【作者】 熊付；

【导师】 冯敢生； 郑传胜；

【作者基本信息】 华中科技大学 ， 影像医学与核医学， 2013， 博士

【摘要】 目的：选取兔VX2细胞和人肝癌细胞HepG2,体外研究羟基喜树碱脂质体对肝肿瘤细胞HIF-1a表达的影响。材料与方法：体外培养人HepG2肝癌细胞和兔VX2瘤细胞,分别在常氧(21%02)和缺氧(1%02)条件下采用不同浓度的羟喜树碱脂质体处理两种肿瘤细胞。采用CCK-8试剂盒检测羟基喜树碱脂质体对两种肿瘤细胞的毒性效应。采用RT-PCR检测两种细胞HIF-1αmRNA的表达,采用Western blot检测相应蛋白的表达。结果：在常氧和缺氧两种状态下,HCPT-lipo对VX2和HepG2两种细胞的毒性作用无差异。缺氧不影响HIF-lamRNA表达,但是可以上调HIF-1α蛋白表达。随着HCPT-lipo浓度增高,其对HIF-1α蛋白抑制作用加强。结论：HCPT-lipo能够抑制缺氧诱导的HepG2和VX2细胞HIF-1α蛋白表达,该抑制效应发生在转录后水平；HCPT-lipo对肿瘤细胞HIF-1α蛋白表达抑制作用与药物本身的细胞毒效应不具有相关性；该抑制作用具有剂量依赖性。目的：研究肝癌动物模型介入栓塞术后缺氧诱导因子1α的表达及其与血管生成VEGF、多重耐药MDR1、浸润和转移MMP-2的关系。材料与方法：建立20只VX2兔肝癌模型,实验组(n=10)经导管肝动脉栓塞处理,栓塞材料为150-250μm大小聚乙烯醇(PVA)。对照组(n=10)经导管肝动脉推注生理盐水处理。于干预后6小时,3天处死动物模型。采用RT-PCR检测缺氧诱导因子1(HIF-1α)、血管内皮生长因子(VEGF)、多重耐药(MDR1)、浸润和转移(MMP-2) mRNA的表达；采用免疫组化染色检测HIF-1α、VEGF、MMP-2和G-pg蛋白表达。结果：成功建立兔肝癌模型,MR检测肿瘤大小,组间差别无统计学意义(P=0.524)。实验组HIF-1a蛋白表达明显高于对照组(P=0.001)。HIF-1α、VEGF、MDR1、MMP-2mRNA表达实验组均高于对照组(分别为P=0.00、P=0.00、P=0.00),并且HIF-1α蛋白表达水平与VEGF、MMP-2、MDR1mRNA和蛋白表达存在相关性(mRNA分别为r=0.635、r=0.773、r=0.758；蛋白分别为r=0.818、r=0.634、r=0.683)。结论：肝癌介入栓塞后缺氧,残存肿瘤细胞感受缺氧并诱导HIF-1a表达,并且上调VEGF、MMP-2、MDRl转录活性,与相应蛋白表达具有显著相关性。目的：研究经导管肝动脉内给药羟基喜树碱脂质体对肝癌动物模型介入栓塞术后缺氧应答的抑制作用。材料与方法：将40只兔VX2肝癌模型随机分为4组,每组10只。干预方式及分组：A组：HCPT-lipo与碘油混悬后经导管动脉灌注+聚乙烯醇(PVA)栓塞；B组：PVA经导管动脉栓塞；C组：HCPT-lipo经导管动脉灌注；D组：生理盐水经导管灌注。分别于干预后6小时,3天处死动物模型。采用免疫组化染色检测HIF-1α、VEGF、、MMP-2多重耐药蛋白G-gp蛋白表达。结果：B组HIF-1a、VEGF、MMP-2、G-gp蛋白表达水平明显高于A、C、D组(P<0.05),A、C、D三组之间比较无显著差异(P>0.05)。结论：经导管动脉灌注HCPT-lipo能够抑制肝癌TAE后HIF-1α表达以及血管生成、肿瘤侵袭转移和多重耐药。更多还原

【Abstract】 Purpose:To determine the inhibitory effect of10-hydroxycamptothecin liposome on hypoxia-induced factor-la (HIF-la) in rabbit VX2cells and human HepG2cells in vitro.Materials and Methods:Rabbit VX2cells and human HepG2cells were cultured under normoxic (21%O2) and hypoxic conditions respectively (1%O2). Both of the two kinds of cells were treated with different HCPT-lipo concentrations for16hours. Cell Counting Kit-8assay was used to determine the cell viability. Real-time PCR was performed to examine the expression of HIF-1a mRNA of the cells. Western blot analysis was used to examine the expression of HIF-1α protein.Results:There was no significant difference in cytotoxicity caused by HCPT-lipo was noted between normoxic and hypoxic in VX2cells and HepaG2cells. Hypoxia, promote expression of HIF-la protein but not happened in its mRNA. With the increasing concentrations of HCPT-lipo, the inhibition ability of the drug enhanced.Conclusions:HCPT-lipo inhibits the expression of HIF-la protein induced by hypoxia in the two kinds of tumor cells, and it occurs at post-transcriptional level. The inhibition is dose-dependent.Purpose:To evaluate expression of hypoxia-inducible factor-la in animal models of liver tumors after transcatheter arterial embolization, and the correlation of hypoxia-inducible factor-la with angiogenesis, drug resistance, and metastasis.Materials and Methods:Twenty rabbit’s implanted VX2liver tumors were established. The experimental group(n=10) were treated by transcatheter hepatic arterial embolization(TAE) with polyvinyl alcohol(PVA) particles sized150～250μm; The control group were treated underwent sham embolization with normal saline. Six hours and3days treatment, animals were sacrificed, and samples were harvested. Expression of HIF-la, VEGF, and G-gp and MMP-2proteins was examined immunohistochemically. Real-time PCR was used to examine the HIF-la, VEGF, MDR1, MMP-2mRNA levels.Results:The animal models were established successfully, the sizes of tumors were detected by MR (P=0.524). The lever of HIF-la protein was significantly higher in experimental group than that in control group(P=0.001).So as the expression of mRNA of VEGF、MDR1、MMP-2(P=0.00、P=0.00、P=0.00,respectively). And HIF-1α protein level was significantly correlated with levels of mRNA and proteins of VEGF、MDR1、MMP-2(r=0.635, P=0.001、 r=0.773,P=0.00、r=0.758, P=0.012respectively)Conclusions:After TAE the tumor hypoxia becomes worse, the residual cells experience and induce the expression of HIF-1α, and HIF-1α generates a overexpression of VEGF、MDR1、MMP-2, which allow the tumors to survive and even evolve.Purpose:To study the inhibitory effect of transcatheter hepatic artery administration of10-hydroxycamptothecin liposome on tumor hypoxia response in animal models after transcatheter arterial embolization.Materials and Methods:Forty rabbits with implanted VX2liver tumor were randomly divided into4groups with10of each. After a microcatheter was placed into the hepatic artery, the treatments were performed by injecting HCPT-lipo mixed lipiodol PVA particles (group A), PVA (group B), HCPT-lipo (group C) and normal saline (group D), respectively. Six hours or3days after the injection, animals were sacrificed. The mRNA levels of HIF-1α, VEGF、MDR1and MMP-2was detected by TR-PCR. Immunohistochemical staining was used to show the protein levels of HIF-1α, VEGF、MDR1and MMP-2.Results:The levels of HIF-1α and VEGF, MMP-2, G-gp in tumors of group B were significantly higher than those of group A, C and D (P<0.05). There was no significant differences were noted among group A, C and D (P>0.05). The protein level of HIF-la was significantly correlated with that of VEGF、MMP-2、G-gp(r=0.816,r=0.655,r=0.677,respectively)Conclusion:Transcatheter infusion of HCPT-lipo has inhibitory effect on HIF-1α expression and angiogenesis, multidrug resistant, invasion and metastasis in liver tumors after transcatheter arterial embolization.更多还原