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

从分离的肾小球培养了三种人类弥赛亚细胞（HMC），这些细胞是通过筛分技术从三名肾肿瘤患者的正常肾皮质中获得的。通过RT-PCR和Northern印迹分析（NB）检查了这些培养的​​HMC中Fcalpha受体（FCALPHAR）mRNA的表达。通过RT-PCR（而非NB）在仅在一条培养的HMC线上检测到FCalphar mRNA表达。通过RT-PCR和核糖核酸酶保护测定法检查了通过用细胞因子刺激的FCALPHAR mRNA表达的调节。在存在或不存在IL-1，IL-6，IL-8，TNF-ALPHA，PDGF，TGF-BETA和热量聚集的IgA的情况下，将HMC孵育12或24小时。尽管IL-1，IL-6和TNF-Alpha增强了HMC上的FCalphar mRNA表达，但这些变化并不显着。每种细胞因子和孵育时间的剂量不会影响HMC上的LSOF FCALPHAR mRNA水平。FCALPHAR的细胞质多肽表示为E.…更多大肠杆菌中的谷胱甘肽S-转移酶（GST）的融合蛋白。通过用谷胱甘肽Sepharose柱抽象纯化带有GST的融合蛋白。 FCALPHAR的胞质多肽的多克隆抗体是由将重组蛋白免疫的Menae制成的。通过对HMC裂解物和该抗体的免疫沉淀检测到60 kDa。该结果可能表明沉淀物是HMC上的FCALPHAR蛋白。而人类肾脏活检标本是使用这种抗体进行间接免疫荧光染色的，但在膜膜中并未明显地染色FCALPHAR蛋白。 RT-PCR的HMC。因此，在45例IGA肾病和30名健康志愿者中，通过Southern印迹分析检查了FCALPHAR的限制片段长度多态性（RFLP）。 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 HMC上的FCALPHAR可能比中膜中IgA免疫复合物的分解代谢，在IgA介导的信号通路中起着更重要的作用。较少的