Efficient The Mice Cd40sirna Gene Fragment Screening And Lymphocyte Cd40 Gene Expression

Objective:To select highly effective mouse CD40 small interfering RNA (siRNA) by the technology of interfering RNA in the mediation of Lipofectamine RNAiMAX via reverse transfection. For preventing and limiting occurrence and progress of the atherosclerosis and atherosclerosis complication by CD40siRNA.Methods:(1) spleen lymphocytes of mouse were collected and culturedTo select 8-10 week female BALB/c spleen in asepsis condition, after grinding and filtering lightly, collected lymphocytes with density gradient centrifugation by Ficoll.And then ablution lymphocytes by PBS twice. Cultured cell in RPMI-1640 contained 10% calf serum, To count the number of cells and cytoactive were measured by trypan blue dyeing.(2) designed and chemically synthesized siRNAsOn the basis of the CD40 cDNA of mouse provided with GenBank (GenelD: 21939),We applied routine siRNA design methed to bolting three target sequence which have not homology with other gene by GenBank Blast respectively.And then chemically synthesized three siRNAs with 19nt and dTdT3'targeting on CD40 of mouse and a nonspecific siRNA which have not mouse gene was synthesized in the same way and was served as control; meanwhile, Cy3-siRNA was synthesized and would be detected transfection efficiency.(3) Choice the transfection methed can raise the transfection efficiencyChemically synthesized siRNAs marking Cy3 were transfected into lymphocytes with Lipofectamine RNAi-MAX via reverse transfection and forward transfection.After 24h of transfection, cytoactive and cytes survival rate of each group was observation via microscope respectively, while transfection efficiency was assessed by fluorescence microscope and flow cytometry for comparing with the transfection efficiency of two ways and searching the more efficient transfection method.(4) highly effective selection of mouse CD40 siRNA There are five groups in our experiment, three siRNAs targeting on CD40 of mouse siCD40-1,siCD40-2 and siCD40-3 served as experimental groups, while nonspecific siRNA and untreated cell served as the control separately. We transfected siRNAs into lymphocytes with Lipofectamine RNAi-MAX via reverse transfection which was selected in prophase experiment.(5) The effect of siRNA on the expression of CD40 in mouse lymphocytesThe expression of CD40 and CD40 mRNA in lymphocytes were measured by flow cytometry and Real-time fluorogentic quantitative PCR (RFQ-PCR) respectively after 24h of transfection. The expression of CD40 protein levels were assessed by Western blot after 48h of transfection, To select highly effective mouse CD40 siRNA which could specificly down-regulate the expression of CD40 mRNA and protein in mouse lymphocytes.Results:(1) cytoactive and cytes survival rateAfter 24h of transfection, trypan blue dyeing showed cytoactive of reverse transfection,forward transfection and untreated cell group were 89.5%,84.5% and 94.9% respectively; meanwhile, cytes survival rate of each group was observation via microscope respectively The results showed there was no significant difference in every group.(2) Assessed transfection efficiency of various methodThere were red fluorescence can be found in reverse transfection and the forward transfection After 24h of transfection,as the blank group was not viewed red fluorescence.The transfection rate of reverse transfection and the forward transfection were 66.5% and 34.5% respectively by fluorescence microscope, and were 63%and 29% via flow cytometry, the difference between the two groups was significant.(P< 0.05).(3) The expression of CD40 were measured by flow cytometryCompared with the experimental and controls, siRNA groups showed significantly down-regulation of CD40 by FCM.The inhibitory rate of CD40 expression for siCD40-2 was 85%, siCD40-1 and siCD40-3 were 79% and 41% respectively. There was no significant difference found in controls.(4) The expression CD40 mRNA were evaluated by QRT-PCR The remarkably decrease of the expression CD40 mRNA for siCD40-2 was 70%, as siCD40-1 and siCD40-3 were 62%,32% respectively.the highly effect siRNA is siCD40-2. Whereas the expression of CD40 mRNA of controls were not remarkably degression(5) The expression of CD40 protein levels were detected via western blotAfter 24h of siRNA transfection, siRNA group showed significantly down-regulation of CD40 protein in lymphocytes, The total inhibitory rate of CD40 expression for siCD40-1,siCD40-2,siCD40-3 were 65%,71% and 46%, however,CD40 protein in the lymphocytes which transfected siRNA-co and untreated were not changed.Conclusions:(1) Compared with the Forward transfection, Reverse transfection could obviously improve transfection efficiency in mouse lymphocytes and was more conveniently.meanwhile, cytoactive and cytes survival rate was no significant difference in every group.(2) Chemically synthesized siRNA targeting on CD40 of mouse could specificly down-regulate the expression of CD40,CD40 mRNA and protein in mouse lymphocytes. siRNA-2 has the highly effective inhibition.