中华妇产科杂志
中華婦產科雜誌
중화부산과잡지
CHINESE JOUNAL OF OBSTETRICS AND GYNECOLOGY
2014年
8期
616-620
,共5页
沈晓燕%韩冰%沈芸%杨隽钧%任彤%沙桂华%向阳
瀋曉燕%韓冰%瀋蕓%楊雋鈞%任彤%沙桂華%嚮暘
침효연%한빙%침예%양준균%임동%사계화%향양
绒毛膜癌%载体蛋白质类%线粒体蛋白质类%细胞系,肿瘤%抗药性,肿瘤%RNA干扰
絨毛膜癌%載體蛋白質類%線粒體蛋白質類%細胞繫,腫瘤%抗藥性,腫瘤%RNA榦擾
융모막암%재체단백질류%선립체단백질류%세포계,종류%항약성,종류%RNA간우
Choriocarcinoma%Carrier proteins%Mitochondrial proteins%Cell line,tumor%Drug resistance,neoplasm%RNA interference
目的 检测补体成分1Q亚成分结合蛋白(C1QBP)基因在绒毛膜癌(绒癌)耐药细胞株及其亲本细胞株中的表达差异,探讨以C1QBP基因为靶向的RNA干扰能否有效逆转绒癌耐药细胞株对相应化疗药物的耐药性.方法 (1)通过实时荧光定量PCR技术、蛋白印迹法和细胞免疫荧光法检测绒癌耐药细胞株,即氟脲嘧啶脱氧核苷(FUDR)耐药细胞株JeG-3/FUDR、甲氨蝶呤(MTX)耐药细胞株JeG-3/MTX、依托泊苷(VP-16)耐药细胞株JeG-3/VP、放线菌素D(ACTD,又称KSM)耐药细胞株JeG-3/KSM细胞,以及亲本细胞株JeG-3细胞中C1QBP mRNA和蛋白的表达及蛋白定位.(2)靶向构建C1QBP基因的短发夹状RNA(shRNA),包装成C1QBP基因RNA干扰(RNAi)慢病毒表达载体,即C 1QBP-RNAi-LV,转染绒癌耐药细胞,实时荧光定量PCR技术及蛋白印迹法检测转染后绒癌耐药细胞株JeG-3/FUDR细胞和亲本细胞株JeG-3细胞中C1QBP mRNA和蛋白的表达,活细胞计数(CCK-8)法检测各绒癌耐药细胞的药物敏感性的变化.结果 (1)转染前绒癌耐药细胞株JeG-3/FUDR、JeG-3/MTX、JeG-3/VP、JeG-3/KSM细胞中C1QBP mRNA表达水平分别为2.520±0.680、1.770±0.230、1.940±0.090、1.740±0.350,均明显高于亲本细胞株JeG-3细胞(为1.000),差异均有统计学意义(P＜0.05).4种绒癌耐药细胞中C1QBP蛋白表达强度均高于亲本细胞株JeG-3细胞;绒癌耐药细胞和亲本细胞中均存在C1QBP蛋白的表达,均定位于细胞内的线粒体.(2)转染后绒癌耐药细胞株JeG-3/FUDR细胞中C1QBP mRNA表达水平下调了93.1％(P＜0.01),C1QBP蛋白表达完全被抑制.(3)转染后绒癌耐药细胞株JeG-3/FUDR、JeG-3/MTX、JeG-3/VP和JeG-3/KSM细胞的耐药指数较其相应RNAi阴性对照分别下降了86.3％、93.9％、92.8％和89.9％,差异均有统计学意义(P＜0.05).结论 C1QBP基因在绒癌耐药细胞株中表达明显增高,沉默C1QBP基因表达可以有效逆转绒癌耐药细胞株对相应化疗药物的耐药性.
目的 檢測補體成分1Q亞成分結閤蛋白(C1QBP)基因在絨毛膜癌(絨癌)耐藥細胞株及其親本細胞株中的錶達差異,探討以C1QBP基因為靶嚮的RNA榦擾能否有效逆轉絨癌耐藥細胞株對相應化療藥物的耐藥性.方法 (1)通過實時熒光定量PCR技術、蛋白印跡法和細胞免疫熒光法檢測絨癌耐藥細胞株,即氟脲嘧啶脫氧覈苷(FUDR)耐藥細胞株JeG-3/FUDR、甲氨蝶呤(MTX)耐藥細胞株JeG-3/MTX、依託泊苷(VP-16)耐藥細胞株JeG-3/VP、放線菌素D(ACTD,又稱KSM)耐藥細胞株JeG-3/KSM細胞,以及親本細胞株JeG-3細胞中C1QBP mRNA和蛋白的錶達及蛋白定位.(2)靶嚮構建C1QBP基因的短髮夾狀RNA(shRNA),包裝成C1QBP基因RNA榦擾(RNAi)慢病毒錶達載體,即C 1QBP-RNAi-LV,轉染絨癌耐藥細胞,實時熒光定量PCR技術及蛋白印跡法檢測轉染後絨癌耐藥細胞株JeG-3/FUDR細胞和親本細胞株JeG-3細胞中C1QBP mRNA和蛋白的錶達,活細胞計數(CCK-8)法檢測各絨癌耐藥細胞的藥物敏感性的變化.結果 (1)轉染前絨癌耐藥細胞株JeG-3/FUDR、JeG-3/MTX、JeG-3/VP、JeG-3/KSM細胞中C1QBP mRNA錶達水平分彆為2.520±0.680、1.770±0.230、1.940±0.090、1.740±0.350,均明顯高于親本細胞株JeG-3細胞(為1.000),差異均有統計學意義(P＜0.05).4種絨癌耐藥細胞中C1QBP蛋白錶達彊度均高于親本細胞株JeG-3細胞;絨癌耐藥細胞和親本細胞中均存在C1QBP蛋白的錶達,均定位于細胞內的線粒體.(2)轉染後絨癌耐藥細胞株JeG-3/FUDR細胞中C1QBP mRNA錶達水平下調瞭93.1％(P＜0.01),C1QBP蛋白錶達完全被抑製.(3)轉染後絨癌耐藥細胞株JeG-3/FUDR、JeG-3/MTX、JeG-3/VP和JeG-3/KSM細胞的耐藥指數較其相應RNAi陰性對照分彆下降瞭86.3％、93.9％、92.8％和89.9％,差異均有統計學意義(P＜0.05).結論 C1QBP基因在絨癌耐藥細胞株中錶達明顯增高,沉默C1QBP基因錶達可以有效逆轉絨癌耐藥細胞株對相應化療藥物的耐藥性.
목적 검측보체성분1Q아성분결합단백(C1QBP)기인재융모막암(융암)내약세포주급기친본세포주중적표체차이,탐토이C1QBP기인위파향적RNA간우능부유효역전융암내약세포주대상응화료약물적내약성.방법 (1)통과실시형광정량PCR기술、단백인적법화세포면역형광법검측융암내약세포주,즉불뇨밀정탈양핵감(FUDR)내약세포주JeG-3/FUDR、갑안접령(MTX)내약세포주JeG-3/MTX、의탁박감(VP-16)내약세포주JeG-3/VP、방선균소D(ACTD,우칭KSM)내약세포주JeG-3/KSM세포,이급친본세포주JeG-3세포중C1QBP mRNA화단백적표체급단백정위.(2)파향구건C1QBP기인적단발협상RNA(shRNA),포장성C1QBP기인RNA간우(RNAi)만병독표체재체,즉C 1QBP-RNAi-LV,전염융암내약세포,실시형광정량PCR기술급단백인적법검측전염후융암내약세포주JeG-3/FUDR세포화친본세포주JeG-3세포중C1QBP mRNA화단백적표체,활세포계수(CCK-8)법검측각융암내약세포적약물민감성적변화.결과 (1)전염전융암내약세포주JeG-3/FUDR、JeG-3/MTX、JeG-3/VP、JeG-3/KSM세포중C1QBP mRNA표체수평분별위2.520±0.680、1.770±0.230、1.940±0.090、1.740±0.350,균명현고우친본세포주JeG-3세포(위1.000),차이균유통계학의의(P＜0.05).4충융암내약세포중C1QBP단백표체강도균고우친본세포주JeG-3세포;융암내약세포화친본세포중균존재C1QBP단백적표체,균정위우세포내적선립체.(2)전염후융암내약세포주JeG-3/FUDR세포중C1QBP mRNA표체수평하조료93.1％(P＜0.01),C1QBP단백표체완전피억제.(3)전염후융암내약세포주JeG-3/FUDR、JeG-3/MTX、JeG-3/VP화JeG-3/KSM세포적내약지수교기상응RNAi음성대조분별하강료86.3％、93.9％、92.8％화89.9％,차이균유통계학의의(P＜0.05).결론 C1QBP기인재융암내약세포주중표체명현증고,침묵C1QBP기인표체가이유효역전융암내약세포주대상응화료약물적내약성.
Objective To examine the complement component 1 Q subcomponent-binding protein (C1QBP) gene expression in human resistance choriocarcinoma cell lines and its parental cell line JeG-3,and to investigate whether silence C 1QBP by small interference RNA could reverse the resistance of human resistance choriocarcinoma cell lines to its relevant chemotherapy drugs.Methods Expression of C1QBP mRNA and protein in cells were detected by real-time fluorogenic quantitative PCR and western blot,respectively.The difference of C 1QBP expression was compared between human resistance choriocarcinoma cell lines and its parental cell line JeG-3.Sub-cellular location was proved by confocal immunofluorescence microscopy.A lentiviral vector containing short hairpin RNA (shRNA) targeting C 1QBP was constructed and cotransfected with the packaging plasmid mixture into 293T cells by lipofectamine 2000.The human resistance choriocarcinoma cell lines were infected with the packaged lentivirus.Real-time fluorogenic quantitative PCR and western blot were used to validate whether the C 1QBP gene expression was silenced.The cell counting kit 8(CCK8)was used to determine the drug sensitivity.Results (1)The C1QBP mRNA expression levels among four human resistance choriocarcinoma cell lines[JeG-3/floxuridiuum (FUDR),JeG-3/methotrexate (MTX),JeG-3/etoposide (VP),JeG-3/dactinomycin (KSM)] were 2.520±0.680,1.770±0.230,1.940±0.090 and 1.740±0.350 folds compared to that in JeG-3 cells.The C1QBP protein was higher expression level in human resistance choriocarcinoma cell lines than that in JeG-3.The immunofluorescence methods and confocal analysis showed that C1QBP localized predominantly in the mitochondrial matrix.(2)The C1QBP mRNA expression in JeG-3/FUDR cells after infected with lentiviral vector were decreased by 93.1％ (P＜0.01).The protein expression of C 1QBP in JeG-3/FUDR cells after infected with lentiviral vector were almost completely suppressed.The resistance indexes of four human resistance choriocarcinoma cell lines(JeG-3/FUDR,JeG-3/MTX,JeG-3/VP,JeG-3/KSM) were respectively 86.3％,93.9％,92.8％ and 89.9％,which were decreased remarkably by knockdown the C 1QBP expression (P＜0.05).Conclusions C1QBP is overexpressed in human resistance choriocarcinoma cell lines compared with parental cell line JeG-3.Inhibition of C 1QBP by lentivirus-mediated small interference RNA could effectively reverses the resistance of human resistance choriocarcinoma cell lines to its relevant chemotherapy drugs.
