关键词: 人肾小球系膜细胞, 免疫球蛋白A, 蛋白质谱, DNA测序

Abstract: Objective： To investigate the expression of immunoglobulin A (IgA) in human mesangial cells (HMCs). Methods: The HMCs were cultured. The subcellular location of IgA 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 IgA could be secreted out of the cell and the protein was further analyzed by mass spectrometry after being purified by affinity chromato-graphy 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 IgA 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 λ mRNA 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 fin-dings suggested that HMCs could synthesize and secret IgA.

Key words: Human mesangial cell, Immunoglobulin A, Mass spectrometry, DNA sequencing