| In this study, small hairpin RNA (shRNA) plasmid vectors targeting rat classâ…¡major histocompability complex transactivator (Câ…¡TA) gene were constructed. Invitro transfection of rat myeloid dendritic cell (DC) and in vivo transfection of ratspleen were performed to investigate the inhibiting effect on classâ…¡majorhistocompability complex (MHC-â…¡) expression by shRNA interfering Câ…¡TA. AshRNA plasmid with efficient inhibitory effect was chosen for further study. Genetransfection efficiency was confirmed by the direct injection of naked plasmid throughportal vein. High responder model of rat orthotopic liver transplantation wasestablished. Graft with lower immunogenicity for liver transplantation was acquiredfrom the donor that received shRNA plasmid delivered by portal vein injection. Theinhibitory effect of shRNA interfering Câ…¡TA on acute rejection in high respondermodel of rat orthotopic liver transplantation and the underlying 'mechanism wereinvestigated.Partâ… Construction and in vitro Characterization of Câ…¡TAshRNA Plasmid VectorObjective: To construct shRNA plasmid vector targeting rat Câ…¡TA gene anddetermine the inhibiting effect of shRNA on Câ…¡TA and the regulating effect onMHC-â…¡expression by Câ…¡TA-shRNA plasmid in vitro. Methods: In vitro culture of the rat myeloid DC was performed and three shRNA plasmids targeting Câ…¡TA genewere constructed. There were totally 6 groups for DC transfection: threepCâ…¡TA-shRNA experimental groups, control group, pHK-shRNA control group andLipofectamine group. Real time QRT-PCR and flow cytometry (FCM) wereperformed to analyze the expression changes of Câ…¡TA and MHC-â…¡in DC aftertransfection. Results" Sufficient rat myeloid DC were obtained by in vitro culture andCâ…¡TA-shRNA plasmids were successfully constructed and confirmed by DNAsequencing. Compared to control group, pHK-shRNA control group andLipofectamine group, the transcription levels of Câ…¡TA and MHC-â…¡and theexpression level of MHC-â…¡were significantly inhibited in all three pCâ…¡TA-shRNAexperimental groups (P＜0.01). There was positive correlation between the expressionof Câ…¡TA and MHC-â…¡. Among the three pCâ…¡TA-shRNA groups, p Câ…¡TA1-shRNAshowed the strongest inhibiting effect on targeting gene expression. The transcriptionlevels of Câ…¡TA and MHC-â…¡were 10.75±3.31% and 15.25±5.73% and theexpression level of MHC-â…¡was 25.01±5.16%. Conclusion: The expressions ofCâ…¡TA and MHC-â…¡in rat DC are inhibited after pCâ…¡TA-shRNA transfection. Theregulating effect on MHC-â…¡expression by Câ…¡TA were confirmed by the significantpositive correlation between them. shRNA interfering Câ…¡TA significantlydownregulates the MHC-â…¡expression, pCâ…¡TA1-shRNA is more effective ininterfering Câ…¡TA and could be applied for further studies.Partâ…¡In vivo study of the effect of shRNA Plasmid Vectorthrough Portal Vein InjectionObjective: To evaluate the transfection efficiency to liver by direct portal veininjection of naked plasmid vector and determine the inhibiting effect of shRNA onCâ…¡TA and MHC-â…¡expression by Câ…¡TA-shRNA plasmid in vivo. Methods: Normal saline group and plasmid vector groups were established for comparison anddirect portal vein injection was performed to transfect liver. Liver specimens wereharvested on day 1 and day 3 after transfection. Spectrofluorometer was used to detectthe enhanced green fluorescent protein (EGFP) intensity and EGFP expression wasalso determined by fluorescence microscope. Totally 5 groups, including three pCâ…¡TA-shRNA groups, normal saline control group and pHK-shRNA control group,received splenic transfection by portal vein injection. Real time QRT-PCR and FCMwere performed to determine the expression of Câ…¡TA and MHC-â…¡in spleen 3 daysafter transfection. Results: The EGFP intensities in liver of plasmid vectortransfection groups were significantly higher than normal saline group in the twoexperimental time points (P＜0.01) and the intensity showed higher in the day 1than in the day 3 for the plasmid groups (P＜0.01). Compared to normal saline controlgroup and pHK-shRNA control group, the transcription levels of Câ…¡TA and MHC-â…¡and the expression level of MHC-â…¡were significantly inhibited in all three pCâ…¡TA-shRNA groups (P＜0.01). There was apparent positive correlation between theexpression of Câ…¡TA and MHC-â…¡. Among the three pCâ…¡TA-shRNA groups, pCâ…¡TAI-shRNA showed the strongest inhibitory effect on targeting gene expression. Thetranscription levels of Câ…¡TA and MHC-â…¡were 9.241±1.12ï¼…and 17.81±3.61ï¼…andthe expression level of MHC-â…¡was 23.87±5.45ï¼…. Conelusion: Highly efficientliver transfection can be achieved by direct portal vein injection of naked plasmid.The expressions of Câ…¡TA and MHC-â…¡in rat spleen are significantly inhibited afterpCâ…¡TA-shRNA were transfected to spleen in vivo. Câ…¡TA strictly regulates MHC-â…¡expression indicated by the significant positive correlation between them. shRNAinterfering Câ…¡TA significantly downregulates the MHC-â…¡expression in vivo. pCâ…¡TAI-shRNA is the most efficient plasmid here and could be applied in further study. Partâ…¢The study of the Effect of shRNA Interfering Câ…¡TAin High Responder Model of Rat Orthotopic Liver TransplantationObjective: To determine the effect of shRNA interfering Câ…¡TA of graft on acutetransplantation rejection in high responder model of rat orthotopic livertransplantation and investigate its anti-rejection mechanisln. Methods: The highresponder model of rat orthotopic liver transplantation was established. There were 4groups: normal saline control group, pHK-shRNA control group, pCâ…¡TA1-shRNAtreating group and ciclosporin A (CsA) treating group. The donors in the first 3 groupsreceived portal vein injection of normal saline or shRNA plasmid 3 days beforetransplantation. 6 recipients in each group were randomly chosen and sacrificed fortheir liver as specimen 4 days after transplantation and the remaining 6 recipients'were monitored for survival. The data of pathological rejection grading, MHC-â…¡expression level, transcription levels of Câ…¡TA, MHC-â…¡,IL-2 and IFN-γinspecimens were collected by pathological examination, immunohistochemistrystaining and real time QRT-PCR. Results: The high responder, model of rat orthotopicliver transplantation was stably established. Compared to normal saline control groupand pHK-shRNA control group, the survival time of pCâ…¡TA1-shRNA groupextended significantly (median survival time 8 days vs 5 days and 5 days, P＜0.01),pathological rejection grading significantly depressed (P＜0.01), MHC-â…¡expressionlevel significantly decreased (P＜0.01) and transcription levels of Câ…¡TA,MHC-â…¡,IL-2 and IFN-γalso significantly decreased (P＜0.01). All recipients ofCsA treating group showed long-term survival (more than 100 days). Conclusion:High responder model of rat orthotopic liver transplantation is an ideal animal modelfor the study of liver transplantation rejection. The expressions of Câ…¡TA andMHC-â…¡show apparent positive correlation with pathological rejection grading ofliver transplantation. Acute rejection reaction can be significantly relieved and survival time is extended by pretreatment of graft and donor with portal vein injectionof Câ…¡TA-shRNA plasmid. The anti-rejection mechanism of shRNA interfering Câ…¡TAcould be the inhibition of direct immunological recognition due to reduced graftimmunogenicity and depressed secretions of rejection related cytokines.
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