CAJ | 학술논문

目的采用RNA干扰(RNAi)技术沉默转化生长因子β1(TGF-β1)基因,观察RNAi的基因沉默效应及对人膀胱癌细胞株(EJ细胞株)中血管内皮生长因子(VEGF)表达的影响.方法构建基因TGF-β1特异性小干扰RNA (siRNA)表达载体,反转录(RT)-PCR及ELISA筛选出抑制效率最高的靶序列,分成EJ细胞组、对照组(TGF-β1组)、重组质粒组(TGF-β1siRNA表达载体组)3组转染,Western印迹检测各组细胞VEGF蛋白表达水平.结果成功构建基因TGF-β1特异性siRNA表达载体,RT-PCR及ELISA筛选出最佳抑制效果的序列[TGF-β1相对mRNA表达水平为0.92-0.19;ELISA检测的蛋白表达水平为(50 ±6) pg/ml];转染后各组的VEGF蛋白表达水平为EJ细胞组(0.86 ±0.18) pg/ml,对照组(1.15 ±0.29) pg/ml,重组质粒组(0.45 ±0.16) pg/ml,重组质粒组明显低于EJ细胞组和对照组(均P＜0.05).结论抑制TGF-β1基因能下调VEGF基因的表达,TGF-β1基因可能通过诱导VEGF基因的表达实现对膀胱肿瘤血管生成的调控.
목적 채용RNA간우(RNAi)기술침묵전화생장인자β1(TGF-β1)기인,관찰RNAi적기인침묵효응급대인방광암세포주(EJ세포주)중혈관내피생장인자(VEGF)표체적영향.방법 구건기인TGF-β1특이성소간우RNA (siRNA)표체재체,반전록(RT)-PCR급ELISA사선출억제효솔최고적파서렬,분성EJ세포조、대조조(TGF-β1조)、중조질립조(TGF-β1siRNA표체재체조)3조전염,Western인적검측각조세포VEGF단백표체수평.결과 성공구건기인TGF-β1특이성siRNA표체재체,RT-PCR급ELISA사선출최가억제효과적서렬[TGF-β1상대mRNA표체수평위0.92-0.19;ELISA검측적단백표체수평위(50 ±6) pg/ml];전염후각조적VEGF단백표체수평위EJ세포조(0.86 ±0.18) pg/ml,대조조(1.15 ±0.29) pg/ml,중조질립조(0.45 ±0.16) pg/ml,중조질립조명현저우EJ세포조화대조조(균P＜0.05).결론 억제TGF-β1기인능하조VEGF기인적표체,TGF-β1기인가능통과유도VEGF기인적표체실현대방광종류혈관생성적조공.
Objective To employ RNA interference technology to silence transforming growth factor-β1 (TGF-β1) gene to examine the gene silencing effects of RNAi on the expression of vascular endothelial growth factor (VEGF) in human bladder cancer cell lines (EJ).Methods The TGF-β1 genespecific siRNA expression vector was constructed.And the most efficiently suppressed target sequences were screened through reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA).The samples were divided into 3 groups of EJ,control (TGF-β1) and recombinant plasmid (TGF-β1 siRNA expression vector).And the expression level of VEGF protein was detected by Western blot.Results TGF-β1 gene-specific siRNA expression vector was constructed successfully.TGF-β1 relative mRNA expression was 0.92 ± 0.19 and the protein expression level (50 ±6) pg/ml.The protein expression level of EJ group after transfection was (0.86 ± 0.18) pg/ml,control group (1.15 ± 0.29) pg/ml and recombinant plasmid group (0.45 ±0.16) pg/ml (both P＜0.05).Conclusions An inhibition of TGF-β1 gene down-regulates the expression of VEGF.And TGF-β1 may regulate angiogenesis of bladder tumor through an induction of VEGF gene expression.