论文部分内容阅读
目的研究环氧合酶(COX)-2表达与肿瘤细胞增殖和凋亡的关系,探讨利用RNA干扰技术作为肿瘤基因治疗的方法。方法构建靶向COX-2的短发夹状双链RNA(shRNA)的真核表达质粒WBH1转染人胃癌细胞系SGC-7901,采用逆转录聚合酶链反应(RT-PCR)和Western印迹分别从mRNA和蛋白质水平检测抑制效果。四甲基偶氮唑蓝(MTT)和流式细胞仪检测COX-2表达被抑制后细胞的增殖和凋亡情况。15只裸鼠随机分为3组,每组5只,皮下接种胃癌细胞。抑制组接种转染抑制质粒的胃癌细胞;正常对照组接种未转染的胃癌细胞;阴性对照组接种转染阴性对照质粒的胃癌细胞。4周后观察比较各组裸鼠皮下形成瘤体的大体和病理情况并计算抑瘤率。结果WBH1高效特异地抑制了人COX-2的表达,抑制率达70.42%。COX-2表达被抑制后,细胞增殖受到明显抑制(P=0.002),细胞凋亡率明显增高,与未转染和转染阴性对照质粒的胃癌细胞的凋亡率相比有统计学意义(52.28%±17.91%、0.52%±0.27%、0.54%±0.16%,P=0.009,实验重复5次)。正常对照组和阴性对照组所有裸鼠均有瘤体形成,平均瘤重分别为(0.490g±0.017g,5只)和(0.490g±0.013g,5只)。抑制组仅2只有瘤体形成平均瘤重0.050g±0.003g,与正常对照组相比差异有统计学意义(P<0.01),抑瘤率为89.8%。结论COX-2与肿瘤细胞的增殖和凋亡密切相关,抑制细胞中COX-2的表达可以抑制肿瘤细胞增殖,促进肿瘤细胞凋亡。通过构建靶向COX-2的shRNA真核表达载体导入细胞可以高效特异地抑制人胃癌细胞中COX-2的表达。
Objective To study the relationship between cyclooxygenase (COX) -2 expression and tumor cell proliferation and apoptosis, and to explore the use of RNA interference as a method for gene therapy of tumors. Methods Human gastric cancer cell line SGC-7901 was transfected with the eukaryotic expression plasmid WBH1 of short hairpin double stranded RNA (shRNA) targeting COX-2. The expression of COX-2 was detected by reverse transcription polymerase chain reaction (RT-PCR) The inhibitory effect is detected from mRNA and protein levels. MTT and flow cytometry were used to detect the proliferation and apoptosis of the cells after COX-2 expression was inhibited. Fifteen nude mice were randomly divided into 3 groups with 5 mice in each group, which were inoculated subcutaneously with gastric cancer cells. The control group was inoculated with the gastric cancer cells transfected with the suppressor plasmid; the untreated gastric cancer cells were inoculated in the normal control group; the negative control group was inoculated with the gastric cancer cells transfected with the negative control plasmid. After 4 weeks, the general and pathological conditions of subcutaneous tumor formation in nude mice in each group were observed and compared. Results WBH1 efficiently and specifically inhibited the expression of human COX-2 with an inhibitory rate of 70.42%. After COX-2 expression was inhibited, cell proliferation was significantly inhibited (P = 0.002), apoptosis rate was significantly increased compared with untransfected and transfected negative control plasmid of gastric cancer cell apoptosis rate was statistically significant (P < 52.28% ± 17.91%, 0.52% ± 0.27%, 0.54% ± 0.16%, P = 0.009, experiment repeated 5 times). All nude mice in normal control group and negative control group had tumor formation with mean tumor weights (0.490g ± 0.017g, 5pcs) and (0.490g ± 0.013g, 5pcs) respectively. In the inhibition group, only 2 tumors formed a mean tumor weight of 0.050g ± 0.003g, which was significantly different from the normal control group (P <0.01). The tumor inhibition rate was 89.8%. Conclusions COX-2 is closely related to the proliferation and apoptosis of tumor cells. Inhibiting the expression of COX-2 can inhibit the proliferation of tumor cells and promote the apoptosis of tumor cells. Introduction of COX-2 shRNA targeting eukaryotic expression vector into cells can efficiently and specifically inhibit the expression of COX-2 in human gastric cancer cells.