ROLE OF Fcalpha-RECEPTOR IN IgA IMMUNE COMPLEX DEPOSITS ON MESANGIUM

Fcalpha 受体在系膜上 IgA 免疫复合物沉积中的作用

• 批准号: 05670961
• 负责人: MATSUMURA Osamu
• 金额: $ 1.41万
• 依托单位: SAITAMA MEDICAL SCHOOL
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 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/en/grant/KAKENHI-PROJECT-05670961/
• 关键词: Mesangial Cell; Fcalpha-receptor; IgA immune-complexes; RT-PCR; Fusion Protein with GST; RFLP; Fcα-receptor; 糸球体メサンギウム細胞; RPA法; RT-PCR法; Fcalpha-receptor; サイトカイン

Three lines of human mesangial cells (HMC) were cultured from isolated glomeruli, which were obtained from the normal renal cortices of three patient with renal tumor by sieving techniques. Fcalpha-receptor (FcalphaR) mRNA expression in these cultured HMC was examined by RT-PCR and northern blot analysis (NB). The FcalphaR mRNA expression was detected on an only one line of cultured HMC by RT-PCR but not NB.Modulation of the FcalphaR mRNA expresson by stimulation with cytokines was examined by RT-PCR and ribonuclease protection assay. HMC were incubated in the presence or absence of IL-1, IL-6, IL-8, TNF-alpha, PDGF,TGF-beta and heat aggregated IgA for 12 or 24 hours. Although IL-1, IL-6 and TNF-alpha enhanced the FcalphaR mRNA expression on HMC,these changes were not significant. Doses of each cytokine and incubated times did not affect the leve lsof FcalphaR mRNA on HMC.The cytoplasmic polyeptide of FcalphaR was expressed as a fusion protein with glutathione S-transferase (GST) in E. … More coli. The fusion protein with GST was purified by absorption with glutathione sepharose column. A polyclonal antibody to the cytoplasmic polypeptde of FcalphaR was made by menae of immunazed the recombinant protein into a white rabbit. The 60 kDa band was detected by immunoprecipitation of HMC's lysates and this antibody. This result may suggest that the precipitates were the FcalphaR proteins on HMC.While human renal biopsy specimens were performed indirect immunofluorescent staining using this antibody, the FcalphaR protein was not distinctly stained in mesangium.A silent one point mutation (C*T) was found on sty I site (670 base) of FcalphaR cDNA fragments, which amplified from cultured HMC by RT-PCR.Therefore, restriction fragment length polymorphism (RFLP) of FcalphaR were examined by southern blot analysis in 45 patients with IgA nephropathy and 30 healthy volunteers. However, RFLP of FcalphaR with Sty I digestion were not recognized.In this study, the expression of FcalphaR gene and protein were determined in cultured HMC.The expression of FcalphaR on HMC was scanty and unstable, and cytokines did not change the FcalphaR mRNA levels on HMC.It is suggested that HMC express constitutively low levels of FcalphaR mRNA,and FcalphaR on HMC may play more important roles of the IgA-mediated signaling pathways than the catabolism of IgA immune complexes in mesangium. Less

采用筛分法从3例肾肿瘤患者的正常肾皮质分离肾小球，培养人系膜细胞（HMC）。RT-PCR和北方印迹分析（NB）检测FcalphaR mRNA的表达。RT-PCR检测到FcalphaR mRNA的表达，而NB检测不到。RT-PCR和核糖核酸酶保护试验检测了细胞因子刺激对FcalphaR mRNA表达的调节。将HMC在存在或不存在IL-1、IL-6、IL-8、TNF-α、PDGF、TGF-β和热聚集伊加的情况下孵育12或24小时。IL-1、IL-6和TNF-α均能促进HMC FcalphaR mRNA的表达，但这些变化并不显著。FcalphaR的胞质多肽在E. coli中表达为与谷胱甘肽S-转移酶（GST）的融合蛋白。 ...更多信息 杆菌GST融合蛋白经谷胱甘肽琼脂糖凝胶柱层析纯化。将重组蛋白免疫白色家兔，制备了抗FcalphaR胞浆多肽的多克隆抗体。通过HMC裂解物和该抗体的免疫沉淀检测60 kDa条带。用该抗体对人肾活检标本进行间接免疫荧光染色，发现系膜区FcalphaR蛋白染色不明显，在sty Ⅰ位点发现一个沉默的单点突变（C*T（670个碱基）的FcalphaR cDNA片段，其通过RT-PCR从培养的HMC扩增。应用Southern印迹法对45例伊加肾病患者和30例正常人的FcalphaR基因进行了限制性片段长度多态性（RFLP）分析。本研究测定了体外培养的HMC中FcalphaR基因和蛋白的表达，结果表明，HMC中FcalphaR基因表达量很少且不稳定，细胞因子不能改变HMC中FcalphaR mRNA的表达水平，提示HMC组成性低表达FcalphaR mRNA，FcalphaR在IgA介导的信号通路中的作用可能比伊加免疫复合物在系膜区的催化作用更重要。少