【作者】 蒋文文；

【导师】 徐存栓；

【作者基本信息】 河南师范大学 ， 细胞生物学， 2012， 硕士

【摘要】 丝氨酸蛋白酶抑制剂Kazal Ⅲ型（serine protease inhibitor, Kazal type3, SPINK3）是一种胰蛋白酶抑制剂，在胰腺癌、大肠癌，乳腺癌、前列腺癌、肝癌等多种肿瘤中高表达，但其对肝癌细胞株的作用研究尚未见报道。为此，本文构建了spink3基因的表达载体（pEGFP-N1-spink3），干涉载体（pGenesil-1.0-spink3(123)和pGenesil-1.0-spink3(143)）和检验载体（pGenesil-1.0-spink3(123)-spink3、spink3(143)-spink3和-HK-spink3）。将各检验载体进行尾静脉液压转基因至小鼠肝脏，用荧光显微技术观察与SPINK3融合表达的增强型绿色荧光蛋白（enhanced greenfluorescent protein, EGFP）的阳性细胞率，筛选出最佳干涉载体。采用脂质体介导法将4种质粒（表达载体pEGFP-N1-spink3及其对照pEGFP-N1，最佳干涉载体pGenesil-1.0-spink3(123)及其对照pGenesil-1.0-HK）转染肝癌细胞株BEL-7402，并用G418选择性培养基筛选出稳定转染细胞株。对正常BEL-7402细胞株和上述筛选出来的四种稳定细胞株用下列方法进行检测：通过HE染色观察细胞形态结构；荧光显微镜下观察细胞形态以及EGFP分布；通过计数法和普通MTT法绘制细胞的生长曲线；通过PCNA细胞免疫化学检测细胞中PCNA的表达变化；通过Hoechst33258染色检测细胞凋亡；通过甲基纤维素半固体培养基检测集落形成率；通过药物MTT法检测细胞的耐药性。结果表明： SPINK3-EGFP融合蛋白均匀分布在细胞质中，细胞核无荧光，并且在细胞核外侧核膜周围表达量较高，为典型的分泌性表达模式；spink3能抑制肝癌细胞株BEL-7402增殖，但不引起细胞凋亡；spink3能够降低肝癌细胞株BEL-7402恶性程度，但对其耐药性无明显影响。更多还原

【Abstract】 The serine protease inhibitor, Kazal type3(SPINK3) is a trypsin inhibitor which is highly expressed inpancreatic cancer, colorectal cancer, breast cancer, prostate cancer, liver cancer and other tumors, but theresearch on the effect of spink3on hepatic cancer cell strain has not been reported.Therefore, the expression vector (pEGFP-N1-spink3), interference vectors (pGenesil-1.0-spink3(123) and pGenesil-1.0-spink3(143)) and test vectors (pGenesil-1.0-spink3(123)-spink3, pGenesil-1.0-spink3(143)-spink3and pGenesil-1.0-HK-spink3) were established. The constructed test vectors wereadministrated using a hydrodynamics-based procedure in the tail vein of little rat to select the bestrecombinant interference plasmid using fluorescence microscopy technique to observe enhanced greenfluorescent protein (EGFP)-positive cell rate. Next, the expression vector pEGFP-N1-spink3and its controlpEGFP-N1, interference vector pGenesil-1.0-spink3(123) and its control pGenesil-1.0-HK were stablytransfected into hepatic cancer cell strain BEL-7402in vitro by lipofectamine and their stably transfectedcell strains were selected by G418. Then, the hepatic cancer cell strain BEL-7402and the four kinds ofstably transfected cell strains above were detected using the following methods, including cell morphologyobserved by HE staining, cell morphology and EGFP distribution observed under a fluorescencemicroscope, cell growth curve drew by cell counting and MTT assay, PCNA expression changes detectedby PCNA immuneocytochemistry, cell apoptosis detected by Hoechst33258staining, cell colony formationrate detected by Methyl cellulose semi-solid medium, and cell resistance to drugs detected by MTT assay.The results show that the SPINK3-EGFP fusion protein was evenly distributed in the cytoplasm, nofluorescence in the nucleus, more expression surrounding the outer nuclear membrane which is a typicalsecretory expression pattern, that spink3can inhibit hepatic cancer cell strain BEL-7402proliferation, butnot cause apoptosis, and that spink3can reduce the degree of malignancy of cells, but had no significanteffect on the resistance to drugs.更多还原