Effect On T Cells Proliferation Of Recombinant Adnovirus-mediated HLA-G In Macaca Mulatta Immature Dendritic Cells

Objects:Expression and identification of recombinant adnovirus-mediate macca mulatta HLA-G in macaca mulatta imumature dendritci cells. And to explore the influence on cells proliferation caused by recombinant adnovirus-mediated HLA-G in macaca mulatta immature dendritic cells.Methods:After collecting the a macaca mulatta’s marrow blood, separate mononuclear from the extracted blood by using monkey Ficoll-Hypaque solution. In order to sort and purify positive CD34~+ cells, we use the techonology ofimmunogametic beads sorting with the cultured cells of CD34 positive. We add the culture media to offer cells the nutrition, surely with the help of little does of GM-CSF and IL-4 as well, after 7-days of culturtivation, we get the immature dendritic cells. The above immature dendritic cells were inoculated into different wells with 2.5 x10~5 cells per well. Based on different MOI values, drop the virus into the wells respecetively. At the meanwhile, add cytokines and culture media, observe the the green fluorescent expressions of the target protein in 2 days. Obtain the same immature dendritic cells infected by recombinant adenovirus of HLA-G at the 500～600 MOI and then extract the protein to detect and analysis the expressions of the target protein by Western Blot detection technology. Dividing the simulated cells into five groups,like group DC, group imDC, group LPS~+imDC, group imDC infected by recombinant adnovirus, and group LPS~+ imDC infected by recombinant adnovirus. And add the reacted cells with the different proportion on the basis of simulated cells:reacted cells. Observe and analysis the proliferations of the cells in the wells in the way of statistics at last.Results:By using use the MACS, we get the CD34~+ with high purity and acquire the immature dendritic cells in seven days with the help of nutrition and cytokines as well. These acquired cells are transfected by recombinant adnovirus, we found thatwhen the MOI is between 500 and 600, recombinant adnovirus stimulates stably the immature dendritic cells of macaca mulatta. The specificit stripe, as the same as the target protein, both proved the successfully and stably expression of protein in immature DC. By reserving, we could draw a conclusion that the target protein expression of groups of HLA-G is much higher,as 85.77% and 86.57, when cpmpared with the blank controller. We also can draw a conclusion by analysising the statistics of MLR, applying the pairing T test, which the ability of group imDC infected by recombinant adnovirus, to stimulate T cells is clearly weaker than group mature DCs’, group immature DCs’, group LPS~+imDCs’, let alone group LPS~+ imDC infected by recombinant adnovirus.Conclution:First of all, HLA-G, which was mediated by ecombinant adnovirus, could entered successfully and expressed stably in the immature dendritic cells of the macaca mulatta. And immature dendritic cells could inhinbit the proliferation of T cells better than mature dendrtic cells, while under the help of the LPS, immature ones could better work than the ones without LPS. And because of the infection caused by ecombinant adnovirus, the immature dendritic cells stimulate the T cells to grow more weakly than other groups as we hope. In addition, with LPS, immature DCs infected by ecombinant adnovirus could inhibit the proliferation of T cells too.