本文選題：SMOC2 + 原發(fā)性肝癌��； 參考：《廣東藥科大學(xué)》2017年碩士論文

【摘要】：研究背景及目的肝癌是全球范圍內(nèi)高發(fā)的一類(lèi)惡性腫瘤,其中大約有90%的肝癌為原發(fā)性肝癌(HCC)。據(jù)全球癌癥調(diào)查資料顯示,肝癌發(fā)病率在所有腫瘤中排列第五,因肝癌而死亡的人數(shù)在所有惡性腫瘤中高居第三位。在中國(guó),肝癌更是威脅我國(guó)公民生命健康的主要腫瘤之一。據(jù)中國(guó)2015年癌癥統(tǒng)計(jì)數(shù)據(jù)顯示,肝癌已成為我國(guó)實(shí)體瘤中第四位高發(fā)病率和第三位死因的癌癥。目前研究發(fā)現(xiàn)原發(fā)性肝癌的分子發(fā)病機(jī)制包括多基因及表觀遺傳的改變、染色體畸變、基因突變等,但關(guān)于HCC具體發(fā)病機(jī)制仍不明確。因此,探索與HCC發(fā)生發(fā)展相關(guān)的潛在基因并研究其在HCC發(fā)生進(jìn)展過(guò)程中的作用,對(duì)于人們更好地了解HCC發(fā)生發(fā)展的分子機(jī)制具有重要意義。模塊化鈣結(jié)合蛋白2(SMOC2)屬于SPARC蛋白家族成員之一。有研究表明,SMOC2參與調(diào)節(jié)多種細(xì)胞生物學(xué)功能,如細(xì)胞周期、細(xì)胞增殖、遷移等,但關(guān)于其在腫瘤中作用的相關(guān)研究仍較少。目前,有研究利用微陣列分析技術(shù)發(fā)現(xiàn)SMOC2在多種腫瘤中呈現(xiàn)低表達(dá),包括卵巢癌、胰腺癌等,由此可推斷SMOC2在多數(shù)腫瘤中可能為抑癌基因,但目前仍未見(jiàn)關(guān)于SMOC2在HCC中的表達(dá)、預(yù)防診治意義及作用的研究報(bào)道。本研究欲通過(guò)檢測(cè)SMOC2在HCC中的表達(dá)情況,結(jié)合肝癌患者病歷及隨訪資料來(lái)探討SMOC2表達(dá)與HCC患者臨床病理參數(shù)及預(yù)后的關(guān)系,并進(jìn)一步探究SMOC2對(duì)HCC發(fā)展過(guò)程中的細(xì)胞生物學(xué)功能的影響,從而來(lái)明確SMOC2在HCC中的臨床診治、預(yù)防意義及生物學(xué)功能。研究方法實(shí)驗(yàn)方法:采用熒光定量RT-PCR技術(shù)檢測(cè)SMOC2在肝癌組織中的轉(zhuǎn)錄水平,利用western blotting和免疫組織化學(xué)技術(shù)檢測(cè)SMOC2蛋白在肝癌標(biāo)本和肝癌細(xì)胞中的表達(dá),探討SMOC2與肝癌患者腫瘤相關(guān)指標(biāo)的關(guān)系,并分析其表達(dá)情況與患者預(yù)后的關(guān)系。通過(guò)慢病毒轉(zhuǎn)染,構(gòu)建過(guò)表達(dá)SMOC2的HepG2和Bel-7402細(xì)胞穩(wěn)株,研究SMOC2過(guò)表達(dá)后對(duì)肝癌細(xì)胞的生長(zhǎng)活力、運(yùn)動(dòng)能力等的影響。統(tǒng)計(jì)方法:肝癌組織和癌旁對(duì)照組織中的SMOC2在轉(zhuǎn)錄水平、蛋白水平上的差異性比較及相關(guān)細(xì)胞生物學(xué)功能指標(biāo)的差異性比較使用符號(hào)秩和檢驗(yàn);卡方檢驗(yàn)用于分析SMOC2表達(dá)水平與HCC患者臨床病理參數(shù)間關(guān)系;生存分析用于比較SMOC2高表達(dá)組與低表達(dá)組之間總體生存期及無(wú)病進(jìn)展生存期的差異;單因素、多因素Cox比例風(fēng)險(xiǎn)回歸分析用于研究SMOC2表達(dá)及臨床病理指標(biāo)對(duì)HCC患者總體生存期及無(wú)病進(jìn)展生存期的影響。研究結(jié)果1、肝癌標(biāo)本中SMOC2 mRNA轉(zhuǎn)錄水平(n=40,P0.0001)和蛋白表達(dá)水平(n=40,P=0.0101)明顯低于癌旁對(duì)照組織;較正常肝細(xì)胞系L02,SMOC2在肝癌細(xì)胞系中(Bel-7402,HepG2,QGY-7701,SK-Hep1,SMMC-7721)的表達(dá)水平下調(diào)。2、免疫組化結(jié)果顯示SMOC2免疫陽(yáng)性染色主要處于胞漿中;卡方檢驗(yàn)結(jié)果表明SMOC2表達(dá)水平的高低在腫瘤大小(χ~2=9.156,P=0.002)、腫瘤數(shù)量(χ~2=4.896,P=0.027)、TNM分期(χ~2=16.356,P0.001)及是否發(fā)生轉(zhuǎn)移(χ~2=6.141,P=0.013)中的分布情況存在著差異;生存分析發(fā)現(xiàn)SMOC2高表達(dá)組的總生存期(OS,P0.001)和無(wú)病進(jìn)展生存期(DFS,P=0.001)明顯長(zhǎng)于低表達(dá)組;多因素Cox回歸分析顯示SMOC2表達(dá)水平是影響患者預(yù)后的獨(dú)立因素。3、亞組分析結(jié)果顯示在AFP≤25ng/ml或400ng/ml、腫瘤長(zhǎng)徑5cm或≥5cm及TNM分期為Ⅰ-Ⅱ的HCC患者中,SMOC2高表達(dá)組總生存率及無(wú)病進(jìn)展生存率均比低表達(dá)組高(P0.05)。4、體外實(shí)驗(yàn)顯示:肝癌細(xì)胞的SMOC2表達(dá)水平上調(diào)后,細(xì)胞的增殖、克隆形成以及遷移侵襲能力受到抑制,并且會(huì)阻礙肝癌細(xì)胞周期進(jìn)程,誘導(dǎo)細(xì)胞周期阻滯于G0/G1期。5、小鼠成瘤實(shí)驗(yàn)顯示皮下注射過(guò)表達(dá)SMOC2肝癌細(xì)胞的裸鼠腫瘤生長(zhǎng)速度明顯低于對(duì)照組(P0.05),皮下注射過(guò)表達(dá)SMOC2肝癌細(xì)胞的裸鼠長(zhǎng)出的腫瘤體積及重量也明顯小于對(duì)照組(P0.05)。結(jié)論與HCC發(fā)展相關(guān)的細(xì)胞生物學(xué)進(jìn)程中,SMOC2發(fā)揮著抑癌基因的作用。它可作為預(yù)測(cè)HCC患者預(yù)后的有效指標(biāo),也可能作為HCC分子靶向治療的潛在靶點(diǎn)。此外,肝癌患者SMOC2的表達(dá)情況還可作為為患者制定三級(jí)預(yù)防方案的參考依據(jù)。
[Abstract]:Background and objective liver cancer is a worldwide type of high incidence of malignant tumors, of which about 90% of the liver cancer is primary liver cancer (HCC). According to the global cancer survey data, the incidence of liver cancer is fifth in all tumors. The number of deaths caused by liver cancer is third in all malignant tumors. In China, liver cancer is a threat. According to China's 2015 cancer statistics, liver cancer has become one of the fourth high incidence and third causes of death in China's solid tumors. The molecular pathogenesis of primary liver cancer includes polygene and epigenetic changes, chromosomal aberrations and gene mutations. The specific pathogenesis of HCC is still unclear. Therefore, exploring the potential genes associated with the development of HCC and studying its role in the progress of HCC are of great significance for people to better understand the molecular mechanism of HCC development. Modularized calcium binding protein 2 (SMOC2) is one of the members of the SPARC family. SMOC2 is involved in regulating a variety of cellular biological functions, such as cell cycle, cell proliferation, migration, etc. but there are still few related studies on its role in the tumor. At present, microarray analysis techniques have been used to detect the low expression of SMOC2 in a variety of tumors, including nesting and pancreatic cancer, and thus infer that SMOC2 is in most of the tumors. It may be a tumor suppressor gene, but there is still no study on the expression of SMOC2 in HCC, the significance and the role of prevention and treatment. This study intends to explore the relationship between the expression of SMOC2 in HCC, the case history and the follow-up data of the patients with liver cancer, to explore the relationship between the expression of SMOC2 and the clinical pathophysiological parameters and prognosis of the patients with HCC, and to further explore the SMOC2 The effect of HCC on the cell biological function in the process of development, so as to clarify the clinical diagnosis, the preventive significance and the biological function of SMOC2 in HCC. The method of research is to detect the transcriptional level of SMOC2 in the liver cancer tissues by fluorescence quantitative RT-PCR technique, and to detect SMOC2 protein by Western blotting and immunohistochemical technique. The relationship between SMOC2 and the tumor related indexes of HCC patients and the relationship between the expression of HCC and the prognosis of the patients were analyzed. The expression of HepG2 and Bel-7402 cells expressing SMOC2 were constructed by lentivirus transfection. The effects of SMOC2 overexpression on the growth activity and exercise ability of hepatoma cells were studied. Statistical methods: the difference in the transcriptional level, the protein level and the related cell biological function indexes of the SMOC2 in the liver cancer tissue and the paracancerous control tissue were compared with the sign rank sum test, and the chi square test was used to analyze the relationship between the SMOC2 expression level and the clinical pathophysiological parameters of the HCC patients; the survival analysis was used to compare the high SMOC2 level. The difference between the overall life period and the progression free survival period between the expression group and the low expression group; the single factor, multi factor Cox proportional risk regression analysis was used to study the effect of SMOC2 expression and clinicopathological indexes on the overall survival and the progression free survival of HCC patients. Results 1, the SMOC2 mRNA transcriptional level (n=40, P0.0001) in the specimens of liver cancer. The expression level of protein (n=40, P=0.0101) was significantly lower than that of the paracancerous control tissue, and the expression level of SMOC2 in the hepatocellular carcinoma cell line (Bel-7402, HepG2, QGY-7701, SK-Hep1, SMMC-7721) was down regulated by L02, and the immunohistochemical results showed that SMOC2 immunoreactive staining was mainly in the cytoplasm. The chi square test showed that the level of SMOC2 expression was high. There were differences in the tumor size (chi ~2=9.156, P=0.002), the number of tumor (x ~2=4.896, P=0.027), TNM staging (chi ~2=16.356, P0.001) and the occurrence of metastasis (chi ~2=6.141, P=0.013). The survival analysis found that the total survival time (OS, P0.001) and the progression free survival period of the high expression group of SMOC2 were significantly longer than those of low expression groups. Multiple factor Cox regression analysis showed that the expression of SMOC2 was an independent factor affecting the prognosis of the patients.3. The results of subgroup analysis showed that in AFP < 25ng/ml or 400ng/ml, the total survival rate and the progression free survival rate of SMOC2 high expression group were higher than those of low expression group (P0.05) in the AFP < 25ng/ml or 400ng/ml. The proliferation, clone formation and migration and invasion ability of hepatoma cells were inhibited, and the cell cycle progression was inhibited and the cell cycle was blocked at G0/G1.5. The tumor growth rate of nude mice subcutaneously expressed by subcutaneous injection of SMOC2 liver cancer cells was significantly lower than that of the control group, after the SMOC2 expression level of hepatoma cells was up-regulated. (P0.05) the tumor size and weight of the nude mice expressed by subcutaneous injection of SMOC2 hepatoma cells were also significantly smaller than those of the control group (P0.05). Conclusion SMOC2 plays a role in the tumor suppressor gene in the process of cell biology related to the development of HCC. It can be used as an effective indicator for predicting the prognosis of HCC patients, and may also be the potential for targeting therapy of HCC molecules. In addition, the expression of SMOC2 in patients with liver cancer can also be used as a reference for patients to develop a three level prevention plan.

【學(xué)位授予單位】：廣東藥科大學(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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本文編號(hào)：1806188