【作者】 罗先润；

【导师】 李建生；

【作者基本信息】 郑州大学 ， 内科学， 2012， 博士

【摘要】 胃癌是临床最常见的消化道恶性肿瘤之一,全世界每年大约有100多万新发胃癌患者,在我国其发病率和病死率均为全身恶性肿瘤的首位,在国内某些地区已居全部恶性肿瘤死亡原因之首。近年来,随着胃癌相关研究的不断深入,人们在胃癌的发病机制和临床诊治方面取得了大量的成果,但目前,胃癌的诊治仍面临许多困难,整体治疗效果仍不理想,其临床特点依然表现为三低一高,即早期诊断率低(约10%),手术切除率低(<70%),5年生存率低(约40%),根治术后复发转移率高(约50%)。因此,积极寻找新的胃癌治疗手段,提高胃癌的治疗水平,具有积极而重要的现实意义。肿瘤基因治疗通常是指把正常的目的基因利用载体及基因转化方法导入患者体内,利用基因的功能抑制肿瘤细胞生长或杀死肿瘤细胞。自杀基因疗法又称为基因介导的酶前药治疗——GDE(Gene-directed enzyne prodrug treatment),已经成为手术治疗、放射治疗、化疗等传统肿瘤疗法后的一种新的抗肿瘤治疗措施。自杀基因(suicide gene)是指来自病毒和细菌等的一类可以将生物前药转换成有毒产物的基因。此类基因编码的特异性酶类,可以将对细胞本来没有毒或毒性极低的药物前体,在肿瘤细胞内代谢成毒性产物,杀死肿瘤细胞,导致这些细胞自杀。来源于大肠杆菌的胞嘧啶脱氨酶基因(E.coli cytosine deaminase, CD)及来源于病毒的单纯疱疹病毒胸苷激酶基因(herpes simplex virus thymidine kinase, HSV-TK)是目前研究应用较多的两类自杀基因。大肠杆菌胞嘧啶脱氨酶基因编码的酶为胞嘧啶脱氨酶(cytosine deaminase),可激活5-氟胞嘧啶,变成5-氟尿嘧啶,从而抑制DNA和RNA的合成,发挥细胞毒作用。单纯疱疹病毒胸苷激酶基因编码胸苷激酶,可将核苷类似物代谢为二磷酸化物,进一步代谢为对细胞有毒性的三磷酸化合物,进而抑制DNA聚合酶发挥抗肿瘤作用。胃癌自杀基因治疗是指将自杀基因克隆至适当表达载体,然后导入胃癌细胞,自杀基因编码的特异性酶可以将无毒的药物前体代谢成毒性产物,达到杀死胃癌细胞的目的。在肿瘤基因治疗的研究中,基因的靶向性表达是亟需解决的关键问题之一。靶向性表达是指目的基因仅在肿瘤细胞表达,在正常细胞中不表达,从而减少对正常细胞的影响。有些基因的启动子能在肿瘤细胞中特异表达,而在正常细胞中不表达,利用这些启动子序列构建能在肿瘤细胞中特异表达的载体,让目的基因在肿瘤细胞中的特异表达,特异杀死肿瘤细胞,是目前肿瘤基因治疗靶向性的一个有效途径。目前在肿瘤研究中应用的启动子主要有：端粒酶基因启动子、癌胚抗原(CEA)启动子等,但这些启动子存在明显的缺点,如调控作用弱、应用范围窄、特异性不高等。Survivin是凋亡抑制蛋白家族中的一员,它在多数肿瘤中高表达,但是在成人正常终末分化组织中不表达或者是低表达,具有明显的肿瘤特异性。因此利用survivn特异启动子可以驱动下游目的基因在肿瘤细胞中的特异表达,从而避免对正常细胞的损伤。鉴于上述,在本实验中我们克隆了Survivin启动子,构建了Survivin启动子介导的CD/TK表达载体,转染至胃癌细胞,建立3ALB/C裸鼠胃癌模型,给予一定浓度的5-FC、GCV,证实了Survivin-CD/TK系统对裸鼠的胃癌皮下移植瘤有明显的靶向杀伤作用。本论文分为四部分：第一部分Survivin启动子的克隆及在胃癌细胞中的特异表达目的利用分子克隆技术,构建survivin介导的EGFP基因重组真核表达质粒,转染胃癌细胞,鉴定survivin启动子在胃癌细胞的特异表达。方法1.从人外周血白细胞中提取基因组DNA,设计合适引物,通过PCR扩增出survivin启动子并与pMD18-T载体连接,经筛选得到重组质粒pMD-survivin,进行酶切鉴定及测序。2.提取质粒pMD-survivin,酶切获得survivin启动子片段。采用同样酶切真核表达载体pEGFP-Cl,连接,转化细菌,构建重组真核表达载体pEGFP-Su,酶切鉴定。3.脂质体转化胃癌SGC7901细胞及正常胃上皮细胞株GES-1,72小时后荧光显微镜下观察EGFP的表达情况。结果1.经酶切和测序证实成功获得survivin启动子(约910bp)。2.经酶切鉴定成功构建重组质粒pEGFP-Su。3.荧光显微镜下观察,survivin启动子介导的EGFP能特异在胃癌SGC7901细胞表达,而在正常的胃上皮GES-1细胞没有活性。第二部分重组pEGS-CD/TK双自杀基因载体的构建与鉴定目的利用分子克隆技术,构建pEGS-CD/TK双自杀基因载体。方法1.提取JE.coli BL21基因组DNA,设计合适引物,通过PCR扩增出CD基因并与pMD18-T载体连接,经筛选得到重组质粒pMD-CD,进行酶切鉴定及测序。2.提取pORF-HSVtk质粒,设计合适引物,通过PCR扩增出TK基因并与PMD18-T载体连接,经筛选得到重组质粒pMD-TK,进行酶切鉴定及测序。3.将质粒pMD-CD进行双酶切,获得CD基因片段,连接与相同酶切的真核表达载体pEGFP-Su上,构建重组真核表达载体pEGS-CD,酶切鉴定。4.再将质粒pMD-TK进行双酶切,获得TK基因片段,连接与相同酶切的真核表达载体pEGS-CD上,构建重组真核表达载体pEGS-CD/TK,酶切鉴定。结果1.经酶切和测序证实成功获得CD基因(约1281bp),TK基因(约1161bp)。2.经酶切鉴定成功构建重组质粒pEGS-CD。3.经酶切鉴定成功构建重组质粒pEGS-CD/TK。第三部分pEGS-CD/TK双自杀基因载体对胃癌SGC7901细胞的体外杀伤作用目的利用细胞转染技术,研究survivin启动子介导的CD/TK双自杀基因系统对胃癌细胞SGC7901的体外杀伤作用。方法1.利用脂质体转染技术,将pEGS-CD/TK双自杀基因质粒转染胃癌SGC7901细胞、GES-1细胞。2. SGC790、GES-1细胞加入不同浓度的5-FC、GCV前体药物。MTT方法测定吸收值,计算细胞存活率。3.转染和未转染pEGS-CD/TK的SGC7901细胞混合,加入5-FC+GCV二者混合液,观察有无旁观者效应。4.应用RT-PCR检测转染细胞中CD、TK基因mRNA的表达；Western blot法检测转染细胞CD、TK蛋白的表达。5.流式细胞术检测细胞凋亡率。结果1.在给予5-FC、GCV后并达到一定浓度时,5-FC和GCV对SGC7901细胞存活率随着药物浓度的升高而呈现下降趋势,其体外杀伤作用具有浓度依赖性,且联合用药对SGC7901细胞体外的杀伤作用要强于二者单独应用；而不同浓度梯度的5-FC和GCV对GES-1细胞的细胞存活率改变不大。2.转染的SGC7901细胞即可引起大部分未转染的细胞发生凋亡,旁观者效应较为明显。3.通过RT-PCR和(?) Western blot检测证实CD,TK基因在SGC7901细胞中成功表达。4.转基因SGC7901细胞在给予前药处理24h后,治疗组SGC7901细胞凋亡率明显高于对照组凋亡率(P<0.01)。第四部分pEGS-CD/TK双自杀基因系统对胃癌SGC7901细胞裸鼠模型的体内抑瘤效应目的观察survivin启动子介导的CD/TK双自杀基因系统对胃癌SGC7901细胞裸鼠模型的体内抑瘤效应。方法1.动物模型的建立：小鼠左腋下皮下接种2×106SGC7901细胞,第三天复种一次,建立胃癌动物模型。2. SGC7901皮下移植术后第10天,实验分为4组,每组10支。分组方法：A组：注射重组pEGS-CD/TK与前药5-FC与GCV；B组：仅注射重组pEGS-CD/TK;C组：仅注射前药5-FC与GCV；D组：空白对照,不施加任何处理。每3天测量一次皮下移植瘤的大小,计算瘤体体积。处死裸小鼠,HE染色,观察肿瘤的病理变化。3. RT-PCR检测(?)CD/TK基因的表达。结果1.接种肿瘤细胞后10天左右,出现皮下肿瘤结节。裸鼠成瘤达0.5cm后开始治疗。注射重组pEGS-CD/TK与前药5-FC与GCV组与仅注射pEGS-CD/TK,前药GCV和5-FC,空白对照组相比较,移植瘤体积明显缩小,有统计学意义(P<0.05)。2.治疗组切片中可见肿瘤细胞数目稀少,胞浆少,细胞核较小且染色较深,细胞分裂相少,核和胞浆凝缩及坏死区散在分布,纤维血管较少,可见炎性细胞浸润3.治疗组瘤体内RT-PCR可检测至(?)CD/TK基因表达结论1. Survivin启动子能在胃癌肿瘤细胞中特异表达。2.经酶切和测序鉴定,利用分子克隆技术成功获得CD和TK基因。3.成功构建重组Survivin启动子介导的CD/TK双自杀载体系统。4.含CD和TK融合基因的双自杀基因载体对SGC7901细胞具有强烈的杀伤作用,并且存在明显的旁观者效应。治疗组SGC7901细胞凋亡率明显高于对照组凋亡率(P<0.01)。5. RT-PCR及Western blot检测结果CD/TK基因在SGC7901细胞中成功表达。6.应用SGC7901细胞可成功建立致瘤BALB/C裸小鼠。7. Survivin启动子介导的CD/TK双自杀基因能在胃癌小鼠的瘤体内特异表达。8. Survivin启动子介导的CD/TK双自杀基因在前体药物GCV和5-FC作用下,能抑制胃癌瘤体的生长,具备体内抑瘤效应。更多还原

【Abstract】 Gastric cancer is one of the most common digestive system malignancies in clinical practice. There are more than10million new cancers worldwide every year-Gastric cancer has become the first cause of death of all malignant tumors in some areas of China. In recent years, with the deepening of the stomach cancer research, the pathogenesis and treatment of gastric cancer have made a lot of achievements. However, the diagnosis and treatment of gastric cancer still faces many difficulties, and the overall treatment effect is still not ideal. The clinical characteristics shows "three low and one high", namely the diagnosis is low (about10%), surgical excision rate low (＜70%),5years of survival rate low (about40%), radical surgery relapse and metastasis rate high (about50%).Therefore, to find a new stomach cancer prevention and control method, will have a positive and important practical significance to further improve the overall prevention level and cure of gastric cancer.Cancer gene therapy is usually transferring the exogenous genes into patients using expression vector and different methods, which produce the product to inhibit and kill cancer cells. Suicide gene therapy, also called gene-directed enzyme prodrug treatment, has become a new anti-tumor therapeutic approach after the traditional surgical resection, radiotherapy, chemical drug treatment.Suicide gene generally refers to the prodrug conversion gene or cytotoxic receptor gene from prokaryote or lower organisms (viruses and bacteria),which encode the specificity enzyme can convert the nontoxic prodrug (or cytotoxic factor) into toxic product (or cytotoxic factor) to achieve the purpose of killing cancer cells. At present, a variety of suicide genes have been found, including Escherichia coli cytosine deaminase (CD), herpes simplex virus-thymidine kinase (HSV-TK). Escherichia coli cytosine deaminase gene encodes the cytosine deaminase, which can convert the cytosine into uracil, change5-Fc into cells toxic metabolites5-Fu, to play cytotoxic effects through inhibiting RNA and DNA synthesis. HSV-TK gene encodes thymidine kinase, which can convert the nucleoside analogue (NA) into bisphosphate iodide, further turn into triphosphate compound to inhibit the DNA polymerases to play antitumor function.In cancer gene therapy, one of key issues is to improve the targeting expression of therapeutic gene. The targeted expression, is to make the gene function as in tumor cells as possible, and to reduce the influence to normal cells. Specifically expressed promoter sequence in tumor cells has been widely used, which can drive the downstream gene to express in tumor cells specifically, is an effective way to solve the problems of tumor targeted gene therapy. At present, the embryo carcinoma antigen (CEA) promoter, telomerase gene promoter, etc have been used as the regulated element for tumor target gene therapy, but the promoters have some disadvantages, such as application is limited, regulation effect is weak. Survivin is a member of apoptosis inhibiting protein family, it express highly in most tumors, but do not express or lower express in adult normal terminal differentiation tissue, has the obvious tumour specific. Therefore, survivn specific promoter can be used to drive the gene express specific in the tumor cells, to avoid the damage to normal cells.In the current study, we cloned the survivin promoter and constructed the CD/TK expression vector mediated by survivin promoter, then transfected into gastric cancers cells, established nude BALB/C mouse gastric cancers model, gave certain concentration of5-FC, GCV. We demonstrated that survivin-CD/TK systems have obvious target killing function to gastric cancer subcutaneous BALB/C rats.Our studies include the following four parts. Part I:Cloning of survivin promoter and express specific in the gastric cancer cellObjective:The aim of this study was to construct the recombinant EGFP expression vector driven by survivin promoter, and identify the promoter activity specific in gastric cancer cells.Methods:1. Human genome DNA was extracted from white blood cells. Primers were designed according to the sequence of survivin promoter. Survivin gene promoter was obtained by PCR technology, and ligated into the pMD18-T vector, followed by digestion with restriction endonuclease and sequencing.2. pMD-survivin vector was digested by double restriction endonuclease to release the survivin fragment, and ligated into the pEGFP-C1vector to generate the recombinant pEGFP-Su, and then identified by enzyme digestion.3. pEGFP-Su plasmid was transfected into gastric cancer SGC7901cells and normal gastric epithelial GES-1cell, and EGFP gene expression was detected by fluorescence microscope.Results:1. Human survivin promoter of910bp long was obtained via PCR method.2. We constructed the recombinant pEGFP-Su plasmid successfully.3. EGFP driven by survivin promoter can express specific in SGC7901gastric cancer cells, but not in the normal gastric epithelium GES-1cell.Part II:Construction and identification of the recombinant pEGS-CD/TK double suicide genes vectorObjective:To construct and identify the recombinant pEGS-CD/TK plasmid using molecular cloning technology Methods:1. E.coli BL21DNA was extracted. Primers were designed according to the sequence of CD gene. CD gene was obtained by PCR technology, and ligated into the pMD18-T vector, followed by digestion with restriction endonuclease and sequencing.2. pORF-HSVtk was extracted and used as PCR template. Primers were designed according to the sequence of TK gene. TK gene was obtained by PCR technology, and ligated into the pMD18-T vector, followed by digestion with restriction endonuclease and sequencing.3. pMD-CD vector was digested by double restriction endonuclease to release the CD gene fragment,and ligated into the pEGFP-Su vector to generate the recombinant pEGS-CD, then identified them by enzyme digestion.4. pMD-TK vector was digested by double restriction endonuclease to release the TK gene fragment, and ligated into the pEGS-CD vector to generate the recombinant pEGS-CD/TK, then identified them by enzyme digestion.Results:1. CD gene (1281bp) and TK gene (1161bp) were obtained through PCR method.2. We constructed the recombinant pEGS-CD plasmid successfully.3. We constructed the recombinant pEGS-CD/TK plasmid successfully.Part Ⅲ Killing effect of pEGS-CD/TK double suicide gene vector on the Gastric Cancer Cells in vitroObjective:To study the killing effect of CD/TK double suicide genes driven by survivin promoter on the gastric cancer cells in vitro.Methods:1. pEGS-CD/TK plasmid was transfected into SGC7901, GES-1cells using the liposome. 2. The prodrug5-FC and GCV were added into the SGC7901, GES-1cells, and then the cell survival rate was determined by MTT methods.3. The untransfected and transfected SGC7901were mixed, followed by adding the prodrug5-FC, GCV. The bystander effect was observed through MTT methods.4. CD, TK gene expression was detected by RT-PCR and Western blot.5. Cell apoptosis rate was detected by flow cytometry.Results:1. SGC7901cell survival rate decreased with the concentration5-FC, GCV increased, and the killing effect in vitro showed concentration dependent.The killing effect of drug combination on SGC7901cells was stronger than the drug used alone in vitro. However, the5-FC and GCV had little effect on GES-1cells survival rate.2. The transfected SGC7901cells can cause the untranfected cells apoptosis, showed the obvious bystander effect.3. CD, TK gene can express in the transfected in the SGC7901cell through RT-PCR and Western blot assay.4.The transfected SGC7901cells was treated by the prodrug for24h, SGC7901cell apoptosis rate of the treatment group was significantly higher than that of the control group (P<0.01).Part IV:Killing effect of pEGS-CD/TK double suicide gene on the nude mice model of gastric cancer cells in vivoObjective:To investigate the killing effect of CD/TK double suicide genes on the nude mice model of gastric cancer cells in vivo.Methods:1. The nude mice were subcutaneously inoculated by5×106gastric cancer cells, re inoculated at the third day to establish the gastric cancer nude mice model.2. After10days of SGC7901subcutaneous transplantation, the mice were divided into4groups (n=10). Group A:injected with recombinant pEGS-CD/TK and prodrug5-FC and GCV; Group B:only injected with recombinant pEGS-CD/TK; Group C:only injected prodrug5-FC and GCV; Group D:blank control, no any treatment. The size of the subcutaneous transplantation tumor was measured once every3days. The nude mice was killed and stained by HE method, the tumor pathological changes were observed.3. CD/TK gene expression was detected by RT-PCR method.Results:1.Ten days after tumor cells inoculation, subcutaneous tumor nodules appeared.The treatment began after the nude mice tumor achieved0.5cm.Compared with the only injected pEGS-CD/TK, prodrug GCV and5-FC and blank control group, the transplantable tumor size in the injected with pEGS-CD/TK and prodrug GCV and5-FC group was sigficantly reduced, and showed statistically significant (P＜0.05).2.Tumor cells number in the treatment group were visible scarce, had scant cytoplasm, nuclear was smaller and staining was deeper, the cell division was fewer, nuclear and cytoplasm condensed and necrotic area were scattered, fiber blood vessels were fewer, inflammatory cells infiltration was visible.3. CD, TK gene expression could be detected by RT-PCR in the treatment group.Conclusions1. Survivin promoter expresses specific in the gastric cancer cells.2. CD and TK genes have been obtained successfully by gene cloning technology.3. CD/TK double suicide genes plasmid driven by survivin promoter has been constructed.4. CD and TK double suicide genes have stronger killing effect on the SGC7901cells, and show the obvious bystander effect.SGC7901cell apoptosis rate of the treatment group is significantly higher than that of the control group (P＜0.01).5. CD/TK gene express in SGC7901cells successfully.6. Tumorigenic BALB/C nude mice have been successful established using SGC7901cells. 7. CD/TK double suicide gene driven by Survivin promoter can specifilly express in the gastric cancer cells.8. CD/TK double suicide gene driven by survivin promoter can inhibit the growth of gastric cancer in the presence of prodrug drug GCV and5-FC, has an inhibitory effect in vivo.更多还原