【作者】 肖朝文；

【导师】 薛新波；

【作者基本信息】 华中科技大学 ， 外科学， 2011， 博士

【摘要】 第一部分携带人mda-7/IL-24基因的溶瘤腺病毒SG600-IL24载体的构建及表达鉴定目的：本文通过构建携带人MDA-7/IL-24基因的溶瘤腺病毒SG600-IL24载体,观察其在肝癌细胞和正常肝细胞中的表达,探讨其对肝癌基因治疗的理论价值。方法：酶切质粒ZD55-IL24得到IL24连接到pClon9-INS-IL24,将pClon9-INS-IL24共酶切得到INS+IL24片段,装入SG502-△CR2载体中,获得重组质粒pSG600-IL24,以Polyfect介导将其与腺病毒骨架质粒ppE3共转染293细胞,通过细胞内同源重组,生成携带人MDA-7/IL-24基因的溶瘤腺病毒SG600-IL24,同样的方法构建空载体SG600-EGFP。将其感染人肝癌细胞株HepG2、Hep3B、SMMC7721和正常的肝细胞L02, RT-PCR验证mda-7/IL-24基因的表达。结果：可见感染后的HEK293细胞出现特征性CPE,细胞变大、变圆、漂浮,呈葡萄串聚集。取克隆1和克隆2的病毒DNA各2μ1为模板,通过PCR方法扩增出约640bp的特异性片段,其大小与阳性对照相符,而阴性对照SG600-EGFP则无片段扩增。RT-PCR结果提示该载体能介导外源基因mda-7/IL-24在人肝癌细胞和正常肝细胞中高效表达。结论：成功构建了携带人mda-7/IL-24基因的溶瘤腺病毒SG600-IL24载体,该载体能介导mda-7/IL-24基因在人肝癌细胞和正常肝细胞中高效表达。目的：观察携带人：mda-7/IL-24基因的溶瘤腺病毒SG600-IL24对肝癌细胞株HepG2. Hep3B、SMMC7721、MHCC97L、HCCLM3和正常的肝细胞L02的选择性抗肿瘤作用,为肝癌的基因治疗提供理论基础。方法：将携带人mda-7/IL-24基因的溶瘤腺病毒SG600-IL24感染肝癌细胞株HepG2、Hep3B、SMMC7721、MHCC97L、HCCLM3和正常的肝细胞L02。通过逆转录-聚合酶链式反应(RT-PCR)、酶联免疫吸附试验(ELISA)、Western-blot观察MDA-7/IL-24基因的表达,四甲基偶氮哩蓝染色法(MTT)观察肝癌细胞的生长抑制,Hoechst33258染色观察其对肝癌细胞的抗肿瘤作用,Annexin-V和PI双染后流式细胞仪检测细胞的凋亡,以及利用PI单染法流式细胞仪检测SG600-IL24对细胞周期的影响。结果：RT-PCR及Western-blot提示溶瘤腺病毒SG600-IL24能介导外源基因MDA-7/IL-24在肝癌细胞株HepG2、Hep3B、SMMC7721、MHCC97L、HCCLM3和正常的肝细胞L02中高效表达,ELISA检测结果提示细胞培养上清液中MDA-7/IL-24蛋白也明显增加,MTT结果表明SG600-IL24能明显抑制各种肝癌细胞的生长,Hoechst33258染色提示SG600-IL24促进肝癌细胞的凋亡,流式细胞仪提示SG600-IL24能选择性杀伤肝癌细胞,细胞周期分析提示MDA-7/IL-24阻滞肝癌细胞在G2/M期,而对正常的肝细胞没有明显的促凋亡作用和增殖阻滞作用。结论：携带人mda-7/IL-24基因的溶瘤腺病毒SG600-IL24能特异性杀伤不同转移潜能的肝癌细胞和诱导凋亡,促进肝癌细胞增殖阻滞,而对正常肝细胞无明显的促进凋亡和增殖阻滞作用。第三部分溶瘤腺病毒SG600-IL24选择性杀伤肝癌细胞的机理探讨目的：探讨携带人mda-7/IL-24基因的溶瘤腺病毒SG600-IL24对肝癌细胞株HepG2、HCCLM3的杀伤机制,为肝癌的基因治疗提供理论基础。方法：用溶瘤腺病毒SG600-IL24干预肝癌细胞株HepG2、HCCLM3和正常肝细胞株LO2。通过RT-PCR和Western-blot蛋白印迹检测SG600-IL24干预后,肝癌细胞株HepG2、HCCLM3和正常肝细胞株LO2内不同时间点总STAT3以及信号通路下游信号分子C-myc、bax. bcl-2、bcl-xl、CyclinD2、Survivin、XIAP、OPN、MMP-2、MMP-9和VEGF基因和蛋白的表达变化,同时检测各细胞株在溶瘤腺病毒SG600-IL24处理后,磷酸化STAT3蛋白的表达变化。结果：RT-PCR和Western-blot结果提示STAT3以及信号通路下游信号分子C-myc、Bcl-xl、bcl-2、CyclinD2、Survivin、MMP-2、MMP-9、XIAP、OPN、VEGF基因和蛋白表达水平下调,而Bax基因和蛋白表达水平在溶瘤腺病毒SG600-IL24感染后上调。而磷酸化STAT3蛋白在感染后先上升后下降,在感染2小时达到峰值。.结论：溶瘤腺病毒SG600-IL24可能通过抑制STAT3及下游的信号通路来达到抗肝癌的作用,而正常肝细胞几乎不受此影响,使得溶瘤腺病毒SG600-IL24具有显著的选择性抗肿瘤效应。第四部分溶瘤腺病毒SG600-IL24和干扰素联合治疗裸鼠肝癌的研究目的：利用携带人mda-7/IL-24基因的溶瘤腺病毒SG600-IL24与干扰素(Interferon, IFN)联合治疗肝癌裸鼠移植瘤,探讨病毒基因治疗和新辅助治疗相结合治疗肝癌的新方法。方法：首先建立裸鼠皮下种植肝癌模型,当肿瘤体积生长至100-150mm3，计为第0天,并将裸鼠随机分为4组,每组8只：(1)对照组：正常荷瘤裸鼠,每只裸鼠给予生理盐水0.3ml,右侧腹腔内注射,每天一次,共3次。(2)新辅助治疗组(IFN组)：IFN1.5×104IU/g/只,注射于右侧腹腔,每天一次,共10次。(3)基因治疗组(SG600-IL24组)：给予溶瘤腺病毒(SG600-IL24)治疗,肿瘤局部多点注射,每只2×108PFU/次,共5次,隔日一次。(4)基因治疗+新辅助治疗组(SG600-IL24+IFN组)：IFN的用法同前,SG600-IL24在IFN治疗结束后一天进行,SG600-IL24的用法同基因治疗组。并在干预30d后检测肿瘤组织中mda-7/IL-24基因和蛋白的表达,随后观察各组裸鼠生存时间及肿瘤体积的变化。结果：溶瘤腺病毒SG600-IL24介导的外源基因MDA-7/IL-24在基因治疗组和联合治疗组裸鼠体内表达明显增高,SG600-IL24+IFN联合治疗组裸鼠有3只生存时间超过120d,达到长期生存,与其他三组比较生存期明显延长(P<0.01); SG600-IL24+IFN组裸鼠肝癌体积也明显小于SG600-IL24组或IFN(P<0.01)。结论：溶瘤腺病毒SG600-IL24联合干扰素对裸鼠人肝癌种植模型有很好的协同抗肿瘤作用,为肝癌的基因病毒治疗提供了很好的前景。更多还原

【Abstract】 Construction and identification of Oncolytic Adenovirus SG600-IL24 Expressing Human MDA-7/IL-24Objective:We constructed oncolytic adenovirus SG600-IL24 vector which carrying human MDA-7/IL-24, and observed its expression in hepatocellular carcinoma cell lines and the normal liver cell lines, in order to investigate its theoretical value of gene therapy in hepatocellular carcinoma.Methods:Digesting pZD55-IL24 to get IL24, then connected to pClon9-INS-IL24, and digesting pClon9-INS-IL24 to get INS+IL24, loading into SG502-ACR2 vector, and then to get recombinant plasmid pSG600-IL24, Using pSG600-IL24 and adenovirus plasmid backbone ppE3 to polyfect mediated cotransfected into 293 cells, to obtain oncolytic adenovirus SG600-IL24 vector which carrying human MDA-7/IL-24 by intracellular homologous recombination. The vector SG600-EGFP was constructed using the same methods. The human hepatocullular carcinoma lines HepG2, Hep3B, SMMC7721 and normal liver cell line L02 were infected with oncolytic adenovirus SG600-IL24, the expression of mda-7/IL-24 gene was verified by RT-PCR.Results:HEK293 cells was appeared characteristic CPE after infection, HEK293 Cells become large and round, floating, were gathered like grapes. To take the virus DNA in Clone 1 and clone 2 each 2μl as a template, the specific fragments of PCR amplification is about 640bp, and its size is consistent with the positive control, while the negative control SG600-EGFP has no PCR amplification products. RT-PCR results suggest that the exogenous MDA-7/IL-24 gene was highly expressed in hepatocullular carcinoma lines HepG2, Hep3B, SMMC7721 and normal liver cell line L02 after infected with SG600-IL24.Conclusion:We constructed oncolytic adenovirus SG600-IL24 vector which carrying human MDA-7/IL-24 successfully. SG600-IL24 vector which carrying MDA-7/IL-24 gene was highly expressed in human hepatocullular carcinoma lines and normal liver cell line.Oncolytic adenovirus SG600-IL24 selectively kills hepatocellular carcinoma cell lines in vitroObjective:We investigate the selective antitumor activity of oncolytic adenovirus SG600-IL24 vector which carrying human MDA-7/IL-24 on human hepatocullular carcinoma lines HepG2, Hep3B, SMMC7721, MHCC97L, HCCLM3 and normal liver cell line L02 in vitro, to provide a theoretical basis for hepatocullular carcinoma gene therapy.Methods:The human hepatocullular carcinoma lines HepG2, Hep3B, SMMC7721, MHCC97L, HCCLM3 and normal liver cell line L02 were infected with oncolytic adenovirus SG600-IL24. MDA-7/IL-24 mRNA and protein expressions in infected cells were detected by reverse transcription-polymerase chain reaction (RT-PCR), enzymelinked immunosorbent assay (ELISA), and Western-blot respectively. MTT assay was used to investigate the proliferation of HCC cell lines and normal liver cell lines. Antitumor activity of SG600-IL24 on HCC cell lines was detected by Hoechst33258 staining. Annexin-V and PI staining was studied to indicate the apoptosis effect and the flow cytometry was used to assess the cell cycle.Results:RT-PCR and Western blotting showed that the exogenous MDA-7/IL-24 gene was highly expressed in hepatocullular carcinoma lines HepG2, Hep3B, SMMC7721, MHCC97L, HCCLM3 and normal liver cell line L02 after infected with SG600-IL24. The protein product was confirmed by assay the supernatant with ELISA. MTT and Hoechst33258 staining indicated that SG600-IL24 induced growth suppression, promoted apoptosis, and blocked cancer cell lines in the G2/M phase in hepatocellular carcinoma cell lines but not in the normal liver cell line.Conclusion:Oncolytic adenovirus SG600-IL24 vector which carrying human MDA-7/IL-24 can selectively kill different metastatic potential HCC cell lines and induce apoptosis, block cell proliferation but not normal liver cell line L02.The mechanism of Oncolytic adenovirus SG600-IL24 selectively kills hepatocullular carcinoma cell linesObjective:To explore the mechanism of oncolytic adenovirus SG600-IL24 selectively kills hepatocullular carcinoma cell lines HepG2 and HCCLM3 in vitro, and in order to provide a theoretical basis for hepatocullular carcinoma gene therapy.Methods:The human hepatocullular carcinoma cell lines HepG2, HCCLM3 and normal liver cell line L02 were intervened by oncolytic adenovirus SG600-IL24. RT-PCR and Western-blot were used to detected the protein expressions of total STAT3 and the its signaling pathway downstream signal molecule C-myc, bax, bcl-2, bcl-xl, CyclinD2, Survivin, XIAP, OPN, MMP-2, MMP-9 and VEGF in HepG2, HCCLM3 and normal liver cell line L02 after intervened by oncolytic adenovirus SG600-IL24, also examined the expression of p-STAT3 after infection with SG600-IL24.Results:It was confirmed by RT-PCR and western-blot method that the STAT3 and the its signaling pathway downstream signal molecule C-myc, Bcl-xl, bcl-2, CyclinD2, Survivin, MMP-2, MMP-9, XIAP, OPN, VEGF were downregulated while upregulation of Bax. The phosphorylation of STAT3 after infection was increased and then decreased, and reach to the peak in 2 hours.Conclusion:Oncolytic adenovirus SG600-IL24 may achieve the role of anti-hepatoma through inhibiting STAT3 and the its signaling pathway downstream signal molecule while not normal liver cell line, which make oncolytic adenovirus SG600-IL24 has significant selectively antitumor effects. p-STAT3.Treatment of hepatoma in nude mice combined oncolytic adenovirus SG600-IL24 with InterferonObjective:To study the effects of oncolytic adenovirus SG600-IL24 which carrying human MDA-7/IL-24 and Interferon on hepatoma xenografts in nude mice and explore a new way for hepatoma gene therapy combined with neoadjuvant therapy.Methods:First established subcutaneous hepatoma nude mice model, when the tumors had grown to 100-150 mm3(day 0), the animals were randomized into four groups(each group has eight nude mice) as follows:(1) Control group:Saline 0.3 ml, intraperitoneal injection to the right side,1/d,3 times. (2) Neoadjuvant therapy group (IFN group):IFN 1.5 x104 IU/g, intraperitoneal injection to the right side,1/d,10 times. (3) SG600-IL24 group:SG600-IL24 2 x 108 PFU, multi-point injection to the local tumor,5 times every other day. (4) SG600-IL24+IFN group:IFN 1.5 x104 IU/g, intraperitoneal injection to the right side,1/d, 10 times, and the next day SG600-IL242x108 PFU, multi-point injection to the local tumor, 5 times every other day. And the expression of mda-7/IL-24 gene and protein was detected in tumor tissue after intervented for 30d, the survival time and the tumor volume of the nude mice were observed in each group subsequently.Results:The exogenous MDA-7/IL-24 gene was highly expressed in nude mice after treatment with SG600-IL24 or SG600-IL24 plus IFN. The mice treated with SG600-IL24 plus Interferon had significantly longer survival time (3 mice>120) days and smaller tumor volume than in other groups(P<0.01).Conclusion:Oncolytic adenovirus SG600-IL24 has stronger synergistic antitumor effect on metastastic hepatoma nude mice model combined with Interferon, and pave a new way for HCC virus gene therapy.更多还原