【作者】 于爱芳；

【导师】 辛晓燕；

【作者基本信息】 第四军医大学 ， 妇产科学， 2004， 硕士

【摘要】 目的：通过检测COX-2蛋白在上皮性卵巢肿瘤组织和卵巢癌细胞系中的表达，探讨COX-2蛋白在上皮性卵巢肿瘤发生、进展中的作用以及COX-2表达水平与肿瘤临床病理参数间的关系。利用基因重组技术构建COX-2启动子调控促凋亡基因BAX的重组质粒，体外转染实验检测COX-2启动子在卵巢癌基因治疗中的应用价值。 方法：(1)采用SP免疫组化染色法，对118例原发性上皮性卵巢肿瘤石蜡切片及5种卵巢癌细胞系的COX-2蛋白表达进行检测，分析卵巢浆液性囊腺癌中的COX-2蛋白表达与临床病理参数之间的关系。(2)用基因重组技术构建重组质粒COX-2-BAX和CMV-Luc，并酶切签定。(3)脂质体法分别把质粒COX-2-Luc、CMV-Luc瞬时转染COX-2阳性表达的卵巢癌细胞系SKOV-3和COX-2阴性表达的结肠癌细胞系SW480，用光照度计检测荧光素酶报告基因的表达水平。(4)脂质体法分别把质粒COX-2-BAX、CMV-BAX瞬时转染卵巢癌细胞系SKOV-3和结肠癌细胞系SW480，流式细胞仪检测细胞凋亡率。 结果：(1)COX-2蛋白在卵巢恶性、交界性和良性上皮性肿瘤石蜡切片中的表达率分别为44％(37／84)、32％(6／19)和0％(0／15)，结果有显著性差异(P=0.006)。(2)COX-2蛋白在所检测的5种卵巢癌细胞系中有4种呈阳性表达。(3)在68例卵巢浆液性囊腺癌组织中，不同临床分期、不同病理分级的COX-2表达无显著性差异(分别P=0.716及P=0.700)。(4)成功构建重组质粒COX-2-BAX和CMV-Luc，经测序及酶切鉴定均含目的序列。(5)COX-2-Luc转染SKOV3和SW480细胞24h后，荧光素酶.-~~~~-二日臼公日涟匕华理毕尝全‘--- 报告基因活性分别为1554士86.5和53.7士10.9.C柳一Luc转染SKOV3和 SW48O细胞24h后，荧光素酶报告基因活性分别为9851.7士129.5和 8831.0士167.3。在同一种细胞，C柳启动子的转录效率分别是Cox一2启 动子的6.3倍(SKOV3)和164.5倍(SW480)。(6)COX一2一BAX转染SKOV一3 和SW480细胞36h后，细胞凋亡率分别为10.4%，3.7%;C柳一BAx转 染SKOV一3和SW480细胞36后，细胞凋亡率分别为21.7%，25.6%. 结论:(1) COx--2蛋白在上皮性卵巢癌中表达明显上调。(2)在卵巢 浆液性囊腺癌，COX一2蛋白的表达水平与肿瘤临床病理参数无明显相关。 (3)COX一2启动子有一定的肿瘤特异性，但其转录效率明显低于C袱启 动子。用COX一2启动子构建的重组载体需经合理修饰后才可用于卵巢癌 的基因治疗。更多还原

【Abstract】 OBJECTIVES: To explore the role of COX-2 in the generation and progression of epithelial ovarian neoplasm and the correlation between the COX-2 expression levels and the clinical pathologic parameters of the tumor tissues through examining the expression of COX-2 in human epithelial ovarian neoplasm tissues and ovarian carcinoma cell lines. Moreover, to verify the value of the COX-2 promoter in gene therapy of ovarian cancer through transfection tests in vitro using the recombination plasmids contained COX-2 promoter drivening pro-apoptosis BAX gene.METHODS: (1) Immunohistochemical staining (SP) was used to detect the expression of COX-2 protein in the paraffine slice of 118 primary ovarian epithelial neoplasm tissues and in S kinds of ovarian carcinoma cell lines. Furthermore, the relationship between COX-2 expression levels and the clinical pathologic parameters was analyzed in ovarian serous cystadenocarcinoma tissues. ( 2 ) The recombinant plasmids named COX-2-BAX and CMV-Luc were constructed by molecular cloning technique and identified by enzyme digestion. (3 ) COX-2-positive ovarian cancer cell line - SKOV3 and COX-2-negative colon cancer cell line-SW480 were transfected liposome-mediated with the recombinant plasmids COX-2-Luc and CMV-Luc , respectively. Luciferase Assay System was used to measure the expression of Luciferase reporter gene. (4) SKOV-3 and SW480 were transfected with C0X-2-BAX and CMV-BAX, respectively. Flow cytometrywas used to measure their apoptosis-inducing effectRESULTS: (1) The positive rates of COX-2 expression in malignant, borderline and benign ovarian epithelial neoplasm were 44% (37/84 ), 32%(6/19) and 0% (0/15) , respectively. The expressions of COX-2 had significant difference(P=0.006) in different ovarian epithelial neoplasm. (2 ) The COX-2 expressions had no correlation to FIGO stage and tumor grade( P=0.716 . and P=0.700, respectively ) in 68 ovarian serous cystadenocarcinoma tissues. (3) COX-2 protein expression could be observed in 4/5 ovarian cancer cell lines examined. (4) The recombinant plasmids named COX-2-BAX and CMV-Luc were constructed successfully and identified by DNA sequencing and enzyme digestion. ( 5 ) The expression efficiency of luciferase reporter gene was 1554+86.5 in SKOV3 and 53.7+ 10.9 in SW480 after 24 hours transfected with COX-2-Luc, 9851.7+129.5 in SKOV3 and 8831.0 +167.3 in SW480 after 24 hours transfected with CMV-Luc, respectively. But in the same cell line, the efficiency of CMV promoter is 6.3 times (in SKOV3) and 164.5 times (SW480) of COX-2 promoter’s. (6) The apoptosis rate was 10.4% in SKOV3 and 3.7% in SW480 after 36 hours transfected with COX-2-BAX, 21.7% in SKOV3 and 25.6% in SW480 after 36 hours transfected with CMV -BAX.CONCLUSION: ( 1 ) The expression of COX-2 is obviously up-regulated in epithelial ovarian carcinoma. (2) There is no significant correlation between COX-2 expression and clinical pathologic parameters in ovarian serous cystadenocarcinoma. (3) COX-2 promoter is partial tumor specific, but its transcription efficiency was lower than CMV promoter’s. With proper modification, COX-2 promoter is useful in gene therapy of ovarian cancers.更多还原