本文選題：CCN3　切入點(diǎn)：肝細(xì)胞肝癌　出處：《南京醫(yī)科大學(xué)》2015年碩士論文

【摘要】：[目的]CCN3(NOV,Nephroblastoma overexpressed gene,腎母細(xì)胞瘤過(guò)表達(dá)基因)是CCN家族的一個(gè)成員,參與多種細(xì)胞功能的發(fā)生和發(fā)展,包括細(xì)胞生長(zhǎng),分化,血管生成,粘附等。目前研究者們發(fā)現(xiàn)CCN3在多種惡性腫瘤中高表達(dá),調(diào)控多種腫瘤的發(fā)生與發(fā)展,如骨肉瘤,食管癌等。然而CCN3在肝細(xì)胞肝癌中的相關(guān)研究尚未報(bào)道。在本研究中,我們對(duì)CCN3在肝癌組織及細(xì)胞中的表達(dá)量進(jìn)行檢測(cè),并進(jìn)行相應(yīng)的功能實(shí)驗(yàn),探討相應(yīng)機(jī)制。[方法]采用實(shí)時(shí)定量聚合酶鏈?zhǔn)椒磻?yīng)(RT-PCR)實(shí)驗(yàn)和蛋白免疫印跡法(Western bolt)檢測(cè)50對(duì)肝癌組織及其癌旁組織中CCN3的表達(dá)水平,統(tǒng)計(jì)分析CCN3的表達(dá)水平和臨床肝癌患者病例資料的相關(guān)性。同時(shí)檢測(cè)肝癌細(xì)胞株MHCC-97H,SMMC-7721,Hep G2,Huh7,MHCC-97L及正常人類肝細(xì)胞L02中CCN3的m RNA表達(dá)水平,構(gòu)建CCN3過(guò)表達(dá)質(zhì)粒,并通過(guò)脂質(zhì)體轉(zhuǎn)染并建立CCN3過(guò)表達(dá)的肝癌細(xì)胞株。利用transwell細(xì)胞侵襲實(shí)驗(yàn)和細(xì)胞劃痕遷移實(shí)驗(yàn)檢測(cè)CCN3對(duì)肝癌細(xì)胞侵襲遷移能力的影響。Western blot檢測(cè)肝癌細(xì)胞株COX-2的表達(dá)變化來(lái)探討相應(yīng)機(jī)制。[結(jié)果]研究發(fā)現(xiàn)CCN3的RNA和蛋白水平在人類肝癌組織中均表達(dá)上調(diào),并且其表達(dá)水平與臨床肝癌患者的轉(zhuǎn)移有一定相關(guān)性。各個(gè)肝癌細(xì)胞株間的CCN3表達(dá)有差異,但均高于人類正常肝細(xì)胞。成功建立了CCN3過(guò)表達(dá)的肝癌細(xì)胞株,并通過(guò)Western bolt驗(yàn)證CCN3蛋白表達(dá)水平。通過(guò)體外細(xì)胞功能實(shí)驗(yàn)transwell細(xì)胞侵襲實(shí)驗(yàn)和細(xì)胞劃痕遷移實(shí)驗(yàn)證實(shí)了CCN3可以上調(diào)COX-2從而促進(jìn)肝癌細(xì)胞的的侵襲與轉(zhuǎn)移。[結(jié)論]CCN3在肝癌組織中表達(dá)異常,并可以通過(guò)COX-2表達(dá)的上調(diào)從而促進(jìn)肝癌的的侵襲與轉(zhuǎn)移,這些研究為CCN3將來(lái)成為肝癌預(yù)后指標(biāo)及肝癌的分子靶向治療提供了新的思路。
[Abstract]:[objective] CCN3 Nov Nephroblastoma overexpressed gene is a member of the CCN family and is involved in the development and development of many cell functions, including cell growth, differentiation, angiogenesis, adhesion and so on.At present, researchers have found that CCN3 is highly expressed in many kinds of malignant tumors and regulates the occurrence and development of many kinds of tumors, such as osteosarcoma, esophageal cancer and so on.However, the study of CCN3 in hepatocellular carcinoma has not been reported.In this study, we detected the expression of CCN3 in liver cancer tissues and cells, and carried out corresponding functional experiments to explore the corresponding mechanism.[methods] Real-time quantitative polymerase chain reaction (RT-PCR) assay and Western blot were used to detect the expression of CCN3 in hepatocellular carcinoma and its adjacent tissues.The correlation between the expression of CCN3 and the clinical data of patients with liver cancer was analyzed statistically.At the same time, the expression level of m RNA of CCN3 in the hepatoma cell line MHCC-97H SMMC-7721 Hep G2H2HH7MHCC-97L and normal human hepatocytes L02 was detected. The CCN3 overexpression plasmid was constructed, and the CCN3 overexpressed hepatoma cell line was transfected and established by liposome.Transwell cell invasion assay and cell scratch migration assay were used to detect the effect of CCN3 on the invasion and migration of hepatoma cells. Western blot was used to detect the expression of COX-2 in hepatoma cells.[results] it was found that the expression of RNA and protein of CCN3 was up-regulated in human liver cancer tissues, and its expression level was correlated with the metastasis of clinical HCC patients.The expression of CCN3 was higher than that of normal hepatocytes.CCN3 overexpression hepatoma cell line was successfully established, and the expression of CCN3 protein was verified by Western bolt.In vitro, transwell cell invasion assay and cell scratch migration assay confirmed that CCN3 can up-regulate COX-2 and promote the invasion and metastasis of hepatoma cells.[conclusion] the abnormal expression of CCN3 in HCC tissues can promote the invasion and metastasis of HCC through the up-regulation of COX-2 expression. These studies provide a new idea for CCN3 to be a prognostic indicator of HCC and a molecular targeted therapy for HCC in the future.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R735.7

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