本文關(guān)鍵詞： 肝癌 SMMC-7721細(xì)胞 巰基氧化酶1 RNA干擾　出處：《廣西醫(yī)科大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：背景與目的:肝癌是我國(guó)常見(jiàn)腫瘤之一,肝癌發(fā)病機(jī)制錯(cuò)綜復(fù)雜,遺傳、飲食、病毒和寄生蟲(chóng)感染等均可與肝癌發(fā)生發(fā)展有關(guān)。隨著分子生物學(xué)發(fā)展,有多種肝癌相關(guān)基因經(jīng)過(guò)相同或不同研究者多次研究,被認(rèn)為可能參與到肝癌的發(fā)病過(guò)程中。Araujo等發(fā)現(xiàn)QSOX1在高分化的神經(jīng)母細(xì)胞瘤腫瘤組織中高表達(dá),且在復(fù)發(fā)率高的病人中也出現(xiàn)高表達(dá)。另外,他們還發(fā)現(xiàn)QSOX1在神經(jīng)母細(xì)胞瘤的分化和侵襲中起重要作用。QSOX1也被認(rèn)為可能參與到乳腺癌、前列腺癌等的發(fā)生發(fā)展過(guò)程。目前關(guān)于QSOX1與肝癌的發(fā)生發(fā)展間關(guān)系的研究報(bào)道甚少,本研究進(jìn)一步驗(yàn)證QSOX1在肝癌細(xì)胞中的表達(dá)及其對(duì)肝癌細(xì)胞生物學(xué)功能的影響,揭示QSOX1對(duì)肝癌細(xì)胞生物學(xué)功能所發(fā)揮的作用,為肝癌早診和治療提供理論基礎(chǔ)和實(shí)驗(yàn)依據(jù)。方法:針對(duì)QSOX1的shRNA慢病毒載體的構(gòu)建由上海吉?jiǎng)P基因技術(shù)有限公司完成。將針對(duì)QSOX1的shRNA慢病毒載體,以及空病毒載體(帶有陰性對(duì)照病毒)各轉(zhuǎn)染至人SMMC-7721肝癌細(xì)胞,即分別為轉(zhuǎn)染組和陰性對(duì)照組�？瞻讓�(duì)照組為不做任何轉(zhuǎn)染的目的細(xì)胞。經(jīng)篩選出穩(wěn)定表達(dá)株后擴(kuò)大培養(yǎng),采用實(shí)時(shí)熒光定量核酸擴(kuò)增(qPCR)檢測(cè)系統(tǒng),及免疫印跡法(Western-blot)分別檢測(cè)細(xì)胞中QSOX1基因mRNA和蛋白質(zhì)在肝癌細(xì)胞中的表達(dá)情況;運(yùn)用cck-8法通過(guò)檢測(cè)細(xì)胞活力以驗(yàn)證細(xì)胞增殖情況;采用細(xì)胞克隆形成實(shí)驗(yàn)測(cè)定細(xì)胞成瘤能力;利用流式細(xì)胞技術(shù)檢測(cè)細(xì)胞周期;運(yùn)用細(xì)胞transwell實(shí)驗(yàn)和劃痕實(shí)驗(yàn)分別測(cè)定細(xì)胞轉(zhuǎn)移和遷移能力。通過(guò)spss17.0統(tǒng)計(jì)學(xué)軟件進(jìn)行統(tǒng)計(jì)分析。結(jié)果:慢病毒轉(zhuǎn)染組和對(duì)照病毒轉(zhuǎn)染組均成功轉(zhuǎn)染;慢病毒轉(zhuǎn)染組qsox1基因mrna和蛋白質(zhì)表達(dá)均分別明顯低于空病毒轉(zhuǎn)染組和空白對(duì)照組;經(jīng)cck-8細(xì)胞增殖實(shí)驗(yàn)發(fā)現(xiàn),慢病毒轉(zhuǎn)染組細(xì)胞增殖相對(duì)于其他對(duì)照組明顯減緩(p0.05);慢病毒轉(zhuǎn)染組細(xì)胞克隆數(shù)量分別明顯低于其他對(duì)照組(p0.05);流式細(xì)胞術(shù)分析表明,慢病毒轉(zhuǎn)染組與空細(xì)胞組比較,慢病毒轉(zhuǎn)染組g1期細(xì)胞所占比例明顯減少(p0.001),而s期明顯增多,在g2/m期無(wú)明顯變化;慢病毒轉(zhuǎn)染組與空病毒轉(zhuǎn)染組細(xì)胞相比,慢病毒轉(zhuǎn)染組g1期細(xì)胞也明顯減少,而s期細(xì)胞無(wú)顯著變化,g2/m期細(xì)胞明顯增多。通過(guò)細(xì)胞劃痕實(shí)驗(yàn)觀察細(xì)胞在0小時(shí)、8小時(shí)、24小時(shí)的遷移情況,結(jié)果顯示,慢病毒轉(zhuǎn)染組遷移能力在24小時(shí)明顯降低(p0.05)。細(xì)胞transwell轉(zhuǎn)移實(shí)驗(yàn)顯示,空細(xì)胞組和空病毒組細(xì)胞轉(zhuǎn)移數(shù)無(wú)明顯差異,而慢病毒轉(zhuǎn)染組細(xì)胞轉(zhuǎn)移數(shù)明顯減少(p0.05)。結(jié)論:通過(guò)慢病毒轉(zhuǎn)染,干擾qsox1基因在smmc-7721細(xì)胞中的表達(dá),能降低smmc-7721肝癌細(xì)胞增殖能力,克隆形成實(shí)驗(yàn)亦表明腫瘤細(xì)胞成瘤能力明顯減弱�；蚯脺p后,根據(jù)各組細(xì)胞周期,結(jié)合細(xì)胞增殖速度的結(jié)果,說(shuō)明細(xì)胞周期可能被阻滯在s期或g2期。這些結(jié)果表明,QSOX1基因在肝癌進(jìn)展中起重要調(diào)控作用,可能作為一個(gè)靶向指標(biāo),指導(dǎo)肝癌的診斷和治療。
[Abstract]:Background & objective: liver cancer is one of the common tumors in China. The pathogenesis of liver cancer is complicated. Heredity, diet, virus and parasite infection are all related to the occurrence and development of liver cancer. With the development of molecular biology. Many genes related to liver cancer have been studied many times by the same or different researchers. It is believed that QSOX1 may be involved in the pathogenesis of HCC. Araujo et al have found that QSOX1 is highly expressed in well-differentiated neuroblastoma. They also found that QSOX1 plays an important role in the differentiation and invasion of neuroblastoma. QSOX1 is also thought to be involved in breast cancer. There are few reports on the relationship between QSOX1 and the occurrence and development of liver cancer. This study further verified the expression of QSOX1 in HCC cells and its effect on the biological function of HCC cells, and revealed the role of QSOX1 in the biological function of HCC cells. Methods: to provide theoretical and experimental basis for early diagnosis and treatment of liver cancer. The construction of shRNA lentivirus vector for QSOX1 was completed by Shanghai Qikai Gene Technology Co., Ltd. ShRNA lentivirus vector for QSOX1 will be constructed. The empty virus vector (with negative control virus) was transfected into human SMMC-7721 hepatoma cells. That is the transfection group and the negative control group respectively. The blank control group is the target cell without any transfection. The stable expression strain was screened out and the culture was expanded. The real-time fluorescence quantitative nucleic acid amplification system was used. The expression of QSOX1 gene mRNA and protein in hepatoma cells was detected by Western blotting. Cck-8 method was used to test cell viability to verify cell proliferation. The tumor-forming ability of the cells was measured by cell clone forming assay. Cell cycle was detected by flow cytometry. Cell metastasis and migration were measured by cell transwell test and scratch test respectively. Statistical analysis was carried out by spss17.0 software. Results:. The lentivirus transfection group and the control virus transfection group were successfully transfected. The expression of qsox1 gene mrna and protein in lentivirus transfection group was significantly lower than that in empty virus transfection group and blank control group. Cck-8 cell proliferation assay showed that the proliferation of lentivirus transfected group was significantly slower than that of other control groups. The number of cell clones in lentivirus transfection group was significantly lower than that in other control groups (P 0.05). The results of flow cytometry showed that the percentage of g1 phase cells in lentivirus transfected group was significantly lower than that in blank cell group, while the percentage of g1 phase cells in lentivirus transfection group was significantly lower than that in blank cell group. There was no significant change at g 2 / m; In lentivirus transfection group, compared with empty virus transfection group, g1 phase cells in lentivirus transfection group were significantly reduced, but no significant changes were found in s phase cells. The cell migration was observed at 0 h, 8 h and 24 h by cell scratch assay. The migration ability of lentivirus transfection group decreased significantly at 24 hours. Cell transwell transfer assay showed that there was no significant difference between empty cell group and empty virus group. Conclusion: lentivirus transfection interferes with the expression of qsox1 gene in smmc-7721 cells. After gene knockout, according to the cell cycle of each group, combined with the results of cell proliferation rate. These results suggest that QSOX1 gene plays an important role in the progression of HCC and may be used as a target marker to guide the diagnosis and treatment of HCC.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R735.7

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