【作者】 柯昌兴；

【导师】 王剑松； 徐鸿毅； 杨德林； 左毅刚；

【作者基本信息】 昆明医学院 ， 泌尿外科， 2010， 博士

【摘要】 研究表明,第三代溶瘤单纯疱疹病毒G47δ（Herpes simplex Virus G47delta,简称G47δ）,具有较第二代溶瘤单纯疱疹病毒G207特异性地杀伤肿瘤细胞的能力更强、速度更快。内源性血管生长抑制因子可抑制肿瘤原发灶和转移灶的生长,具有无免疫源性,不良反应轻等优点。丝裂霉素C（Mitomycin C,MMC）广泛用于膀胱尿路上皮癌（简称膀胱癌）术后的膀胱灌注化疗。联合应用G47δ与内源性血管生长抑制因子或MMC抗膀胱癌,可能发挥协同作用。研究目的1.探讨能否从人膀胱癌组织克隆出Kringle 5基因（简称K5）,并鉴定其原核表达蛋白有无生物学活性。2.探讨G47δ、pcDNA3.1/K5对膀胱癌细胞的增殖抑制作用,二者联合应用有无协同作用。3.探讨联合应用G47δ和MMC对膀胱癌细胞的协同杀伤作用。4.探讨能否从人膀胱癌组织克隆出Endostatin基因（简称ES）,并把ES与K5拼接构建成融合基因的技术和方法。5.探讨膀胱癌患者血、尿及癌组织中血管生成因子和抑制因子的表达水平与膀胱癌分级、分期的相关性,为运用内源性血管生成抑制因子抗膀胱癌治疗提供依据。研究内容与方法1.K5基因的克隆、优化表达、纯化及活性鉴定:从人膀胱癌组织中提取总RNA,RT-PCR扩增K5基因;构建原核表达载体pGEX-5X-1/K5;运用工程菌表达蛋白,并对表达条件参数进行优化;带谷胱甘肽巯基转移酶（GST）标签琼脂糖4B亲和柱层析、纯化蛋白;CellTiter 96 AQ_ueous细胞增殖检测法（MTS）检测K5蛋白对人脐静脉内皮细胞ECV304增殖的影响。2.pcDNA3.1/K5联合G47δ抗膀胱癌效应研究:提取病毒DNA,PCR扩增目的片段,进行琼脂糖凝胶电泳和基因测序鉴定G47δ构建的正确性;蚀斑实验测定G47δ的蚀斑形成单位;培养人膀胱癌细胞株BIU-87（浅表性膀胱癌）和EJ（侵润性膀胱癌）细胞;构建重组真核表达质粒pcDNA3.1/K5,用脂质体PolyFect介导pcDNA3.1/K5转染BIU-87和EJ细胞,再用G47δ感染癌细胞,MTS法检测癌细胞增殖情况;构建pEGFP-n2/K5,用PolyFect介导pEGFP-n2/K5转染癌细胞,再用G47δ感染癌细胞,荧光显微镜观测K5在癌细胞中的定位变化;蚀斑实验检测pcDNA3.1/K5-PolyFect对G47δ的繁殖影响;pcDNA3.1/K5转染癌细胞后,RT-PCR检测癌细胞内K5 mRNA水平。3.G47δ联合MMC抗膀胱癌效应研究:G47δ感染癌细胞1h后,加MMC,MTS法检测癌细胞增殖情况,Chou-Talalay法推算联合用药指数并用Isobologram法分析联合用药效应;比较G47δ与野生型HSV-1对癌细胞增殖抑制作用;蚀斑实验检测MMC对G47δ病毒繁殖有无影响。4.ES基因的克隆、ES-K5融合基因拼接及其真核表达质粒的构建:从人膀胱癌组织中提取总RNA,RT-PCR扩增ES基因;重叠延伸拼接法（SOE）构建ES-K5融合基因;构建真核表达质粒pcDNA3.1/ES、pcDNA3.1/ES-K5、pEGFP-n2/ES-K5,基因测序鉴定。5.膀胱癌患者血、尿及癌组织中血管生成因子和抑制因子的检测:酶联免疫吸附试验（ELISA）检测膀胱癌患者血清、尿液中VEGF（vascularendothelial growth factor）、Angiostatin（AS）、ES、K5表达水平,免疫组织化学法（IHC）检测膀胱癌组织标本中VEGF、AS、ES、K5表达情况,分析它们与膀胱癌分级、分期的相关性。实验结果1.K5基因的克隆、优化表达、纯化及活性鉴定:PCR扩增得到282 bp基因片段,并成功插入pGEX-5X-1质粒;诱导温度为37℃,IPTG浓度为1mmol/L,时间为6h,pGEX-5X-1/K5在BL21中表达最佳;获得特异性融合蛋白,经凝血酶酶切后得到分子量约为12kD的重组K5蛋白（rK5）;rK5能特异性抑制ECV304细胞增殖,抑制率最高蛋白浓度为5μg/ml。2.pcDNA3.1/K5联合G47δ抗膀胱癌效应研究:G47δ能在Vero细胞中繁殖,并出现特征性的CPE病变;琼脂糖凝胶电泳PCR扩增的G47δ目的片段条带宽亮,位于250bp、500bp之间,而PCR扩增的HSV-1目的片段位于500bp、750bp之间,病毒DNA序列测序均正确,表明所重组G47δ构建正确;蚀斑实验计算G47δ蚀斑形成单位为2.5×10⁷pfu/ml;pcDNA3.1/K5、G47δ对BIU-87、EJ细胞增殖有抑制作用,而且抑制率与G47δ剂量、作用时间正相关,联合作用抑制率比其单一作用要强;荧光显微镜观测,K5-EGFP融合蛋白在癌细胞胞浆、胞核中均有表达,G47δ对其定位并无影响;pcDNA3.1/K5对G47δ繁殖无明显影响,蚀斑形成单位数基本相同;pcDNA3.1/K5转染癌细胞后,RT-PCR扩增K5 mRNA条带位于250bp、500bp之间,符合K5核苷酸大小。3.G47δ联合MMC抗膀胱癌效应研究:G47δ、MMC对BIU-87、EJ细胞增殖均有抑制作用,而且抑制率与G47δ、MMC的剂量和作用时间正相关,Chou-Talalay法分析合用指数CI_x＜1,Isobologram法分析q＜1,表明二者具有协同作用;G47δ对癌细胞的增殖抑制作用比野生型HSV-1明显要强;MMC对G47δ繁殖无明显影响,蚀斑形成单位数基本相同。4.ES基因的克隆、ES-K5融合基因拼接及其真核表达质粒的构建:PCR扩增得到552bp基因片段,并成功插入pcDNA3.1质粒,基因测序正确;SOE法所构建融合基因ES与K5中间的Linker为（Gly4Ser）3,ES-K5融合基因成功插入pcDNA3.1和pEGFP-n2真核表达质粒,基因测序正确。5.膀胱癌患者血、尿及癌组织中血管生成因子和抑制因子的检测:膀胱癌患者血清、尿液中VEGF、AS、ES、K5表达水平与膀胱癌分级、分期正相关;VEGF、AS、ES在癌组织中的表达也与分期正相关。结论1.K5基因的克隆、优化表达、纯化及活性鉴定:1)从膀胱癌组织能克隆出Kringle5基因。2)对蛋白表达条件参数优化后,确定诱导温度为37℃,IPTG浓度为1mmol/L,时间为6h,pGEX-5X-1/K5在BL21中表达最佳。3)含重组质粒pGEX-5X-1/K5大肠杆菌表达产物量较多且易纯化。2.pcDNA3.1/K5联合G47δ抗膀胱癌效应研究:1) pcDNA3.1/K5、G47δ对膀胱癌细胞增殖均有抑制作用,而且抑制率与G47δ剂量、作用时间正相关,pcDNA3.1/K5与G47δ联合作用抑制率比其单一作用要强。2) pcDNh3.1/K5对G47δ病毒繁殖无明显影响。3) pcDNA3.1/K5转染癌细胞后,能在细胞内稳定表达。3.G47δ联合MMC抗膀胱癌效应研究:1) G47δ、MMC对膀胱癌细胞增殖均有抑制作用,抑制率与G47δ、MMC的剂量和作用时间正相关,二者合用具有协同作用。2) G47δ对癌细胞的增殖抑制作用比野生型HSV-1明显要强。3) MMC对G47δ繁殖无明显影响。4.ES基因的克隆、ES-K5融合基因拼接及其真核表达质粒的构建:1)从膀胱癌组织能克隆出Endostatin基因。2) SOE法所构建Linker能把ES与K5拼接起来,并能成功插入到真核表达质粒中。5.膀胱癌患者血、尿及癌组织中血管生成因子和抑制因子的检测:1) VEGF、AS、ES、K5在膀胱癌中分泌表达与分级、分期正相关。2)肿瘤血管新生正性和负性调控平衡被打破的原因可能是由于血管生成因子与血管生成抑制因子表达分泌均增多、增强,但以前者更为显著。3)去除原发肿瘤后微转移灶迅速生长的原因是由于微转移灶中血管生成因子与由原发肿瘤所分泌的血管生成抑制因子来源减少而使调控平衡被打破,但这一机制不能合理解释血管生成抑制因子表达量与肿瘤分级、分期正相关的现象,有待于进一步研究。更多还原

【Abstract】 The third-generation oncolytic herpes simplex virus G47δ(G47δ) can kill cancer cells more stronger and faster than the second-generation oncolytic HSV G207,it has already been proved by results of experimental research.The growth of primary and metastatic cancers can be restrained by endogenous angiogenesis inhibitors with merits of little immunogenicity or adverse reactions.Mitomycin C(MMC) as an intravesical chemotherapy drug is widely used to treat urothelial carcinoma of bladder after transurethral resection of bladder tumor(TURBt).It is possible to exert drug synergism to combine oncolytic herpes simplex virus G47δwith angiogenesis inhibitor or MMC on urothelial carcinoma of bladder.Objective1.To investigate whether the Kringle 5(K5) gene could be cloned from the human bladder carcinoma tissue,and to identify biological activity of bacterial expressed recombinant K5 protein.2.To investigate the killing effect of G47δor pcDNA3.1/K5 on bladder cancer cells,and to evaluate the drug synergism of the combination of the two drugs.3.To investigate the drug synergism of G47δand MMC on killing bladder cancer cells.4.To investigate whether the Endostatin(ES) gene could be cloned from the human bladder carcinoma tissue,and the method of ES fused with K5 gene.5.To investigate the correlation of pathologic grade and clinical stage with expression level of VEGF(vascular endothelial growth factor),Angiostatin(AS), ES,K5 in serum,urine,or cancer tissue.Materials and Methods1.Cloning,optimal expression,purification and activity identification of K 5: Total RNA was extracted from human bladder carcinoma tissue,then the K5 cDNA was amplified by RT-PCR.A recombinant prokaryotic expression vector pGEX-5X-1/K5 was constructed and the K5 protein was expressed in engineering bacteria.K5 was purified by tagged GST agarose 4B affinity column chromatography,the anti-proliferative effects of K5 on ECV304 were examined by MTS assay. 2.Combining pcDNA3.1/K5 with G47δon killing bladder cancer cells:DNA were extracted from G47δand wild-type HSV-1,then the target fragments amplified by PCR were identified by agarose gel electrophoresis and gene sequencing technologies; Virus titer was determined by virus plaque test;A eukaryotic expression vector pcDNA3.1/K5 was constructed and pcDNA3.1/K5 was transferred into human urinary bladder carcinoma cell line BIU-87 or EJ by polyfect liposome,subsequently,the cells were infected with G47δ,then the anti-proliferative effects of pcDNA3.1/K5 and G47δon BIU-87 or EJ cell were examined by MTS assay;Recombinant plasmid of pEGFP-n2/K5 was constructed and the plasmid was transferred into cancer cells,and the cells were infected with G47δsubsequently,the location of K5 in cells was observed through fluorescence microscope;The effects of pcDNA3.1/K5 on replication of G47δwas assessed using virus plaque test;K5 mRNA was detected by RT-PCR after pcDNA3.1/K5 was transferred into cancer cells by PolyFect liposome.3.Combining MMC with G47δon killing bladder cancer cells:The cancer cells were infected with G47δ,1h later,MMC was added,the anti-proliferative effects of MMC and G47δon BIU-87 or EJ cells were examined by MTS assay,the combination index (CI)-isobologram of Chou-Talalay analysis was used to analyze virus and drug combinations;The anti-proliferative effects of wild-type HSV-1 and G47δon cancer cell were assessed by MTS assay;The effects of MMC on replication of G47δwas assessed using virus plaque test.4.Cloning of ES,ES fused with K5 gene and eukaryotic expression vector with ES-K5 was constructed:Total RNA was extracted from human bladder carcinoma tissue,then the ES cDNA was amplified by RT-PCR;The fusion gene of ES-K5 was artificially synthesized by splicing by overlap extension(SOE) method and inserted into eukaryotic expression vectors such as pcDNA3.1,pEGFP-n2,then the recombinant plasmids were identified by gene sequencing technologies.5.Detection of the expression level of VEGF,AS,ES,K5 in serum,urine,or cancer tissue:Expression level of VEGF,AS,ES,K5 in serum and urine were detected with ELISA,and expression level of them in cancer tissue was detected with IHC,then correlation of pathologic grade and clinical stage with the expression level of VEGF,AS, ES,K5 was analyzed.Results1.Cloning,optimal expression,purification and activity identification of K 5: The acquired gene was 282bp,and it was inserted into pGEX-5X-1 plasmid successfully; The optimal parameters of expressing K5 in prokaryotie expression system pGEX-5X-1 are: engineering bacteria for E.coli BL21,temperature for 37℃,concentration of IPTG for 1 mmol/L,time for 6h;K5 protein,molecular weight of 12 kD,was obtained after digested by thrombin;K5 protein showed effective in specifically inhibiting proliferation of ECV304 and its optimum concentration was 5μg/ml.2.Combining pcDNA3.1/K5 with G47δon killing bladder cancer cells:G47δvirus can incubate very rapidly in Vero cell;Agarose electrophoresis shows:the target fragment of G47δamplified by PCR was between 250bp and 500bp,but the target fragment of wild-type HSV-1 was between 500bp and 750bp,DNA sequences of two viruses were right by gene sequencing analysis,these indicated that the recombinant virus of G47δwas constructed successfully;The virus titer was 2.5×10~7 pfu/ml by virus plaque test;Both pcDNA3.1/K5 and G47δcould restrain the proliferation of BIU-87 and EJ cells, the inhibition effects were positively correlated with their action time and dosage,and combining pcDNA3.1/K5 with G47δcould cause the greater inhibition than one of them; G47δdid not disturb the location of K5 in cytoplasm and karyon;pcDNA3.1/K5 hardly disturbed on replication of G47δ,the number of plaque forming unit was same;K5 mRNA was between 250bp and 500bp,which was accorded with the molecular weight of K5.3.Combining MMC with G47δon killing bladder cancer cells:Both MMC and G47δcould restrain the proliferation of BIU-87 and EJ cells,the inhibition effects were positively correlated with their action time and dosage,and combination index<1 which indicated the drug synergism;G47δcaused the greater inhibition than wild-type HSV-1; MMC hardly disturbed on replication of G47δ,the number of plaque forming unit was same.4.Cloning of ES,ES fused with K5 gene and eukaryotie expression vector with ES-K5 was constructed:The acquired gene was 552bp,and it was inserted into pcDNA3.1 plasmid successfully;The fusion gene of ES-K5 was constructed successfully by the method of SOE,the linker was(Gly4Ser)3,and ES-K5 was inserted into pcDNA3.1 and pEGFP-n2 plasmids successfully.5.Detection of the expression level of VEGF,AS,ES,K5 in serum,urine,or cancer tissue:Expression level of VEGF,AS,ES,K5 in serum and urine were positively correlated with pathologic grade and clinical stage;expression level of them in cancer tissue was positively correlated with pathologic grade too.Conclusions1.Cloning,optimal expression,purification and activity identification of K5:1) K5 gene was cloned successfully from human bladder carcinoma tissue.2) The optimal parameters of expressing K5 in prokaryotic expression system pGEX-5X-1 are:engineering bacteria for E.coli BL21,temperature for 37℃, concentration of IPTG for 1 mmol/L,time for 6h. 3) K5 protein has a large expression level and easy to purify by way of E.coli containing recombinant plasmid pGEX-5X-1/K5.2.Combining pcDNA3.1/K5 with G47δon killing bladder cancer cells:1) pcDNA3.1/K5 and G47δcould restrain the proliferation of bladder cancer cell,the inhibition effects were positively correlated with their action time and dosage,and combining pcDNA3.1/K5 with G47δcaused the greater inhibition than one of them.2) pcDNA3.1/K5 hardly disturbed on replication of G47δ.3) pcDNA3.1/K5 can stably express in cells3.Combining MMC with G47δon killing bladder cancer cells:1) MMC and G47δcould restrain the proliferation of bladder cancer cells,the inhibition effects were positively correlated with their action time and dosage,and combination can produce synergistc effect.2) G47δcaused greater inhibition than HSV-1.3) MMC hardly disturbed on replication of G47δ.4.Cloning of ES,ES fused with K5 gene and eukaryotic expression vector with ES-K5 was constructed:1) ES gene was cloned successfully from human bladder carcinoma tissue.2) Fusion gene of ES-K5 could be inserted into eukaryotic expression vectors.5.Detection of the expression level of VEGF,AS,ES,K5 in serum,urine,or cancer tissue:1) Expression level of VEGF,AS,ES,K5 in bladder cancer was positively correlated with pathologic grade and clinical stage.2) Positive and negative regulation control angiogenesis,and the balance is broken in tumor angiogenesis,the possible reason was that it increased the expression and secretion of both angiogenesis factors and angiogenesis inhibitors,but biological activity of angiogenesis factors is stronger.3) The reason of a rapid progression of metastasis after surgical resection of the primary tumor is that the reduction of production and secretion of antimitotic factors by the cells of the primary tumor that inhibit the proliferation of metastatic cells,but it can not reasonably explain that expression level of angiogenesis inhibitor were positively correlated with pathologic grade and clinical stage.更多还原