【作者】 耿广勇；

【导师】 汤志刚；

【作者基本信息】 安徽医科大学 ， 外科学， 2010， 硕士

【摘要】 胰腺癌恶性程度最高的肿瘤之一,易侵犯神经、血管、淋巴管,加之解剖位置深在、隐蔽及早期缺乏特异性症状等特点,故现有检查很难早期发现。而且,胰腺癌对现有的放疗、化疗又极为不敏感,手术切除率低(仅为15-30%)、转移复发率高、预后差[1]。因此,常规治疗方法对胰腺癌的治疗效果很不理想,积极探索新的治疗方法,抑制胰腺癌的侵袭转移,改善其预后已成为目前亟待解决的关键问题。得益于DNA重组技术的发展,肿瘤是基因异常持续积累结果的认可,针对恶性肿瘤所采取的生物治疗技术日趋成为肿瘤治疗的重要手段之一。理论上通过细胞的基因治疗,纠正相应的基因异常,可以逆转肿瘤细胞的恶性属性。体外实验证明:将组织金属蛋白酶抑制物(tissue inhibitor of metalloproteinase, TIMP)转染胰腺癌细胞Panc-1后,可显著抑制胰腺癌细胞裸鼠接种的成功率、接种瘤的生长、血管生成和转移,并增加Panc-1细胞的凋亡率,并显著延长裸鼠负瘤生存期[2,3]。定位于人类染色体7q22的TFPI-2基因,其编码产物组织因子途径抑制物-2(tissue factor pathway inhibitor 2, TFPI-2)是广谱丝氨酸蛋白酶抑制物,广泛分布于人类肝、胰腺、肾、心、骨骼肌和前列腺等正常组织,体外可强烈抑制纤溶酶、胰蛋白酶,基质金属蛋白酶1(matrix metalloproteinases, MMP-1)和3(MMP-3)的活化[4,5],参与细胞外基质(extracellular matrix, ECM)的重塑过程,维持ECM的结构完整,调控肿瘤细胞的侵袭转移,在肿瘤细胞侵袭转移等一系列生理和病理过程中扮演重要角色。现已发现TFPI-2与多种肿瘤如神经胶质瘤、绒毛膜癌、黑色素瘤以及肺癌等的发生、发展有密切关系[6-9]。本研究构建TFPI-2基因的真核表达载体pEGFP-C1-TFPI-2,并将其转染胰腺癌细胞系Panc-1细胞,检测转染细胞、正常胰腺组织和不同时期及不同分化胰腺癌组织中TFPI-2基因的表达,探讨TFPI-2对胰腺癌细胞增殖、凋亡及体内和体外侵袭浸润能力影响及其可能的作用机制,为判断胰腺癌的预后及其基因治疗提供新的实验依据。目的:本研究应用分子生物学、免疫学技术,构建稳定表达TFPI-2的人TFPI-2基因真核表达载体pEGFP-C1-TFPI-2,将其转染胰腺癌细胞系Panc-1细胞,观察TFPI-2基因在胰腺癌细胞系Panc-1、正常胰腺组织和胰腺癌组织中的表达情况,测定并比较TFPI-2表达组与非表达组细胞的生长曲线,利用Boyden小室检测TFPI-2表达组与非表达组胰腺癌细胞的穿膜细胞数,比较TFPI-2表达组与非表达组胰腺癌细胞的裸鼠成瘤大小,以此作为评价胰腺癌细胞体外和体内侵袭浸润能力的指标。利用DNA片段化分析检测TFPI-2表达组与非表达组细胞凋亡率,研究TFPI-2的表达对胰腺癌细胞凋亡的影响。方法:RT-PCR实验:目的基因的扩增及转染后TFPI-2 mRNA检测;正常胰腺组织及胰腺癌组织中TFPI-2 mRNA检测;Western blot实验:转染前后TFPI-2蛋白表达的检测;正常胰腺组织及胰腺癌组织中TFPI-2蛋白表达的检测;细胞培养及转染:采用脂质体Lipofectamine2000介导;Boyden小室侵袭实验:观察TFPI-2对胰腺癌细胞体外迁徙和浸润能力的影响;裸鼠皮下种植瘤模型建立:观察TFPI-2对胰腺癌细胞裸鼠成瘤的影响及体内浸润能力的影响;细胞凋亡检测:采用DNA Ladder带凝胶电泳进行分析。结果:成功构建了人TFPI-2基因真核表达载体pEGFP-C1-TFPI-2;Panc-1细胞无TFPI-2基因表达,脂质体Lipofectamine2000能介导真核表达载体pEGFP-C1-TFPI-2转染Panc-1细胞,并使其稳定表达TFPI-2 mRNA及相应蛋白;TFPI-2能抑制胰腺癌细胞的体外侵袭能力;TFPI-2能抑制胰腺肿瘤的生长及体内浸润转移;TFPI-2能诱导胰腺癌细胞凋亡。结论:TFPI-2能显著抑制胰腺癌细胞的体外、体内侵袭转移能力,其作用可能通过维持ECM的完整性、诱导肿瘤细胞凋亡来实现,为胰腺癌的基因治疗提供了实验依据。更多还原

【Abstract】 Pancreatic carcinoma remains one of the most malignant tumor, which is easy to metastasize to nerves, blood vessel, lymph vessel. Current screen modalities are unable to identify it for inaccessible location, proximity to other vital organs, inherently aggressive pattern of growth, and atypical symptom, which make most patients have local or metastasis spread at the time of presentation that precludes surgical resection. Less than 30% of cases are constitute candidates for resection. Furthermore, pancreatic carcinoma is highly resistant to current available chemotherapy and radiation protocols. Therefore, new modalities in the treatment of this disease to improve its prognosis are required urgently. For the development of DNA recombinant technology, and the consent of accumulated abnormal gene resulting tumorigenesis, biotherapy is becoming one of the most means in oncotherapy. In theory, it can reconvert the malignant attribute of tumor cells by gene therapy. It has been improved in vitro that tumor growth, angiogenesis, and metastasis was inhibited, tumor cell apoptosis was increased, life span was significant prolongation of athymic mice bear to tumor by transfecting tissue inhibitor of metalloproteinase to Panc-1 cell.Human tissue factor pathway inhibitor 2 (TFPI-2) gene, localizated to chromosome 7q22, is a broad-spectrum serine protease inhibitor that inhibits plasmin, trypsin, metrix metalloproteinase 1 and 3 in vitro, which is important to maintain the integrity of extracellular matrix and regulate the migration and invasion of tumor. TFPI-2 transcripts are abundant in various adult human tissue, such as liver, pancreas, kidney, heart, skeletal muscle and prostate gland. It has been discovered that TFPI-2 is related to tumorigenesis and development of multitude tumors such as neuroglioma, chorionic carcinoma, melanoma, and lung cancer.In this research, we construct the eukaryotic expression vector pEGFP-C1-TFPI-2 of TFPI-2 gene, and then transfect it to pancreatic carcinoma cell line Panc-1 cell. TFPI-2 expression in 50 tumor samples, 8 pancreatic tissue samples and Panc-1 cells are detected. Afterwards, we study the influence of TFPI-2 expression on growth curve, invasion and migration in vivo and in vitro, and apoptosis of Panc-1 cell and its potential mechanism. All these studies will provide new experimental basis for prognosis and recidivation of pancreatic carcinoma.Objective:We observe the expression of TFPI-2 in pancreatic carcinoma cell line Panc-1 cell, tumor samples and pancreatic tissue samples, construct the eukaryotic expression vector pEGFP-C1-TFPI-2 of TFPI-2 gene and then transfect it to pancreatic carcinoma cell line Panc-1 cell. Afterwards, we detect and compare the growth curve, invasion and migration and apoptosis in TFPI-2 expression and non-expression group by Boyden chamber, model of subcutaneous explantation tumor in athymic mice and DNA fragmentation assay, study the influence of TFPI-2 expression on growth, invasion and migration in vivo and in vitro, and apoptosis of Panc-1 cell.Methods:Reverse transcriptase polymerase chain reaction (RT-PCR): Amplification of TFPI-2 gene and detection of TFPI-2 mRNA after transfection. Detection of TFPI-2 mRNA of pancreatic normal and tumor tissue.Western blot: Detection of TFPI-2 protein before and after transfection. Detection of TFPI-2 protein of pancreatic normal and tumor tissue.Cell culture and transfection: Lipofectamine2000 was used as a media.Invasion experiment of Boyden chamber: To observe the influence of TFPI-2 on invasion and migration in vitro.Modeling of subcutaneous explantation tumor in athymic mice: To observe the influence of TFPI-2 on invasion and migration in vivo.Detection of cell apoptosis: DNA Ladder electrophoresis and flow cytometry was used.Results:The eukaryotic expression vector pEGFP-C1-TFPI-2 of human TFPI-2 gene was constructed successfully.There was non-expression of TFPI-2 in Panc-1 cell, after transfection, it can express TFPI-2 mRNA and protein stably.TFPI-2 can inhibit invasion of pancreatic carcinoma cell in vitro.TFPI-2 can inhibit growth and invasion of pancreatic carcinoma.TFPI-2 can induce apoptosis of pancreatic carcinoma cell.Conclusion:TFPI-2 expression may obviously inhibit the invasion ability of pancreatic cancer cells in vitro and in vivo by maintaining the integrity of ECM or inducing cell apoptosis, which provides an experimental basis for curing human pancreatic cancer with gene-therapy.更多还原