AQP9过表达对肝癌细胞侵袭转移的影响及分子机制的研究

发布时间：2018-07-05 15:15

本文选题：肝细胞癌 + 水通道蛋白9　；参考：《重庆医科大学》2016年硕士论文

【摘要】：目的:研究水通道蛋白9(Aquaporin9,AQP9)过表达对肝癌细胞侵袭转移的影响及其分子机制研究。方法:(第一部分)实时荧光定量PCR和蛋白免疫印迹法检测正常肝细胞株L02和肝癌细胞株SMMC-7721、HepG2、Hep3B及Huh-7中AQP9表达水平,并筛选出SMMC-7721作为后续实验肝癌细胞株。将重组慢病毒LV-AQP9-GFP(以空载体LV-GFP作为阴性对照)导入SMMC-7721细胞中,构建AQP9过表达SMMC-7721/LV-AQP9重组细胞模型。激光共聚焦显微镜下观察重组慢病毒LV-AQP9-GFP在SMMC-7721细胞中的转染效率,蛋白质印迹法检测AQP9在SMMC-7721/LV-AQP9重组细胞模型中过表达水平。细胞肿胀实验检测AQP9过表达细胞模型中AQP9作为水通道功能活化程度。采用划痕实验、Transwell侵袭实验检验AQP9过表达对SMMC-7721细胞迁移、侵袭的影响。(第二部分)在动物水平,将AQP9过表达重组肝癌SMMC-7721/LV-AQP9细胞和SMMC-7721/LV-GFP细胞接种于裸鼠皮下构建裸鼠皮下移植瘤模型,动态观察2组裸鼠皮下移植瘤生长的差异。同时,通过尾静脉注射重组肝癌smmc-7721/lv-aqp9细胞和smmc-7721/lv-gfp细胞构建肝癌肺转移模型,观察2组裸鼠肺表面癌结节转移数量,并采用he染色观察肺组织中转移瘤的情况。(第三部分)蛋白免疫印迹法检测aqp9过表达重组肝癌smmc-7721/lv-aqp9细胞和smmc-7721/lv-gfp细胞中emt相关蛋白(e-cadherin、n-cadherin、vimentin),pi3k/akt信号通路相关蛋白pi3k、akt、p-akt及基质金属蛋白酶mmp2、mmp9的表达差异。采用免疫组化法检测移植瘤组织中e-cadherin、n-cadherin、vimentin蛋白表达。结果:(第一部分)肝癌细胞株hep3b不表达aqp9,而smmc-7721、hepg2、huh-7中aqp9mrna和aqp9蛋白表达均较正常肝细胞株l02显著降低(p值均0.05),其中smmc-7721中aqp9mrna和aqp9蛋白表达量最低。激光共聚焦显微镜下观察发现,重组慢病毒lv-aqp9在smmc-7721细胞中的转染效率为90%左右,并通过gfp荧光信号定位发现,aqp9-gfp融合蛋白绿色荧光主要分布在smmc-7721/lv-aqp9细胞的细胞膜上。在smmc-7721/lv-aqp9重组细胞模型(aqp9组)中aqp9蛋白表达量较空载对照组smmc-7721/lv-gfp细胞(gfp组)显著增加(p0.01)。在低渗状态下smmc-7721/lv-aqp9重组细胞(aqp9组)膨胀体积较smmc-7721/lv-gfp细胞(gfp组)显著增大(p0.05),而用hgcl2(aqp9阻滞剂)阻滞水通道蛋白活性后,在低渗状态下smmc-7721/lv-aqp9重组细胞(aqp9组)和smmc-7721/lv-gfp细胞(gfp组)膨胀体积无显著差异(p=0.783)。划痕实验显示,aqp9组细胞48h的迁移率(6.38%±1.70%)较gfp组(16.74%±1.40%)下降(p0.01)。transwell实验结果显示,aqp9组48h穿过小室细胞数(57±8)较gfp组(95±11)减少(p0.01)。(第二部分)动物水平,在裸鼠皮下移植瘤模型中,重复测量方差分析2组移植瘤体积提示,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)。在裸鼠肺转移模型中,he染色观察aqp9组裸鼠肺组织中转移瘤体较gfp组更小,aqp9组裸鼠肺表面癌结节转移数量较gfp组显著减少(p0.05)。(第三部分)westernblot检测结果显示aqp9过表达后上皮表型标志物(e-cadherin)、pi3k、p-akt蛋白表达上调(p值均0.05),间皮表型标志物(n-cadherin)表达下调(p0.05),而vimentin、akt、mmp9、mmp2蛋白表达水平较gfp组均无明显变化(p值均0.05)。同样,免疫组化及光密度分析结果显示,aqp9组移植瘤中e-cadherin蛋白表达水平较gfp组上调(p0.05),aqp9组移植瘤中n-cadherin、vimentin蛋白表达水平较gfp组下调(p0.01)。结论:肝癌细胞中aqp9表达水平较正常肝细胞下调。aqp9过表达抑制肝癌smmc-7721细胞侵袭转移及上皮间质转化,而pi3k/akt信号通路可能参与这一调控过程。此外,aqp9过表达对基质金属蛋白酶MMP9和MMP2表达无影响。
[Abstract]:Objective: To study the influence and molecular mechanism of the overexpression of Aquaporin9 (Aquaporin9, AQP9) on the invasion and metastasis of hepatoma cells. Methods: (Part I) real-time fluorescence quantitative PCR and protein immunoblotting were used to detect the AQP9 expression level in normal liver cell line L02 and liver cancer cell lines SMMC-7721, HepG2, Hep3B and Huh-7, and to screen out SMMC-7721. As a follow-up experimental liver cancer cell line, the recombinant lentivirus LV-AQP9-GFP (LV-GFP as negative control) was introduced into SMMC-7721 cells to construct a AQP9 overexpressed SMMC-7721/LV-AQP9 recombinant cell model. The transfection efficiency of recombinant lentivirus LV-AQP9-GFP in SMMC-7721 cells was observed under confocal laser scanning microscope, and the Western blot assay was used to detect the transfection efficiency of the recombinant lentivirus in SMMC-7721 cells. The level of overexpression of AQP9 in the SMMC-7721/LV-AQP9 cell model was measured. The cell swelling test was used to detect the function activation of AQP9 in the AQP9 overexpressed cell model. The scratch test was used, the Transwell invasion test was used to test the effect of AQP9 over expression on the migration of SMMC-7721 cells and the impact of the invasion. (second) at animal level, AQP9 over the table. The recombinant hepatoma SMMC-7721/LV-AQP9 cells and SMMC-7721/LV-GFP cells were inoculated subcutaneously in nude mice by subcutaneous transplantation of nude mice. The difference of the growth of the 2 groups of nude mice was observed dynamically. At the same time, the lung metastasis model of liver cancer was constructed by injection of the recombinant hepatoma smmc-7721/lv-aqp9 cells and smmc-7721/ lv-gfp cells by the tail vein, and the 2 groups were observed. The number of metastatic tumor nodules in lung surface of nude mice was observed by HE staining. (third) protein immunoblotting was used to detect AQP9 over expression of EMT associated protein (E-cadherin, N-cadherin, vimentin) in smmc-7721/lv-aqp9 cells and smmc-7721/lv-gfp cells, pi3k/akt signaling pathway related proteins PI3K, Akt, p-. The expression of Akt and matrix metalloproteinase MMP2, MMP9 expression. The expression of E-cadherin, N-cadherin, vimentin protein in the transplanted tumor tissue was detected by immunohistochemistry. Results: (Part 1) the expression of Hep3B did not express AQP9 in the liver cancer cell line Hep3B, and the expression of aqp9mrna and protein in SMMC-7721, HepG2, Huh-7 was significantly lower than that of normal liver cell lines (0 5) the expression of aqp9mrna and AQP9 protein in SMMC-7721 was the lowest. The transfection efficiency of recombinant lentivirus lv-aqp9 in SMMC-7721 cells was about 90% under confocal laser scanning microscope, and the green fluorescence of aqp9-gfp fusion protein was mainly distributed on the cell membrane of smmc-7721/lv-aqp9 cells. The expression of AQP9 protein in the -7721/lv-aqp9 recombinant cell model (group AQP9) was significantly higher than that in the empty control group (Group GFP) (P0.01). In the hypotonic state, the expansion volume of the smmc-7721/lv-aqp9 recombinant cells (AQP9 group) was larger than the smmc-7721/lv-gfp cell (P0.05) (P0.05), and the water channel protein was blocked by HgCl2 (inhibitor). After activity, there was no significant difference in the expansion volume of smmc-7721/lv-aqp9 cells (group AQP9) and smmc-7721/lv-gfp cells (Group GFP) in hypotonic state (p=0.783). The scratch test showed that the mobility of 48h in group AQP9 (6.38% + 1.70%) was lower than that of GFP group (16.74% + 1.40%) (P0.01).Transwell experimental results showed that AQP9 48h passed through the cell number of cells (57 +). 8) compared with group GFP (95 + 11) reduction (P0.01). (second) animal level, in nude mice subcutaneous transplantation tumor model, repeated measurement of variance analysis of 2 groups of transplanted tumor volume suggested that the volume of AQP9 group was significantly smaller than that of group GFP (F group =79.161, P group =0.000), and the growth rate of subcutaneous transplanted tumor in AQP9 group was significantly smaller than that of GFP group (f grouping * time =18.481). X time =0.002). In the lung metastasis model of nude mice, he staining showed that the metastatic tumor in lung tissue of AQP9 group was smaller than that in group GFP. The number of metastatic nodules in lung surface of nude mice decreased significantly in AQP9 group than that in GFP group (P0.05). (third) Westernblot detection results showed that the epithelial phenotype marker (E-cadherin), PI3K, p-Akt protein expression after AQP9 overexpression. Up regulation (P value was 0.05), mesothelial phenotype marker (N-cadherin) expression down (P0.05), while vimentin, Akt, MMP9, MMP2 protein expression level was not significantly changed compared with GFP group (P 0.05). Similarly, immunohistochemical and optical density analysis showed that the expression level of E-cadherin protein in the AQP9 group was higher than that of the GFP group. Adherin, vimentin protein expression level was lower than that of group GFP (P0.01). Conclusion: the expression level of AQP9 in hepatoma cells inhibited the invasion and metastasis of hepatocellular carcinoma SMMC-7721 cells and epithelial mesenchymal transition compared with normal hepatocytes, and pi3k/akt signaling pathway may be involved in this regulatory process. In addition, AQP9 overexpression on matrix metalloproteinase MMP9 and P0.01. The expression of MMP2 has no effect.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.7

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