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目的探讨Beclin-l基因与胰腺癌发生发展的关系。方法 (1)回顾性收集2009年4月至2009年11月期间四川大学华西医院行手术切除的胰腺癌标本25例及正常胰腺组织20例,采用实时荧光定量PCR和免疫组化染色方法分别检测胰腺癌组织及正常胰腺组织中Beclin-1 m RNA及其蛋白的表达水平,并分析胰腺癌组织中Beclin-1蛋白的表达水平与患者临床病理学特征之间的关系。(2)将PANC-1细胞分为2组,转染组细胞转染PLen O-WPI-Beclin-1重组慢病毒载体,未转染组细胞未进行重组慢病毒载体转染,分别于共培养24及48 h时采用PCR法检测Beclin-1 m RNA的表达水平。(3)将PANC-1细胞分为3组,转染组加入PLen O-WPI-Beclin-1重组慢病毒载体,空载体组加入PLen O-WPI载体,空白对照组未加入任何载体,各组细胞于培养1~7 d期间每天采用MTT法检测细胞增殖情况。结果 (1)人胰腺癌中Beclin-1 m RNA及其蛋白的表达水平均低于正常胰腺组织(P<0.05)。在胰腺癌患者中,Beclin-1蛋白的表达水平与患者的年龄、性别、肿瘤直径及分化程度均无关(P>0.05),而与TNM分期及淋巴结转移有关,TNMⅢ期和Ⅳ期患者的Beclin-1蛋白的表达水平低于Ⅰ期或Ⅱ期患者(P<0.05);淋巴结转移阳性患者的Beclin-1蛋白的表达水平低于阴性患者(P=0.011)。(2)转染24 h和48 h时,同时点转染组细胞中Beclin-1 m RNA的表达水平高于未转染组(P<0.05)。(3)MTT实验结果显示,转染1、2及3 d时,转染组、空载体组和空白对照组细胞的OD值比较差异均无统计学意义(P>0.05);从转染4 d开始,至转染7 d期间,同时点转染组细胞的OD值较空白对照组和空载体组均较低(P<0.05)。结论 Beclin-1基因及其蛋白在胰腺癌组织中的表达均下调,其可能是胰腺癌发生发展的原因之一。将Beclin-1基因导入PANC-1细胞后,细胞的增殖能力降低,提示Beclin-1基因可能是胰腺癌基因治疗的目标基因。
Objective To investigate the relationship between Beclin-1 gene and the development of pancreatic cancer. Methods (1) We retrospectively collected 25 cases of pancreatic cancer resected from West China Hospital of Sichuan University from April 2009 to November 2009, and 20 cases of normal pancreatic tissue. Real-time fluorescence quantitative PCR and immunohistochemical staining were used to detect The expression of Beclin-1 m RNA and its protein in pancreatic cancer tissue and normal pancreatic tissue was analyzed. The relationship between the expression of Beclin-1 protein and the clinicopathological features of pancreatic cancer was analyzed. (2) The PANC-1 cells were divided into two groups. The transfected cells were transfected with PLen O-WPI-Beclin-1 recombinant lentiviral vector. The untransfected cells were not transfected with recombinant lentiviral vector, At 24 and 48 h, the expression of Beclin-1 m RNA was detected by PCR. (3) The PANC-1 cells were divided into 3 groups. The transfected group was injected with PLen O-WPI-Beclin-1 recombinant lentiviral vector, the empty vector group was added to PLen O-WPI vector, the blank control group was not added any vector, Cells were cultured daily for 1 to 7 days using MTT assay cell proliferation. Results (1) The expression level of Beclin-1 mRNA and protein in human pancreatic cancer was lower than that in normal pancreatic tissue (P <0.05). In patients with pancreatic cancer, the expression level of Beclin-1 protein had no correlation with the age, sex, tumor diameter and differentiation (P> 0.05), but correlated with TNM staging and lymph node metastasis. Beclin- (P <0.05). The expression of Beclin-1 protein in patients with positive lymph node metastasis was lower than that in negative patients (P = 0.011). (2) At 24 h and 48 h after transfection, the expression of Beclin-1 m RNA in transfected cells was higher than that in untransfected cells (P <0.05). (3) The results of MTT assay showed that there was no significant difference in the OD value between the transfected group, empty vector group and blank control group at 1, 2 and 3 days (P> 0.05) d. At the 7th day after transfection, the OD value of the transfected cells was lower than that of the blank control group and the empty vector group (P <0.05). Conclusion The expression of Beclin-1 gene and its protein in pancreatic cancer tissues are down-regulated, which may be one of the reasons for the development of pancreatic cancer. Beclin-1 gene into PANC-1 cells, the proliferation of cells decreased, suggesting that Beclin-1 gene may be the target gene for pancreatic cancer gene therapy.