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Regulation and analysis of systemic and local effect of TNF

TNF全身和局部效应的调控与分析

基本信息

批准号：
04454189
负责人：
HIGUCHI Yasunori
金额：
$ 3.97万
依托单位：
Oita Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04454189/
关键词：
CD14 TNF transgene recombinant diabetes beta-cell cDNA beta-cell 〓DNA transgenic mouse TNF-α shock

项目摘要

I.We analyzed the structure of the 5' end of the mouse CD14 (mCD14) gene. Sequentially 5'-deleted, chimeric and point mutated clones were tested for their ability to stimulate chroramphnicol acetyltransferase (CAT). An 8-base pair sequence, TGATTCAC, at position -255 enhanced the expression of the CAT gene in macrophage cell lines. The 5' upstream of the human CD14 gene were sequenced and compared with that of mouse. II.Recombinant mCD14 protein was deleted at carboxyterminus was produced in E.coli. We made monoclonal antibodies (mAb) against the protein. Immunohistochemical staining using mAb showed that CD14-positive Kupffer cells (KC) were small in number in the liver in nonstimulated mice. The number of stained KC gradually increased with time after intraperitoneal injection of LPS, peaked 6 h after injection, and returned to normal by 20 h after injection. Slight increase in mCD14 expression was observed in peritoneal macrophages 2 h after LPS administration in vivo using flow cytometric analysis. Unique features of mCD14 expression in KC were also confirmed by the expression of mCD14 mRNA which peaked at 3 h after LPS administration. Mice bearing a mCD14 transgene controlled by an metallotionein promoter (MTTG) were produced. Northen blot analysis of MTTG showed that the mice strongly expressed mCD14 mRNA in the liver, small intestine and testis. III.Mice bearing a TNFalpha transgene controlled by an insulin promoter developed an increasingly severe insulitis. Diabetes never divelopped, even though a number of potentially inducing conditions were used, including the prolonged perfusion of TFNgamma and the permanent expression of a non-tolerogenic viral protein on beta cells.

我们分析了小鼠CD14 （mCD14）基因5'端的结构。对5'序列缺失、嵌合和点突变克隆刺激铬酚乙酰转移酶（CAT）的能力进行了测试。一个位于-255位的8碱基对序列TGATTCAC增强了巨噬细胞中CAT基因的表达。测定了人CD14基因上游5′序列，并与小鼠进行了比较。2。在大肠杆菌中产生了羧基端缺失的重组mCD14蛋白。我们制备了针对该蛋白的单克隆抗体（mAb）。单克隆抗体免疫组化染色显示，未刺激小鼠肝脏中cd14阳性KC细胞数量较少。腹腔注射LPS后，染色的KC数量随时间逐渐增加，注射后6 h达到峰值，注射后20 h恢复正常。流式细胞术分析显示，LPS给药2 h后，腹腔巨噬细胞mCD14表达略有升高。mCD14 mRNA的表达在LPS处理后3 h达到峰值，证实了mCD14在KC中表达的独特特征。产生了携带由金属化蛋白启动子（MTTG）控制的mCD14转基因小鼠。MTTG的northern blot分析显示，小鼠肝脏、小肠和睾丸中mCD14 mRNA表达强烈。3。携带由胰岛素启动子控制的TNFalpha转基因的小鼠发展为日益严重的胰岛素炎。尽管使用了许多潜在的诱导条件，包括长时间灌注TFNgamma和在β细胞上永久表达非耐受性病毒蛋白，但糖尿病从未发生。