HIF-1α基因沉默對(duì)人脈絡(luò)膜黑色素瘤細(xì)胞上皮—間質(zhì)轉(zhuǎn)化的影響.pdf

1、HIF1a基因沉默對(duì)人脈絡(luò)膜黑色素瘤細(xì)胞上皮一間質(zhì)轉(zhuǎn)化的影響摘要目的：以特異性小干擾RNA(siRNA)沉默缺氧培養(yǎng)的人眼脈絡(luò)膜黑色瘤細(xì)胞(OCM一1)中缺氧誘導(dǎo)因子一10【(HIF—la)基因，觀察其對(duì)細(xì)胞骨架分子波形蛋白(Vimentin)表達(dá)的影響，以初步探討HIF1a對(duì)人眼脈絡(luò)膜黑色瘤上皮一間質(zhì)轉(zhuǎn)化(EMT)的調(diào)控作用。方法：在正常培養(yǎng)基qbJJll21009mol／L的氯化鈷(CoCl2)以模擬腫瘤內(nèi)部缺氧微環(huán)境，構(gòu)建靶向HI

2、F—ls的siRNA干擾載體，同時(shí)設(shè)立6一actinljH性對(duì)照、無義寡核苷酸陰性對(duì)照及空載脂質(zhì)體對(duì)照組，以LipofectamineTM2000為載體介導(dǎo)siRNA轉(zhuǎn)染缺氧培養(yǎng)的OCM1細(xì)胞，以RTPCR和Westernblot方法檢測(cè)缺氧培養(yǎng)前后及轉(zhuǎn)染前后HIF1、VimentinmRNA和蛋白的表達(dá)情況。結(jié)果：OCM一1細(xì)胞經(jīng)缺氧培養(yǎng)后可由上皮細(xì)胞樣形態(tài)轉(zhuǎn)變成間質(zhì)細(xì)胞樣形態(tài)。與常氧組相比，單純?nèi)毖踅MHIF1amRNA表達(dá)無明顯差異

3、(PO05)，蛋白表達(dá)顯著升高(PO05)。結(jié)論：1缺氧培養(yǎng)可使OCM1細(xì)胞由上皮細(xì)胞樣形態(tài)轉(zhuǎn)變成間質(zhì)細(xì)胞樣形態(tài)，提示缺氧微環(huán)境可促使OCM一1細(xì)胞發(fā)生EMT。2缺氧可在蛋白水平上調(diào)OCM一1細(xì)胞中HIF一10【的表達(dá)，提示缺氧對(duì)HIF一10【的調(diào)控主要發(fā)生在轉(zhuǎn)錄后水平。3HIF—ls可通過轉(zhuǎn)錄激活Vimentin，使其表達(dá)上調(diào)，提示HIFla可能參與調(diào)控OCM1細(xì)胞的EMT，進(jìn)而影響其侵襲、轉(zhuǎn)移能力。4可成功利用Lipofectami

4、neTM2000為載體將siRNA轉(zhuǎn)入OCM一1細(xì)胞內(nèi)。EffectofHIFlaGeneSilencingonEMTInMalignantMelanomaoftheHumanChoroidCellAbstractObjectives：Toinvestigatetheeffectofspecificsilencinghypoxiainduciblefactor1alpha(HIFla)bysmallinterferenceRNA(siR

5、NA)onexpressionofVimentininmalignantmelanomaofthehumanchoroidcelllines(OCM1)underhypoxia，inordertodiscusstheroleofHIF一1Qonepithelial—mesenchymaltransition(EMT)inmalignantmelanomaofthehumanchoroidMethods：Chemicalhypoxiain

6、ducerCobaltchloride(COCl2)wereaddedintonutrientmediumattheconcentrationoflOOgmol／LtosimulatehypoxiamicroenvironmentinsidetumorHIF一1Q—siRNAweredesignedandsynthetizedinvitroLipofectamineTM2000wastakentotransfectsiRNAintoOC

7、M1cellsunderhypoxiaAndhypoxicgroupcellsweredividedintofivegroups：simplehypoxicgroup，HIF1Qinterferencegroup，13actinpositivecontrolgroup，negativecontrolgroupandliposomegroupRTPCRandWesternblotwereusedtochecktheexpressionst

8、atusofHIF—laandVimentinonmRNAandproteinlevelsbeforeandafterhypoxiacultureandcelltransfectionResults：Afterhypoxiaculture，OCM1cellscouldtransformfromepitheliallikeintomesenchymallikeinmorphologyComparedwithnormoxiagroup，th

9、erewasnoobviouschangeontheexpressionlevelofmRNAofHIF一1Qinsimplehypoxiagroup伊005)，whiletheexpressionlevelofitsproteinincreasedobviously(P005)Conelusion：1Afterhypoxiaculture，OCM一1cellscouldtransformfromepitheliallikeintome