Expression And Identification Of Recombinant Adenovirus Mediated Gene Silencing MyD88siRNA In Macaca Mulatta Immature Dendritic Cells

[Object]Expression and identification of macaca mulatta MyD88gene silencing siRNA recombinant adenovirus vector in macaca mulatta immature dendritic cells in the expression and to explore the influence of rhesus MyD88gene silencing siRNA adenovirus carrier infected immature dendritic cells of rhesus to T cell immune response.[Methods]In the case of ntravenous anesthesia,extract marrow blood of rhesus.Using lymphocyte separation medium separate mononuclear from the extracted marrow blood.Then using immune magnetic bead kits and the magnetic poles positive sorting positive CD34+cells in order to purify it.Add medium with cytokine GM-CSF and cytokine IL-4to the cells. The above positive CD34+cells were cultured and induced to7days,were inoculated in24pore plates with2.5×105cells per pore.Then add the virus respectively according to the MOI for100,200,300,400,500and600.Observe expressions of the green fluorescent protein in48hours.Culture and induce the first part of positive CD34+cells to the7th day,infect them with recombinant adenovirus for48hours according to MOI500-600and then extract the protein.Detect expressions of the protein by Western Blot detection technology. Let the lymphocyte cells mixed and stimulate them in different ways.Divide the stimulated cells as group DC,group imDC,group gene silencing imDC,group LPS+imDC and group LPS+gene silencing imDC. Detect T cells,which were used as reacted cells,by Flow cytometry.Then add the reacted cells with the proportion of1:5,1:10,1:20and1:40on the basis of stimulated cells:reacted cells. Finally,analysis proliferations of T cells through the statistics.[Result]The immune magnetic bead method has a higher purity, and imDC will be matured in4days through stimulated by cytokine. The MOI of recombinant adenovirus carrier,witch is stable stimulate in the immature dendritic cells of rhesus is500-600. The appearing specificity stripe in33kD by Western Blot detection and the appearing expression of β-actin in43kD both have proved the successful expression of protein in imDC and the protein expression of recombinant adenovirus carrier in imDC is significantly lower than that in blank controller and negative controller. By analysis the result,we have found that the protein expression of MyD88disturb controller dropped73.5%compared with the blank controller, and dropped80.6%compared with the negative controller. It is meaningful that the purify of T cell separated by Nylon Wool Fiber column method was more than80%, which can be reacted cells to do experiment. In addition, the conclusion by statistical results of mixed lymphocyte culture has shown that ability of group imDC witch stimulated T cells is obviously lower than group mDC, and the imDC,witch infected by recombinant adenovirus,has the lowest ability to stimulate T cells combined with any other groups.[Conclution]DC can be purified by immuno magnetic bead method, the differentiation from DC into imDC was induced by cytokines; When MOI is500-600, Recombinant adenovirus vector has the best infection efficiency in the imDC of Rhesus Macaque; The protein expression of rhesus MyD88gene silencing siRNA was significantly inhibited in the mucaca mulatta imDC; Recombinant adenovirus infected imDC inhibits the proliferation of T cells.[Prospect]Infect the immature dendritic cells by using recombinant adenovirus, then inject the infected imDC into the body through portal vein during the vivo experiment. In this way, we hope it can induce the immune tolerance, and then create a new path to transplant filed.