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细胞分为李白对照组（转染空载体）、无关对照组（转染尤关对照质粒）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、GLUT1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50d后处死裸鼠，实验组裸鼠的肿瘤重量为（1．90±0．28）g，也明显轻于窄白对照组[（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-1αmRNA和蛋白的相对表达量分别为0．45±0．04和1．25±0．92，GLUT1 mRNA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HKⅡ 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。
Objective To observe the anti-tumor effect of silencing the expression of HIF-1α on cervical cancer in nude mice and to explore its mechanism of action. Methods Human cervical cancer cell line Siha cells were divided into 3 groups： mock control group, control group transfected with scrambled sequence plasmid, and experimental group transfected with pU-HIF-1α-shRNA eukaryotic expression plasmid. Cultured cells of the three groups were inoculated in nude mice to establish cervical cancer-bearing nude mice. HIF-1α RNAi assay was performed to evaluate the tumor-suppressive effect of HIF-1α silencing on cervical cancer-bearing nude mice. Immunohistochemistry and Western blot were used to observe the distribution and protein expression of HIF-1α and GLUT1, while RT-PCR was adopted to detect the gene expression of HIF-1α, 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 weight 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 GLUT1 were 0.32 ± 0.02 and 1.25 ± 0.48, respectively. Both indicators in HIF-1α 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 therapy by siRNA targeted silencing of HIF-1α may down-regulate its downstream genes CLUTI and HK Ⅱ expression, therefore, to reduce the tumor glycolysis activity and promote tumor cell apoptosis, and exert a tumor-suppressing effect in vivo.
出处
《中华肿瘤杂志》
CAS
CSCD
北大核心
2009年第11期820-825,共6页
Chinese Journal of Oncology
