摘要
原发性肝癌是人类最常见的恶性肿瘤之一,其死亡率高,在恶性肿瘤中列第三位。原发性肝癌主要包括肝细胞癌和胆管细胞癌,其中90%为肝细胞癌,胆管细胞癌仅占7%~10%。两者都具有起病隐匿、进展快、侵袭性强、易转移、预后差的特点,且胆管细胞癌的预后更差。本课题即以肝细胞癌和胆管细胞癌为研究对象,分成两部分内容进行研究。第一部分通过回顾性调查,分析骨桥蛋白在肝细胞癌组织中的表达情况,并结合患者的临床资料及长期随访结果,分析肝癌组织中骨桥蛋白表达与肝癌肝移植术后肿瘤复发和转移的相关性,探讨骨桥蛋白表达在预测肿瘤复发与转移的临床意义。同时还就骨桥蛋白表达在肝癌肝移植受体纳入标准中的意义进行了分析,期待制定更加适合的肝癌肝移植纳入标准,以提高肝细胞癌患者的疗效。本研究的第二部分是关于胆管细胞癌反义基因治疗的体外试验,采用脂质体包裹转染技术,用Mcl-1 反义寡核苷酸转染胆管细胞癌细胞株,同时加用化疗药物治疗胆管细胞癌,探讨通过降低凋亡抑制基因Mcl-1 表达来增强胆管细胞癌对化疗药物敏感性的可能性,从而为应用反义技术治疗难治性胆管细胞癌提供重要依据。
Part 1: Significance of Osteopontin Overexpression in Hepatocellular Carcinoma with Liver Transplantation
Object: Hepatocellular carcinoma (HCC) is the most common primary liver malignancy worldwide, being responsible for more than 1 million deaths annually. HCC occurs mainly in chronic liver diseases and progression often indicates vascular invasion and intrahepatic metastasis. The extremely poor prognosis of HCC is largely the result of a high rate of recurrence after surgery and HCC often leads to a grave prognosis. Orthotopic liver transplantation (OLT) is the only potentially curable treatment in selected cases of small tumors. It is important for tumor control to identify the factors that predispose patients to tumor recurrence. A recent report showed that osteopontin mRNA overexpression predicts a higher early recurrence rate, and osteopontin expression could serve as an unfavorable prognostic factor and a useful marker for predicting early recurrence in early-stage HCC. However, osteopontin expression has rarely been mentioned and its role is not clearly understood in patients with HCC undergoing liver transplantation. We wanted to examine whether osteopontin expression in HCC-tissue would be a prognostic factor for prediction of HCC recurrence a retrospective analysis of all HCC’s that underwent OLT in Vienna. We also wanted to assess whether
addition of OPN to the Mazzaferro-criteria would be of help for HCC patient selection for OLT. Methods: The expression of osteopontin was investigated immunohistochemically in surgically resected specimens from 125 HCC patients underwent OLT. We examined the correlation between the expression of osteopontin and the clinicopathological features, early tumor recurrence, prognosis and overall survival, including Mazzaferro-criteria, in patients with HCC underwent OLT. Results: We detected the positive expression of osteopontin in 39 of 125 (31.2%--tumor margin part) and in 27 of 125 (21.6%--tumor center part) of the primary HCCs. The percentage of osteopontin-positive cancer cells were 17.56±25.45/HPF (mean±SD) and 14.44±24.69/HPF, in tumor margin part and center part, respectively. It seemed that osteopontin expression in tumor center part had more significance. Osteopontin positive expression was correlated with tumor recurrence and metastasis, and the RR=0.217 (P=0.015). Outside Mazzaferro-criteria the correlation is more tight (RR=0.297, P=0.007). 7 parameters were found to correlate with HCC recurrence, which included explant pTNM stage, the lobar distribution of the tumors, multifocality, tumor size, pathological vascular involvement and with cirrhosis. With survival analysis, the overall survival rates for 1-year, 2-year, 3-year and 5-year were 73%、60%、48% and 42%, respectively. The median survival time was 34.72 months. Overall survival was significantly lower among the patients with a negative expression of osteopontin than it was among those with a positive expression of osteopontin (P<0.05). The survival rates for 1-year, 2-year, 3-year and 5-year of patients with osteopontin negative expression were 77%、67%、54% and 48%, respectively. The median survival time was 53 months. Those of patients with positive expression were 59%、33%、26% and 22%, respectively. The median survival time was 19.5 months. Outside Mazzaferro-criteria, the patients with osteopontin negative expression had better prognosis than those with positive expression (P=0.002). The survival rates for 1-year, 2-year, 3-year and 5-year of patients outside Mazzaferro-criteria with osteopontin negative expression were 73%、63%、48% and 39%, respectively. The median survival time was 38.27 months. Those of patients with positive expression were 53%、21%、11% and 5%, respectively. The median survival time was 16.5 months. Conclusion: We detected the positive expression of osteopontin in HCC patients underwent OLT. There was different staining between
tumor center part and margin part and it seemed that osteopontin expression in tumor center part had more significance. Osteopontin positive expression was correlated with tumor recurrence and metastasis, especially in patients outside Mazzaferro criteria. It is important to detect osteopontin in these patients and osteopontin expression has prognostic significance. 7 parameters were found to correlate with HCC recurrence and metastasis. Explant pTNM stage and pathological vascular invasion had more intensive correlation. Survival analysis indicated that osteopontin staining had prognostic significance. So osteopontin was regarded as marker for tumor recurrence and it was helpful to detect osteopontin in HCC patients before liver transplantation.
Part II: Mcl-1 Antisense Therapy Chemosensitizes Cholangiocarcinoma in vitro Research Object: Cholangiocarcinoma is the second most common primary malignancy of the liver. Despite advances in diagnosis, surgery offers the only possible chance for long term survival, but only in a minority of cases. The five year survival rate after curative resection is between 0% and 30%. Currently available chemotherapeutic agents and radiation therapy are ineffective at improving survival. Chemotherapy and radiotherapy used in the treatment of a wide variety of malignancies induces apoptosis in responsive cells. Failure to initiate apoptosis is a major factor limiting the efficacy of these treatments. The Bcl-2 protein family plays a central part in the control of apoptosis. Several clinical studies have provided evidence for the hypothesis that high level expression of antiapoptotic Bcl-2 family members, such as Bcl-2, Bcl-xL, and Mcl-1, contribute to chemoresistance in a number of human malignancies, including cholangiocarcinoma. Antisense oligonucleotides (ASO) are designed to bind to their complementary mRNA sequence once they get inside the cell, thereby inhibiting expression of the encoded protein. Based on the available evidence, Mcl-1 seems to be a suitable molecular target to enhance chemosensitivity in human cholangiocarcinoma. In this study we used Mcl-1 ASO to downregulate Mcl-1 protein expression in human cholangiocarcinoma cells in vitro. The downregulation chemosensitized human cholangiocarcinoma to subsequent treatment with cisplatin. Methods: One kind of human cholangiocarcinoma cell line—NEC cells growing in culture were used to perform in vitro experiments over 24 hours following treatment. NEC cells were incubated for 4 h with the indicated oligonucleotides or vehicle control in the presence of Lipofectemin2000. Twenty-four hours later, protein was extracted and analyzed for Mcl-1 and β-tubulin expression by Western blotting. At same time to detect viability of NEC cells treated with Mcl-1 antisense oligonucleotide plus cisplatin. 24h after the transfection cisplatin was added. Cells count was analyzed at time point of 0h, 24 h, 48h, 72h after transfection. Results: Mcl-1 expression was down regulated to 44.57% and to 64.46% in NEC cells treated with ASO 200nM and 100nM respectively. A significant reduction of Mcl-1 protein levels compared to MM control (91.88% and 99.69% respectively, P<0.05). Treatment with Mcl-1 ASOs
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