本文選題：肝細(xì)胞癌 + 水通道蛋白9　； 參考：《重慶醫(yī)科大學(xué)》2016年碩士論文

【摘要】：目的:研究水通道蛋白9(Aquaporin9,AQP9)過表達(dá)對肝癌細(xì)胞侵襲轉(zhuǎn)移的影響及其分子機(jī)制研究。方法:(第一部分)實時熒光定量PCR和蛋白免疫印跡法檢測正常肝細(xì)胞株L02和肝癌細(xì)胞株SMMC-7721、HepG2、Hep3B及Huh-7中AQP9表達(dá)水平,并篩選出SMMC-7721作為后續(xù)實驗肝癌細(xì)胞株。將重組慢病毒LV-AQP9-GFP(以空載體LV-GFP作為陰性對照)導(dǎo)入SMMC-7721細(xì)胞中,構(gòu)建AQP9過表達(dá)SMMC-7721/LV-AQP9重組細(xì)胞模型。激光共聚焦顯微鏡下觀察重組慢病毒LV-AQP9-GFP在SMMC-7721細(xì)胞中的轉(zhuǎn)染效率,蛋白質(zhì)印跡法檢測AQP9在SMMC-7721/LV-AQP9重組細(xì)胞模型中過表達(dá)水平。細(xì)胞腫脹實驗檢測AQP9過表達(dá)細(xì)胞模型中AQP9作為水通道功能活化程度。采用劃痕實驗、Transwell侵襲實驗檢驗AQP9過表達(dá)對SMMC-7721細(xì)胞遷移、侵襲的影響。(第二部分)在動物水平,將AQP9過表達(dá)重組肝癌SMMC-7721/LV-AQP9細(xì)胞和SMMC-7721/LV-GFP細(xì)胞接種于裸鼠皮下構(gòu)建裸鼠皮下移植瘤模型,動態(tài)觀察2組裸鼠皮下移植瘤生長的差異。同時,通過尾靜脈注射重組肝癌smmc-7721/lv-aqp9細(xì)胞和smmc-7721/lv-gfp細(xì)胞構(gòu)建肝癌肺轉(zhuǎn)移模型,觀察2組裸鼠肺表面癌結(jié)節(jié)轉(zhuǎn)移數(shù)量,并采用he染色觀察肺組織中轉(zhuǎn)移瘤的情況。(第三部分)蛋白免疫印跡法檢測aqp9過表達(dá)重組肝癌smmc-7721/lv-aqp9細(xì)胞和smmc-7721/lv-gfp細(xì)胞中emt相關(guān)蛋白(e-cadherin、n-cadherin、vimentin),pi3k/akt信號通路相關(guān)蛋白pi3k、akt、p-akt及基質(zhì)金屬蛋白酶mmp2、mmp9的表達(dá)差異。采用免疫組化法檢測移植瘤組織中e-cadherin、n-cadherin、vimentin蛋白表達(dá)。結(jié)果:(第一部分)肝癌細(xì)胞株hep3b不表達(dá)aqp9,而smmc-7721、hepg2、huh-7中aqp9mrna和aqp9蛋白表達(dá)均較正常肝細(xì)胞株l02顯著降低(p值均0.05),其中smmc-7721中aqp9mrna和aqp9蛋白表達(dá)量最低。激光共聚焦顯微鏡下觀察發(fā)現(xiàn),重組慢病毒lv-aqp9在smmc-7721細(xì)胞中的轉(zhuǎn)染效率為90%左右,并通過gfp熒光信號定位發(fā)現(xiàn),aqp9-gfp融合蛋白綠色熒光主要分布在smmc-7721/lv-aqp9細(xì)胞的細(xì)胞膜上。在smmc-7721/lv-aqp9重組細(xì)胞模型(aqp9組)中aqp9蛋白表達(dá)量較空載對照組smmc-7721/lv-gfp細(xì)胞(gfp組)顯著增加(p0.01)。在低滲狀態(tài)下smmc-7721/lv-aqp9重組細(xì)胞(aqp9組)膨脹體積較smmc-7721/lv-gfp細(xì)胞(gfp組)顯著增大(p0.05),而用hgcl2(aqp9阻滯劑)阻滯水通道蛋白活性后,在低滲狀態(tài)下smmc-7721/lv-aqp9重組細(xì)胞(aqp9組)和smmc-7721/lv-gfp細(xì)胞(gfp組)膨脹體積無顯著差異(p=0.783)。劃痕實驗顯示,aqp9組細(xì)胞48h的遷移率(6.38%±1.70%)較gfp組(16.74%±1.40%)下降(p0.01)。transwell實驗結(jié)果顯示,aqp9組48h穿過小室細(xì)胞數(shù)(57±8)較gfp組(95±11)減少(p0.01)。(第二部分)動物水平,在裸鼠皮下移植瘤模型中,重復(fù)測量方差分析2組移植瘤體積提示,aqp9組移植瘤體積顯著小于gfp組(f分組=79.161,p分組=0.000),aqp9組皮下移植瘤生長速度明顯小于gfp組(f分組×?xí)r間=18.481,p分組×?xí)r間=0.002)。在裸鼠肺轉(zhuǎn)移模型中,he染色觀察aqp9組裸鼠肺組織中轉(zhuǎn)移瘤體較gfp組更小,aqp9組裸鼠肺表面癌結(jié)節(jié)轉(zhuǎn)移數(shù)量較gfp組顯著減少(p0.05)。(第三部分)westernblot檢測結(jié)果顯示aqp9過表達(dá)后上皮表型標(biāo)志物(e-cadherin)、pi3k、p-akt蛋白表達(dá)上調(diào)(p值均0.05),間皮表型標(biāo)志物(n-cadherin)表達(dá)下調(diào)(p0.05),而vimentin、akt、mmp9、mmp2蛋白表達(dá)水平較gfp組均無明顯變化(p值均0.05)。同樣,免疫組化及光密度分析結(jié)果顯示,aqp9組移植瘤中e-cadherin蛋白表達(dá)水平較gfp組上調(diào)(p0.05),aqp9組移植瘤中n-cadherin、vimentin蛋白表達(dá)水平較gfp組下調(diào)(p0.01)。結(jié)論:肝癌細(xì)胞中aqp9表達(dá)水平較正常肝細(xì)胞下調(diào)。aqp9過表達(dá)抑制肝癌smmc-7721細(xì)胞侵襲轉(zhuǎn)移及上皮間質(zhì)轉(zhuǎn)化,而pi3k/akt信號通路可能參與這一調(diào)控過程。此外,aqp9過表達(dá)對基質(zhì)金屬蛋白酶MMP9和MMP2表達(dá)無影響。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R735.7

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