【作者】 夏向文；

【导师】 冯敢生；

【作者基本信息】 华中科技大学 ， 影像医学与核医学， 2010， 博士

【摘要】 目的构建携带增强型绿色荧光蛋白(EGFP)和人白细胞介素12双亚基目的基因(hIL-12,P35,P40)的真核表达质粒pEGFP-C1_IL-12,研究其体外抗肿瘤作用。分离、纯化白芨多糖,制备阳离子型白芨多糖,研究其表征,探讨其在体内外作为基因递送载体的可行性研究动脉内白细胞介素12基因治疗联合化疗栓塞术治疗VX2肝癌的抗肿瘤及抗肿瘤血管生成的疗效,探讨其可能的机制。材料与方法提取脂多糖(LPS)诱导后的人脐带血淋巴细胞总RNA,RT-PCR法扩增人白细胞介素12的双亚基P35和P40的全长cDNA,采用重叠PCR法连接两个亚基,双酶切双亚基片段及载体pEGFP-C1,T4DNA连接酶连接,重组质粒转化大肠埃希菌,挑取阳性克隆,PCR、双酶切及测序鉴定。借助脂质体将重组质粒转染人肝癌细胞系HepG2,通过荧光显微镜检测报告基因的表达,RT-PCR及Western blot去检测目的基因的表达。MTT法检测肝癌细胞的生长情况,RT-PCR法检测肝癌细胞表达血管内皮生长因子(VEGF)的变化。经热水抽提,乙醇沉淀,Sevag法脱蛋白,经阴离子交换纤维素柱(DE-52)和凝胶柱(Sephadex G-100)层析,分离纯化得白芨多糖,通过高碘酸钾氧化得到含醛基的还原性多糖,该还原性多糖与精胺在碱性条件下形成希夫碱式共轭物,经硼氢化钠还原得含伯胺基的阳离子型多糖,分别通过盐酸羟胺法和三硝基苯磺酸法测醛基与伯胺基的产量。考察氧化反应时间对反应产物的影响。运用琼脂糖凝胶电泳检测该阳离子型多糖对基因的结合与保护作用。将阳离子型白芨多糖载基因复合物与体外生长的肝癌细胞系HepG2共培养,研究转染效率。进一步通过介入途径经肝动脉导入复合物,以GFP作为报告基因检测复合物在体内对活体兔肝组织细胞的转染,同时检测其血清转氨酶水平及肾功能指标以评估体内毒性。32只生长有VX2肝癌的新西兰大白兔随机分为四组,每组八只,开腹后使用自制穿刺针置入肝动脉行介入治疗。A组行生理盐水灌注(对照组),B组行常规化疗栓塞,C组行动脉内基因灌注治疗,D组行动脉内基因灌注治疗+化疗栓塞。治疗前及治疗后第14天行螺旋CT扫描检测肿瘤大小,计算肿瘤相对生长速率；于治疗前及治疗后第3、7、14天采静脉血检测循环血中IFN-γ的水平；于第14天处死所有实验兔,免疫组化法检测瘤组织VEGF表达水平、微血管密度(MVD值)及肿瘤细胞凋亡指数。结果PCR及测序鉴定证实重组质粒pEGFP-C1_IL-12构建成功,RT-PCR及Westernblot法证实重组质粒在HepG2细胞内正常表达。转染重组质粒后的肝癌细胞生长未受明显影响,表达VEGF能力无显著变化。所提纯的白芨多糖为一种不含阳离子基团的非还原性多糖,通过胺化还原反应的方法可成功制备出含伯胺基的阳离子型多糖,红外光谱证实了多糖上伯胺基的存在。该阳离子型多糖可以结合并保护质粒DNA免受DNA酶的降解,体外实验证实该复合物可以转染入体外培养的肝癌细胞,以阳离子白芨多糖作为转染载体时转染效率要低于脂质体,差异具有统计学意义(28.87%±3.27% vs 36.64%±6.87%,t检验,P<0.05)。采用介入方法经肝动脉给药时复合物能靶向转染入活体兔肝细胞内并实现表达,阳离子白芨多糖未显示出明显体内毒性。体内实验中,C、D组于第3、7天出现高水平IFN-γ的表达,明显高于治疗前水平及同期A、B组(Turkey HSD法方差分析,P<0.05),至第14天血清IFN-γ水平恢复至治疗前水平。两个基因治疗组(C组和D组)的瘤组织VEGF和MVD水平均下降,但单纯基因治疗组(C组)的肿瘤生长速度及肿瘤细胞凋亡率与对照组相比均无显著差异(Turkey HSD法方差分析,P>0.05),而基因治疗与化疗栓塞联合组(D组)肿瘤生长速度减慢,肿瘤细胞凋亡率增加,与对照组及化疗栓塞组相比均有显著差异。结论成功构建IL-12双亚基共表达质粒pEGFP-C1_IL-12,该质粒转染入肝癌细胞后,表达产物对体外培养的肝癌细胞的生长及表达VEGF的能力无显著影响。通过胺化还原法制备的阳离子型白芨多糖可以结合并保护质粒DNA,该阳离子白芨多糖能作为基因递送载体携载质粒pEGFP-C1_IL-12转染入体外培养的肝癌细胞及体内生长的肝细胞。经动脉IL-12基因治疗与化疗栓塞术联合应用能对VX2肝癌产生明显的抗肿瘤作用及抗肿瘤血管生成作用,联合应用的疗效明显优于化疗栓塞单独应用。更多还原

【Abstract】 ObjectiveThe first purpose of this study was to construct a recombinant eukaryotic expression plasmid pEGFP-C1_IL-12 containing report gene (EGFP) and recombinant human interleukin 12 double-submit(P40 and P35), and to research its anti-tumor effect in vitro. The second purpose was to prepare the cationic product of a polysaccharide isolated from Chinese medicinal herb Bletilla striata, and to investigate the characterization and the possibility of being a non-viral gene vector in vitro and in vivo. The third purpose was to evaluate the anti-tumor and anti-angiogenesis effect of intraarterial IL-12 gene therapy alone and in combination with transarterial chemoemboliztion (TACE) in a rabbit VX2 liver cancer model.Methods and MaterialsTotal RNA was extracted from human umbilical cord blood lymphocytes, from which the total cDNA of human interleukin 12 double-submit was amplified by RT-PCR. The linkaged double-submit connected by overlap PCR was inserted into the MCS of vector pEGFP-C1 to construct a recombinant plasmid, which was identified by PCR and sequencing analysis. Then the recombinant plasmid was transfected into HepG2 cells lipidosome and its expression was detected by fluorescent microscopy, RT-PCR and Western blot.Bletilla striata polysaccharide was prepared by ethanol precipitation followed the hot water extraction Bletilla striata stem and deproteinized with Sevag method. It was further isolated by ion-exchange chromatography on DE-52 column and gel filtrationon Sephadex G-100 column. Cationic polysaccharide was synthesized by conjugation of spermine to oxidized polysaccharide. The ability of the caionic polysaccharide to incorporate and protect plasmid DNA was measured. The ability of the BSP+/pDNA complex to transfect into HepG2 cells was measured. Then, we investigated the transfected effect of the BSP+/pDNA complex in vivo using green fluorescence protein (GFP) gene as a reporter gene administered through the hepatic artery. Rabbits developed VX2 tumor in the liver were randomized into four groups, eight of each. After laparotomy and placement of a self-made 30-gauge needle into the proper liver artery, the following protocols of the interventional procedure were applied: 0.9% saline solution (group A), TACE (group B), intraarterial interlukin-12 gene infusion (group C) and intraarterial interlukin-12 gene infusion in combination with TACE (group D). Growth ratio was estimated by computed tomography. Interferon-gamma level of blood was analyzed by ELISA before and 3day,7day,14day after intervention. All animals were sacrificed 14day after procedure, and the tumor tissue were explanted to analyze vessel endothelial growth factor (VEGF), microvessel density (MVD) and apoptotic index of tumor tissues using immunohistochemistry.ResultsIdentification of pEGFP-C1_IL-12 by PCR and sequencing analysis showed that the length, inserted location and direction was correct, and the expression of reported gene and target gene was observed by fluorescent microscopy, RT-PCR and Western blot.There is no cationoid primary amine and reductive carbonyl in the polysaccharide isolated from the Chinese medicinal herb Bletilla striata. The structure of cationic Bletilla striata polysaccharide contained primary amine group was confirmed by FT-IR. The synthesized cationic product incorporated and protected plasmid DNA to avoidenzymolysis.Tumor growth and angiogenesis were significantly suppressed in group D compared with other groups, characterized by lower tumor growth rate, VEGF, MVD, and higher apoptotic index of tumor cells. Interferon-gamma were higher in group C and group D 3day and 7day after intervention compared to group A and group B, but there is no significantly difference between group C and group D.ConclusionThe cationic Bletilla striata polysaccharide synthesized by grafted spermine to the polysaccharide isolated from the Chinese medicinal herb Bletilla striata could incorporate and protect plasmid DNA, which could be an ideal non-viral gene vector in gene transduction. The BSP+/pDNA complex could target transfect into liver cell in vivo when administered through the hepatic artery using the interventional method, which could produce a marked effect as a new type polycation gene vector in gene therapy.Intraarterial interlukin-12 encoded gene delivery could induce interferon-gamma expression, which may contributed to anti-angiogenesis effect of interlukin-12. Intraarterial interlukin-12 encoded gene delivery combined with chemoembolization could inhibit rabbit VX2 hepatocellular growth significantly, perhaps the anti-tumor and anti-angiogenesis effect were associated with high concentration IL-12 of tumor local when combined with chemoembolization.更多还原