【作者】 兰英华；

【导师】 任红；

【作者基本信息】 重庆医科大学 ， 内科学， 2004， 博士

【摘要】 目的：由于原发性肝癌（HCC）存在突变、免疫逃避、抗原性弱、血供丰富等因素导致其死亡率、复发率较高，现有治疗方案并不能改善不能手术的患者的预后。近年来基因疫苗被运用在肿瘤的防治研究上，取得了较大进展，部分肿瘤基因疫苗已经进入临床试验阶段。本实验以与人AFP变化十分近似的小鼠为研究对象，以HCC高表达的肿瘤相关性抗原（Tumor Associated Antigen,TAA）-甲胎蛋白（AFP）作为基因治疗的靶抗原，融合有载体分子效应和佐剂作用的Mt.HSP70的基因，构建融合DNA疫苗，以裸DNA免疫荷瘤鼠以研究该种DNA疫苗的有效性和靶向性；另构建表达mAFP的含荧光蛋白的真核 载体，导入减毒的沙门菌株，以活菌免疫小鼠，研究活菌苗在小鼠体内分布和表达mAFP情况，为进一步研究融合基因疫苗和口服减毒菌苗奠定基础。方法：1．hAFP和mAFP在肝癌细胞中的表达：以RT-PCR方法鉴定所用小鼠细胞系EL4、Hepa1-6及C57BL/6J幼鼠肝脏是否表达mAFP，<WP=10>以HE染色和免疫组织化学法鉴定荷瘤鼠模型是否成功，以免疫组织化学法检测人肝癌组织中hAFP表达情况；2．mAFP与Mt.HSP70融合真核表达质粒的构建和表达： 以RT-PCR方法从Hepa1-6细胞株中获取mAFP基因，应用分子克隆技术构建了系列载体包括pcDNA3.1-AFP、pcDNA3.1-HSP70、pcDNA3.1-AFP -HSP70。经酶切和DNA测序鉴定正确后以脂质体导入COS-7细胞，用RT-PCR和免疫细胞化学检测重组质粒在真核细胞中的表达，将DNA注射小鼠肌肉以免疫组织化学法检测重组质粒在动物体内的表达；3．mAFP与 Mt.HSP70融合真核表达质粒诱导小鼠的免疫应答：免疫前三天以0.25% 布比卡因和0.1%对羟基苯甲酸甲酯多点注射右股四头肌，以重组质粒DNA 100μg 注射小鼠同一部位，一月后加强免疫一次。最后一次免疫2周后取眼眶静脉血测肝功能；分离脾细胞，以ELISA检测经超声破碎的EL4/EL4（mAFP）细胞刺激后分泌的IL-4、IFN-γ，用乳酸脱氢酶释放法（LDH）检测AFP特异性CTL活性。以同样方法免疫Hepa1-6荷瘤小鼠，测量肿瘤大小、观察小鼠存活情况评价重组DNA疫苗抗肝癌作用。4．以减毒沙门菌为载体的mAFP DNA疫苗在小鼠体内的分布和表达：以分子克隆技术构建含荧光蛋白的mAFP真核表达质粒，经酶切鉴定正确后，转染COS-7细胞考查质粒在真核细胞中的表达；电转化减毒沙门菌SL5928(敲除鞭毛)，构建重组活菌苗。体外连续传50代，<WP=11>抽提质粒并酶切鉴定重组菌的稳定性；以多种CFU数灌胃免疫小鼠，观察小鼠生长、精神、腹泻、死亡情况等考查疫苗的安全性；灌胃免疫小鼠后4、7、14、21天取肝、脾、回盲部结肠作冰冻切片，荧光显微镜下观察，考查活菌苗在组织中的表达。结果：1．hAFP和mAFP在肝癌细胞中的表达：小鼠细胞系Hepa1-6、C57BL/6J小鼠肝脏RT-PCR可扩增出预期条带，EL4无目的条带，以Hepa1-6成瘤的肿瘤组织HE染色符合肿瘤组织特征，mAFP染色阳性，人HCC组织标本hAFP染色大部分细胞阳性；2．mAFP与Mt.HSP70融合真核表达质粒的构建和表达：重组质粒pcDNA3.1-AFP、pcDNA3.1-HSP70、pcDNA3.1-AFP-HSP70经酶切和测序鉴定符合Genbank中相应序列，转染细胞后免疫细胞化学染色mAFP和/或HSP70阳性，肌肉注射免疫后肌肉免疫组织化学染色mAFP和/或HSP70阳性；3．mAFP与 Mt.HSP70融合真核表达质粒诱导小鼠的免疫应答：3.1加强免疫后各组小鼠肝功能指标ALB、ALT、AST无统计学差异（p>0.05）；3.2各组脾细胞均未检测到IL-4分泌，经pcDNA3.1-AFP、pcDNA3.1-HSP70、pcDNA3.1-AFP-HSP70免疫的脾细胞受EL4/ EL4(mAFP)刺激后分泌IFN-γ分别为50.18±0.815/123.83±0.815pg/mL，57.65±0.534/35.65±0.815pg/mL，163.32±0.774/197.48<WP=12>±0.815pg/mL，各组分泌的IFN-γ有差别(p<0.05)；3.3效/靶比为25：1时pcDNA3.1-AFP、pcDNA3.1-HSP70、pcDNA3.1-AFP-HSP70三组杀伤率（％）分别4.813±0.259/15.567±0.176、15.590±0.392/15.287±0.425、4.613±0.680/28.983±0.266；效/靶比为50：1时分别为6.447±0.303/17.163±0.162、18.773±0.220/19.287±0.425、6.767±0.231/32.650±0.431。两种效/靶比时，除pcDNA3.1-HSP70组受EL4/EL4(mAFP)刺激的两组间杀伤率无差别外（p>0.05），其余各组间都有差别（p<0.05）；3.4重组质粒免疫Hepa1-6荷瘤7d的小鼠后，荷瘤第28d、35d时， pcDNA3.1-AFP组、pcDNA3.1-AFP-HSP70组肿瘤较PBS组、pcDNA3.1组肿瘤体积小。28d后， PBS组、pcDNA3.1组小鼠开始死亡，至42d两组小鼠全部死亡，共观察80d，pcDNA3.1-AFP-HSP70 组肿瘤较pcDNA3.1-AFP组小。4．含EGFP的mAFP表达载体构建正确，在体外转染真核细胞有表达；将重组质粒电转化导入减毒沙门菌，重组菌在含/不含抗生素的培养基上体外连续传50代，均无质粒丢失；口服免疫小鼠，毒性实验中除操作失误致小鼠死亡外，无明显不良反应；口服后2周内，小鼠肝、脾、结肠LN中均有细菌分布，并且有EGFP的表达。结论：1．Hepa1-6细胞系、C57BL/6J幼鼠均有mAFP表达，EL4中无表达，人HCC中有hAFP表达，选用C57BL/6J作为实验动物、Hepa1-6<WP=13>为荷瘤细胞与人HCC?更多还原

【Abstract】 Objective：The death and recurrence rates of HCC is rather high as its high mutation rate、immunity escaption、weak antigenicity and abundant blood supplement. None of available therapies has significantly improved the dismal prognosis of patients with unresectable HCC. DNA vaccine application in prophylaxis and therapies has made a big progress and some of them is being on trial. To aim to AFP, TAA frequently expressed at high level in HCC, we constructed chimeric DNA vaccine of mAFP and chaperon of Mt.HSP70 for researching its targeting effectiveness. C57BL/6J mice were used for object as the level and change of AFP in serum just as human being. Moreover, mAFP eukaryotic expressed plamid with EGFP was constructed and transformed into Salmonella. For investigating distribution and mAFP expression of recombinant attenuated <WP=15>vaccine in vivo, oral immunization was performed in mice. These were groundworks for exploring immunity arosing by fused DNA vaccine and oral live vaccine. Methods：1．hAFP and mAFPexpression in HCC cells: RT-PCR was used to identify whether mAFP was expressed in EL4、Hepa1-6 and liver of immature C57BL/6J. Implanted tumor was confirmed by HE staining and immunohistochemical staining. Immunohistochemical staining was performed to detect hAFP in HCC tissue； 2．Construction and expression of chimeric plasmids of mAFP and Mt.HSP70: mAFP gene was gained by RT-PCR from Hepa1-6 cell line. pcDNA3.1-AFP、pcDNA3.1-HSP70、pcDNA3.1-AFP-HSP70 were constructed by molecular clone techniques. Plasmids were transfected into COS-7 cells with Lipofectamine 2000 after being confirmed by restriction enzyme digestion and DNA sequencing. Protein expressed in cells was detected by RT-PCR and immunocytochemical staining. Furthermore, immunohistochemical staining was performed to assay protein expressed in muscles after i.m. nude DNA； 3. Immune response induced by chimeric plasmids of mAFP and Mt.HSP70: Facilitated DNA immunization was performed by injecting plasmids into quadriceps muscles of mice at 3 different sites in a final <WP=16>volume of 100μL 0.25% Bupivacaine and 0.1% Methylphydroxy- benzoate. 100μg nude DNA was injected into the same sites 3 days after pretreatment. Booster immunization was performed as the first 1 month later. Venous blood was collected from orbital vein for assaying hepatic function. Splenocytes derived from immunized mice were stimulated by EL4/EL4（mAFP）inactivated by ultrasound. IL-4、IFN-γin supernatant were assayed by ELISA. AFP specific CTL activity was assayed by LDH method. Anti-tumor effect was evaluated by tumor size after injecting recombinant DNA into mice bearing Hepa1-6 tumor； 4. Distribution and expression of DNA vaccine carried by attenuated Salmonella typhimurium in mice: mAFP eukaryotic plasmids contained EGFP were constructed by molecular clone techniques. These plasmids were transfected into COS-7 cells with Lipofectamine 2000 after confirming by restriction enzyme digestion. They were introduced into attenuated Salmonella typhimurium-SL5928 (flagellin knocked out) by electroporation. Stability of vaccine was inspected by comparing restriction enzyme digestion charts of plasmids purified from Salmonella after being cultivated 50 generations continuously in medium containing/or no antibiotics. Growth、spirit、diarrhea、death were acted as markers to verdict its safety in mice after taking bacteria with serial CFU counts. 4、7、14、21 days after oral，liver、spleen、ileocecal colon were sectioned and <WP=17>observed by fluorescence microscope. Whether mAFP was expressed in tissues was judged by observing fluorescence. Results：hAFP and mAFPexpression in HCC cells: Specific bands could be amplified from total RNA of Hepa1-6 cells and liver of C57BL/6J mice by RT-PCR. No bands from EL4 cells. HE staining of tumor implanted with Hepa1-6 accorded with characteristics of tumor tissue and immunohistochemical staining for AFP was positive. It was also positive in HCC tissue.Construction and expression of chime更多还原