Fundamental studies of regularion for endotoxin shock by CD14

CD14对内毒素休克调节的基础研究

• 批准号: 05557057
• 负责人: HIGUCHI Yasunori
• 金额: $ 11.58万
• 依托单位: Oita Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-05557057/
• 关键词: CD14; shock; LPS; TNF-alpha; IL-1beta; transgene; Kupffer cells; mAb; Kupffer; transgeve; recombinant; transgene; TNF; monoclonal antibody; metallotionein

1.Separation of rabbit and rat CD14 cDNA : Rabbit and rat CD14 (rabCD14 and ratCD14) cDNAs were separated and sequenced. 2.Production of recombinant CD14 (rCD14) : Full length and N-terninal side encoding amino acid position from 1 to 71 of mouse CD14 (mCD14) and the latter of rabCD14 and ratCD14 were expressed in E.coil. 3.Preparation of anti-CD14 antibodies : Polyclonal (pAb) and monoclonal (mAb) anti-mCD14 antibodies were prepared. Each mAb detected different epitopes. mAb designated rmC5-3 detected most carboxyterminal epitope of mCD14.4.Functional features of mAbs : rmC5-3 enhanced LPS-induced TNF-alpha production in vitro. mAbs designated rmA4-2, rmF4-3 and rmA12-10, all of which recognized epitopes of middle area of mCD14, slightly inhibited LPS-induced TNF-alpha production in vitro. Because of the low inhibitory effect of mAbs. we are continuing to produce mAbs using the N-terminal protein described above. 5.Effect of pAb and mAbs on LPS-induced TNF-alpha production in vivo : A … More dministration of F (ab') 2 of pAb significantly reduced LPS-induced TNF-alpha production in vivo. However, effect of mAbs were unclear. 6.Analysis of soluble mCD14 (smCD14) : Serum levels of smCD14. TNF-alpha and IL-1beta were analyed by Western blotting using mAbs and ELISA befoce and after administration of LPS.7.Features of expression of mCD14 and cytokines including TNF-alpha, IL-1beta and IL-6 in Kupffer cells (KC) and peritoneal macrophages (Mphi) after LPS stimulation in vivo : mRNA and protein levels of mCD14 and cytokines including TNF-alpha, IL-1beta and IL-6 in Kupffer cells (KC) and peritoneal macrophages (Mphi) after LPS stimulation in vivo TNF-alpha, IL-1beta and IL-6 in Kupffer cells (KC) and peritoneal macrophages (Mphi) after LPS stimulation in vivo were analyzed. Signal for cytokine production could be triggered by small numbers of mCD14.8.Close localization of mCD14 and F_<CY>RII/III and possible involvement of F_<CY>RII/III in LPS triggering. 9.Establishment of mCD14 transgenic mice : Transgenic mice expressing membrane or soluble forms of mCD14 mice designated M14M and M14S respectively were established. M14S mice showed significantly higher levels of smCD14 than M14M and control mice. Levels of TNF-alpha and IL-1beta in M14S mice were significantly lowere than those of M14M and control mice. However, lethality in M14S mice after a high dose of LPS administration was indistinguishable from that in M14M and control mice. 10.Production of mCD14 knock out mice : Production of mCD14 knock out mice is now underway. Less

1.兔和大鼠CD 14 cDNA的分离：分离兔和大鼠CD 14（rabCD 14和ratCD 14）cDNA并测序。2.重组CD 14（rCD 14）的制备：在大肠杆菌中表达小鼠CD 14（mCD 14）全长和N端编码氨基酸1 - 71位，以及rabCD 14和大鼠CD 14的后者。3.抗-CD 14抗体的制备：制备多克隆（pAb）和单克隆（mAb）抗-mCD 14抗体。每种mAb检测不同的表位。单克隆抗体rmC 5 -3检测到mCD 14.4的大部分羧基端抗原表位。单克隆抗体的功能特点：rmC 5 -3体外增强LPS诱导的TNF-α的产生。分别命名为rmA 4 -2、rmF 4 -3和rmA 12 -10的单克隆抗体均识别mCD 14中间区的表位，在体外对LPS诱导的TNF-α产生有轻微的抑制作用。由于单克隆抗体的抑制作用较低。我们继续使用上述N-末端蛋白生产单克隆抗体。5. pAb和mAb对体内LPS诱导的TNF-α产生的影响：A ...更多信息 pAb的F（ab '）2的施用显著降低体内LPS诱导的TNF-α产生。然而，mAb的作用尚不清楚。6.可溶性mCD 14（smCD 14）分析：血清smCD 14水平。用单克隆抗体和ELISA法检测LPS刺激前后Kupffer细胞（KC）和腹腔巨噬细胞（Mphi）中mCD 14和细胞因子TNF-α、IL-1 β和IL-6的表达特征：体内LPS刺激后枯否细胞（KC）和腹腔巨噬细胞（Mphi）中mCD 14和细胞因子（包括TNF-α、IL-1 β和IL-6）的mRNA和蛋白水平TNF-α，在LPS刺激后，分析体内枯否细胞（KC）和腹腔巨噬细胞（Mphi）中的IL-1 β和IL-6。少量mCD 14.8可触发细胞因子产生信号。mCD 14与F_<CY>RII/III定位密切，F_<CY>RII/III可能参与LPS触发。9. mCD 14转基因小鼠的建立：建立了分别表达膜或可溶形式mCD 14的转基因小鼠，命名为M14 M和M14 S。M14 S小鼠的smCD 14水平显著高于M14 M和对照小鼠。M14 S小鼠TNF-α和IL-1 β水平显著低于M14 M和对照小鼠。然而，高剂量LPS给药后M14 S小鼠的致死率与M14 M和对照小鼠的致死率无法区分。10. mCD 14敲除小鼠的生产：mCD 14敲除小鼠的生产目前正在进行中。少

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