本文選題：GP73抗體 + 陽(yáng)離子脂質(zhì)體　； 參考：《山西醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:1.篩選肝癌細(xì)胞和其他非肝癌細(xì)胞表面高爾基體膜蛋白73(GP73)陽(yáng)性表達(dá)率,確定GP73在肝癌細(xì)胞表面表達(dá)的特異性;2.制備普通陽(yáng)離子脂質(zhì)體;3.將普通陽(yáng)離子脂質(zhì)體(lipoplexes)與GP73抗體偶聯(lián),構(gòu)建一種新型的由GP73抗體修飾的能夠特異性結(jié)合肝癌細(xì)胞的靶向治療載體;4.制備不同劑量的GP73抗體修飾脂質(zhì)體,轉(zhuǎn)染Hep G2肝癌細(xì)胞,流式細(xì)胞術(shù)篩選轉(zhuǎn)染率最高的GP73抗體修飾組,確定脂質(zhì)體中GP73抗體的最佳負(fù)載量;5.比較GP73抗體修飾的脂質(zhì)體和普通陽(yáng)離子脂質(zhì)體對(duì)人肝癌細(xì)胞株Hep G2、Bel-7402、SMMC-7721和人正常肝細(xì)胞HL-7702以及人非肝癌細(xì)胞SW480和正常人胚腎細(xì)胞HEK293T的轉(zhuǎn)染效率;6.研究由GP73抗體修飾的陽(yáng)離子脂質(zhì)體介導(dǎo)的HSVtk自殺基因聯(lián)合更昔洛韋(GCV)對(duì)肝癌細(xì)胞的體外殺傷作用;7.研究由GP73抗體修飾的陽(yáng)離子脂質(zhì)體介導(dǎo)的HSVtk自殺基因聯(lián)合GCV對(duì)肝癌荷瘤裸鼠體內(nèi)腫瘤的殺傷作用。方法:1.用GP73的一抗(Anti-GOLPH2)連接二抗FITC分別孵育細(xì)胞Hep G2、SMMC-7721、Bel-7402、HL-7702、Hela、SW480和HEK293T,通過(guò)流式細(xì)胞儀檢測(cè)細(xì)胞表面GP73的陽(yáng)性表達(dá)率。2.乙醇注入法和擠壓法聯(lián)合制備普通陽(yáng)離子脂質(zhì)體(實(shí)驗(yàn)室前期制備方法)。3.將GP73抗體與陽(yáng)離子脂質(zhì)體偶聯(lián),制備GP73抗體修飾的脂質(zhì)體。4.將普通陽(yáng)離子脂質(zhì)體和GP73抗體修飾脂質(zhì)體分別與p Genesil-1質(zhì)�；靹颉⒎跤�,確定最佳的包裹比例。5.制備含量不同的GP73抗體修飾的陽(yáng)離子脂質(zhì)體,轉(zhuǎn)染Hep G2肝癌細(xì)胞,流式細(xì)胞術(shù)篩選轉(zhuǎn)染率最高的GP73抗體修飾脂質(zhì)體組,確定脂質(zhì)體中GP73的最佳負(fù)載量。6.分別使用GP73抗體修飾的陽(yáng)離子脂質(zhì)體轉(zhuǎn)染p Genesil-1質(zhì)粒,流式細(xì)胞儀檢測(cè)Hep G2、Bel-7402、SMMC-7721、HL-7702、Hela、SW480、HEK293T細(xì)胞的報(bào)告基因綠色熒光蛋白(EGFP)的表達(dá)。7.用GP73抗體修飾的陽(yáng)離子脂質(zhì)體攜帶p Survivin-TK-PBI-CMV2質(zhì)粒分別轉(zhuǎn)染細(xì)胞Hep G2、Bel-7402和HL-7702,48小時(shí)后提取細(xì)胞的總RNA和總蛋白,并通過(guò)RT-PCR、Westernblot技術(shù)方法檢測(cè)細(xì)胞中HSVtk基因的m RNA和蛋白表達(dá)水平。8.用GP73抗體修飾的陽(yáng)離子脂質(zhì)體攜帶p Survivin-TK-PBI-CMV2質(zhì)粒分別轉(zhuǎn)染肝癌細(xì)胞Hep G2、Bel-7402和肝正常細(xì)胞HL-7702,加入濃度為150μg/m L的前體藥物GCV處理24 h,經(jīng)流式細(xì)胞儀檢測(cè)細(xì)胞凋亡情況。9.梯度劑量的GCV處理轉(zhuǎn)染后的肝癌細(xì)胞48小時(shí),MTT法檢測(cè)HSVtk自殺基因系統(tǒng)對(duì)肝(癌)細(xì)胞生存率的影響情況。10.用生長(zhǎng)狀態(tài)良好的Hep G2肝癌細(xì)胞接種裸鼠成瘤,利用公式V=ab2/2,(a代表瘤體最長(zhǎng)徑,b代表瘤體最短徑)計(jì)算瘤體積。11.HE染色檢測(cè)干預(yù)后三組腫瘤組織壞死情況。12.TUNEL凋亡試劑盒檢測(cè)干預(yù)后三組腫瘤組織細(xì)胞凋亡情況。13.裸鼠開始治療后,觀察裸鼠生長(zhǎng)狀態(tài),并持續(xù)40天記錄裸鼠存活狀態(tài),用Graphpad Prism v 6.0軟件分析數(shù)據(jù),繪制生長(zhǎng)曲線圖譜,比較三組荷瘤裸鼠生存期。結(jié)果:1.通過(guò)對(duì)細(xì)胞表面GP73陽(yáng)性表達(dá)率的篩選,肝癌細(xì)胞Hep G2、Bel-7402、SMMC-7721細(xì)胞表面GP73陽(yáng)性率高于HL-7702、Hela、SW480、HEK293T,差異有統(tǒng)計(jì)學(xué)意義(P0.05),表明了GP73在肝癌細(xì)胞表面表達(dá)有特異性;2.制備了一種普通陽(yáng)離子脂質(zhì)體;3.制備了一種GP73抗體修飾的能夠特異性結(jié)合肝癌細(xì)胞的靶向治療載體(Anti GP73-lipoplexes);4.GP73抗體修飾的脂質(zhì)體對(duì)人肝癌細(xì)胞株Hep G2、Bel-7402和SMMC-7721的轉(zhuǎn)染效率明顯高于肝正常細(xì)胞和非肝癌細(xì)胞(P0.05);5.基因和蛋白水平檢測(cè)表明,HSVtk基因在Hep G2和Bel-7402肝癌細(xì)胞中有表達(dá),而在肝正常細(xì)胞HL-7702中未見表達(dá);6.細(xì)胞增殖實(shí)驗(yàn)(MTT)結(jié)果可見,Hep G2和Bel-7402肝癌細(xì)胞轉(zhuǎn)染組在GCV濃度不斷增加的情況下,細(xì)胞存活率不斷下降,與未轉(zhuǎn)染加藥組和正常肝細(xì)胞HL-7702相比有統(tǒng)計(jì)學(xué)差異(P0.05);7.流式細(xì)胞儀(FC 500)檢測(cè)凋亡結(jié)果表明Hep G2、Bel-7402肝癌細(xì)胞死亡率明顯高于肝正常細(xì)胞組(P0.05);8.肝腫瘤裸鼠造模成功,腫瘤體積達(dá)160~200 mm3;9.免疫組化和TUNEL檢測(cè)腫瘤組織細(xì)胞凋亡結(jié)果表明,Anti GP73-lipoplexes運(yùn)載HSVtk基因聯(lián)合GCV治療組腫瘤組織細(xì)胞凋亡明顯,而lipoplexes+HSVtk+GCV組有少量細(xì)胞凋亡,空載組(lipoplexes+Vector+GCV)幾乎未見凋亡細(xì)胞;10.生存曲線分析表明Anti GP73-lipoplexes+HSVtk+GCV治療組裸鼠生存期明顯長(zhǎng)于對(duì)照組(P0.05)。結(jié)論:1.肝癌細(xì)胞Hep G2、Bel-7402和SMMC-7721表面GP73陽(yáng)性表達(dá)率高于肝正常細(xì)胞和非肝癌細(xì)胞。2.GP73修飾的陽(yáng)離子脂質(zhì)體(GP73-lipoplexes)載體運(yùn)輸系統(tǒng)成功構(gòu)建。3.Anti GP73-lipoplexes對(duì)肝癌細(xì)胞具有較高的轉(zhuǎn)染效率。4.Anti GP73-lipoplexes介導(dǎo)自殺基因真核表達(dá)質(zhì)粒p Survivin-TK-PBI-CMV2在GP73陽(yáng)性高表達(dá)的Hep G2、Bel-7402肝癌細(xì)胞內(nèi)成功表達(dá)。5.在細(xì)胞水平:Anti GP73-lipoplexes介導(dǎo)SUR啟動(dòng)子調(diào)控的HSVtk/GCV自殺基因系統(tǒng)對(duì)Hep G2、Bel-7402肝癌細(xì)胞具有選擇性殺傷作用,對(duì)正常肝細(xì)胞HL-7702沒(méi)有殺傷作用。6.Hep G2肝癌細(xì)胞異種移植瘤裸鼠接種成功。7.在動(dòng)物水平:Anti GP73-lipoplexes介導(dǎo)SUR啟動(dòng)子調(diào)控的HSVtk/GCV自殺基因系統(tǒng)對(duì)Hep G2肝癌細(xì)胞異種移植瘤裸鼠腫瘤的生長(zhǎng)與增殖有抑制作用,而且能夠延長(zhǎng)治療組荷瘤裸鼠的生存期。
[Abstract]:Objective: 1. screening of hepatoma cells and other non HCC cell surface Golgi membrane protein 73 (GP73) positive expression rate, determine the specificity of GP73 expression in hepatocellular carcinoma cell surface; 2. the preparation of cationic liposome; 3. common cationic liposome (Lipoplexes) coupled with GP73 antibody, construct a novel by GP73 the modified antibody could specifically bind to hepatoma cells targeting vector; GP73 antibody modified liposome preparation of 4. different doses of Hep transfected with G2 hepatoma cells, the transfection efficiency of screening GP73 antibody modified group the highest flow cytometry, determine the optimal loading of GP73 antibody in liposome; Bel-7402 liposomes and cations 5. comparison of GP73 liposome modified antibody on human hepatocellular carcinoma cell line Hep, G2, SMMC-7721 and human normal liver cell HL-7702 and human hepatoma cells SW480 and non normal human embryonic kidney cells HEK293T transfection efficiency; 6. study by G Cationic liposome P73 antibody modified by HSVtk gene combined with ganciclovir (GCV) Dutch act killing effect on hepatocarcinoma cells in vitro; cytotoxicity 7. study by cationic lipid GP73 antibody modified body mediated HSVtk gene combined with GCV Dutch act on hepatocellular carcinoma tumor. Methods: 1. GP73 - (Anti-GOLPH2) connecting the two anti FITC were incubated with Hep, G2, SMMC-7721, Bel-7402, HL-7702, Hela, SW480 and HEK293T, the positive expression of GP73 cell surface was detected by flow cytometry.2. ethanol injection method and extrusion method combined with the preparation of cationic liposome (Lab preparation method).3. antibody and GP73 cationic liposome conjugated.4. liposome preparation of GP73 antibody modified ordinary cationic liposome and GP73 antibody modified liposome and plasmid Genesil-1 P were mixed and incubated to determine the best preparation for the proportion of.5. package Cationic lipid GP73 antibody modified with different content of the body, transfection of Hep G2 cells, the transfection efficiency of screening GP73 antibody modified liposome group by flow cytometry. The determination of GP73 liposome in the best load amount of.6. respectively using cationic lipid GP73 antibody modified P transfected Genesil-1 plasmid, Hep G2 detection, flow cytometry SMMC-7721, HL-7702, Bel-7402, Hela, SW480, reporter gene of HEK293T cells with green fluorescent protein (EGFP) expression of.7. using GP73 antibody modified cationic liposome with P Survivin-TK-PBI-CMV2 plasmid were transfected into Hep cells G2, Bel-7402 and HL-7702,48 hours after the extraction of total RNA cells and total protein, and the expression of m by RT-PCR. RNA and HSVtk protein gene Westernblot method of detecting the expression level of.8. with cationic lipid GP73 antibody modified body carrying P Survivin-TK-PBI-CMV2 plasmid respectively transfectedinto fine Hep cell G2, Bel-7402 and normal liver cells HL-7702, the concentration of 150 g/m L prodrugs of GCV treatment for 24 h, the cell apoptosis was detected by flow cytometry.9. doses of GCV treatment of hepatocellular carcinoma cells after transfection for 48 hours, MTT method for detection of HSVtk gene on liver Dutch Act (cancer) growth state good Hep G2 hepatocellular carcinoma cells in nude mice inoculated with.10. on cell survival rate, using the formula V=ab2/2 (a represents the longest diameter of the tumor, the tumor B represents the shortest path) to start treatment of the three groups of tumor cell apoptosis..13. nude mice tumor volume calculation.11.HE staining of tumor tissue necrosis after the intervention of three groups.12.TUNEL apoptosis detection kit after intervention, to observe the growth state of nude mice, and lasted for 40 days to record the survival status of nude mice, analysis of data using Graphpad Prism v 6 software, drawing the growth curves, compared with three groups of nude mice survival. Results: 1. through the screening of cell surface GP73 positive expression rate of Hep cells, G2, Bel-7402, SMMC-7721 positive rate of cell surface GP73 is higher than HL-7702, Hela, SW480, HEK293T, the difference was statistically significant (P0.05), showed that GP73 specific expression in HCC cell surface; a common cationic liposome preparation 2.; 3. were prepared by a modified GP73 antibody could specifically bind to hepatoma cells targeting vector (Anti GP73-lipoplexes); 4.GP73 antibody modified liposome on human hepatocellular carcinoma cell line Hep G2, Bel-7402 SMMC-7721 and the transfection efficiency was significantly higher than that of normal liver cells and non liver cancer cells (P0.05) show; detection of 5. gene and protein level, HSVtk gene expression in Hep G2 and Bel-7402 in HCC cells, whereas in normal liver cells HL-7702 no expression; 6. cell proliferation assay (MTT) showed that Hep, G2 and Bel-7402 hepatoma cells transfected group In the case of increasing the concentration of GCV, cell survival rate decreased, there was significant difference compared with untransfected dosing group and normal liver cells (P0.05 HL-7702 7.); flow cytometry (FC 500) apoptosis detection results showed that Hep G2, Bel-7402 cell mortality was significantly higher than normal liver cells group (P0.05); 8. liver cancer nude mice model, the tumor volume reached 160~200 mm3; 9. TUNEL and immunohistochemical detection of tumor cell apoptosis. The results showed that the Anti GP73-lipoplexes carrying HSVtk gene combined with GCV on the apoptosis of tumor cells was significantly, while the lipoplexes+HSVtk+GCV group has a small amount of apoptosis vectorgroup (lipoplexes+Vector+GCV) almost no apoptotic cells; 10. survival curves Anti analysis showed that GP73-lipoplexes+HSVtk+GCV treatment group was significantly longer than the control group of nude mice survival (P0.05). Conclusion: the liver cancer cell Hep 1. G2, Bel-7402 and GP73 on the surface of SMMC-7721 Cationic lipid positive expression rate is higher than the normal liver cells and non liver cells modified with.2.GP73 body (GP73-lipoplexes) carrier transport system was successfully constructed.4.Anti GP73-lipoplexes.3.Anti GP73-lipoplexes has high transfection efficiency on hepatoma cells by Dutch act gene eukaryotic expression plasmid P Survivin-TK-PBI-CMV2 in GP73 positive expression of Hep G2 and Bel-7402.5. cells in hepatoma cells the expression level of success: Dutch act system of Hep G2 Anti gene GP73-lipoplexes mediated by SUR promoter HSVtk/GCV, Bel-7402 cells with selective cytotoxicity on normal liver cells, HL-7702 had no killing effect of.6.Hep G2 cell xenografts in nude mice successfully.7. in animal level: Anti GP73-lipoplexes mediated by SUR promoter of HSVtk/GCV gene Dutch act the system of Hep G2 cell xenografts in nude mice and tumor growth Proliferation has inhibitory effect and can prolong the survival period of nude mice with tumor bearing.

【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

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