原癌基因Pim-3对烧伤及内毒素引起的肠黏膜损伤保护作用的实验研究  

Protective role of Pim-3 gene in intestinal mucosa damaged by burn or lipopolysaccharide

作　　者：陈建勇[1] 沈学文[1] 张吉翔[1] 

机构地区：[1]南昌大学第二附属医院消化科 江西省分子医学重点实验室,330006

出　　处：《中华医学杂志》2007年第42期2960-2964,共5页National Medical Journal of China

基　　金：国家自然科学基金(30360037);江西省卫生厅科研基金(0301068)

摘　　要：目的研究 Pim-3基因在烧伤及内毒素引起的肠黏膜损伤中的保护作用。方法体内实验在成年 Wistar 大鼠中进行,分为 A 组(烧伤组),B 组(脂多糖组),C 组(正常对照组)。各组依次在处理后3、6、12、24、48 h 取小肠,提取总 RNA 及蛋白质,并进行逆转录(RT)-PCR 及 Western 印迹分别检测内源性 Pim-3、细胞间黏附分子(ICAM-1)、紧密连接蛋白闭锁蛋白(occludin)在肠道的表达。观察烧伤和内毒素对内源性 Pim-3基因在肠道表达的影响及 Pim-3基因与肠黏膜损伤的相关性。体外实验部分,首先原代肠上皮细胞分离培养,再重组质粒 pEGFP-N_2/Pim-3转染和内毒素处理原代肠上皮细胞。实验分为6组:①正常对照+内毒素处理组(5μg/ml);②空载质粒 pEGFP-N_2转染+内毒素处理组(5μg/ml);③重组质粒 pEGFP-N_2/Pim-3转染+内毒素处理组(5μg/ml);④正常对照组;⑤空载质粒 pEGFP-N_2转染组;⑥重组质粒 pEGFP-N_2/Pim-3转染组;内毒素处理6 h 后,提取各组总 RNA,利用逆转录(RT)-PCR 检测各指标,并利用流式细胞仪观察各组细胞凋亡情况。结果(1)体内实验部分:A、B 组内源性 Pim-3基因在3 h 表达开始增多,分别为 C 组的(12.25±1.01)和(15.08±1.07)倍,6 h 到达顶点,分别为 C 组的(21.13±1.78)和(25.24±1.80)倍,以后逐渐减少。A、B 组 ICAM-1在6 h 后一直处于高表达的位置,12 h 达高峰,分别为 C 组的(88±7)和(58±5)倍,差异有统计学意义(P<0.05)。A、B 组闭锁蛋白在3 h 表达开始增多,6 h 达顶点,分别为 C 组的(6.65±0.24)和(6.25±0.26)倍,差异均有统计学意义(均 P<0.05)。(2)体外实验部分:⑥组的Pim-3及闭锁蛋白的表达与④⑤组之间的差异均有统计学意义(均 P<0.05),而④⑤⑥组之间 ICAM-1表达及细胞凋亡差异无统计学意义。③组 Pim-3、闭锁蛋白和 ICAM-1的表达及细胞凋亡与①②组之间的差异有统计学意义(P<0.05)。另外,①②③组的 ICAM-1表达与④⑤⑥组之间的差异有�Objective To explore the protective role of Pim-3 gene in intestinal mucosa damaged by burn or lipopolysaccharide （LPS）. Methods （1） Ninety Wister mice were randomly divided into 3 equal groups： Group A, undergoing 30% grade Ⅲ burning on the back; Group B, undergoing intraperitoneal injection of LPS ; and Group C, undergoing intraperitoneal injection of normal saline. 3, 6, 12, 24, and 48 hours after the treatment 5 rats from each group were killed with their small intestine tissues taken out. RTPCR was used to detect the mRNA expression of Pim-3, a serine/threonine kinase, occludin, intercellular adhesion molecule （ICAM）-1, and Western blotting was used to detect the protein expression of Pim-3 and occludin. （2） Intestinal endothelial cells （IECs） of newborn Wistar rats were collected, cultured, and divided into 6 groups ： Group （1）, treated with LPS （ endotoxin）, Group （2）, transfected with blank plasmid pEGFP-N2 and treated with LPS, Group （3）, transfected with recombinant pEGFP-N2/Pim-3and treated with LPS, Group （4）, as normal control group, Group （5）, transfected with blank plasmid pEGFP-N2, and Group （6）, transfected with recombinant plasmid pEGFP-N2/Pim-3. Six hours later RT-PCR was used to detect the mRNA expression of Pim-3, ICAM-1, and occludin. The apoptosis of the cells was examined by flow cytometry. Results （1）In Groups A and B the mRNA expression of Pim-3 began to increase 3 h later,peaked 6 h later, and then gradually decreased. The Pim-3 mRNA expression of Group C, however, remained always at a low level. The ICAM-1 mRNA expression levels of Groups A and B were constantly up-regulated 6 h later, all significantly higher than those of Group C （ all P 〈 0.01 ）. The occludin mRNA expression levels of Groups A and B began to increase 3 hours later, and peaked 12 hours later, all significantly higher than those of Group C （ all P 〈 0. 05 ）. （ 2 ） The mRNA expression levels of Pim-3 and occludin of Group （6） was significantl

关 键 词：细胞培养 烧伤 脂多糖类 肠黏膜 PIM-3 

分 类 号：R644[医药卫生—外科学]