UTMD转染HIF-1α shRNA联合TAE治疗肝癌的实验研究

发布时间：2018-06-09 13:18

  本文选题：超声靶向微泡破灭 + 缺氧诱导因子1α　； 参考：《华中科技大学》2014年博士论文

【摘要】：背景缺氧诱导因子la(Hypoxia-inducible factor-1alpha, HIF-la)基因的激活和过表达是经导管肝动脉栓塞术(transcatheter arterial embolization, TAE)后肝癌复发和转移的重要因素,提高TAE的治疗效果则必须抑制HIF-1α的高表达。 目的超声转染HIF-1α shRNA至肝癌细胞内,抑制HIF-1α的表达,提高TAE的治疗效果。 方法1、体外缺氧培养大鼠肝癌细胞walker256,将靶向针对HIF-1α的短发夹状RNA(small hairpin RNA, shRNA)即HIF-1α shRNA表达质粒与超声微泡Sonovue混合后,利用超声靶向微泡破坏(ultrasound targeted microbubbles destruction, UTMD)技术将质粒转染至细胞内,观察HIF-1α的沉默效果。2、评估UTMD体内转染时不同条件对大鼠的损伤效应；评估安全的转染条件下介导eGFP肝内转染时的转染效果,有助于选择安全、有效的转染条件。3、构建wistar大鼠肝癌模型。经大鼠尾静脉注入shRNA表达质粒与SonoVue的混合液,同时在大鼠肝癌部位用合适的超声条件击碎微泡,将shRNA质粒释放入细胞,观察沉默HIF-1α基因表达的效果和肿瘤大小的变化；超声辐照完毕后行TAE。研究中设置不同的实验组：对照(control)组、UTMD组、TAE组、UTMD+TAE组,对比分析各组对肝癌的治疗效果。 结果1、体外实验证实HIF-1α shRNA能够有效抑制缺氧条件下walker256肝癌细胞内HIF-1α的表达；2、UTMD介导基因肝内转染时,SonoVue剂量为4ul/g条件下,辐照条件为1MHz,20%DC,辐照6min时,超声强度≤1.5w/cm2对大鼠无明显损伤,1.5w/cm2辐照6min可以获得较好的转染效果。3、体内实验结果显示：control组肿瘤持续增大,TAE组肝癌前期肿瘤生长明显受抑制,后期出现复发,UTMD组肝癌生长抑制较明显,TAE联合UTMD组肿瘤生长抑制显著,体积明显缩小,达到治愈肝癌的效果。 结论UTMD能有效介导HIF-la shRNA转染至细胞内,沉默HIF-1α的表达,联合TAE能显著抑制肿瘤生长,癌细胞坏死彻底,能够达到治愈肝癌、防止复发的目的,为肝癌治疗提供新的方法和思路。
[Abstract]:Background the activation and overexpression of Hypoxia-inducible factor-1 alpha (HIF-la-1) gene is an important factor in the recurrence and metastasis of hepatocellular carcinoma (HCC) after transcatheter arterial embolization (TAEs). To increase the therapeutic effect of Tae, we must inhibit the high expression of HIF-1 伪. Objective to transfect HIF-1 伪 shRNA into hepatoma cells and inhibit the expression of HIF-1 伪. Methods 1. Rat hepatoma cell line walker 256 was cultured under hypoxia. The short hairpin targeting HIF-1 伪, HIF-1 伪 shRNA expression plasmid, was mixed with ultrasound microbubble Sonovue. The silencing effect of HIF-1 伪 was observed by ultrasound targeted microbubble destruction ultrasound targeted microbubbles destruction, UTMD-mediated transfection into the cells, and the damage effects of different transfection conditions on the rats were evaluated by observing the silencing effect of HIF-1 伪. To evaluate the transfection effect of intrahepatic transfection of eGFP under the safe transfection condition, it is helpful to select the safe and effective transfection condition. 3. To construct the hepatoma model of wistar rats. The mixture of shRNA expression plasmid and SonoVue was injected into the tail vein of rats. At the same time, the shRNA plasmid was released into the cells with appropriate ultrasound condition. The effect of silencing HIF-1 伪 gene expression and the change of tumor size were observed. After ultrasonic irradiation, TAE was used. In the study, different experimental groups were set up: control group (control group), UTMD group (Tae group), Results 1. In vitro experiments showed that HIF-1 伪 shRNA could effectively inhibit the expression of HIF-1 伪 in walker256 hepatoma cells under hypoxia. When 6min was irradiated, ultrasound intensity 鈮，

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