發(fā)布時(shí)間：2018-01-17 17:12

  本文關(guān)鍵詞：干預(yù)磷脂酰肌醇蛋白多糖3基因轉(zhuǎn)錄聯(lián)合抗腫瘤藥物抑制肝癌細(xì)胞增殖的協(xié)同作用　出處：《臨床肝膽病雜志》2016年12期 　論文類型：期刊論文

  更多相關(guān)文章： 肝腫瘤 磷脂酰肌醇蛋白聚糖類 抗腫瘤藥 基因 抑制 藥物協(xié)同作用

【摘要】：目的探討干預(yù)磷脂酰肌醇蛋白多糖(GPC)3基因轉(zhuǎn)錄和聯(lián)合抗腫瘤藥物對(duì)肝癌細(xì)胞增殖的抑制效果。方法構(gòu)建4種GPC-3-shRNA質(zhì)粒轉(zhuǎn)染肝癌HepG2細(xì)胞,以realtime-PCR、Western Blot法觀察GPC-3基因及蛋白表達(dá)水平,分析其與肝癌細(xì)胞增殖、凋亡的關(guān)系。計(jì)量資料兩組間比較采用獨(dú)立樣本t檢驗(yàn),多組間比較采用單因素方差分析。結(jié)果 4種轉(zhuǎn)染質(zhì)粒中shRNA1轉(zhuǎn)染HepG2細(xì)胞效率85%,在mRNA水平的沉默效率為89.3%,GPC-3蛋白表達(dá)顯著抑制(P0.01)。shRNA1干擾組72 h HepG2細(xì)胞抑制率71.1%,與陰性組比較差異有統(tǒng)計(jì)學(xué)意義(t=18.092,P0.001);shRNA1干擾組肝癌細(xì)胞發(fā)生生物學(xué)特性改變,遷移抑制率為89.1%,明顯慢于陰性組,差異有統(tǒng)計(jì)學(xué)意義(t=8.326,P0.001);HepG2細(xì)胞運(yùn)動(dòng)抑制率為53.6%、侵襲抑制率60.1%,與陰性組比較差異均有統(tǒng)計(jì)學(xué)意義(t值分別為52.400、48.245,P值均0.001)。shRNA1干擾組β-catenin mRNA抑制率為46.9%,Gli1 mRNA上調(diào)率為7.4%,與對(duì)照組比較差異均有統(tǒng)計(jì)學(xué)意義(t值分別為30.108、-3.551,P值分別為0.001、0.009)。在24 h時(shí),10μmol/L索拉非尼與shRNA1聯(lián)合,對(duì)肝癌細(xì)胞抑制率為52.6%,100μmol/L索拉非尼與shRNA1聯(lián)合,對(duì)肝癌細(xì)胞的抑制率為79.5%,與對(duì)照組比較差異有統(tǒng)計(jì)學(xué)意義(t值分別為23.314、50.352,P值均0.001)。索拉非尼對(duì)HepG2細(xì)胞的半數(shù)抑制濃度(IC_(50))值為(4.67±1.20)μmol/L、雷帕霉素為(7.85±2.00)nmol/L、厄洛替尼為(18.36±0.56)μmol/L,與shRNA1聯(lián)合使用后HepG2細(xì)胞抑制率達(dá)95.11%。結(jié)論特異性shRNA干預(yù)GPC-3轉(zhuǎn)錄可抑制肝癌細(xì)胞增殖、遷移運(yùn)動(dòng)和侵襲能力,并誘導(dǎo)肝癌細(xì)胞凋亡,與抗腫瘤藥物協(xié)同抑制癌細(xì)胞增殖,提示GPC-3可能是肝癌治療有效靶點(diǎn),聯(lián)合靶向治療將為肝癌提供更佳治療策略。
[Abstract]:Objective to investigate the intervention of phosphatidylinositol proteoglycan (GPC). 3 the inhibitory effect of gene transcription and antitumor drugs on the proliferation of hepatoma cells. Methods four kinds of GPC-3-shRNA plasmids were constructed and transfected into HCC HepG2 cells. The expression levels of GPC-3 gene and protein were detected by realtime-PCR Western Blot assay, and the relationship between the expression of GPC-3 gene and the proliferation of hepatoma cells was analyzed. The correlation of apoptosis. The comparison between the two groups was carried out by independent sample t-test. Results among the four transfection plasmids, the efficiency of shRNA1 transfection into HepG2 cells was 85%, and the silencing efficiency at mRNA level was 89.3%. The expression of GPC-3 protein significantly inhibited the inhibition rate of HepG2 cells in the interference group of P0.01and shRNA1 for 72 h, and the inhibition rate was 71.1%. Compared with the negative group, the difference was statistically significant (P 0.001). The biological characteristics of hepatoma cells in shRNA1 interference group were changed, the inhibition rate of migration was 89.1, which was significantly slower than that in negative group, and the difference was statistically significant (P 0.001). The inhibition rate of HepG2 cell motion and invasion was 53.6% and 60.1% respectively. The difference was statistically significant compared with the negative group (52.400% 48.245). The inhibition rate of 尾 -catenin mRNA was 46.9% and the up-regulation rate of Gli1 mRNA was 7.4%. Compared with the control group, the difference was statistically significant (t = 30.108) -3.551 ~ (-1) P = 0.001 ~ (0.009), respectively, at 24 h. The inhibitory rate of 10 渭 mol/L sorafenib combined with shRNA1 was 52.6 渭 mol/L and 100 渭 mol/L with shRNA1. The inhibition rate of hepatoma cells was 79.5%, and the difference was statistically significant compared with the control group (23.314% 50.352). The median inhibitory concentration of Solafenib on HepG2 cells was 4.67 鹵1.20 渭 mol/L. Rapamycin was 7.85 鹵2.00 nmol / L, and erlotinib was 18.36 鹵0.56 渭 mol/L. The inhibition rate of HepG2 cells in combination with shRNA1 was 95.110.Conclusion specific shRNA intervention in GPC-3 transcription can inhibit the proliferation, migration and invasion of hepatoma cells. It also induced apoptosis of hepatoma cells and inhibited the proliferation of cancer cells in cooperation with antitumor drugs, suggesting that GPC-3 may be an effective target for the treatment of HCC, and the combination of targeted therapy will provide a better treatment strategy for HCC.
【作者單位】： 南通大學(xué)藥學(xué)院;南通大學(xué)醫(yī)學(xué)院;南通大學(xué)附屬醫(yī)院;
【基金】：國家自然科學(xué)基金(81673241) 江蘇省“六大人才高峰”項(xiàng)目(2014-YY-028)
【分類號(hào)】：R735.7
【正文快照】： 肝細(xì)胞癌(HCC)是我國長江口地區(qū)常見的惡性腫瘤之一[1-2]。近年來發(fā)現(xiàn),成人正常肝組織中磷脂酰肌醇蛋白聚糖(glypican,GPC)3未見明顯表達(dá),胎肝和肝癌組織則高表達(dá)。GPC-3通過糖基磷脂酰肌醇錨定于細(xì)胞膜上[3-4],且位于Wnt/β-catenin和Hedgehog(Hh)信號(hào)通路上游[5-6],導(dǎo)致Wnt/

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本文編號(hào)：1437172