Study Of The Effects Of CD59, CD55 On Lipid Rafts-mediated Singnal Transduction Of T Cell

Objectives:To construct recombinant vector pSUPER-siCD55 pSUPER-siCD59 with siRNA technique. Detect the changes of T cell signal after transfected with pSUPER-siCD55 or pSUPER-siCD59, and investigate the synergistic effects of glycosylphosphatidyl inositol (GPI)-anchored protein CD59, CD55 in lipid raft-mediated transduction.Methods:recombinant vector pSUPER-siCD55 pSUPER-siCD59 was constructed and subsequently identified by PCR, restrictiion endonuclease reaction and gene-sequencing methods. Jurkat cells were transfected with blank plasmid (Ⅱgroup),pSUPER-siCD59 (Ⅲgroup) and pSUPER-siCD55 (Ⅳgroup) respectively using Lipofectamne2000. Stable expression clones were selected by the addition of G418. RT-PCR were used to detect CD59 and CD55 mRNA level in untransfected Jurkat cells (Ⅰgroup),Ⅱ.ⅢandⅣgroup. After crosslinking of CD55 and CD59 antibodies, T cell proliferation was measured by MTT assay (lymphocyte transformation test), the phosphorylation levels of Src family of protein tyrosine kinase (Src PTK) were investigated by Western Blot, and the kinetic changes of [Ca2+] in T cell endochylema were detemined by Laser scanning confocal microscope imaging.Results:The result of electrophoresis of PCR, enzyme digestion and sequence demonstrated that the target sequence had been inserted into the vector. Jurkat cells transfected with pSUPER-siCD59 and pSUPER-siCD55 expressed green fluorescent protein. Resistant cells were obtained under the pressure of G418. RT-PCR demonstrated mRNA expression of CD59 inⅡgroup and CD55 inⅢgroup were significantly inhibited(P<0.05), Compared withⅠ,Ⅱgroup. After crosslinking anti-CD55 mAb and anti-CD59 mAb, the proliferation of lymphocyte, Src PTK protein phosphorylation bands gray value and the degree of the [Ca2+]ⅠinⅠgroup was evidently increased compareing withⅡ,Ⅲgroup (P< 0.05), but that inⅡgroup was lower thanⅢgroup (P<0.05). There was no difference betweenⅠgroup andⅡgroup (P>0.05).Conclusions:Recombinant vector pSUPER-siCD55 and pSUPER-siCD59 respectively inhibited the expression of CD55 and CD59. Experiment demonstrated that co-expression of two proteins(CD59,CD55) are more effective in cell proliferation,Src phosphorylation and inducing an acute rise in [Ca2+]ⅰ. otherwise, CD59 play an important role. Hence, CD59 and CD55 have synergistic effects in signal transduction of T cell activation, which may provide a very promising strategy to study the mechanism of GPI(glycosylphosphatidyl inositol)anchored protein-mediated T cell signal transduction. The research opens up a new way for gene targeting treatment of T cell leukemia that has important theoretical significance and clinical application prospects.