HBV对ANG的体外调控作用及机制研究

发布时间：2018-10-31 16:51

【摘要】：研究目的观察HBV对ANG的调控作用及其机制,并观察抑制ANG对细胞增殖的影响。研究方法利用HepG2.2.15细胞模型及质粒pcDNA3.1-HBx转染的HepG2细胞,通过Real-time PCR检测ANG mRNA及Elisa, Western Blot检测ANG蛋白变化,观察HBV/HBx对ANG的调控作用。通过Western Blot及Elisa检测HBV/HBx对IL-6的调控作用,并检测IL-6刺激及抗体封闭IL-6活性后ANG细胞内外蛋白含量的变化。Western Blot检测HBV/HBx对HIF-1α的调控作用,并Western Blot检测HIF-1α siRNA沉默HIF-1α基因后,ANG蛋白表达的变化。采用免疫荧光方法检测HBV对ANG核转移的影响。通过ANG siRNA(50nM)转染HepG2.2.15细胞及使用ANG核转移抑制剂neomycine, MTT检测细胞抑制率,并检测ANG基因沉默后核糖体RNA45S RNA的合成变化。研究结果 Real-time PCR及Elisa, Western Blot结果显示在HBV病毒稳定复制的HepG2.2.15细胞及转染X蛋白的HepG2-HBx细胞中,ANG mRNA及蛋白水平均高于对照组。Real-time PCR结果显示在HepG2-HBx细胞中ANG水平升高45SRNA转录水平升高,ANG基因沉默后其转录水平降低。Western Blot结果显示,HepG2.2.15细胞及HepG-HBx细胞中IL-6及HIF-1α蛋白水平均高于对照组。Real-time PCR及Elisa结果显示IL-6刺激后ANG mRNA及蛋白水平均明显升高,抗体封闭IL-6活性后,ANG蛋白表达降低。Western Blot及Elisa结果显示,HIF-1α基因沉默后,ANG蛋白表达降低。免疫荧光结果显示较于对照,2215细胞中核转移明显被促进。MTT结果显示,HepG2.2.15细胞中ANG基因沉默或加入核转移抑制剂后细胞增殖明显抑制。研究结论 HBV/HBx可上调ANG,这种上调作用可能是通过其对IL-6及HIF-1α的上调实现；HBV可促进上调的ANG核转移；ANG活性被抑制后,可抑制细胞增殖。
[Abstract]:Objective to investigate the regulatory effect of HBV on ANG and its mechanism, and to observe the effect of inhibiting ANG on cell proliferation. Methods HepG2.2.15 cell model and HepG2 cells transfected with plasmid pcDNA3.1-HBx were used to detect the changes of ANG protein by Real-time PCR and Elisa, Western Blot, and the effect of HBV/HBx on ANG was observed. The regulatory effect of HBV/HBx on IL-6 was detected by Western Blot and Elisa, and the changes of protein content in ANG cells after IL-6 stimulation and blocking of IL-6 activity by antibody were detected. The effect of HBV/HBx on HIF-1 伪 was detected by. Western Blot. The expression of ANG protein was detected by Western Blot after HIF-1 伪 siRNA silenced HIF-1 伪 gene. The effect of HBV on nuclear metastasis of ANG was detected by immunofluorescence method. ANG siRNA (50nM) was used to transfect HepG2.2.15 cells and ANG nuclear transfer inhibitor (neomycine, MTT) was used to detect the inhibition rate and the synthesis of ribosomal RNA45S RNA after ANG gene silencing. Results the results of Real-time PCR and Elisa, Western Blot showed that in the stable replication of HBV virus HepG2.2.15 cells and X protein transfected HepG2-HBx cells, The levels of ANG mRNA and protein were higher than those of the control group. Real-time PCR results showed that the ANG level increased in HepG2-HBx cells, and the transcription level of 45SRNA increased after ANG gene silencing. The results showed that the ANG level decreased after ANG gene silencing. The levels of IL-6 and HIF-1 伪 protein in HepG2.2.15 cells and HepG-HBx cells were higher than those in the control group. The results of Real-time PCR and Elisa showed that ANG mRNA and protein levels increased significantly after IL-6 stimulation, and the antibody blocked IL-6 activity. The results of. Western Blot and Elisa showed that the expression of ANG protein decreased after HIF-1 伪 gene silencing. The results of immunofluorescence showed that nuclear metastasis was significantly promoted in 2215 cells compared with the control. MTT results showed that ANG gene silencing in HepG2.2.15 cells or the addition of nuclear transfer inhibitors significantly inhibited cell proliferation. Conclusion the upregulation of ANG, by HBV/HBx may be due to its up-regulation of IL-6 and HIF-1 伪, HBV can promote the up-regulation of ANG nuclear metastasis, and ANG can inhibit the proliferation of cells when the activity of ANG is inhibited.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.62

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