RNA干扰CⅡTA控制大鼠肝移植急性排斥反应的实验研究
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摘要
本实验构建针对大鼠Ⅱ类主要组织相容性复合物反式转录激活因子(classⅡmajor histocompability complex transactivator，CⅡTA)基因的短发夹状RNA(small hairpin RNA，shRNA)质粒载体，通过体外转染大鼠骨髓源树突状细胞(dendritic cell，DC)和体内转染脾脏的方法，研究RNA干扰CⅡTA对于Ⅱ类主要组织相容性复合物(classⅡmajor histocompability complex，MHC-Ⅱ)基因表达的抑制效果，并筛选相对高效shRNA质粒；采用经门静脉注射质粒载体的方法体内转染肝脏研究基因转染效果；建立稳定的高应答大鼠原位肝移植模型，采用门静脉注射质粒载体方法干预供体，获得免疫原性减低的移植物用于肝移植，研究RNA干扰CⅡTA在高应答大鼠原位肝移植模型中控制移植排斥反应的作用并探讨其机制。
第一部分CⅡTA-shRNA质粒载体构建及体外研究
目的：构建针对大鼠CⅡTA基因的shRNA质粒载体，探讨体外RNA干扰CⅡTA的抑制效果和对于MHC-Ⅱ基因表达的调控作用，并筛选相对高效shRNA质粒。方法：体外培养大鼠骨髓源DC，构建针对大鼠CⅡTA基因的三个shRNA质粒载体，设置空白对照组、HK-shRNA阴性质粒对照组、转染剂Lipofectamine组和三个CⅡTA-shRNA质粒干预组，分别转染DC，以荧光实时定量PCR和流式细胞术检测DC转染后CⅡTA和MHCⅡ的表达变化。结果：大鼠骨髓源DC培养成功，CⅡTA-shRNA质粒载体构建成功；与空白对照组、HK-shRNA阴性质粒对照组和Lipofectamine组相比，三个CⅡTA-shRNA质粒干预组DC转染后的CⅡTA和MHC-Ⅱ的mRNA转录水平及MHC-Ⅱ抗原表达水平均显著性减低(P＜0.01)，CⅡTA与MHC-Ⅱ基因表达具有明显正相关；pCⅡTA1-shRNA干预组对目的基因表达抑制最为明显，CⅡTA mRNA转录水平为10.75±3.31％，MHC-ⅡmRNA转录水平为15.25±5.73％，MHC-Ⅱ抗原表达水平为25.01±5.16％。
结论：应用成功构建的CⅡTA-shRNA质粒载体转染大鼠骨髓源DC，可显著抑制DC的CⅡTA和MHC-Ⅱ基因表达；CⅡTA与MHC-Ⅱ的基因表达具有明显正相关，说明CⅡTA严格调控MHC-Ⅱ表达，RNA干扰CⅡTA可明显抑制MHC-Ⅱ基因表达：pCⅡTA1-shRNA为相对高效的shRNA质粒，可进一步应用于体内实验和动物实验。
第二部分经门静脉注射质粒载体的体内研究
目的：评价经门静脉注射质粒载体方法体内转染肝脏的转染效果，探讨体内RNA干扰CⅡTA的抑制效果和对于MHC-Ⅱ基因表达的调控作用，并筛选相对高效shRNA质粒。方法：设置生理盐水对照组和质粒载体组，经门静脉注射方法体内干预大鼠肝脏，采取转染后第一天和第三天肝脏标本，应用全波长酶标仪检测和荧光显微镜观察的方法评价体内肝脏转染效果；设置生理盐水对照组、HK-shRNA阴性质粒对照组和三个CⅡTA-shRNA质粒干预组，通过经门静脉注射方法体内干预大鼠脾脏，转染3天后取脾脏标本，以荧光实时定量PCR和流式细胞术检测脾脏转染后CⅡTA和MHC-Ⅱ的表达变化。结果：第一天和第三天质粒载体组的增强型绿荧光蛋白(enhanced green fluorescent protein，EGFP)荧光强度均显著强于盐水对照组(P＜0.01)，质粒载体组第三天荧光强度强于第一天(P＜0.01)；与盐水对照组和HK-shRNA阴性质粒对照组相比，三个CⅡTA-shRNA质粒干预组脾脏转染后的CⅡTA和MHC-Ⅱ的mRNA转录水平及MHC-Ⅱ的抗原表达水平均显著性减低(P＜0.01)，CⅡTA与MHC-Ⅱ基因表达具有明显正相关；pCⅡTA1-shRNA干预组对目的基因表达抑制最为明显，CⅡTA mRNA转录水平为9.24±1.12％，MHC-ⅡmRNA转录水平为17.81±3.61％，MHC-Ⅱ抗原表达水平为23.87±5.45％。结论：经门静脉注射质粒载体方法可使shRNA质粒在体内肝脏获得高效转染；应用成功构建的CⅡTA-shRNA质粒载体体内转染大鼠脾脏，可显著抑制脾脏的CⅡTA和MHC-Ⅱ基因表达，CⅡTA与MHC-Ⅱ基因表达具有明显正相关，说明CⅡTA严格调控MHC-Ⅱ表达，在体内采用RNA干扰技术抑制CⅡTA可明显抑制MHC-Ⅱ基因表达；pCⅡTA1-shRNA为相对高效的shRNA质粒，可进一步应用于动物实验。第三部分RNA干扰CⅡTA在高应答大鼠原位肝移植模型中的实验研究
目的：观察经门静脉注射CⅡTA-shRNA质粒方法干预供体移植物对高应答大鼠原位肝移植模型中移植排斥反应的影响，探讨其抗排斥机制。方法：建立高应答大鼠原位肝移植模型；采用经门静脉注射pCⅡTA1-shRNA方法干预供体大鼠肝脏，干预后3天取移植物进行肝移植，同时设置盐水对照组、HK-shRNA阴性质粒对照组和环孢素A治疗组，每组各12只大鼠，于移植术后第4天随机选取6只大鼠取出移植肝脏，病理学检查评定排斥反应分级，荧光实时定量PCR方法检测标本中CⅡTA、MHC-Ⅱ、IL-2和IFN-γ的mRNA转录水平变化，免疫组化方法检测MHC-Ⅱ抗原表达变化，其余6只用于观察存活期。结果：稳定建立高应答大鼠原位肝移植模型；与盐水对照组和HK-shRNA阴性质粒对照组相比，CⅡTA1-shRNA质粒干预组大鼠存活时间明显延长(中位生存期8天vs5天和5天，P＜0.01)，排斥反应病理分级明显降低(P＜0.01)，CⅡTA、MHC-Ⅱ、IL-2和IFN-γ的mRNA转录水平均明显降低(P＜0.01)，MHC-Ⅱ抗原表达水平明显降低(P＜0.01)。环孢素A治疗组大鼠均获得了长期存活(超过100天)。
结论：高应答大鼠原位肝移植模型是研究肝移植排斥反应的理想动物模型；CⅡTA与MHC-Ⅱ基因的表达与肝移植排斥反应病理分级具有明显正相关，采用经门静脉注射CⅡTA-shRNA质粒方法干预供体移植物可明显减轻肝移植排斥反应，延长存活时间；其机制为移植物免疫原性减低抑制了受体对移植物的直接识别，从而减少排斥反应相关细胞因子的分泌，降低了排斥反应强度。
In this study, small hairpin RNA (shRNA) plasmid vectors targeting rat classⅡmajor histocompability complex transactivator (CⅡTA) 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 classⅡmajorhistocompability complex (MHC-Ⅱ) expression by shRNA interfering CⅡTA. 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 CⅡTA on acute rejection in high respondermodel of rat orthotopic liver transplantation and the underlying 'mechanism wereinvestigated.
PartⅠConstruction and in vitro Characterization of CⅡTAshRNA Plasmid Vector
Objective: To construct shRNA plasmid vector targeting rat CⅡTA gene anddetermine the inhibiting effect of shRNA on CⅡTA and the regulating effect onMHC-Ⅱexpression by CⅡTA-shRNA plasmid in vitro. Methods: In vitro culture of the rat myeloid DC was performed and three shRNA plasmids targeting CⅡTA genewere constructed. There were totally 6 groups for DC transfection: threepCⅡTA-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 CⅡTA and MHC-Ⅱin DC aftertransfection. Results" Sufficient rat myeloid DC were obtained by in vitro culture andCⅡTA-shRNA plasmids were successfully constructed and confirmed by DNAsequencing. Compared to control group, pHK-shRNA control group andLipofectamine group, the transcription levels of CⅡTA and MHC-Ⅱand theexpression level of MHC-Ⅱwere significantly inhibited in all three pCⅡTA-shRNAexperimental groups (P＜0.01). There was positive correlation between the expressionof CⅡTA and MHC-Ⅱ. Among the three pCⅡTA-shRNA groups, p CⅡTA1-shRNAshowed the strongest inhibiting effect on targeting gene expression. The transcriptionlevels of CⅡTA and MHC-Ⅱwere 10.75±3.31% and 15.25±5.73% and theexpression level of MHC-Ⅱwas 25.01±5.16%. Conclusion: The expressions ofCⅡTA and MHC-Ⅱin rat DC are inhibited after pCⅡTA-shRNA transfection. Theregulating effect on MHC-Ⅱexpression by CⅡTA were confirmed by the significantpositive correlation between them. shRNA interfering CⅡTA significantlydownregulates the MHC-Ⅱexpression, pCⅡTA1-shRNA is more effective ininterfering CⅡTA and could be applied for further studies.
PartⅡIn vivo study of the effect of shRNA Plasmid Vectorthrough Portal Vein Injection
Objective: To evaluate the transfection efficiency to liver by direct portal veininjection of naked plasmid vector and determine the inhibiting effect of shRNA onCⅡTA and MHC-Ⅱexpression by CⅡTA-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 pCⅡTA-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 CⅡTA and MHC-Ⅱin 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 CⅡTA and MHC-Ⅱand the expression level of MHC-Ⅱwere significantly inhibited in all three pCⅡTA-shRNA groups (P＜0.01). There was apparent positive correlation between theexpression of CⅡTA and MHC-Ⅱ. Among the three pCⅡTA-shRNA groups, pCⅡTAI-shRNA showed the strongest inhibitory effect on targeting gene expression. Thetranscription levels of CⅡTA and MHC-Ⅱwere 9.241±1.12％and 17.81±3.61％andthe expression level of MHC-Ⅱwas 23.87±5.45％. Conelusion: Highly efficientliver transfection can be achieved by direct portal vein injection of naked plasmid.The expressions of CⅡTA and MHC-Ⅱin rat spleen are significantly inhibited afterpCⅡTA-shRNA were transfected to spleen in vivo. CⅡTA strictly regulates MHC-Ⅱexpression indicated by the significant positive correlation between them. shRNAinterfering CⅡTA significantly downregulates the MHC-Ⅱexpression in vivo. pCⅡTAI-shRNA is the most efficient plasmid here and could be applied in further study.
PartⅢThe study of the Effect of shRNA Interfering CⅡTAin High Responder Model of Rat Orthotopic Liver Transplantation
Objective: To determine the effect of shRNA interfering CⅡTA 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, pCⅡTA1-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-Ⅱexpression level, transcription levels of CⅡTA, MHC-Ⅱ,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 pCⅡTA1-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-Ⅱexpressionlevel significantly decreased (P＜0.01) and transcription levels of CⅡTA,MHC-Ⅱ,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 CⅡTA andMHC-Ⅱshow 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 CⅡTA-shRNA plasmid. The anti-rejection mechanism of shRNA interfering CⅡTAcould be the inhibition of direct immunological recognition due to reduced graftimmunogenicity and depressed secretions of rejection related cytokines.
第一部分CⅡTA-shRNA质粒载体构建及体外研究
目的：构建针对大鼠CⅡTA基因的shRNA质粒载体，探讨体外RNA干扰CⅡTA的抑制效果和对于MHC-Ⅱ基因表达的调控作用，并筛选相对高效shRNA质粒。方法：体外培养大鼠骨髓源DC，构建针对大鼠CⅡTA基因的三个shRNA质粒载体，设置空白对照组、HK-shRNA阴性质粒对照组、转染剂Lipofectamine组和三个CⅡTA-shRNA质粒干预组，分别转染DC，以荧光实时定量PCR和流式细胞术检测DC转染后CⅡTA和MHCⅡ的表达变化。结果：大鼠骨髓源DC培养成功，CⅡTA-shRNA质粒载体构建成功；与空白对照组、HK-shRNA阴性质粒对照组和Lipofectamine组相比，三个CⅡTA-shRNA质粒干预组DC转染后的CⅡTA和MHC-Ⅱ的mRNA转录水平及MHC-Ⅱ抗原表达水平均显著性减低(P＜0.01)，CⅡTA与MHC-Ⅱ基因表达具有明显正相关；pCⅡTA1-shRNA干预组对目的基因表达抑制最为明显，CⅡTA mRNA转录水平为10.75±3.31％，MHC-ⅡmRNA转录水平为15.25±5.73％，MHC-Ⅱ抗原表达水平为25.01±5.16％。
结论：应用成功构建的CⅡTA-shRNA质粒载体转染大鼠骨髓源DC，可显著抑制DC的CⅡTA和MHC-Ⅱ基因表达；CⅡTA与MHC-Ⅱ的基因表达具有明显正相关，说明CⅡTA严格调控MHC-Ⅱ表达，RNA干扰CⅡTA可明显抑制MHC-Ⅱ基因表达：pCⅡTA1-shRNA为相对高效的shRNA质粒，可进一步应用于体内实验和动物实验。
第二部分经门静脉注射质粒载体的体内研究
目的：评价经门静脉注射质粒载体方法体内转染肝脏的转染效果，探讨体内RNA干扰CⅡTA的抑制效果和对于MHC-Ⅱ基因表达的调控作用，并筛选相对高效shRNA质粒。方法：设置生理盐水对照组和质粒载体组，经门静脉注射方法体内干预大鼠肝脏，采取转染后第一天和第三天肝脏标本，应用全波长酶标仪检测和荧光显微镜观察的方法评价体内肝脏转染效果；设置生理盐水对照组、HK-shRNA阴性质粒对照组和三个CⅡTA-shRNA质粒干预组，通过经门静脉注射方法体内干预大鼠脾脏，转染3天后取脾脏标本，以荧光实时定量PCR和流式细胞术检测脾脏转染后CⅡTA和MHC-Ⅱ的表达变化。结果：第一天和第三天质粒载体组的增强型绿荧光蛋白(enhanced green fluorescent protein，EGFP)荧光强度均显著强于盐水对照组(P＜0.01)，质粒载体组第三天荧光强度强于第一天(P＜0.01)；与盐水对照组和HK-shRNA阴性质粒对照组相比，三个CⅡTA-shRNA质粒干预组脾脏转染后的CⅡTA和MHC-Ⅱ的mRNA转录水平及MHC-Ⅱ的抗原表达水平均显著性减低(P＜0.01)，CⅡTA与MHC-Ⅱ基因表达具有明显正相关；pCⅡTA1-shRNA干预组对目的基因表达抑制最为明显，CⅡTA mRNA转录水平为9.24±1.12％，MHC-ⅡmRNA转录水平为17.81±3.61％，MHC-Ⅱ抗原表达水平为23.87±5.45％。结论：经门静脉注射质粒载体方法可使shRNA质粒在体内肝脏获得高效转染；应用成功构建的CⅡTA-shRNA质粒载体体内转染大鼠脾脏，可显著抑制脾脏的CⅡTA和MHC-Ⅱ基因表达，CⅡTA与MHC-Ⅱ基因表达具有明显正相关，说明CⅡTA严格调控MHC-Ⅱ表达，在体内采用RNA干扰技术抑制CⅡTA可明显抑制MHC-Ⅱ基因表达；pCⅡTA1-shRNA为相对高效的shRNA质粒，可进一步应用于动物实验。第三部分RNA干扰CⅡTA在高应答大鼠原位肝移植模型中的实验研究
目的：观察经门静脉注射CⅡTA-shRNA质粒方法干预供体移植物对高应答大鼠原位肝移植模型中移植排斥反应的影响，探讨其抗排斥机制。方法：建立高应答大鼠原位肝移植模型；采用经门静脉注射pCⅡTA1-shRNA方法干预供体大鼠肝脏，干预后3天取移植物进行肝移植，同时设置盐水对照组、HK-shRNA阴性质粒对照组和环孢素A治疗组，每组各12只大鼠，于移植术后第4天随机选取6只大鼠取出移植肝脏，病理学检查评定排斥反应分级，荧光实时定量PCR方法检测标本中CⅡTA、MHC-Ⅱ、IL-2和IFN-γ的mRNA转录水平变化，免疫组化方法检测MHC-Ⅱ抗原表达变化，其余6只用于观察存活期。结果：稳定建立高应答大鼠原位肝移植模型；与盐水对照组和HK-shRNA阴性质粒对照组相比，CⅡTA1-shRNA质粒干预组大鼠存活时间明显延长(中位生存期8天vs5天和5天，P＜0.01)，排斥反应病理分级明显降低(P＜0.01)，CⅡTA、MHC-Ⅱ、IL-2和IFN-γ的mRNA转录水平均明显降低(P＜0.01)，MHC-Ⅱ抗原表达水平明显降低(P＜0.01)。环孢素A治疗组大鼠均获得了长期存活(超过100天)。
结论：高应答大鼠原位肝移植模型是研究肝移植排斥反应的理想动物模型；CⅡTA与MHC-Ⅱ基因的表达与肝移植排斥反应病理分级具有明显正相关，采用经门静脉注射CⅡTA-shRNA质粒方法干预供体移植物可明显减轻肝移植排斥反应，延长存活时间；其机制为移植物免疫原性减低抑制了受体对移植物的直接识别，从而减少排斥反应相关细胞因子的分泌，降低了排斥反应强度。
In this study, small hairpin RNA (shRNA) plasmid vectors targeting rat classⅡmajor histocompability complex transactivator (CⅡTA) 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 classⅡmajorhistocompability complex (MHC-Ⅱ) expression by shRNA interfering CⅡTA. 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 CⅡTA on acute rejection in high respondermodel of rat orthotopic liver transplantation and the underlying 'mechanism wereinvestigated.
PartⅠConstruction and in vitro Characterization of CⅡTAshRNA Plasmid Vector
Objective: To construct shRNA plasmid vector targeting rat CⅡTA gene anddetermine the inhibiting effect of shRNA on CⅡTA and the regulating effect onMHC-Ⅱexpression by CⅡTA-shRNA plasmid in vitro. Methods: In vitro culture of the rat myeloid DC was performed and three shRNA plasmids targeting CⅡTA genewere constructed. There were totally 6 groups for DC transfection: threepCⅡTA-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 CⅡTA and MHC-Ⅱin DC aftertransfection. Results" Sufficient rat myeloid DC were obtained by in vitro culture andCⅡTA-shRNA plasmids were successfully constructed and confirmed by DNAsequencing. Compared to control group, pHK-shRNA control group andLipofectamine group, the transcription levels of CⅡTA and MHC-Ⅱand theexpression level of MHC-Ⅱwere significantly inhibited in all three pCⅡTA-shRNAexperimental groups (P＜0.01). There was positive correlation between the expressionof CⅡTA and MHC-Ⅱ. Among the three pCⅡTA-shRNA groups, p CⅡTA1-shRNAshowed the strongest inhibiting effect on targeting gene expression. The transcriptionlevels of CⅡTA and MHC-Ⅱwere 10.75±3.31% and 15.25±5.73% and theexpression level of MHC-Ⅱwas 25.01±5.16%. Conclusion: The expressions ofCⅡTA and MHC-Ⅱin rat DC are inhibited after pCⅡTA-shRNA transfection. Theregulating effect on MHC-Ⅱexpression by CⅡTA were confirmed by the significantpositive correlation between them. shRNA interfering CⅡTA significantlydownregulates the MHC-Ⅱexpression, pCⅡTA1-shRNA is more effective ininterfering CⅡTA and could be applied for further studies.
PartⅡIn vivo study of the effect of shRNA Plasmid Vectorthrough Portal Vein Injection
Objective: To evaluate the transfection efficiency to liver by direct portal veininjection of naked plasmid vector and determine the inhibiting effect of shRNA onCⅡTA and MHC-Ⅱexpression by CⅡTA-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 pCⅡTA-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 CⅡTA and MHC-Ⅱin 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 CⅡTA and MHC-Ⅱand the expression level of MHC-Ⅱwere significantly inhibited in all three pCⅡTA-shRNA groups (P＜0.01). There was apparent positive correlation between theexpression of CⅡTA and MHC-Ⅱ. Among the three pCⅡTA-shRNA groups, pCⅡTAI-shRNA showed the strongest inhibitory effect on targeting gene expression. Thetranscription levels of CⅡTA and MHC-Ⅱwere 9.241±1.12％and 17.81±3.61％andthe expression level of MHC-Ⅱwas 23.87±5.45％. Conelusion: Highly efficientliver transfection can be achieved by direct portal vein injection of naked plasmid.The expressions of CⅡTA and MHC-Ⅱin rat spleen are significantly inhibited afterpCⅡTA-shRNA were transfected to spleen in vivo. CⅡTA strictly regulates MHC-Ⅱexpression indicated by the significant positive correlation between them. shRNAinterfering CⅡTA significantly downregulates the MHC-Ⅱexpression in vivo. pCⅡTAI-shRNA is the most efficient plasmid here and could be applied in further study.
PartⅢThe study of the Effect of shRNA Interfering CⅡTAin High Responder Model of Rat Orthotopic Liver Transplantation
Objective: To determine the effect of shRNA interfering CⅡTA 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, pCⅡTA1-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-Ⅱexpression level, transcription levels of CⅡTA, MHC-Ⅱ,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 pCⅡTA1-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-Ⅱexpressionlevel significantly decreased (P＜0.01) and transcription levels of CⅡTA,MHC-Ⅱ,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 CⅡTA andMHC-Ⅱshow 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 CⅡTA-shRNA plasmid. The anti-rejection mechanism of shRNA interfering CⅡTAcould be the inhibition of direct immunological recognition due to reduced graftimmunogenicity and depressed secretions of rejection related cytokines.
引文
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