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目的探讨慢病毒介导靶向VEGF小干扰RNA(siRNA)联合应用5-氟尿嘧啶(5-FU)对人乳腺癌细胞株MCF-7细胞荷瘤裸鼠的作用。方法 (1)以携带VEGF siRNA序列的慢病毒重组载体(VEGF-RNAi-Lv)及无义序列的慢病毒重组载体(NC-Lv)分别感染正常MCF-7细胞,Western blot检测各组细胞VEGF蛋白表达量。(2)30只SPF级雌性裸鼠随机分成五组:正常对照组(接种正常MCF-7细胞)、NC-Lv阴性组(接种已感染NC-Lv的MCF-7细胞)、VEGF-siRNA组(接种已感染VEGF-RNAi-Lv的MCF-7细胞)、5-FU组(接种正常MCF-7细胞,腹腔注射5-FU20mg·kg~(-1)·3d~(-1))及VEGF-siRNA+5-FU组(接种已感染VEGF-RNAi-Lv的MCF-7细胞,腹腔注射5-FU 20mg·kg~(-1)·3d~(-1)),测量肿瘤生长曲线;提取各组移植瘤组织的总蛋白,Western blot检测VEGF、p53和Bcl-2蛋白表达量。结果与正常对照组细胞相比,VEGF-siRNA组细胞VEGF蛋白水平降低(P<0.01),NC-Lv阴性组细胞VEGF蛋白水平相近(P>0.05)。与正常对照组和NC-Lv阴性组裸鼠肿瘤生长速度相比,VEGF-siRNA组、5-FU组和VEGF-siRNA+5-FU组肿瘤生长缓慢(P<0.05或P<0.01)。与正常对照组和NC-Lv阴性组相比,VEGF-siRNA组、5-FU组及VEGF-siRNA+5-FU组VEGF、Bcl-2蛋白水平降低,p53蛋白水平升高(P<0.05或P<0.01)。结论慢病毒介导的siRNA显著降低人乳腺癌细胞VEGF基因的表达,抑制乳腺肿瘤的生长并诱导细胞的凋亡,提高了乳腺癌细胞对5-FU的敏感性。
Objective To investigate the effect of lentivirus mediated targeting of small interfering RNA (siRNA) on 5-fluorouracil (5-FU) on human breast cancer cell line MCF-7 in nude mice. Methods (1) The normal MCF-7 cells were infected by lentiviral recombinant vector (VEGF-RNAi-Lv) carrying VEGF siRNA sequence and non-sense lentiviral vector (NC-Lv) respectively. Protein expression level. (2) Thirty SPF female nude mice were randomly divided into five groups: normal control group (normal MCF-7 cells), NC-Lv negative group (MCF-7 cells infected with NC-Lv), VEGF-siRNA group (Inoculated with MCF-7 cells infected with VEGF-RNAi-Lv), 5-FU group (inoculated with normal MCF-7 cells, intraperitoneal injection of 5-FU20mg · kg -1 · 3d -1) The tumor growth curve was measured by using the siRNA + 5-FU group (inoculated with MCF-7 cells infected with VEGF-RNAi-Lv and injected with 5-FU 20mg · kg -1 · 3d -1 by intraperitoneal injection) The total protein of the xenografts in each group was detected by Western blot, and the protein expression of VEGF, p53 and Bcl-2 were detected. Results Compared with the normal control cells, the VEGF protein level in VEGF-siRNA group decreased (P <0.01), and the VEGF protein level in NC-Lv negative group was similar (P> 0.05). The tumor growth of VEGF-siRNA group, 5-FU group and VEGF-siRNA + 5-FU group was slower than that of normal control group and NC-Lv negative group (P <0.05 or P <0.01). Compared with the normal control group and the NC-Lv negative group, VEGF-siRNA group, 5-FU group and VEGF-siRNA + 5-FU group decreased VEGF and Bcl-2 protein levels and increased p53 protein levels P <0.01). Conclusion Lentivirus-mediated siRNA can significantly reduce the expression of VEGF gene in human breast cancer cells, inhibit the growth of breast cancer cells and induce cell apoptosis, and improve the sensitivity of breast cancer cells to 5-FU.