抑制ALCAM基因表达对肝癌HepG2细胞增殖和侵袭的影响

发布时间：2018-05-20 10:53

本文选题：ALCAM + 肝细胞癌　；参考：《重庆医科大学》2016年硕士论文

【摘要】：背景活化白细胞黏附分子(Activated Leukocyte Cell Adhesion Molecule,ALCAM)是免疫球蛋白基因超家族成员,主要表达在白细胞和胸腺上皮细胞。ALCAM为I型跨膜蛋白,其通过异嗜性黏附形式(ALCAM-CD6)和同嗜性黏附形式(ALCAM-ALCAM)介导细胞间黏附。既往研究表明ALCAM与肿瘤侵袭与转移密切相关,参与多种肿瘤的发生发展。目的本研究探讨ALCAM表达与肝细胞癌的相关性以及其在肝细胞癌发生发展中的作用。方法1.RT-PCR及免疫组化方法检测ALCAM基因在52例肝细胞癌组织及相应癌旁组织中的表达,分析ALCAM的表达与肝细胞癌临床病理特征的相关性。2.利用p SES-HUS系统构建特异性ALCAM基因的si RNA腺病毒载体p SES-ALCAM-si RNA,测序证实基因序列正确后包装成腺病毒si RNA并转染肝癌细胞株HepG2,应用RT-PCR和Western blot检测si RNA对肝癌细胞HepG2中ALCAM基因的干扰效果。3.MTT及流式细胞技术检测干扰ALCAM表达后HepG2细胞增殖及凋亡情况;Transwell实验检测HepG2迁移、侵袭能力改变。结果1.RT-PCR结果显示与癌旁正常组织相比较,ALCAM在肝细胞癌组织中的高表达(P0.01)。免疫组化与临床资料统计分析进一步证实ALCAM与肿瘤分化程度(P=0.024),肝内转移(P=0.019)呈负相关,肝癌组织中ALCAM异常低表达提示肿瘤分化程度低,易转移;而与患者性别,年龄,术前AFP值,肿瘤直径及临床TNM分期无明显相关性(P0.05)。2.构建腺病毒p SES-ALCAM-si RNA并转染HepG2,RT-PCR和Western blot显示ALCAM在基因及蛋白表达水平均被明显抑制。3.通过si RNA干扰肝癌细胞株HepG2中ALCAM基因表达,MTT及流式细胞术检测表明干扰ALCAM表达后肝癌细胞株HepG2增殖受到抑制(P0.05),凋亡率增加(P0.05);而Transwell实验发现ALCAM干扰后HepG2迁移、侵袭能力上升(P0.05)。结论1.ALCAM在肝细胞癌组织中的高表达,并且与肝癌分化程度,肝内转移有相关性。这些数据表明ALCAM可能作为一个新指标诊断肝癌并且提示肿瘤分化程度及转移风险。2.腺病毒si RNA成功构建并可特异性干扰HepG2肝癌细胞株ALCAM基因表达,为后继实验进一步揭示ALCAM与肝癌的关系做准备。3.干扰ALCAM基因可抑制肝癌细胞HepG2增殖,促进其凋亡;然而HepG2肝癌细胞在ALCAM基因表达下调后体外迁移和侵袭能力增强,这意味着ALCAM基因在肝癌发生发展中有着多方面的作用,有重要研究价值。
[Abstract]:The background activated leukocyte adhesion molecule (Activated Leukocyte Cell Adhesion Molecule, ALCAM) is a member of the immunoglobulin gene superfamily, mainly expressed in the leukocyte and thymic epithelial cells.ALCAM as a I type transmembrane protein, which mediates the intercellular adhesion through the heterophil adhesion form (ALCAM-CD6) and the isophil adhesion form (ALCAM-ALCAM). The study shows that ALCAM is closely related to tumor invasion and metastasis and participates in the occurrence and development of various tumors. Objective to investigate the relationship between ALCAM expression and hepatocellular carcinoma and its role in the development of hepatocellular carcinoma. Methods 1.RT-PCR and immunohistochemical methods were used to detect the ALCAM gene in 52 hepatocellular carcinoma tissues and corresponding Para cancerous tissues. The correlation between the expression of ALCAM and the clinicopathological features of hepatocellular carcinoma.2. using the P SES-HUS system to construct the Si RNA adenovirus vector p SES-ALCAM-si RNA of the specific ALCAM gene, and the sequence of DNA sequencing confirmed that the gene sequence was correctly packaged into adenovirus Si RNA and transfected to the liver cancer cell line. Interference effect on ALCAM gene in hepatoma cell HepG2.3.MTT and flow cytometry to detect the proliferation and apoptosis of HepG2 cells after interference of ALCAM expression; Transwell test was used to detect HepG2 migration and invasion ability. Results 1.RT-PCR results showed high expression of ALCAM in hepatocellular carcinoma tissues compared with normal tissues adjacent to cancer (P0.01). The statistical analysis of histochemical and clinical data further confirmed that ALCAM was negatively correlated with the degree of tumor differentiation (P=0.024) and intrahepatic metastasis (P=0.019). The abnormal low expression of ALCAM in the hepatocellular carcinoma showed that the tumor differentiation was low and easy to transfer, but there was no significant correlation between the sex, age, preoperative AFP, the diameter of the tumor and the clinical TNM staging (P0.05).2. construction gland. The virus P SES-ALCAM-si RNA was transfected with HepG2, RT-PCR and Western blot showed that the expression level of ALCAM in the gene and protein expression was obviously inhibited by Si RNA interference in the hepatocellular carcinoma cell line, and the proliferation of the hepatocellular carcinoma cell line was inhibited and the apoptosis rate increased after the flow cytometry. 05); and the Transwell experiment found that HepG2 migration and invasion ability increased after ALCAM interference (P0.05). Conclusion 1.ALCAM is highly expressed in hepatocellular carcinoma and is associated with the degree of differentiation and intrahepatic metastasis of HCC. These data suggest that ALCAM may be a new marker for the diagnosis of liver cancer and that the degree of differentiation and metastasis risk of.2. adenosis may be suggested. Si RNA was successfully constructed and specifically interfered with the expression of ALCAM gene in HepG2 cell line, which further revealed the relationship between ALCAM and HCC by preparing.3. interference ALCAM gene to inhibit the proliferation of hepatoma cell HepG2 and promote its apoptosis. However, the migration and invasion ability of HepG2 hepatoma cells was enhanced in vitro after the expression of ALCAM gene was down. This means that ALCAM gene plays an important role in the occurrence and development of liver cancer and has important research value.
【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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