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目的 :观察水通道蛋白9(aquaporin 9,AQP9)对人肝癌SMMC-7721细胞裸鼠皮下移植瘤生长的影响,并初步探讨其可能的机制。方法 :将重组慢病毒LV-AQP9(以空载体LV-GFP作为阴性对照)导入SMMC-7721细胞中,构建SMMC-7721/LV-AQP9重组细胞。激光共聚焦显微镜下观察重组慢病毒LV-AQP9在SMMC-7721细胞中的感染效率,实时荧光定量PCR和蛋白质印迹法检测AQP9 m RNA和蛋白在SMMC-7721细胞中表达的改变。采用FCM法和DAPI染色检测细胞的凋亡情况。CCK-8法检测SMMC-7721细胞增殖能力的变化。将重组肝癌SMMC-7721/LV-AQP9细胞和SMMC-7721/LV-GFP细胞分别接种于裸鼠皮下,观察2组小鼠中移植瘤生长的情况,并采用HE染色观察移植瘤及肺组织病理学的情况,免疫组织化学法检测移植瘤中增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)的表达水平。结果:激光共聚焦显微镜下观察发现,重组慢病毒LV-AQP9在SMMC-7721细胞中的感染效率为90%左右,且AQP9 m RNA以及蛋白在SMMC-7721细胞中的表达水平显著增加(P值均<0.01)。与空白对照组和阴性对照组(GFP组)相比,AQP9过表达组(AQP9组)细胞的总凋亡率显著增加(P<0.01);而AQP9组细胞在48~96 h各时间点的细胞增殖能力均受到抑制(P值均<0.05)。重复测量方差分析比较2组移植瘤体积的结果提示,AQP9组移植瘤体积显著小于GFP组(F分组=79.161,P分组=0.000),AQP9组皮下移植瘤生长速度明显小于GFP组(F分组×时间=18.481,P分组×时间=0.002)。AQP9组移植瘤中PCNA蛋白表达水平较GFP组下调。结论 :AQP9过表达抑制人肝癌SMMC-7721细胞在裸鼠皮下生长,并可能通过促进凋亡及抑制增殖两种途径参与调控,为深入研究分子机制奠定基础,且有望成为肿瘤治疗的新靶点。
OBJECTIVE: To observe the effect of aquaporin 9 (AQP9) on the growth of subcutaneous xenografts in human hepatocellular carcinoma SMMC-7721 cells and to explore its possible mechanism. Methods: Recombinant lentivirus LV-AQP9 (empty vector LV-GFP as a negative control) was introduced into SMMC-7721 cells to construct SMMC-7721 / LV-AQP9 recombinant cells. The infection efficiency of recombinant lentivirus LV-AQP9 in SMMC-7721 cells was observed by laser confocal microscopy. The expression of AQP9 mRNA and protein in SMMC-7721 cells was detected by real-time fluorescence quantitative PCR and Western blotting. Cell apoptosis was detected by FCM and DAPI staining. CCK-8 assay SMMC-7721 cell proliferation. The hepatocellular carcinoma SMMC-7721 / LV-AQP9 cells and SMMC-7721 / LV-GFP cells were inoculated subcutaneously in nude mice respectively to observe the growth of the transplanted tumor in the two groups of mice. HE staining was used to observe the transplanted tumors and lung tissues In the case of Neutropenia, the expression of proliferating cell nuclear antigen (PCNA) was detected by immunohistochemistry. Results: Confocal laser scanning microscopy showed that the infection efficiency of recombinant lentivirus LV-AQP9 in SMMC-7721 cells was about 90%, and the expression of AQP9 m RNA and protein in SMMC-7721 cells was significantly increased (P value All <0.01). Compared with the blank control group and the negative control group (GFP group), the total apoptosis rate of AQP9 overexpression group (AQP9 group) was significantly increased (P <0.01), while the AQP9 group cells at 48-96 h Cell proliferation was inhibited (P value <0.05). Repeated measures analysis of variance (ANOVA) showed that the volume of transplanted tumor in AQP9 group was significantly smaller than that in GFP group (F group = 79.161, P group = 0.000). The growth rate of subcutaneous tumor in AQP9 group was significantly less than that in GFP group Time = 18.481, P packet × time = 0.002). The expression of PCNA protein in AQP9 group was lower than that in GFP group. CONCLUSIONS: AQP9 overexpression inhibits the growth of human hepatocellular carcinoma SMMC-7721 cells in nude mice, and may be involved in the regulation through promoting apoptosis and inhibiting proliferation, laying a solid foundation for further study of molecular mechanisms and is expected to become a new target for cancer therapy .