【作者】 唐勇；

【导师】 刘长安；

【作者基本信息】 重庆医科大学 ， 外科学， 2010， 硕士

【摘要】 基因治疗开辟了肝癌治疗的新途径,成为肝癌治疗的有效方式之一,越来越受到人们的重视,是目前医学生物学界研究的热点。基因治疗的关键之一是获得一种载体安全有效地介导基因转染靶细胞。然而目前应用的病毒载体和非病毒载体均存在局限性,无法同时具备安全性,靶向性和有效性,因此临床应用受到限制。目前,自杀基因治疗是肿瘤基因治疗中最有成效的方案之一,其中单纯疱疹病毒Ⅰ型胸苷激酶基因/丙氧鸟苷(HSV1-TK/GCV)自杀基因系统在肝癌基因治疗中最为成熟。其原理是向肿瘤细胞导入HSV1-TK基因,通过表达其编码的胸苷激酶,将低毒的抗病毒药物更昔洛韦磷酸化,从而阻碍细胞DNA的合成,杀灭肿瘤细胞。由于该系统缺乏特异性的靶向载体,单独应用转染率极低,疗效不佳。最近研究发现,超声靶向破坏微泡能增强基因的转染,其原理是利用超声监测并击碎到达靶组织内的微泡,释放目的基因,实现基因治疗的精确性和靶向性。超声波作用于微泡产生的空化效应和机械效应使靶区组织细胞膜性结构通透性增加,并致直径≤7μm的微血管破裂,血管内皮细胞间隙增宽,使得外源基因更容易穿透毛细血管进入组织和细胞内;而微泡作为基因转染的远程靶向促进剂,同时也可在基因传输过程中能保护基因;并且低频超声辐照条件下,待转染基因不会被破坏。因此,本实验通过构建增强型绿色荧光蛋白(EGFP)与单纯疱疹病毒Ⅰ型胸苷激酶基因(HSV1-TK)的真核共表达质粒载体(pIRES2-EGFP-TK),然后运用低频超声辐照破坏微泡介导HSV1-TK基因转染肝癌细胞,观察TK基因在肝癌细胞中的转染率和表达情况,以及超声微泡介导HSV1-TK/GCV自杀基因系统对肝癌细胞的杀伤效果,以期为肝癌的靶向基因治疗提供实验依据。本课题研究分为以下两部分内容。第一部分质粒pIRES2-EGFP-TK的构建及其在人肝癌细胞株HepG2中的表达目的构建增强型绿色荧光蛋白与单纯疱疹病毒Ⅰ型胸苷激酶真核共表达质粒载体(pIRES2-EGFP-TK),并检测它在肝癌细胞株HepG2中转染和表达。方法从含有目的基因cDNA的质粒pORF-HSV1tk中获得HSV1-TK基因片段,连接到载体pIRES2-EGFP上,得到重组质粒pIRES2-EGFP-TK并进行酶切鉴定、测序。将重组质粒用脂质体法转染肝癌细胞株HepG2,荧光显微镜下观察EGFP在细胞内的表达,Western Blot法检测TK蛋白表达情况。结果重组质粒酶切和测序结果与预期完全相符,单纯疱疹病毒Ⅰ型胸苷激酶基因HSV1-TK序列与GenBank报告的序列一致,且插入方向正确。重组质粒转染肿瘤细胞后在荧光显微镜下可观察到有明亮的绿色荧光,与空白质粒pIRES2-EGFP转染后的荧光表达无明显差异。Western-blot结果表明,重组质粒转染HepG2组HSV1-TK基因高表达,而对照组几乎没有TK表达,两者比较有统计学差异,P<0.01。结论本实验成功构建EGFP和TK真核共表达质粒,在肝癌细胞能有效表达,为肝癌基因治疗的下一步研究奠定了实验基础。第二部分超声微泡介导HSV1-TK/GCV自杀基因系统对人肝癌细胞株HepG2的杀伤作用目的探讨超声微泡介导HSV1-TK/GCV自杀基因系统对人肝癌细胞株HepG2的杀伤效率,为肝癌靶向基因治疗提供实验依据。方法将HepG2接种在培养板中,随机分为以下5组:(1)空白对照组;(2)单纯质粒组;(3)超声微泡+质粒组;(4)超声+质粒组;(5)超声+超声微泡+质粒组。转染后24h,荧光显微镜观察各组细胞绿色荧光的表达情况,流式细胞仪检测各组细胞的转染效率,MTT法检测转染方法对HepG2活性的影响,Western Blot法检测各组细胞的TK蛋白表达情况;转染后24h,各组细胞加入不同浓度梯度的更昔洛韦(GCV),72h后MTT法检测各组细胞的存活率。结果超声+超声微泡+质粒组的绿色荧光强度、细胞转染率及TK蛋白的表达均高于其他各组(p<0.05);转染方法对HepG2细胞活性无明显影响(p>0.05);GCV浓度在50ug/mL以上培养72h后,低频超声+超声微泡+质粒组细胞几乎全部被杀灭,杀伤效应最强(p<0.05),其余各组几乎无杀伤作用。结论超声破坏微泡造影剂可提高HSV1-TK基因在HepG2中的转染和表达,增强HSV1-TK/GCV自杀基因系统对肝癌细胞的杀伤效应,而超声辐照和微泡造影剂在转染过程中对细胞的活性无影响。本实验结果为肝癌靶向基因治疗提供了良好的基础。更多还原

【Abstract】 The advent and development of gene therapy has provided new insights into the treatment for liver cancer. Being a“hot spot”of biomedical researches, gene therapy, especially that mediated by HSV1-TK/GCV suicide gene system, has now become one of the most effective approaches for the treatment of liver cancer and attracted more and more attentions. The key problem of gene therapy is to choose a reliable and effective vector for transfecting gene of interest into the target cell. However, existing viral or non-viral vectors have drawbacks, such as hepatic toxicity, low tissue specificity and lack of security, which limit their clinical appliance.Recent studies have showed that ultrasound-targeted microbubble destruction (UTMD) were able to increase membrane permeability and enhance exogenous genetic materials into targeted cells by mechanical and cavitation effects of UTMD, ultrasound-targeted microbubble destruction is a new noninvasive gene transfer technology, providing a new method of gene therapy for liver cancer. In the current study, we prepare to construct vector pIRES2-EGFP-TK that co-expressing enhanced green fluorescent protein and (EGFP) and herpes simplex virus type 1 thymidine kinase (HSV1-TK). And by transfecting the recombinant plasmid into the HepG2 cell line by ultrasound-targeted microbubble destruction, we seek to observe the expression of the EGFP and TK protein in hepatocarcinoma cells and .explore the specific lethal effectiveness of treating hepatoma carcinoma in vitro using ultrasound-targeted microbubble destruction mediated by HSV1-TK/GCV suicide gene system. Our study can provide reliable experimental evidences for the research of targeted gene therapy for hepatoma carcinoma. The study includes the follwiong two parts:PARTⅠ:CONSTRUCTION OF A VECTOR CO-EXPRESSING EGFP AND HSV1-TK AND ITS EXPRESSION IN HUMAN HEPATOMA CELL LINE HEPG2Objective To construct a vector co-expressing EGFP and HSV1-TK, and detect its expression level in human hepatoma cell line HepG2.The insertion and the direction of the insert were confirmed by polymerase chain reaction (PCR) and restriction enzyme digestion. Methods HSV1-TK gene was isolated from pORF-HSV1tk plasmids and cloned into eukaryotic expression plasmid pIRES2-EGFP. The recombinant plasmid pIRES2-EGFP-TK was identified by restriction endonuclease digestion and DNA sequencing. Then recombinant plasmid was transfected into hepatoma carcinoma cell line HepG2 by liposome reagent,and the expression of EFGP were observed by fluorescence microscopy in cells , TK protein was analyzed by Western blotting . Results The sequence of the cloned DNA fragment was identical to HSV1-TK that was reported on GeneBank and direction of the insert were correct. The recombinant expression plasmid was successfully transfected into HepG2 cell line, and green fluorescent was observed under fluorescent microscope.The protein expression level of TK was high. Conclusion The recombinant eukaryotic co-expression vector of EGFP and HSV1-TK was successfully constructed and can be effectively expressed in HepG2 cell line.PARTⅡ: HSV1-TK/GCV SUICIDE GENE SYSTEM FOR HEPATOCELLULAR CARCINOMA BY ULTRASOUND CONTRAST AGENT MEDIATED IN VITROObjective To explore the specific lethal effectiveness of treating hepatoma carcinoma in vitro using ultrasound-targeted microbubble destruction mediated by HSV1-TK/GCV suicide gene system; and indentify the effects of ultrasound-targeted microbubble destruction treatment on the cell viability and provide experimental basis for further research. Methods HepG2 was seeded in the 24-well plates, and randomly divided into 5 groups, (1) Blank control group. (2) HSV1-TK: add pIRES2-EGFP-TK only ;(3) HSV1-TK+UM: add gene and ultrasound contrast agent but didn’t exposed to pulsed-ultrasound; (4) HSV1-TK+US: add gene and ultrasound contrast agent then exposed to pulsed-ultrasound; (5) HSV1-TK+UM+US: add gene and ultrasound contrast agent and pIRES2-EGFP-TK, then exposed to pulsed-ultrasound. After 24 hours, expression of EGFP was observed on fluorescent microscopy and quantified by FACS analysis. Western Blotting was used to detect the expression of TK protein. 24 hours after transfection, different concentrations of GCV were added into the culture mediums, then cell were cultured for another 72 hours. The viability of HepG2 was measured by MTT assay. Results Gene fluorescence intensity and tansfection efficiency in group of ultrasound contrast agent and ultrasound and pIRES2-EGFP-TK were higher than other groups, TK protein in the fifth group was higher than other groups(P<0.05).The cell viability had no significant difference before add GCV. Almost all HepG2 in the fifth group have been killed when the concentration of GCV is above 50ug/mL, and we observed almost no killing effect in other groups after add different concentrations of GCV. Conclusion Ultrasound-targeted microbubble destruction can enhance the efficiency of gene transfection without obvious damage to cell viability in HepG2.And the method can also enhance the killing effect of HSV1-TK/GCV suicide gene system in vitro. This experimental study provides a good basis for the research of targeted gene therapy for hepatoma carcinoma.更多还原