Effects Of CD147siRNA Transfection On Cell Proliferation,Activation And Adhesion Activity In The Peripheral Blood T Lymphocytes Of Patients With Psoriasis

ObjectiveTo investigate the effects of CD147siRNA transfection on the expression of CD147 and cell proliferation,the potential of activation and adhesion in the peripheral blood T-lymphocytes of patients with psoriasis vulgaris.To further identify the role and mechanism of CD147 in the progression of psoriasis and investigate the feasibility of using gene therapy of siRNA technology to treat psoriasis with CD147 as the target molecule.Methods1.Peripheral blood mononuclear cell(PBMC)were isolated from ten patients with psoriasis vulgaris by density gradient centrifugation and cultured with staphylococcal enterotoxin B(SEB)and human recombinant interleukin 2(rIL-2)for 5 days,greater than 92%cells were CD3 positive, as flow cytometry(FCM)analysis disclosed;2.The chemical synthetic CD147siRNA was electroporated into the peripheral blood T-lymphocytes of patients with psoriasis vulgaris,and the changes of the expression of CD147 mRNA in transfected cells within 96 hours were measured by semi-quantitive reverse transcriptase-poly merase chain reaction(RT-PCR);3.The change of proliferation level of T-lymphocytes at 24-,48-and 72 h after transfection was detected by MTT assay;4.Flow cytometry(FCM)was used to analyse CD25 expression of the tranfected cells at 24-,48- and 72 h;5.The effect of CD147siRNA on T-lymphocytes adhesion avtivity at 24-,48- and 72 h to extracelluar matrix(ECM)in vitro was detected used the fibronectin-coated flat-bottomed 96-well plates and then the absorbance of the samples was measured at 490 nm with ultraviolet-spectrophotometer.Results1.After the transfection with CD147siRNA 1 within 72 hours,the expression of CD147 in the T-lymphocytes was down-regulated(all P＜0.05);with maximal silencing at 48 h(P＜0.001),the interference effects was(62.87±5.5)%;By 96 h,the expression of CD147 on the transfected cells was nearly same as nontransfected cells(P＞0.05).While CD147siRNA 2 exhibited interference effect on the expression of CD147 mRNA at 48 h,the interference effects was(10.2±3.1)%(P＜0.05),no interference effect was found at 24-,72- and 96 h(P＞0.05);2.The proliferation level of T-lymphocytes at 24,48 and 72 h after transfection decreased significantly(all P＜0.001),the decreased rates were(44.5±3.13)%,(50.7±3.5)%,(53.98±4.15)%respectively;3.24-,48- and 72 h post-transfection CD147siRNA,the number of CD25-expressing cells was reduced by(47.23±3.65)%,(31.5±4.22)%,(23.05±4.15)%respectively(all P＜0.001)compared with untransfected T-lymphocyte cells【(80.2±4.8)%,(81.6±3.35)%,(83.5±4.1)%】;4.CD147siRNA transfection significantly downregulated adhesion activity of T-lymphocytes,the decreased rates of transfected cells at 24-, 48-,and 72 h were respectively(29.8±4.66)%,(43.53±3.88)%,(56.5±4.13)%respectively(P＜0.01,＜0.001,＜0.001).Conclusion1.Transient transfected method of human peripheral blood T-lymphocytes with siRNA was established;2.CD147 is correlated with cell proliferation,activation and adhesion of the peripheral blood T-lymphocytes of patients with psoriasis vulgaris,and it might be a new psoriasis therapy target molecule;...