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1、邰伯軍，等．磷脂酰肌醇蛋白多糖一3對(duì)肝癌細(xì)胞增殖和凋亡的影響863論著／肝膽胰腫瘤磷脂酰肌醇蛋白多糖一3對(duì)肝癌細(xì)胞增殖和凋亡的影響邰伯軍，姚敏，顧星，時(shí)運(yùn)，蔚丹丹，陳潔，鄭文杰，姚登福，陸少林(南通大學(xué)附屬醫(yī)院，臨床醫(yī)學(xué)研究中心，江蘇南通226001)摘要：目的探討shRNA干預(yù)磷脂酰肌醇蛋白多糖(GPC)一3基因轉(zhuǎn)錄對(duì)肝癌細(xì)胞增殖和凋亡的影響。方法將GPC一3一shRNA插入pGPU6／GFP／Neo質(zhì)粒，轉(zhuǎn)染人肝癌HepG2細(xì)胞，以WesternBlot和熒光定量PCR分別分析GPC一3蛋白和mRNA表達(dá)；以MTY法分析HepG2細(xì)胞增殖；以流式細(xì)
2、胞術(shù)、Annexin—V—PE／7一AAD及細(xì)胞凋亡一DNAladder等分析細(xì)胞周期及細(xì)胞凋亡率。結(jié)果shRNA1轉(zhuǎn)染HepG2細(xì)胞，GPC一3mRNA沉默效率為89．3％，與GPC一3蛋白下調(diào)一致；以shRNA轉(zhuǎn)染HepG2細(xì)胞，增殖抑制率為71．1％；細(xì)胞周期阻滯在G1期，細(xì)胞凋亡率達(dá)65．6％。結(jié)論資料顯示shRNA干預(yù)GPC一3基因轉(zhuǎn)錄，可顯著抑制肝癌細(xì)胞增殖，促進(jìn)肝癌細(xì)胞凋亡。關(guān)鍵詞：癌，肝細(xì)胞；磷脂酰肌醇蛋白聚糖類；基因沉默；細(xì)胞凋亡；細(xì)胞增殖中圖分類號(hào)：R735．7文獻(xiàn)標(biāo)志碼：A文章編號(hào)：1001—5256(2013)11—0863—0
3、4Efectsofsilencingglypican——3genetranscriptiononproliferationandapoptosisofhepatomacellsTAI＆n，YAOMin，GUXing，eta1．(ResearchCenterofClinicalMedicine，AffiliatedHospitalofNantongUniversity，Nantong226001，China)Abstract：ObjectiveToinvestigatetheeffectsofsilencingglypican一3(GPC一3)genet
4、ranscriptionbyshRNAontheproliferationandapop—tosisofhepatom~cells．MethodsGPC一3一shRNAwasinsertedintopGPU6／GFP／Neovector，andHepG2cellsweretransfectedwiththevector．ThernRNAandproteinexpressionofGPC一3wasmeasuredbyfluorescencequantitativePCRandWesternblot；theproliferationofHepG2cells
5、wasevaluatedbyMTFassay；thecellcycleandapoptosisofHepG2cellswereanalyzedbyflowcflomet~．Annexin—V—PE／7一AADstaining．a(chǎn)ndapoptoticDNAladderkit．Resl
6、ltsAftertheHepG2cellsweretransfectedwithGPC一3一shRNA．theGPC-3mRNAsilencingratewasupto89．3％，inaccordancewiththedown—regulationofGPC一3prote
7、in；theproliferationinhibitionrateofHepG2cellswasashighas71．1％；theHepG2cellswerearrestedinGIphase．a(chǎn)ndtheapoptosisrateofHepG2cellsreached65．6％．ConclusionSilencingGPC一3genetranscriptionbyshRNAcansignificantlyinhibittheproliferationandpromotetheapoptosisofhepatomacells．Keywords：carc
8、inoma，hepatocellular；glypican；genesilencing；apoptosis；cellproliferation磷脂酰肌醇蛋白多糖一3(Glypican一3，GPC一3)基一3基因轉(zhuǎn)錄可抑制肝癌細(xì)胞增殖與進(jìn)展l8J。為此，本因定位于人染色體Xq26．1區(qū)，蛋白分子中含膜性硫酸乙文構(gòu)建GPC一3基因短發(fā)夾型RNA(shorthairpinRNA，酰肝素多糖蛋白，羧基末端與糖基磷脂酰肌醇共價(jià)結(jié)合shRNA)真核表達(dá)載體，經(jīng)篩選、轉(zhuǎn)染人肝癌(HepG2)細(xì)錨定于細(xì)胞膜，與wnt蛋白共受體調(diào)節(jié)Wnt／[3一catenin胞株，探討
9、干預(yù)GPC一3基因轉(zhuǎn)錄對(duì)HepG2細(xì)胞增殖與信號(hào)通路，介導(dǎo)Wnt3a調(diào)節(jié)肝癌細(xì)胞分化與生長¨，
