摘要目的 观察体内沉默缺氧诱导因子1α(HIF-1α)的表达后对宫颈癌的抑瘤效应,并探讨其可能的机制.方法 将实验用宫颈癌Siha细胞分为空白对照组(转染空载体)、无关对照组(转染无关对照质粒)和实验组(稳定转染pU-HIF-1α-shRNA的真核表达载体),接种裸鼠,建立裸鼠荷瘤模型,观测HIF-1α-shRNA对裸鼠皮下移植瘤的生长抑制作用.采用免疫组化SP法和Western blot 法,检测HIF-1α和葡萄糖转运蛋白1(GLUT1)蛋白在肿瘤组织中的表达.采用逆转录聚合酶链反应(RT-PCR)法,检测HIF-1α、GLUTI和己糖激酶Ⅱ(HKⅡ)mRNA的表达.采用酶显色法,检测肿瘤组织中的乳酸含量.采用原位末端标记(TUNEL)法,检测细胞凋亡.结果 实验组裸鼠肿瘤的生长速度较空白对照组和无关对照组明显减慢(P＜0.05).接种50 d后处死裸鼠,实验组裸鼠的肿瘤重量为(1.90±0.28)g,也明显轻于空白对照组[(2.95±0.77)g]和无关对照组[(2.54±0.56)g,P＜0.01].实验组肿瘤组织中HIF-1α mRNA和蛋白的相对表达量分别为0.45±0.04和1.25±0.92,GLUT1mRNA和蛋白的相对表达量分别为0.32±0.02和1.25±0.48,均明显低于空白对照组和无关对照组(均P＜0.05).实验组中HK Ⅱ mRNA和乳酸的含量均明显低于空白对照组和无关对照组(均P＜0.05),但凋亡细胞数较空白对照组和无关对照组明显增多(均P＜0.01).结论 以HIF-1α为靶点的基因治疗,可通过下调靶基因GLUT1和HKⅡ的表达来降低宫颈癌Siha细胞的糖酵解水平,促进肿瘤细胞凋亡,从而发挥抑制宫颈癌生长的作用.

abstractsObjective To observe the anti-tumor effect of silencing the expression of HIF-1α on cervical cancer jn nude mice and to explore its mechanism of action.Methods Human cervical cancer cell line Siha cells were di6ded into 3 groups:mock control group,control group transfected with scrambled sequence plasmid,and experimental group transfected with pU-HIF-1a-shRNA eukaryotic expression plasmid.Cultured cells of tlle three groups were inoculated in nude mice to establish cervical cancer-bearing nude mice.HIF-1a RNAi assay wsa performed to evaluate the tumor-suppressive effect of HIF-1a silencing on cervical cancer-bearing nude mice.Immunohistochemistry and Western blot were used to observe the distilbufion and protein expression of HIF-1a and GLUT1,while RT-PCR was adopted to detect the gene expression of HIF-1a.GLUT1 and HK Ⅱ.The product of glycolysis(lactic acid)and apoptosis in tumor cells were detected by colorimetry and semi-quantitative TUNEL staining,respectively.Results The tumor growth in experimental group was significantly slower than that in the two control groups(P＜0.05).On the 50th day after transplantation.the tumor weisht in the experimental group was(1.90±0.28)g,significantly lower than(2.95±0.77)g in the control group and(2.54±0.56)g in the mock group(P＜0.01).In the experimental group.the gene and protein levels of HIF-1α were 0.45±0.04 and 1.25±0.92.and the levels of GLUTl were 0.32±0.02 and 1.25±0.48.respectively.Both indicators in HIF-1a and GLUT1 were lower than that in the two control groups(P＜0.05).The expression levels of HK Ⅱ gene and lactic acid in the experimental group were lower than that in the two control groups(P＜0.05),but the apoptotic cells were much more numerous in the experimental group than that in matched control groups (P＜0.01).Conclusion The gene therepy by siRNA targeted silencing of HIF-1a may down-regulate its downstream genes GLUT1 and HK Ⅱ expression,therefore,to reduce the tumor glycolysis activity and promote tumor cell apoptosis,and exert a tumor-suppressing effect in vivo.