白细胞介素10基因体外转染大鼠骨髓基质细胞对内毒素休克的干预  

Effect of bone marrow stromal cells in vitro transfected with interleukin-10 gene on endotoxin shock in rats

作　　者：李新强[1] 王燮衡[2] 郭燕妮[1] 朱家源[3] 文杏珠 胡检[1] 陈武鹏[1] 张显文[1] 

机构地区：[1]深圳市宝安区人民医院烧伤科,广东省深圳市518101 [2]深圳市宝安区人民医院检验科,广东省深圳市518101 [3]中山大学附属第一医院烧伤科,广东省广州市510080

出　　处：《中国组织工程研究与临床康复》2008年第21期4054-4059,共6页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：深圳市科技和信息局科研基金(JH200505300509A);深圳市宝安区科学技术局科研基金(2005104)~~

摘　　要：目的：机体内前炎症递质肿瘤坏死因子α、白细胞介素6与抗炎症递质白细胞介素10之间的平衡出现严重紊乱，可导致内毒素休克及多脏器功能衰竭。实验拟进一步探讨含大鼠白细胞介素10基因重组复制缺陷型腺病毒体外转染骨髓基质细胞后，内毒素休克动物模型导入的白细胞介素10基因表达及其对机体肿瘤坏死因子α、白细胞介素6水平的调节。方法：实验于2006—05／2007-10在中山大学附属第一医院外科实验室完成。①材料：清洁级SD雌鼠100只，选取10只用于骨髓基质细胞的分离培养，剩余90只随机数字表法分为细胞转染组、模型组、正常组，30只/组，实验过程中对动物的处置符合动物伦理学标准。含大鼠白细胞介素10基因的重组复制缺陷型腺病毒由中山大学附属第一医院烧伤外科朱家源教授惠赠。内毒素Escherichia coli 0111：B4为美国Sigma公司产品。②实验方法：将含大鼠白细胞介素10基因的重组复制缺陷型腺病毒复苏后，加入到贴壁生长达80％的第3代骨髓基质细胞中，将转染后细胞、未转染细胞按5×10^5／孔接种，采用ELISA法和RT-PCR法检测体外白细胞介素10的含量变化。细胞转染组与模型组大鼠均经左侧股静脉注射内毒素5mg／kg建立休克模型，10min后细胞转染组在同侧股静脉注射转染后培养12h的5×10^8L^-1骨髓基质细胞0.5mL，模型组与正常组均注射未转染的5×10^8L^-1骨髓基质细胞0．5mL，分别于0，3，6，12，24，36，48h采用ELISA法检测体内各组大鼠外周血清白细胞介素10、白细胞介素6和肿瘤坏死因子α水平，并记录以上各时相点大鼠存活数。细胞回输48h后，组织病理学检测大鼠肝、肺、肾的变化。结果：①体外白细胞介素10的表达：体外转染后的骨髓基质细胞培养上清中可检测到白细胞介素10的表达，表达高峰位于转染后36h，于560bp处显示凝胶电泳条AIM： It can cause endotoxin shock and multiple organ dysfunctions when the balance between anterior inflammation transmitter tumor necrosis factor （TNF）- α, intedeukin （IL）-6 and anti-inflammation transmitter IL-10 has become serious disorder in the body. This study aimed to investigate the expression of IL-10 gene which is introduced into animal models of endotoxin shock by bone marrow stromal cells transfected in vitro with replication-deficient rat IL-10 recombinant adenovirus, and study the changes in TNF- α and IL-6 levels in the models. METHODS： The experiment was performed at the laboratory of Department of General Surgery, First Affiliated Hospital of Sun Yat-sen University from May 2006 to October 2007. Ten rats of 100 clean female Sprague Dawley rats were selected for the isolation and culture of bone marrow stromal cells, and the remainings were divided into a cell transfection group, a model group and a normal group by random digits table method, with 30 rats in each group. The disposal to animals during the experiment was accorded with animal ethical standards. Replication-deficient rat IL-10 recombinant adenovirus was provided by professor Zhu from Department of Burn of First Affiliated Hospital of Sun Yat-sen University. Endotoxin Escherichia coli 0111： B4 （Sigma, USA） was used in the study. Replication-deficient rat IL-10 recombinant adenoviruses after recovery were added to the bone marrow stromal cells of the 3^rd generation, which had been adhered to the flask more than eighty percent, The cells transfected and untransfected were all inoculated according to 5× 10^5 per hole. The changes in IL-10 levels were detected by enzyme-labeled immunosorbent assay （ELISA） and reverse transcription polymerase chain reaction （RT-PCR） methods, The rat models of shock were established in the cell transfection group and model group by injecting 5 mg/kg endotoxin into left femoral vein. Ten minutes later, rats in the cell transfection group were injected with 0.5 mL of bone marrow

关 键 词：转染 基因治疗 细胞因子 内毒素休克 腺病毒 骨髓基质细胞 

分 类 号：R394.2[医药卫生—医学遗传学]