ABSTRACT
Objective:To explore the function of tumor derived IgG (tIgG) and whether the tIgG can inhibit T cells activity.Methods:The tIgG was purified from ovarian cancer tissue.The cord blood monocyte cells (CBMC) and cord blood lymphocyte (CBL) were isolate from human umbilical cord blood.The CBMC and CBL were stimulated with phytohaemagg lutinin (PHA) in order to let the CBMC and CBL in the state of proliferation.Carboxyfluorescein succinimidyl amino ester (CFSE) was cultured with CBMC and CBL.CFSE had no cell toxicity,which could penetrate through the cell membrane and combine the intracellular protein.The fluorescence intensity decreased with the proliferation of cells step by step,so the proliferation of these cells could be detected in flow ctytometry.The tlgG which was puri fied from ovarian cancer tissue was divided into three groups,1 mg/L group,10 mg/L group,and 100 mg/L group,and the intravenous immunoglobulin (IVIG) was also divided into three groups too.The CBMC and CBL were treated by tIgG with 1 mg/L,10 mg/L,and 100 mg/L in order to observe the proliferation of T cells.The cells were treated with IVIG as a positive control group,and the cells were treated with phosphate buffer saline (PBS) as a negative control.The proliferation of CD4 + or CD8 + T cells were detected in CBMC and CBL.The proliferation of the T cells in CBMC and CBL after 64 h and 86 h were detected.Results:In the system of CBMC,the tIgG could suppress the proliferation of CD4 + or CD8 + T cells.The results could also be found in the system of CBL.The CD4 + or CD8 + T cells in the group which were treated with PBS were more active than those in the group which were treated with tIgG and IVIG.The suppression in the group which were treated with tIgG,was stronger than that in the group treated with IVIG.In addition,the suppression of T cells in the group which were stimulated with tIgG as 100 mg/L was more effective than that in the group which were stimulated with tIgG as 10 mg/L.This could prove that tIgG had the function of immunomodulation.Conclusion:The tIgG can be involved in immune escape of cancer.
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 Igαt 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 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) data-base.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 findings suggested that HMCs could synthesize and secret IgA.