中华预防医学杂志
中華預防醫學雜誌
중화예방의학잡지
CHINESE JOURNAL OF
2009年
8期
714-717
,共4页
傅娟%蒋义国%居颖%沈月兰%陈学敏
傅娟%蔣義國%居穎%瀋月蘭%陳學敏
부연%장의국%거영%침월란%진학민
7,8-二氢-7,8-二羟苯并(a)芘9,10-氧化物%细胞系,转化%基因,erbB-2%RNA干扰
7,8-二氫-7,8-二羥苯併(a)芘9,10-氧化物%細胞繫,轉化%基因,erbB-2%RNA榦擾
7,8-이경-7,8-이간분병(a)비9,10-양화물%세포계,전화%기인,erbB-2%RNA간우
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide%Cell line,transformed%Genes,erbB-2%RNA interference
目的 在经反式二羟环氧苯并芘(BPDE)恶性转化人支气管上皮细胞基础上建立一种载体介导的小发夹RNA(shRNA)抑制人上皮生长因子受体-2(HER2/neu)表达的稳定细胞株.方法 构建靶向干扰HER2/neu shRNA逆转录病毒载体pSIREN-RetroQ-neu,经酶切及测序鉴定后,将重组表达载体经脂质体介导入反式BPDE恶性转化细胞中,同时以阴性片段重组载体转染细胞(阴性对照)和空白细胞(16HBE-T)做对照,经嘌呤霉素筛选阳性转染细胞株.半定量RT-PCR和Westernblotting技术分别检测分析各组细胞中HER2/neu基因mRNA和蛋白表达差异.结果 获得载体介导靶向抑制反式BPDE恶性转化细胞中HER2/neu基因表达的细胞株.pSIREN-RelroQ-neu阳性转染细胞组分别比较阴性对照和空白细胞组HER2/neu mRNA表达量均下降(平均灰度值分别为0.114±0.003、0.186±0.001、0.182±0.015),其差异有统计学意义(t值分别为39.154、7.564,P值均<0.05),而阴性与空白对照组间相比差异无统计学意义(t=-0.409,P>0.05).pSIREN-RetroQ-neu转染细胞组相比阴性对照和空白细胞组HER2/neu蛋白表达明显下降,其抑制率分别达40%和39%.结论 成功构建pSIREN-RetroQ-neu重组质粒,并能有效抑制反式BPDE恶性转化细胞中HER2/neu表达.
目的 在經反式二羥環氧苯併芘(BPDE)噁性轉化人支氣管上皮細胞基礎上建立一種載體介導的小髮夾RNA(shRNA)抑製人上皮生長因子受體-2(HER2/neu)錶達的穩定細胞株.方法 構建靶嚮榦擾HER2/neu shRNA逆轉錄病毒載體pSIREN-RetroQ-neu,經酶切及測序鑒定後,將重組錶達載體經脂質體介導入反式BPDE噁性轉化細胞中,同時以陰性片段重組載體轉染細胞(陰性對照)和空白細胞(16HBE-T)做對照,經嘌呤黴素篩選暘性轉染細胞株.半定量RT-PCR和Westernblotting技術分彆檢測分析各組細胞中HER2/neu基因mRNA和蛋白錶達差異.結果 穫得載體介導靶嚮抑製反式BPDE噁性轉化細胞中HER2/neu基因錶達的細胞株.pSIREN-RelroQ-neu暘性轉染細胞組分彆比較陰性對照和空白細胞組HER2/neu mRNA錶達量均下降(平均灰度值分彆為0.114±0.003、0.186±0.001、0.182±0.015),其差異有統計學意義(t值分彆為39.154、7.564,P值均<0.05),而陰性與空白對照組間相比差異無統計學意義(t=-0.409,P>0.05).pSIREN-RetroQ-neu轉染細胞組相比陰性對照和空白細胞組HER2/neu蛋白錶達明顯下降,其抑製率分彆達40%和39%.結論 成功構建pSIREN-RetroQ-neu重組質粒,併能有效抑製反式BPDE噁性轉化細胞中HER2/neu錶達.
목적 재경반식이간배양분병비(BPDE)악성전화인지기관상피세포기출상건립일충재체개도적소발협RNA(shRNA)억제인상피생장인자수체-2(HER2/neu)표체적은정세포주.방법 구건파향간우HER2/neu shRNA역전록병독재체pSIREN-RetroQ-neu,경매절급측서감정후,장중조표체재체경지질체개도입반식BPDE악성전화세포중,동시이음성편단중조재체전염세포(음성대조)화공백세포(16HBE-T)주대조,경표령매소사선양성전염세포주.반정량RT-PCR화Westernblotting기술분별검측분석각조세포중HER2/neu기인mRNA화단백표체차이.결과 획득재체개도파향억제반식BPDE악성전화세포중HER2/neu기인표체적세포주.pSIREN-RelroQ-neu양성전염세포조분별비교음성대조화공백세포조HER2/neu mRNA표체량균하강(평균회도치분별위0.114±0.003、0.186±0.001、0.182±0.015),기차이유통계학의의(t치분별위39.154、7.564,P치균<0.05),이음성여공백대조조간상비차이무통계학의의(t=-0.409,P>0.05).pSIREN-RetroQ-neu전염세포조상비음성대조화공백세포조HER2/neu단백표체명현하강,기억제솔분별체40%화39%.결론 성공구건pSIREN-RetroQ-neu중조질립,병능유효억제반식BPDE악성전화세포중HER2/neu표체.
Objective To establish the stable inhibition of HER2/neu expression by vector-mediated small hairpin RNA in malignant transformed human bronchial epithelial cell line induced by anti-benzo( a ) pyrene-trans-7,8-dihydrodiol-9,10-epoxide ( anti-BPDE ). Methods The pSIREN-RetroQ-neu recombinant vector targeting HER2/neu was constructed and confirmed by restriction and sequencing analysis,then it was transfected into anti-BPDE malignant transformed 16HBE cells (16HBE-T) through lipofectamine 2000. The control groups included the 16HBE-T cells transfected with negative control vector (negative control) and 16HBE-T. The cells transfected with vectors were screened by puromycin. The HER2/ neu mRNA and protein expressions in the vector-transfected 16HBE-T cells were detected by RT-PCR and Western blot method respectively. Results The pSIREN-RetroQ-neu recombinant vector which inhibited HER2/neu mRNA and protein expressions in the 16HBE-T was constructed. The level of HER2/neu mRNA in the 16HBE-T cells transfected with pSIREN-RetroQ-neu was significantly reduced as compared to the negative control and blank control cells (0.114 ± 0.003 vs. Blank control 0.186 ± 0. 001, t = 39.154, P < 0. 05;and negative control 0.182 ± 0.015 ,t = 7.564, P < 0.05 ), while its level did not differ significantly between negative control cells and blank control of 16HBE-T (t = -0.409 ,P >0.05 ). HER2/neu protein level in pSIREN-RetroQ-neu transfected cells was inhibited by 40% and 39% respectively. Conclusion Plasmid-based shRNA expression systems targeted against HER2/neu gene were generated successfully, which resulted in down-regulation of HER2/neu gene expression in the 16HBE-T efficiently.
