国际肿瘤学杂志
國際腫瘤學雜誌
국제종류학잡지
JOURNAL OF INTERNATIONAL ONCOLOGY
2008年
11期
869-873
,共5页
饶建锋%陈建勇%罗忠金%张吉翔
饒建鋒%陳建勇%囉忠金%張吉翔
요건봉%진건용%라충금%장길상
癌,肝细胞%DNA修复
癌,肝細胞%DNA脩複
암,간세포%DNA수복
Carcinoma,hepatocellular%DNA repair
目的 将人剪切修复基因XPD稳定转染人SMMC-7721肝癌细胞,观察转染后细胞内野生型p53、XPD、周期素依赖性蛋白激酶(CDK)7、c-myc等基因表达的变化以及对细胞生长的影响,探讨野生型XPD基因与p53、CDK7、c-myc的相互作用及细胞凋亡机制.方法 将表达绿色荧光蛋白并含有人类全长野生型XPD的pEGFP-N2-XPD重组体质粒稳定转染人SMMC-7721肝癌细胞中,选择培养基筛选单克隆稳定转染重组质粒的人SMMC-7721肝癌细胞(SMMC-7721-pEGFP-N2-XPD)和稳定转染空载质粒的人SMMC-7721肝癌细胞(SMMC.7721-pEGFP-N2),并将人SMMC-7721肝癌细胞作为空白对照,利用荧光显微镜观测绿色荧光蛋白表达,用逆转录-聚合酶链反应(RT-PCR)、Westem blot法检测转染XPD基因后细胞内XPD、p53、CDK7、c-myc的表达量变化,并用细胞增殖力检测(MTT)法及流式细胞仪分别检测细胞增殖及凋亡和细胞周期变化.结果 ①免疫荧光显微镜下,SMMC.7721.pEGFP.N2.XPD和SMMC-7721-pEGFP-N2细胞中观察到绿色荧光蛋白表达,说明pEGFP-N2-XPD重组质粒和pEGFP-N2空载质粒成功转染.②RT-PCR检测:SMMC-7721-pEGFP-N2-XPD中p53 mRNA、XPD mR-NA表达量与SMMC-7721-pEGFP-N2和SMMC-7721相比均明显增高(P<0.01),CDK7 mRNA、c-myc mRNA在SMMC-7721-pEGFP-N2-XPD的表达量比两对照组明显降低(P<0.01),而两对照组各个基因的表达没有明显差异(P>0.05).③Western blot检测:SMMC-7721-pEGFP-N2-XPD细胞的p53、XPD蛋白表达最较两对照组升高(P<0.01),CDK7、c-myc的蛋白相对表达量比两对照组降低(P<0.01),两对照组差异没有统计学意义(P>0.05).④M1Tr检测:SMMC-7721-pEGFP-N2-XPD的细胞增殖力较对照组减弱(P<0.05),两对照组筹异没有统计学意义(P>0.05).⑤流式细胞仪检测:SMMC-7721-pEGFP-N2-XPD细胞进入s期出现障碍,停滞在G1期的细胞增多.结论 将XPD成功稳定转染到人SMMC-7721肝癌细胞中,XPD、p53在转录和蛋白水平的表达明显升高,CDK7、c-myc表达明显降低,野生型XPD基因的过表达可能抑制CDK7、c-myc表达,改变细胞周期,并促进p53抑制肝癌细胞增长,促进细胞凋亡.
目的 將人剪切脩複基因XPD穩定轉染人SMMC-7721肝癌細胞,觀察轉染後細胞內野生型p53、XPD、週期素依賴性蛋白激酶(CDK)7、c-myc等基因錶達的變化以及對細胞生長的影響,探討野生型XPD基因與p53、CDK7、c-myc的相互作用及細胞凋亡機製.方法 將錶達綠色熒光蛋白併含有人類全長野生型XPD的pEGFP-N2-XPD重組體質粒穩定轉染人SMMC-7721肝癌細胞中,選擇培養基篩選單剋隆穩定轉染重組質粒的人SMMC-7721肝癌細胞(SMMC-7721-pEGFP-N2-XPD)和穩定轉染空載質粒的人SMMC-7721肝癌細胞(SMMC.7721-pEGFP-N2),併將人SMMC-7721肝癌細胞作為空白對照,利用熒光顯微鏡觀測綠色熒光蛋白錶達,用逆轉錄-聚閤酶鏈反應(RT-PCR)、Westem blot法檢測轉染XPD基因後細胞內XPD、p53、CDK7、c-myc的錶達量變化,併用細胞增殖力檢測(MTT)法及流式細胞儀分彆檢測細胞增殖及凋亡和細胞週期變化.結果 ①免疫熒光顯微鏡下,SMMC.7721.pEGFP.N2.XPD和SMMC-7721-pEGFP-N2細胞中觀察到綠色熒光蛋白錶達,說明pEGFP-N2-XPD重組質粒和pEGFP-N2空載質粒成功轉染.②RT-PCR檢測:SMMC-7721-pEGFP-N2-XPD中p53 mRNA、XPD mR-NA錶達量與SMMC-7721-pEGFP-N2和SMMC-7721相比均明顯增高(P<0.01),CDK7 mRNA、c-myc mRNA在SMMC-7721-pEGFP-N2-XPD的錶達量比兩對照組明顯降低(P<0.01),而兩對照組各箇基因的錶達沒有明顯差異(P>0.05).③Western blot檢測:SMMC-7721-pEGFP-N2-XPD細胞的p53、XPD蛋白錶達最較兩對照組升高(P<0.01),CDK7、c-myc的蛋白相對錶達量比兩對照組降低(P<0.01),兩對照組差異沒有統計學意義(P>0.05).④M1Tr檢測:SMMC-7721-pEGFP-N2-XPD的細胞增殖力較對照組減弱(P<0.05),兩對照組籌異沒有統計學意義(P>0.05).⑤流式細胞儀檢測:SMMC-7721-pEGFP-N2-XPD細胞進入s期齣現障礙,停滯在G1期的細胞增多.結論 將XPD成功穩定轉染到人SMMC-7721肝癌細胞中,XPD、p53在轉錄和蛋白水平的錶達明顯升高,CDK7、c-myc錶達明顯降低,野生型XPD基因的過錶達可能抑製CDK7、c-myc錶達,改變細胞週期,併促進p53抑製肝癌細胞增長,促進細胞凋亡.
목적 장인전절수복기인XPD은정전염인SMMC-7721간암세포,관찰전염후세포내야생형p53、XPD、주기소의뢰성단백격매(CDK)7、c-myc등기인표체적변화이급대세포생장적영향,탐토야생형XPD기인여p53、CDK7、c-myc적상호작용급세포조망궤제.방법 장표체록색형광단백병함유인류전장야생형XPD적pEGFP-N2-XPD중조체질립은정전염인SMMC-7721간암세포중,선택배양기사선단극륭은정전염중조질립적인SMMC-7721간암세포(SMMC-7721-pEGFP-N2-XPD)화은정전염공재질립적인SMMC-7721간암세포(SMMC.7721-pEGFP-N2),병장인SMMC-7721간암세포작위공백대조,이용형광현미경관측록색형광단백표체,용역전록-취합매련반응(RT-PCR)、Westem blot법검측전염XPD기인후세포내XPD、p53、CDK7、c-myc적표체량변화,병용세포증식력검측(MTT)법급류식세포의분별검측세포증식급조망화세포주기변화.결과 ①면역형광현미경하,SMMC.7721.pEGFP.N2.XPD화SMMC-7721-pEGFP-N2세포중관찰도록색형광단백표체,설명pEGFP-N2-XPD중조질립화pEGFP-N2공재질립성공전염.②RT-PCR검측:SMMC-7721-pEGFP-N2-XPD중p53 mRNA、XPD mR-NA표체량여SMMC-7721-pEGFP-N2화SMMC-7721상비균명현증고(P<0.01),CDK7 mRNA、c-myc mRNA재SMMC-7721-pEGFP-N2-XPD적표체량비량대조조명현강저(P<0.01),이량대조조각개기인적표체몰유명현차이(P>0.05).③Western blot검측:SMMC-7721-pEGFP-N2-XPD세포적p53、XPD단백표체최교량대조조승고(P<0.01),CDK7、c-myc적단백상대표체량비량대조조강저(P<0.01),량대조조차이몰유통계학의의(P>0.05).④M1Tr검측:SMMC-7721-pEGFP-N2-XPD적세포증식력교대조조감약(P<0.05),량대조조주이몰유통계학의의(P>0.05).⑤류식세포의검측:SMMC-7721-pEGFP-N2-XPD세포진입s기출현장애,정체재G1기적세포증다.결론 장XPD성공은정전염도인SMMC-7721간암세포중,XPD、p53재전록화단백수평적표체명현승고,CDK7、c-myc표체명현강저,야생형XPD기인적과표체가능억제CDK7、c-myc표체,개변세포주기,병촉진p53억제간암세포증장,촉진세포조망.
Objective To investigate the role of wild-type XPD in SMMC-7721 hepatoma cells,its re-lationship with wild-type p53.CDK7 and c-myc.And the apoptosis mechanism of SMMC-7721 hepatoma cells.Methotis We inserted the human length XPD into the pEGFP-N2 plasmid vector which expresses the green fluorescence protein(GFP).And the pEGFP-N2 and pEGFP-N2-XPD were transfected into SMMC-7721 hepa-toma cell lines stably.Cel lines for omparison were matched on the sanle genetic background and passage.The expression of wild-type XPD,CDK7,p53,c-myc waft detected by RT-PCR,Westem blot.Cell growth wag detet-ed by MTT FCM wag employed for examining the cell cycle and apoptosis of the transfected SMMC-7721 hepa-toms cells.Results ①SMMC-7721-pEGFP-N2-xPD and SMMC-7721-pEGFP-N2 express the green fluores-cence protein which could be detected under the immunoffuorescence microscope.②RT-PCR The relative ex-\pression of p53 mRNA,XPD mRNA in the SMMC-7721,SMMC-7721-pEGFP-N2 and SMMC-7721-pEGFP-N2-XPD,the relative expression of p53 mRNA,XPD mRNA in the SMMC-7721-pEGFP-N2-XPD was signifiantly higher than other two oontrols(P<0.01).Othewise,the relative expression of CDK7 mRNA,c-myc mRNA Wag signifi-antlv lower than other two oontrols(P<0.01),the difference of two controls was not signifiant(P>0.05). ③Western blot The relative expression of p53,XPD protein was signifiantly higher than other two oontrols (P<0.01).Othewise,the relative expression of CDK7,c-Myc protein was signifiantly lower than other two oontrols(P<O.01),the difference of two controls Wag not signifiant(P>0.05).④MTT The ressuh showedthat the proliferative ability of SMMC-7721-pEGFP-N2-XPD was much more descreaged than other two oontrols (P<0.05),the other two oontrols were not different(P>0.05).⑤FCM Wild-type XPD gene could change the cell8 and induce apoptitise in vitro though the result of FCM.Conclusions The pEGFP-N2 and pEGFP-N2-XPD were transfected into SMMC-7721hepatoma cell lines stably,wild-type XPD could decreaSe the expres-sion of CDK7,c-myc and increase the expression of p53.Wild-type.XPD could inhibit the activity of cell growth and change cell cycle,as well as induce cell apoptosis in SMMC-7721 hepatoma cell line in vitro.