In Vitro Investigate The Effection Of Lentivirus-mediated Human DNMT1to CD4~+T Cell In Systemic Lupus Erythematosus

Objective:To study the expression of DNMT1mRNA in CD4+T cell with systemic lupus erythematosus.Methods:Peripheral blood mononuclear cells(PBMCs) from30SLE patients and18healthy controls were isolated by Ficoll-Hypaque density gradient centrifugation, CD4+subsets were isolated by positive selection using Miltenyi beads. Expression of DNMT1mRNA in CD4+T cells was analysed by Real time RT-PCR, the global DNA methylation level was detected by the MethylFlashTM Methylated DNA Quantification Kit (Colorimetric). And the correlation between DNMT1mRNA, global methylation level and systemic lupuserythematosus disease activity index(SLEDAI)were analyzed.Results:The expression of DNMT1mRNA in CD4+T cells from SLE patients were significant diminution than that in control group(p<0.01).Global DNA methylation level in CD4+T cells from SLE patients was lower than the control subjects and inactive SLE(p<0.01). There was direct correlation existed between the expression of DNMT1mRNA and global DNA methylation status (r=0.456, p<0.05) in SLE patients, and a inverse correlation existed between the global DNA methylation level and SLE-DAI(r=-0.45,p<0.05),but no correlation existed between expression of DNMT1mRNA and SLE-DAI (r=-0.196,p>0.05).Conclusions:The espression of DNMT1mRNA were significantly decreased in SLE CD4+T cells, which is associated with demethylation. Objective:To construct a lentiviral epressing vectror carrying human DNMT1gene and then being packaged.Methods:The human DNMT1gene fragment was amplified through PCR, then was connected with lentiviral vector which had been digested and linearized. The resulting lentiviralvector was named pLenti6.3/V5-DNMT1and it was converted by bacterial competent cells.The cloning bacteria was identified by PCR, then the positive plasmids were detected by DNA sequencing.293FT cells were contransfected with lentiviralvector and lentiviralpackaging systems.The titles of concentrated lentivirus was tested by Real-time qPCR.Results:DNA sequencing results demonstrated that the recombinanted lentiviralvector was constructed successfully.The titles of concentrated lentivirus was3.13*1010copies/ml.Conclusion:The recombinanted lentiviral vector was successfully constructed and packaged. Objective:To investigate the effection of pLenti6.3/V5-DNMT1to CD4+T cell from SLE patients in vitro.Methods:The pLenti6.3/V5-DNMTl were transformed into CD4+T cell from SLE patients compare to pLenti6.3/V5-GW/LacZ.Using qPCR, Western blotting, flow cytometry and ELISA to observe the change for expression of DNMT1mRNA, DNMT1protein, the global DNA methylation status and the antibody of IgG. Results:The pLenti6.3/V5-DNMT1showed methylate effect to CD4+T cell in SLE, magnify epression of DNMT1mRNA, DNMT1proteins and the global DNA methylation status,and reduce the antibody of IgG。Conclusions:Overexpression DNMT1can upregulate global methylation and reduce autoreactivity in SLE CD4+T cells.the pLenti6.3/V5-DNMT1can play methylate effect to CD4+T cell in SLE.