本文選題：肝癌 + ADAM10　； 參考：《吉林大學(xué)》2015年博士論文

【摘要】：肝癌是最常見的惡性腫瘤之一，全球發(fā)病率居惡性腫瘤第5位，死亡率居第9位，據(jù)估計每年肝癌可導(dǎo)致50余萬人死亡。目前肝癌的治療效果不佳，迫切需要尋求新的治療手段提高療效。研究肝癌發(fā)生的分子機制，對尋找敏感性和特異性強的分子標記及開發(fā)新型治療手段具有重要意義。ADAMs(Adisintegrin and metalloprotease)家族是一類同時具有解整合素和金屬蛋白酶兩種結(jié)構(gòu)域的I型跨膜蛋白家族，廣泛存在于各種組織細胞中。ADAM10是ADAMs家族的重要成員，研究表明，ADAM10在多種腫瘤中高表達，且促進腫瘤細胞的增殖、侵襲和遷移，但在肝癌中相關(guān)研究較少。本研究主要開展以下三部分試驗：第一部分利用免疫組化和熒光定量PCR技術(shù)檢測了ADAM10表達量與肝癌臨床病理參數(shù)的關(guān)系，同時評價了ADAM10在肝癌上的預(yù)后價值。第二部分利用一系列體內(nèi)和體外實驗研究了沉默ADAM10基因表達對肝癌細胞的增殖、遷移和侵襲及腫瘤生長的影響。第三部分是在前兩部分的基礎(chǔ)上，利用基因共表達技術(shù)進一步探討了沉默ADAM10和過表達GRIM-19聯(lián)合應(yīng)用對肝癌細胞增殖、細胞周期、細胞遷移、侵襲和凋亡及腫瘤生長的影響。 主要研究結(jié)果如下： 1．ADAM10表達與肝癌臨床病理參數(shù)的關(guān)系 收集98例肝癌患者組織樣本，利用免疫組化和熒光定量PCR技術(shù)從mRNA和蛋白水平上研究了ADAM10表達與肝臟臨床病理參數(shù)的關(guān)系。在98例肝癌患者中，74(75.6%)人表現(xiàn)ADAM10陽性結(jié)果，24人表現(xiàn)ADAM10陰性結(jié)果。腫瘤大于5cm組免疫組化陽性比例達到90.63%；腫瘤轉(zhuǎn)移組免疫組化陽性比例達100%；ADAM10免疫染色結(jié)果與患者的性別和年齡相關(guān)性不顯著（P＞0.05）。在TNM分期系統(tǒng)中，TNM分級對ADAM10免疫染色結(jié)果影響顯著（P＜0.05），腫瘤變異對ADAM10免疫染色結(jié)果有顯著影響（P＜0.01）。直徑大于5cm的腫瘤，其ADAM10免疫染色陽性顯著高于直徑小于5cm的腫瘤（P＜0.05）。熒光定量PCR結(jié)果表明，ADAM10mRNA在肝癌組織中表達水平顯著高于癌旁組織（P＜0.01)。ADAM10陽性的患者生存指數(shù)明顯低于ADAM10陰性的肝癌患者(P＜0.01），上述研究結(jié)果提示，ADAM10與肝癌密切相關(guān)。 2.沉默ADAM10基因?qū)Ω伟┘毎鲋场⑦w移和侵襲及腫瘤生長的影響 構(gòu)建了ADAM10-RNAi質(zhì)粒，成功轉(zhuǎn)染到肝癌細胞HepG2中。MTT和缺口末端標記實驗結(jié)果表明，沉默ADAM10基因可顯著抑制肝癌細胞的增殖和促進其凋亡(P＜0.01)；western blot實驗表明，沉默ADAM10基因可顯著下調(diào)肝癌細胞中凋亡抑制基因Survivin和Bcl-2的表達水平和AKT和PI3K的磷酸化(P＜0.01)；細胞劃痕實驗和transwell小室細胞體外侵襲實驗結(jié)果表明，沉默ADAM10基因可顯著抑制肝癌細胞的遷移和侵襲,體內(nèi)成瘤實驗也表明，沉默ADAM10基因可顯著抑制腫瘤的生長(P＜0.01)。 3.共表達ADAM10-specific siRNA和GRIM-19對肝癌的影響 構(gòu)建了pSi-ADAM10, pGRIM-19，pSi-ADAM10-GRIM-19質(zhì)粒，并分別轉(zhuǎn)染到肝癌細胞系HepG2中，熒光定量PCR和western blot實驗證明質(zhì)粒轉(zhuǎn)染成功。與pSi-ADAM10和pGRIM-19處理組相比，共表達GRIM-19和ADAM10特異性ShRNA可顯著降低HepG2細胞的增殖能力和S期細胞百分比，提高G1期細胞百分比，上調(diào)細胞周期蛋白P21和下調(diào)細胞周期蛋白cyclinD1的表達。共表達ADAM10-specific siRNA和GRIM-19可進一步促進肝癌細胞凋亡，且顯著上調(diào)caspase-3, caspase-8和caspases-9的活性，而下調(diào)Bcl-2和Survivin的表達。共表達ADAM10-specific siRNA和GRIM-19處理可顯著提高對HepG2細胞遷移和侵襲的抑制，并抑制侵襲相關(guān)蛋白MMP-2和MMP-9的表達。體內(nèi)成瘤實驗結(jié)果表明，共表達ADAM10-specific siRNA和GRIM-19可導(dǎo)致小鼠腫瘤內(nèi)ADAM10基因表達顯著下調(diào)和GRIM-19基因表達顯著上調(diào)，且誘導(dǎo)細胞凋亡和移植腫瘤生長的效果最好(P0.05)。研究結(jié)果提示，與單一治療相比，在肝癌細胞中共表達ADAM10-specific shRNA和GRIM-19可在體內(nèi)外顯著抑制腫瘤細胞的增殖、遷移和侵襲以及腫瘤的生長。 綜合上述結(jié)果，ADAM10在肝癌中高表達，沉默ADAM10可促進肝癌細胞凋亡，，抑制肝癌細胞肝癌細胞的增殖、遷移和侵襲以及腫瘤的生長，共表達ADAM10沉默質(zhì)粒和GRIM-19過表達質(zhì)�？蛇M一步抑制肝癌細胞的增殖、遷移、侵襲以及腫瘤的生長。
[Abstract]:Liver cancer is one of the most common malignant tumors. The global incidence is fifth of the malignant tumors, and the mortality rate is ninth. It is estimated that liver cancer can cause more than 50 million people to die each year. The treatment effect of liver cancer is not good. It is urgent to seek new treatment methods to improve the curative effect. .ADAMs (Adisintegrin and metalloprotease) family is a class of I type transmembrane protein family with two domains of integrin and metalloproteinase..ADAM10 is an important member of the ADAMs family in a variety of tissue cells. The study shows that ADAM10 is in a variety of swelling. The tumor is highly expressed and promotes the proliferation, invasion and migration of tumor cells, but there are few related studies in the liver cancer. This study mainly carried out the following three parts: the first part, the relationship between the expression of ADAM10 and the clinicopathological parameters of liver cancer was detected by immunohistochemistry and fluorescence quantitative PCR, and the prognosis of ADAM10 in the liver cancer was evaluated. The second part uses a series of in vivo and in vitro experiments to investigate the effects of silent ADAM10 gene expression on the proliferation, migration, invasion and tumor growth of hepatoma cells. The third part is on the basis of the first two parts, using gene co expression technology to further explore the combination of silencing ADAM10 and overexpressed GRIM-19 for hepatoma cells. Proliferation, cell cycle, cell migration, invasion and apoptosis, and tumor growth.
The main results are as follows:
Relationship between 1.ADAM10 expression and clinicopathological parameters of hepatocellular carcinoma
The tissue samples of 98 patients with liver cancer were collected. The relationship between the expression of ADAM10 and the clinicopathological parameters of the liver was studied by immunohistochemistry and fluorescence quantitative PCR. In 98 cases of liver cancer, 74 (75.6%) showed ADAM10 positive results and 24 showed ADAM10 negative results. The positive proportion of immunohistochemistry in group 5cm was greater than that of 5cm group. To 90.63%, the positive proportion of immuno histochemistry in the tumor metastasis group was 100%, and the correlation between the results of ADAM10 immunization and the sex and age of the patients was not significant (P > 0.05). In the TNM staging system, the TNM grading had significant influence on the results of ADAM10 immunostaining (P < 0.05), and the tumor variation had a significant influence on the results of ADAM10 immunization (P < 0.01). The diameter was greater than that of the ADAM10. The ADAM10 immunoreaction of 5cm was significantly higher than that of tumor smaller than 5cm (P < 0.05). The fluorescence quantitative PCR showed that the expression level of ADAM10mRNA in the liver cancer tissues was significantly higher than that of the paracancerous tissues (P < 0.01).ADAM10 positive patients were significantly lower than those of the negative liver cancer patients (P < 0.01). These findings suggest A. DAM10 is closely related to liver cancer.
2. effects of silencing ADAM10 gene on proliferation, migration and invasion, and tumor growth of hepatocellular carcinoma cells
The ADAM10-RNAi plasmid was constructed. The results of successful transfection of.MTT and nick end labeling in HepG2 cells showed that the silence of ADAM10 gene could significantly inhibit the proliferation of hepatoma cells and promote its apoptosis (P < 0.01). The Western blot experiment showed that the silence ADAM10 gene could significantly reduce the apoptosis inhibiting gene Survivin and Bcl-2 in the hepatoma cells. The expression level and phosphorylation of AKT and PI3K (P < 0.01); the results of cell scratch test and in vitro invasion test of Transwell cell cells showed that silent ADAM10 gene could significantly inhibit the migration and invasion of hepatoma cells. In vivo tumorigenesis experiment also showed that silent ADAM10 gene could significantly inhibit the growth of tumor (P < 0.01).
3. the effect of co expression of ADAM10-specific siRNA and GRIM-19 on liver cancer
PSi-ADAM10, pGRIM-19 and pSi-ADAM10-GRIM-19 plasmids were constructed and transfected into the hepatoma cell line HepG2 respectively. The fluorescence quantitative PCR and Western blot experiments proved that the plasmid was transfected successfully. Compared with pSi-ADAM10 and pGRIM-19 treatment group, co expression of GRIM-19 and ADAM10 specific ShRNA significantly reduced the proliferation ability of the cells and the percentage of cell percent. Ratio, increase the percentage of G1 cells, up regulation of cyclin P21 and down regulation of the expression of cyclin cyclinD1. Co expression of ADAM10-specific siRNA and GRIM-19 can further promote the apoptosis of hepatoma cells, and significantly up-regulation the activity of Caspase-3, Caspase-8 and caspases-9, and the expression of Bcl-2 and Survivin in the lower modulation. IRNA and GRIM-19 treatments could significantly increase the inhibition of migration and invasion of HepG2 cells and inhibit the expression of invasion related proteins MMP-2 and MMP-9. In vivo tumorigenesis experiment results showed that co expression of ADAM10-specific siRNA and GRIM-19 could lead to a significant downregulation of ADAM10 gene expression in tumor and significantly up-regulated GRIM-19 gene expression in mice, and induce cells to induce cells. The results of apoptotic and transplanted tumor growth are best (P0.05). The results suggest that the expression of ADAM10-specific shRNA and GRIM-19 in hepatoma cells can significantly inhibit the proliferation, migration and invasion of tumor cells and the growth of tumor cells in vivo and in vivo.
Combined with the above results, ADAM10 is highly expressed in liver cancer. Silencing ADAM10 can promote the apoptosis of hepatoma cells, inhibit the proliferation, migration and invasion of HCC cells, and the growth of tumor. Co expression of ADAM10 silencing plasmids and GRIM-19 overexpressed plasmids can further inhibit the proliferation, migration, invasion and tumor growth of hepatoma cells.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R735.7

【參考文獻】

相關(guān)期刊論文 前4條

1 孫雯雯;王哲海;;表皮生長因子受體單克隆抗體療效預(yù)測因素的研究進展[J];中華臨床醫(yī)師雜志(電子版);2013年18期

2 朱艷艷;俞建平;趙瑞力;;喉鱗癌中扭曲基因、上皮型鈣黏附蛋白和神經(jīng)型鈣黏附蛋白基因表達及相關(guān)研究[J];中國眼耳鼻喉科雜志;2014年01期

3 李云;蘇秀蘭;;表皮生長因子受體與乳腺癌關(guān)聯(lián)研究進展[J];中華臨床醫(yī)師雜志(電子版);2013年23期

4 Hong Sun;Man-Sheng Zhu;Wen-Rui Wu;Xiang-De Shi;Lei-Bo Xu;;Role of anti-angiogenesis therapy in the management of hepatocellular carcinoma: The jury is still out[J];World Journal of Hepatology;2014年12期

本文編號：1924523