【作者】 金实；

【导师】 王忠裕；

【作者基本信息】 大连医科大学 ， 中西医结合临床， 2009， 博士

【摘要】 前言:胰腺癌在西方国家居癌症死亡原因的第四位,尽管最近几十年外科手术治疗、化学治疗以及放射治疗取得很大进展,死亡率仍等于发病率,1年生存率约为12%。生长抑素能抑制表达2型生长抑素受体的胰腺癌细胞的生长,对于不表达2型生长抑素受体的胰腺癌细胞则没有作用。2型生长抑素受体在正常的胰腺外分泌细胞中表达,而在胰腺外分泌腺癌、转移癌细胞以及大多数胰腺癌细胞株中很少表达。2型生长抑素受体表达可能是胰腺癌细胞失去的一个重要的肿瘤抑制途径。目的:我们通过构建包含SSTR2基因的腺病毒载体( AdCAG- SSTR2)转染SSTR2丢失的人胰腺癌BxPC-3细胞,观察对胰腺癌细胞肿瘤特性的影响,探讨其作用机制。构建含有COX-2基因启动子和SSTR2基因的腺病毒载体(AdCOX-2L-SSTR2)转染胰腺癌细胞以评估COX-2基因启动子的启动效率。观察苦参碱结合SSTR2基因转染能否增强对细胞增殖的抑制作用和阐明其作用机理。方法:分别应用空载体、AdCAG-SSTR2转染生长抑素受体阴性的人胰腺癌细胞株BxPC-3。RT-PCR和Western印迹法分析检测SSTR2在细胞中的表达。对非转染细胞,转染空载体细胞和转染SSTR2细胞使用血球计数板直接细胞计数测定细胞生长速度。采用流式细胞仪膜联蛋白V/碘化丙啶(Annexin V/PI)标记法检测细胞凋亡。Real Time PCR检测PCNA、Fas、Bax、Bcl-xl、VEGF和MMP9的表达。AdCAG-SSTR2和AdCOX-2-SSTR2重组腺病毒分别转染胰腺癌细胞,RT-PCR和Western印迹法分析检测SSTR2在细胞中的表达。Real Time PCR检测Bax、VEGF的表达。未转染胰腺癌细胞、转染AdCAG-SSTR2细胞、应用苦参碱细胞及转染AdCAG-SSTR2联合应用苦参碱细胞采用流式细胞仪膜联蛋白V/碘化丙啶(Annexin V/PI)标记法检测细胞凋亡。Real Time PCR检测Bax、Bcl-xl的表达。结果:AdCAG-SSTR2基因重组腺病毒转染胰腺癌细胞后可以检测到SSTR2mRNA和蛋白的稳定表达,转染SSTR2后细胞生长受到抑制,与对照组差异有显著性意义(P<0.01)。转染SSTR2后细胞凋亡发生率为9.45%,对照组凋亡发生率分别为0.33%、1.34%,与对照组差异有显著性意义(P<0.01)。同时检测到Fas、Bax的表达量升高,是对照组的1.99倍、3.18倍;Bcl-xl、VEGF、MMP9的表达量降低,分别是对照组的0.34倍、0.23倍及0.3倍。AdCOX-2-SSTR2重组腺病毒转染胰腺癌细胞则未检测到SSTR2mRNA和蛋白明显的表达,Bax、VEGF的表达也无明显变化,分别是对照组的1.23倍、1.12倍。苦参碱组细胞凋亡发生率为5.86±1.01%;AdCAG-SSTR2组细胞凋亡发生率为9.45±0.87%;AdCAG-SSTR2+苦参碱组细胞凋亡发生率为11.92±0.56% ;对照组细胞凋亡发生率为0.33±0.02%。苦参碱组与AdCAG-SSTR2组差异有显著性意义(P<0.01);苦参碱组与对照组、AdCAG-SSTR2+苦参碱组间差异有显著性意义( P< 0.01)。苦参碱组及AdCAG-SSTR2+苦参碱组Bcl-xl的表达下降,分别是对照组的0.67倍和0.19倍;苦参碱组Bax的表达无明显变化,是对照组的1.23倍,AdCAG-SSTR2组与AdCAG-SSTR2+苦参碱组Bax的表达无明显变化,分别是对照组的3.18倍和3.21倍。结论: SSTR2通过诱导胰腺癌细胞凋亡发挥抗肿瘤增殖的作用,其机制可能与上调Fas、Bax的表达,下调Bcl-xl的表达有关。VEGF、MMP9表达下降提示SSTR2可以在胰腺癌的抗血管生成治疗中发挥作用。在胰腺癌细胞株BxPC-3中COX-2启动子驱动SSTR2表达未显示出肿瘤特异性启动子的优势。苦参碱能促进细胞凋亡并增强SSTR2的作用,其机制可能与下调Bcl-xl的表达有关,而与Bax的表达无关。苦参碱在胰腺癌的治疗中显示出潜在的价值。更多还原

【Abstract】 Background:Pancreatic carcinoma is the fourth leading cause of cancer related deaths in Western countries, Despite advances in surgery, chemotherapy, and radiation therapy in recent decades, the mortality rate for pancreatic cancer remains equal to its incidence, with an overall 1-year survival rate of 12% or so. It have been demonstrated that somatostatin inhibits the growth of pancreatic cancers expressing somatostatin receptor subtype 2 (SSTR2) but not receptor-negative cancers. SSTR2 is expressed in normal human endocrine pancreas but not in exocrine pancreatic carcinomas and their metastasis as well as in most of human pancreatic cancer cell lines. SSTR2 expression may be an important tumor suppressor pathway that is lost in human pancreatic cancer.Objectiv:We corrected the SSTR2 defect in human pancreatic cancer BxPC-3 cells by stable transfection with adenoviral vector containing the SSTR2, investigated its effect on tumorigenicity of human pancreatic cancer, elucidated the underlying mechanisms. We evaluated transductional efficiency of adenoviral vector bearing COX-2 promoter and SSTR2 gene in pancreatic cancer. Matrine was combined with SSTR2 gene expression to observe whether it can potentiate the inhibition of cell proliferation and to elucidate the underlying mechanisms.Methods:We used AdCAG-SSTR2 and empty vector transfected so- matostatin receptor-negative human pancreatic cancer cells (BxPC-3) respectively. The expression of SSTR2 were detected by RT-PCR and Western blot. Negative contral, vector control, and SSTR2 transfected cells were cultured and the rate of cell growth was determined by direct cell counting using a hemacytometer. The apoptosis was assessed by flow cytometric Annexin V/PI labelling assay. The expression of PCNA, Fas, Bax, Bcl-xl, VEGF and MMP9 were detected by Real Time PCR.AdCOX-2L-SSTR2 and AdCAG-SSTR2 transfected human pancreatic cancer cells (BxPC-3) respectively. The expression of SSTR2 mRNA and protein was detected by RT-PCR and Western blot. The expression of Bax, VEGF were detected by Real Time PCR.The apoptosis was assessed by flow cytometric Annexin V/PI labelling assay in negative control, AdCAG-SSTR2 transfection, administration of Matrine and AdCAG-SSTR2 transfection combining administration of Matrine respectively. The expression of Bax, Bcl-xl were detected by Real Time PCR.Results:The stable expression of SSTR2 was detected in the cells transfected by AdCAG-SSR2. The significant decrease of pancreatic cancer cell growth was detected in experimental group compared with control groups(P< 0.01).The ratio of apoptosis significantly increased in cells re-expressing SSTR2 (9.45%) than control group (P<0.01).The increase of Fas, Bax(1.99、3.18fold) and decrease of Bcl-xl, VEGF, MMP9(0.34、0.23 & 0.30fold) were detected by Real Time PCR.The stable expression of SSTR2 wasn’t detected in the cells transfected by AdCOX-2L-SSTR2, and no effect on the expression of Bax and VEGF was found.The ratio of apoptosis was 5.86±1.01%, 9.45±0.87%,11.92±0.56% and 0.33±0.02% in administration of Matrine group, AdCAG-SSR2 transfection group, administration of Matrine combining with AdCAG-SSR2 trans- fection group and control group respectively. There was significant di- fference between administration of Matrine group and AdCAG-SSR2 transfection group; the ratio of apoptosis in administration of Matrine combining with AdCAG-SSR2 transfection group was significantly higher than in administration of Matrine group. The decrease of Bcl-xl was detected in administration of Matrine group (0.67 fold) and administration of Matrine combining with AdCAG-SSR2 transfection group (0.19 fold).The expression of Bax hadn’t changed in administration of Matrine group (1.23 fold). There was no significant different expression of Bax between administration of Matrine group and administration of Matrine combining with AdCAG-SSR2 transfection group (3.18 vs 3.21 fold).Conclusions:SSTR2 reversed tumorigenicity of human pancreatic cancer cells. The mechanisms of inducing apoptosis may involved in up- regulating the expression of Fas、Bax and down-regulating the ex- pression of Bcl-xl. The decrease of VEGF and MMP9 may predict response to therapy of anti-angiogenesis in pancreatic carcinoma with SSTR2. COX-2 promoter havn’t represented advantage in promoting the expression of SSTR2 in BxPC-3. Administration of Matrine elevated the ratio of apoptosis and potentiated the effect of AdCAG-SSR2, The mechanisms of inducing apoptosis may be due to the down-regulating the expression of Bcl-xl. Administration of Matrine showed potential therapy effect in pancreatic carcinoma.更多还原