Simultaneous Silencing CD40 Genes in Macrophages and Dendritic Cells Using a Novel siRNA Delivery System for Immunomodulation During Skin Transplantation
作者单位：The First Hospital and Institute of ImmunologyJilin University Department of MedicineColumbia Center for Translational ImmunologyColumbia University College of Physicians and Surgeons
会议名称：《第十一届全国免疫学学术大会》
会议日期：2016年
学科分类：1002[医学-临床医学] 100210[医学-外科学(含：普外、骨外、泌尿外、胸心外、神外、整形、烧伤、野战外)] 10[医学]
关 键 词：CD40 RNAi nanoparticles immunomodulation
摘 要：Objects: Develop a novel siRNA delivery system to down-regulate the expression of CD40 in macrophages and DCs in vivo and prolong the survival of allogeneic skin ***: In our study, PLGA/si CD40 nanoparticles were prepared by a double emulsion-solvent evaporation technique. An aqueous solution of siRNA(0.2 mg) in 25 μL RNase free water was emulsified by sonication for 30 s over an ice bath in 0.5 ml of chloroform containing 1.0 mg DOTAP and 25 mg m PEG5k-PLGA10 k. This primary emulsion was further emulsified in 5 m L dd H2 O by sonication for 1 min to form a water-in-oil-in-water emulsion. Then, the emulsion was moved into round-bottomed flasks to evaporate the chloroform. The PLGA/FAM-siRNA NPs were added into the medium of DC1.2 and RAW264.7 cells and the cells were collected after 48 h for flow cytometry(FCM) and confocal imaging. Furthermore, DC1.2 and RAW264.7 cells were collected for analysis of CD40 expression by quantitative PCR(qP CR) after co-cultured with PLGA/si CD40 NPs for 48 h. The C57BL/6 mice were sacrificed and PBMCs, splenocytes and bone marrow cells were collected for FCM 16 h after PLGA/Cy5-siRNA NPs i.v. injection. We evaluated the biological effects of PLGA/si CD40 NPs in vivo in PLGA NPs i.v. injection Balb/c to C57BL/6 skin graft ***: PEG-PLGA nanoparticles could deliver siRNAinto macrophages and DCs in vitro, which was demonstrated by both FCM results and confocal imaging. Also, we evaluated the efficiency of down regulation of CD40 expression by PLGA/si CD40 NPs both in vitro and in vivo. In the transient parabiosis skin transplantation model, PLGA/si CD40 NPs could prolong the survival of transplanted skin ***: PEG-PLGA nanoparticles was a proper siRNA delivery system for macrophages and DCs, which can effectively down-regulate the expression of CD40 both in vitro and in vivo, and prolonged the survival of allogeneic skin graft.