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.将兔和大鼠CD14 cDNA分离：兔和大鼠CD14（RABCD14和RATCD14）CDNA分离并进行了测序。 2.重组CD14（RCD14）的生产：在E.coil中表达了从小鼠CD14（MCD14）的1到71的全长和N-内部侧面编码氨基酸位置（MCD14）和RABCD14和RatCD14的后者。 3.抗CD14抗体的准备：制备了多克隆（PAB）和单克隆（mAb）抗MCD14抗体。每个mAb都检测到不同的表位。 MAB指定的RMC5-3检测到MCD14.4的大多数羧基终端表位。 MAB的功能特征：RMC5-3在体外增强了LPS诱导的TNF-Alpha产生。 MABS指定RMA4-2，RMF4-3和RMA12-10，所有这些识别MCD14中部区域的表位，在体外略微抑制了LPS诱导的TNF-Alpha产生。由于mAb的抑制作用低。我们将继续使用上述N末端蛋白产生mAb。 5. PAB和MAB在体内对LPS诱导的TNF-Alpha产生的影响：A…对P（AB'）2的更多Dministion在体内显着降低了LPS诱导的TNF-Alpha产生。但是，mAb的作用尚不清楚。 6.固体MCD14的分析（SMCD14）：SMCD14的血清水平。通过使用MABS和ELISA进行蛋白质印迹，分析了TNF-Alpha和IL-1Beta，在施用LPS的前后，分析了MCD14和细胞因子表达和细胞因子的表达和包括TNF-Alpha，IL-1Beta和IL-6的表达的作用。 MCD14和细胞因子在kupffer细胞（KC）中（KC）和腹膜巨噬细胞（MPHI）中的MCD14和细胞因子（包括TNF-Alpha，IL-1Beta和IL-6）在体内TNF-Alpha，IL-1Beta和IL-6中LPS刺激后（MPHI）（MPHI）（KC）和Peroneare的刺激（MPHI）（kC）和iL-6（mphi）（kC）和ir-peroneal maplagages（mphi）（kc）分析。细胞因子产生的信号可能是由少数MCD14.8.MCD14和F_ <cy> RII/III的定位以及F_ <cy> rii/iii可能参与LPS触发的。 9.建立MCD14转基因小鼠：分别建立了指定M14M和M14的MCD14小鼠的转基因小鼠或固体形式。 M14S小鼠的SMCD14水平明显高于M14M和对照小鼠。 M14S小鼠中TNF-Alpha和IL-1BETA的水平明显低于M14M和对照小鼠的水平。然而，高剂量的LPS给药后M14S小鼠的致死性与M14M和对照小鼠的杀伤力是无法区分的。 10. MCD14的生产敲除老鼠：MCD14敲除老鼠的产生正在进行中。较少的

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