The Study On The Effect Of SiRNA To TypeⅡ Endothulial Cell Activation By Inhibition The NF-κB P65 Protein Expression

Background: Allotransplantation is nowadays a generally accepted treatment for organ failure, and xenotransplantation, i.e. transplantation of cells, tissues or organs between individuals of different species, is considered a promising, possible solution to the chronic lack of donor organs. Organs of phylogenetically-distant species are subject to much more severe and irreversible rejection reactions than that in allograft. At present, delayed xenograft rejection(DXR) is the major immunologic barrier in xenotransplantation. During DXR, typeⅡendothelial cell activation induced by xenoreactive antibody plays a key role in the destruction of xenograft. In this study, siRNA targeting NF-κB p65 was transfected to mouse vascular endothelial cells, to observe the effect of siRNA in inhibiting the expression of NF-κB p65 protein, and the activation of endothelial cell induced by TNF-α. By this way, we investigated the application prospectives of RNAi in xenotransplantation. Methods: 1. The aorta was removed from mice in virtue of operation microscope in bacteria free condition. The vessel was opened longitudinally and cut into 1mm×1mm pieces. These pieces were placed on six-well plates with the intima side down, and a few DMEM containing 20%FBS was added to wells. Cells were identified by morphological (growth characteristic) and immunological (cellular markers) criteria. 2. siRNA directed against mouse NF-κB p65 was designed and was chemically synthesized, The transfection reagent with high transfection effect to EOMA cell was filtrated, then, the transfection condition was optimized in tranfecting siRNA to EOMA cell. 3. EOMA cell was devided in to four groups: (1) blank control group, (2) negative control group, (3) NF-κB siRNA group, (4) scramble siRNA group. One day before transfection, EOMA cell was reseeded in 24 well plate with 0.5 ml of growth medium without antibiotics so that they will be 50-70% confluent at the time of transfection. 0h,24h,48h,72h after transfection, Total RNA was isolated from EOMA cells using TRIzol Reagent, the expression level of NF-κB p65 mRNA was evaluated by RT-PCR. Total protein was isolated from EOMA cells, and the expression level of NF-κB p65 protein was evaluated by Western blot. 4. EOMA cell was devided in to seven groups: (A)blank control group, (B)negative control group, (C)TNF group, (D)siRNA group, (E)scramble siRNA group, (F)TNF+siRNA group, (G)TNF+scramble siRNA group. 24h after transfection , the treatment of TNF-αwas performed , and the expression level of NF-κB p65 protein was evaluated by Western blot, 0h,2h,6h,12h,24h,48h after TNF-αtreatment. 5. EOMA cell was devided in to seven groups: (A)blank control group, (B)negative control group, (C)TNF group, (D)siRNA group, (E)scramble siRNA group, (F)TNF+siRNA group, (G)TNF+scramble siRNA group. The treatment of TNF-αwas performed 24h after transfection, and 6h later, the expression level of E-selectin,ICAM-1,VCAM-1,TF,IL-1 mRNA was evaluated by RT-PCR in each groups. Results: 1. After 60h of explant culture, endothelial cells migranting out of the aortic intima could be seen at the edge by a diavert phase-contrast microscope, and formed confluent monolayers which showed a cobblestone shape 10 days later. The growth of passaged cells was fast than primary cells', it would form confluent monolayers 4-5 days later. Characteristic granular cytoplasmic staining pattern were revealed after immunohistochemical labeling for von Willebrand factor. After passaged four generations, Cells became deformed, detached from the dish. 2. Cationic liposome Lipofectamine?2000 has high transfection to EOMA cells than Lipofectamine?-Plus and jetPEI?. By optimization, the effect of transfectingsiRNA to EOMA cell is over 90% by Lipofectamine?2000 reagent. 3. The low level expression of NF-κB p65 mRNA and protein was detectabled in quiescent endothelial cells; The level of mRNA and protein of NF-κB p65 was depressed 24h after siRNA transfection and was undetectable 48h later. But the level of mRNA and protein of NF-κB p65 was increased after transfection in negative control group and scramble siRNA group. 4. In TNF group,TNF+siRNA group,TNF+ scramble siRNA group, the relative expression of NF-κB p65 protein was increased in EOMA cells at 2h,6h,12h,24h and 48h after stimulation of TNF-αthan that before stimulation(0h). The relative expression of NF-κB p65 protein in EOMA cell of blank control group and TNF group is lower than that of negative control group,scramble siRNA group and TNF+scramble siRNA, but higher than that of siRNA group and TNF+siRNA at 0h. The NF-κB p65 protein is disappeared in siRNA group, but, it is higher in other groups than that in blank control group(p<0.001) at 6h. There were no remarkable difference about expression of NF-κB p65 protein among TNF+siRNA group,negative control group and scramble siRNA group, but it was significantly lower in these groups than TNF group and TNF+scramble siRNA group(p<0.001) at 6h. The results at 24h is as same as that at 6h. 5. The levels of mRNA of E-Selectin,ICAM-1,VCAM-1,TF,IL-1 of EOMA were evaluated by RT-PCR in each group. The level of E-selectin,ICAM-1,VCAM-1,TF,IL-1 in negative control group and scramble siRNA group was increased at 6h, but was not in blank control group. The expression of these genes were significantly suppressed in siRNA group. Due to the stimulation of TNF-α, the expression of these genes were remarkably enhanced in TNF group and TNF+scramble siRNA group. In TNF+siRNA group, the levels of mRNA of E-Selectin,ICAM-1,VCAM-1,TF and IL-1 were higher than that in blank control group, but were lower than that in TNF group and TNF+scramble siRNA group(p<0.001). Conclusion: 1. By explant technique, we successfully accomplished in the isolation,primaryculture and passaged culture of mouse aorta vascular endothelial cells. But, the passages of cell culture were limited . 2. By optimizing the paramater, Cationic liposome Lipofectamine? 2000 could transfect chemically synthesized siRNA to EOMA cells with high effect. 3. Introduction of siRNA to EOMA cells could initiate sequence-specific posttranscriptional silencing and abrogate endogenenous gene expression, scramble siRNA had no effect on expression of target genes. 4. Introduction of siRNA to EPMA cells leads to successful inhibition of TNF-αinduced expression of NF-κB p65. 5. Inhibition of activation of NF-κB p65 by siRNA could efficiently suppress the TNF-αinduced expression of NF-κB's target genes, and suppress the activation of endothelial cells.