【摘要】：目的:探討AQP9基因過表達后對人肝癌細胞SMMC-7721增殖的影響及其可能的機制。方法:將空載體慢病毒(LV-Luciferase)和靶向AQP9基因過表達慢病毒(LV-AQP9)分別轉染人肝癌細胞SMMC-7721,并用嘌呤霉素篩選細胞,從而得到空載體穩(wěn)定細胞株(SMMC-7721/LV-Luciferase)和AQP9過表達穩(wěn)定細胞株(SMMC-7721/LV-AQP9),最后通過Western blotting檢測AQP9在肝癌細胞內表達。實驗分組:陰性對照組(NC組)為空載體穩(wěn)定細胞;AQP9過表達組(AQP9組)為AQP9過表達穩(wěn)定細胞。并且運用流式細胞術檢測過表達AQP9基因對人肝癌細胞周期的影響;實時熒光定量PCR檢測SMMC-7721/LV-Luciferase細胞和SMMC-7721/LV-AQP9細胞的周期相關調控因子mRNA水平表達情況;Western blotting技術檢測細胞周期相關調控因子,PCNA,pser675β-catenin,β-catenin蛋白水平表達。同時采用免疫熒光染色觀察PCNA和β-catenin在兩組肝癌細胞中表達情況。兩組數(shù)據(jù)比較采用了獨立樣本t檢驗。結果:Western blotting檢測結果顯示AQP9過表達組較陰性對照組AQP9的蛋白水平有顯著升高,差異有統(tǒng)計學意義(P值0.01)。通過流式細胞技術檢測細胞周期,結果顯示過表達AQP9基因后影響SMMC-7721細胞周期分布,具體地,SMMC-7721細胞G0-G1期細胞比例由52.24%±0.83%升高到65.68%±0.63%(P值0.05);S期細胞比例由34.3%±0.65%降低至16.56%±0.85%(P值0.05);G2-M期由13.46%±0.2%上升到17.4%±1.12%,差異有統(tǒng)計學意義(P值0.05)。對細胞周期調控因子,PCNA,β-catenin分別進行Western blotting檢測,其結果顯示:與陰性對照組相比,過表達AQP9組人肝癌細胞細胞周期調控因子Cyclin D1,CDK2,CDK4表達下調,P27表達上調,差異均有統(tǒng)計學意義(P均值0.05);同時細胞內PCNA,核內β-catenin,pser675-β-catenin表達均降低,差異同樣有統(tǒng)計學意義(P值均0.05)。同樣,細胞免疫熒光染色及光密度分析結果顯示,與陰性對照組相比,AQP9過表達組PCNA蛋白表達水平下調,差異有統(tǒng)計學意義(P0.05)。AQP9過表達后影響β-catenin在細胞內的分布。結論:肝癌細胞中過表達AQP9基因后可抑制人肝癌細胞SMMC-7721細胞增殖,根據(jù)實驗結果分析,其機制可能是AQP9基因下調細胞核內β-catenin的表達水平,進而抑制Cyclin D1蛋白表達水平,使得肝癌細胞阻滯在G1/S期。
[Abstract]:Objective: to investigate the effect of AQP9 gene overexpression on the proliferation of human hepatocellular carcinoma cell line SMMC-7721 and its possible mechanism. Methods: empty vector lentivirus (LV-Luciferase) and target AQP9 gene overexpression lentivirus (LV-AQP9) were transfected into human hepatocellular carcinoma cell line SMMC-7721, and screened by puromycin. The empty vector stable cell line (SMMC-7721/LV-Luciferase) and AQP9 overexpression stable cell line (SMMC-7721/LV-AQP9) were obtained. Finally, the expression of AQP9 in HCC cells was detected by Western blotting. Experimental groups: negative control group (NC group) was empty vector stable cells, AQP9 overexpression group (AQP9 group) was AQP9 overexpression stable cells. Flow cytometry was used to detect the effect of overexpression of AQP9 gene on human hepatocellular carcinoma cell cycle, and real-time fluorescence quantitative PCR was used to detect the expression of mRNA, a cycle-related regulator in SMMC-7721/LV-Luciferase cells and SMMC-7721/LV-AQP9 cells. The expression of cell cycle related regulatory factor and PCNA,pser675 尾-catenin, 尾-catenin protein was detected by Western blotting. At the same time, the expression of PCNA and 尾-catenin in two groups of HCC cells was observed by immunofluorescence staining. The independent sample t test was used to compare the two groups of data. Results the results of: Western blotting showed that the protein level of AQP9 in AQP9 overexpression group was significantly higher than that in negative control group (P < 0.01). The cell cycle was detected by flow cytometry. The results showed that the overexpression of AQP9 gene affected the distribution of SMMC-7721 cell cycle. The proportion of G0-G1 phase cells in SMMC-7721 cells increased from 52.24% 鹵0.83% to 65.68% 鹵0.63% (P < 0.05). The proportion of cells in S phase decreased from 34.3% 鹵0.65% to 16.56% 鹵0.85% (P < 0.05), and the proportion of cells in G2 鈮，

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