免疫球蛋白A在人肾小球系膜细胞中的合成与分泌

Primary investigation of immunoglobulin A synthesis and secretion in human mesangial cells

目的:探讨人肾小球系膜细胞(human mesangial cells,HMCs)中是否表达免疫球蛋白A(immunoglobulin A,Ig A)。方法:培养人HMCs细胞系,以免疫荧光染色检测Igα、Igκ、Igλ在系膜细胞中的表达;以反转录聚合酶链式反应(reverse transcription polymerase chain reaction,RT-PCR)和DNA测序检测HMCs中Igα、Igκ、Igλ恒定区转录本表达;以Western blot方法检测细胞裂解液及培养上清液中Igα、Igκ、Igλ蛋白水平表达;以jacalin为配体的亲和层析纯化上清液蛋白质,以蛋白质谱检测纯化的蛋白氨基酸序列。结果:免疫荧光染色检测到HMCs细胞浆Igα、Igκ、Igλ的阳性表达;RT-PCR检测到细胞内Igα1、Igα2重链、Igκ轻链、Igλ轻链恒定区转录本表达,且其核苷酸序列与美国国立生物技术信息中心(National Center of Biotechnology Information,NCBI)数据库相应的mRNA序列比对,同源性分别达到99%、97%、98%和97%;Western blot检测到细胞内Igα1、Igα2和Igλ蛋白水平的表达,Igκ目前检测为阴性;细胞上清液中可见完整Ig A分子表达;以jacallin为配体亲和层析纯化的培养上清液中的Ig A,蛋白质谱测序所得的氨基酸片段序列与NCBI数据库比对,显示HMCs分泌的蛋白质与B细胞表达的Igα1恒定区52~104之间53个氨基酸、154~221之间68个氨基酸、以及276~327之间52个氨基酸的序列完全相同,与Igα2重链恒定区52~113之间62个氨基酸、151~204之间54个氨基酸、以及251~314之间64个氨基酸的序列完全相同,提示系膜细胞可以将合成的Ig A分泌到胞外。结论:HMCs可能合成并分泌Ig A。

Objective： To investigate the expression of immunoglobulin A（ Ig A） in human mesangial cells（ HMCs）. Methods： The HMCs were cultured. The subcellular location of Ig A was detected by immunofluorescence staining; the transcripts of Ig α,Ig κ and Ig λ constant region were detected by reverse transcription-polymerase chain reaction（ RT-PCR） and further analyzed by DNA sequencing. The expressions of Ig α and Ig λ were detected at transcription level by Western blot after the cytoplasmic protein extraction. The culture supernatant was collected to explore whether Ig A could be secreted out of the cell and the protein was further analyzed by mass spectrometry after being purified by affinity chromatography with jacalin-sepharose. The results of DNA sequencing and mass spectrometry were aligned with the mRNA and amino acid sequences in the National Center of Biotechnology Information（ NCBI） database. Results： By immunofluorescence staining,we detected the presence of Ig A heavy chain Ig α,light chain,both Ig κ and Ig λ in expressions of transcripts of Ig α1,Ig α2,Ig κ and Ig λ in the HMCs and the alignment of the sequences of the RT-PCR products with those of the Ig Cα1,Ig Cα2,Ig κ and Ig λmRNA in the NCBI database exhibited that the similarities were 99%,97%,98% and 97%,respectively. Western blot showed Ig α and Ig λ expressions in the cell lysate and secretion of Ig α1 and Ig α2 heavy chains in cell culture supernatant. To further explore the protein that secreted into the supernatant,after supernatant affinity chromatography with jacalin-sepharose,the proteins were separated by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis（ SDS-PAGE） and the band approximating to 65 000 was cut and sent to mass spectrometry. The results were aligned with the amino acid sequences of Ig α1 and Ig α2 constant region in NCBI database,showing that amino acids between No. 52 and No. 104,amino acids between No. 154 and No. 221,amino acids between No. 276 and No. 327 from Ig Cα1 and amino acids between No. 52 and No. 113,amino acids between No. 151 and No. 204,amino acids between No. 251 and No. 314 from Ig Cα2 were the same with those derived from B cells. Conclusion： Our findings suggested that HMCs could synthesize and secret Ig A.