yī)科大學(xué)》2017年碩士論文
【摘要】：目的:1.將ApoE(載脂蛋白E)與陽離子脂質(zhì)體Lipofectamine 2000偶聯(lián),構(gòu)建能特異轉(zhuǎn)染肝(癌)細(xì)胞的基因運(yùn)載體。2.檢測(cè)在體外細(xì)胞實(shí)驗(yàn)中,ApoE-Lipofectamine 2000的轉(zhuǎn)染特異性及轉(zhuǎn)染效率,并檢測(cè)其對(duì)細(xì)胞的毒性作用。3.通過ApoE-Lipofectamine 2000介導(dǎo)的pcDNA3.1-pSurvivin-TK聯(lián)合更昔洛韋(GCV)系統(tǒng)的體內(nèi)抑制試驗(yàn),觀察該系統(tǒng)對(duì)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(xì)胞實(shí)驗(yàn)中,質(zhì)粒包裹實(shí)驗(yàn)分析脂質(zhì)體與質(zhì)粒的結(jié)合比;脂質(zhì)體攜帶pGenesil-1質(zhì)粒轉(zhuǎn)染肝細(xì)胞HL-7702、肝癌細(xì)胞HepG2、人胚腎細(xì)胞HEK293T、大腸癌細(xì)胞SW480,并通過熒光顯微鏡及流式細(xì)胞儀檢測(cè)轉(zhuǎn)染效率;MTT法分析脂質(zhì)體對(duì)細(xì)胞的毒性作用。3.在體內(nèi)試驗(yàn)中,將HepG2細(xì)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(shí),將荷瘤裸鼠隨機(jī)分為三組;A組:腫瘤空白對(duì)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(jì)算抑瘤率;RT-PCR法與Western blotting法觀察脂質(zhì)體介導(dǎo)的HSV-tk質(zhì)粒在瘤組織中的表達(dá)情況;HE染色與TUNEL法觀察治療后移植瘤的凋亡壞死情況。結(jié)果:1.制備ApoE修飾的Lipofectamine 2000運(yùn)載體,質(zhì)粒包裹實(shí)驗(yàn)顯示質(zhì)粒與Lipofectamine 2000和ApoE修飾的Lipofectamine 2000的質(zhì)量體積比分別為1:2,1:2.5時(shí),可完全包裹質(zhì)粒。2.流式結(jié)果表明,ApoE-Lipofectamine 2000對(duì)HL-7702和HepG2細(xì)胞的轉(zhuǎn)染效率明顯高于Lipofectamine 2000,轉(zhuǎn)染效率有顯著性差異,具有統(tǒng)計(jì)學(xué)意義(P0.05,P=0.03,P=0.001)。在HEK293T、SW480細(xì)胞中,兩種脂質(zhì)體轉(zhuǎn)染效率無明顯差異,無統(tǒng)計(jì)學(xué)意義(P0.05,P=0.93,P=0.55)。3.MTT實(shí)驗(yàn)表明ApoE修飾后的Lipofectamine 2000沒有增加Lipofectamine2000對(duì)HepG2細(xì)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(duì)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(jì)學(xué)意義(P0.05,P=0.007)。5.Western blotting與RT-PCR結(jié)果顯示,在A組未見明顯條帶,B、C兩組均檢測(cè)到HSV-tk質(zhì)粒成功表達(dá)。6.HE染色與TUNEL法顯示,兩種脂質(zhì)體介導(dǎo)的HSV-tk質(zhì)粒聯(lián)合GCV治療對(duì)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(dǎo)的抑制作用更明顯。結(jié)論:1.成功構(gòu)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(duì)肝(癌)細(xì)胞具有較高的親和力,能夠靶向轉(zhuǎn)染肝(癌)細(xì)胞。3.ApoE-Lipofectamine 2000介導(dǎo)的pcDNA3.1-pSurvivin-TK聯(lián)合更昔洛韋(GCV)系統(tǒng)對(duì)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(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.7
【參考文獻(xiàn)】
相關(guān)期刊論文 前8條
1 李勇芳;張英敏;趙娜;孟凡秀;張琪;高然朋;張悅紅;于保鋒;郭睿;王海龍;解軍;徐鈞;;Survivin啟動(dòng)子控制自殺基因HSV-TK真核表達(dá)載體對(duì)肝癌細(xì)胞殺傷活性的研究[J];中華臨床醫(yī)師雜志(電子版);2016年02期
2 陳中華;朱德生;李軍;黃展勤;;非病毒siRNA載體研究進(jìn)展[J];中國藥理學(xué)通報(bào);2015年07期
3 趙海偉;何偉;尹莉芳;;穿膜肽介導(dǎo)紫杉醇脂質(zhì)體對(duì)腫瘤的治療作用[J];藥學(xué)研究;2015年03期
4 熊小麗;陳鵬;趙東赤;;精氨酸-甘氨酸-天冬氨酸肽修飾的脂質(zhì)體雷公藤甲素靶向抗腫瘤效果[J];中華實(shí)驗(yàn)外科雜志;2015年03期
5 李秀英;曾凡;趙曜;居瑞軍;呂萬良;;脂質(zhì)體藥物遞送系統(tǒng)的研究進(jìn)展[J];中國新藥雜志;2014年16期
6 徐少洪;趙斌;閆志強(qiáng);劉璐;何丹農(nóng);;超臨界流體技術(shù)在脂質(zhì)體制備中的應(yīng)用[J];材料導(dǎo)報(bào);2014年05期
7 張德榮;劉福臣;;腫瘤靶向藥物載體系統(tǒng)研究的新進(jìn)展[J];臨床合理用藥雜志;2011年18期
8 張桂紅;劉彥仿;付勇;胡海洋;陳大為;楊守京;趙君;;抗肝癌單鏈抗體免疫脂質(zhì)體的制備及其體外抑瘤實(shí)驗(yàn)[J];醫(yī)學(xué)研究生學(xué)報(bào);2006年01期
，
本文編號(hào)：1678244
本文鏈接：http://sikaile.net/yixuelunwen/zlx/1678244.html
本文選題：載脂蛋白E 切入點(diǎn)：基因治療 出處：《山西醫(yī)科大學(xué)》2017年碩士論文
【摘要】：目的:1.將ApoE(載脂蛋白E)與陽離子脂質(zhì)體Lipofectamine 2000偶聯(lián),構(gòu)建能特異轉(zhuǎn)染肝(癌)細(xì)胞的基因運(yùn)載體。2.檢測(cè)在體外細(xì)胞實(shí)驗(yàn)中,ApoE-Lipofectamine 2000的轉(zhuǎn)染特異性及轉(zhuǎn)染效率,并檢測(cè)其對(duì)細(xì)胞的毒性作用。3.通過ApoE-Lipofectamine 2000介導(dǎo)的pcDNA3.1-pSurvivin-TK聯(lián)合更昔洛韋(GCV)系統(tǒng)的體內(nèi)抑制試驗(yàn),觀察該系統(tǒng)對(duì)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(xì)胞實(shí)驗(yàn)中,質(zhì)粒包裹實(shí)驗(yàn)分析脂質(zhì)體與質(zhì)粒的結(jié)合比;脂質(zhì)體攜帶pGenesil-1質(zhì)粒轉(zhuǎn)染肝細(xì)胞HL-7702、肝癌細(xì)胞HepG2、人胚腎細(xì)胞HEK293T、大腸癌細(xì)胞SW480,并通過熒光顯微鏡及流式細(xì)胞儀檢測(cè)轉(zhuǎn)染效率;MTT法分析脂質(zhì)體對(duì)細(xì)胞的毒性作用。3.在體內(nèi)試驗(yàn)中,將HepG2細(xì)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(shí),將荷瘤裸鼠隨機(jī)分為三組;A組:腫瘤空白對(duì)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(jì)算抑瘤率;RT-PCR法與Western blotting法觀察脂質(zhì)體介導(dǎo)的HSV-tk質(zhì)粒在瘤組織中的表達(dá)情況;HE染色與TUNEL法觀察治療后移植瘤的凋亡壞死情況。結(jié)果:1.制備ApoE修飾的Lipofectamine 2000運(yùn)載體,質(zhì)粒包裹實(shí)驗(yàn)顯示質(zhì)粒與Lipofectamine 2000和ApoE修飾的Lipofectamine 2000的質(zhì)量體積比分別為1:2,1:2.5時(shí),可完全包裹質(zhì)粒。2.流式結(jié)果表明,ApoE-Lipofectamine 2000對(duì)HL-7702和HepG2細(xì)胞的轉(zhuǎn)染效率明顯高于Lipofectamine 2000,轉(zhuǎn)染效率有顯著性差異,具有統(tǒng)計(jì)學(xué)意義(P0.05,P=0.03,P=0.001)。在HEK293T、SW480細(xì)胞中,兩種脂質(zhì)體轉(zhuǎn)染效率無明顯差異,無統(tǒng)計(jì)學(xué)意義(P0.05,P=0.93,P=0.55)。3.MTT實(shí)驗(yàn)表明ApoE修飾后的Lipofectamine 2000沒有增加Lipofectamine2000對(duì)HepG2細(xì)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(duì)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(jì)學(xué)意義(P0.05,P=0.007)。5.Western blotting與RT-PCR結(jié)果顯示,在A組未見明顯條帶,B、C兩組均檢測(cè)到HSV-tk質(zhì)粒成功表達(dá)。6.HE染色與TUNEL法顯示,兩種脂質(zhì)體介導(dǎo)的HSV-tk質(zhì)粒聯(lián)合GCV治療對(duì)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(dǎo)的抑制作用更明顯。結(jié)論:1.成功構(gòu)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(duì)肝(癌)細(xì)胞具有較高的親和力,能夠靶向轉(zhuǎn)染肝(癌)細(xì)胞。3.ApoE-Lipofectamine 2000介導(dǎo)的pcDNA3.1-pSurvivin-TK聯(lián)合更昔洛韋(GCV)系統(tǒng)對(duì)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(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.7
【參考文獻(xiàn)】
相關(guān)期刊論文 前8條
1 李勇芳;張英敏;趙娜;孟凡秀;張琪;高然朋;張悅紅;于保鋒;郭睿;王海龍;解軍;徐鈞;;Survivin啟動(dòng)子控制自殺基因HSV-TK真核表達(dá)載體對(duì)肝癌細(xì)胞殺傷活性的研究[J];中華臨床醫(yī)師雜志(電子版);2016年02期
2 陳中華;朱德生;李軍;黃展勤;;非病毒siRNA載體研究進(jìn)展[J];中國藥理學(xué)通報(bào);2015年07期
3 趙海偉;何偉;尹莉芳;;穿膜肽介導(dǎo)紫杉醇脂質(zhì)體對(duì)腫瘤的治療作用[J];藥學(xué)研究;2015年03期
4 熊小麗;陳鵬;趙東赤;;精氨酸-甘氨酸-天冬氨酸肽修飾的脂質(zhì)體雷公藤甲素靶向抗腫瘤效果[J];中華實(shí)驗(yàn)外科雜志;2015年03期
5 李秀英;曾凡;趙曜;居瑞軍;呂萬良;;脂質(zhì)體藥物遞送系統(tǒng)的研究進(jìn)展[J];中國新藥雜志;2014年16期
6 徐少洪;趙斌;閆志強(qiáng);劉璐;何丹農(nóng);;超臨界流體技術(shù)在脂質(zhì)體制備中的應(yīng)用[J];材料導(dǎo)報(bào);2014年05期
7 張德榮;劉福臣;;腫瘤靶向藥物載體系統(tǒng)研究的新進(jìn)展[J];臨床合理用藥雜志;2011年18期
8 張桂紅;劉彥仿;付勇;胡海洋;陳大為;楊守京;趙君;;抗肝癌單鏈抗體免疫脂質(zhì)體的制備及其體外抑瘤實(shí)驗(yàn)[J];醫(yī)學(xué)研究生學(xué)報(bào);2006年01期
，本文編號(hào)：1678244
本文鏈接：http://sikaile.net/yixuelunwen/zlx/1678244.html
最近更新
教材專著
