小干扰RNA沉默CD柏基因对自身免疫性心肌炎大鼠Th1／Th2与Th17／Treg平衡的影响

Effecof gene silence of CD40 by small interfering RNon balance between Thl/Th2 and Thl7/Treg in ratwith experimental autoimmune myocarditis

目的通过建立自身免疫性心肌炎（EAM）大鼠模型，探讨小干扰RNA（siRNA）沉默CD40基因后对EAM大鼠外周血Th1／Th2与Th17／Treg平衡的影响。方法采用随机数字表法将Lewis大鼠分为正常对照组、EAM组、CD柏siRNA治疗组及siRNA对照组。第0天和第7天，EAM组、CD40siRNA治疗组及siRNA对照组大鼠注射猪心肌肌球蛋白建立EAM模型。第7天，CD40siRNA治疗组大鼠予CD40siRNA治疗。观察各组大鼠基本情况，于第21天记录各组大鼠心电图及超声心动图后处死大鼠，观察心肌组织病理变化并计算病理学积分，应用ELISA法检测大鼠外周血干扰素（IFN）-1、IL-4、IL-17及转化生长因子（TGF）-13水平。结果1．EAM组、CD40siRNA治疗组及siRNA对照组大鼠EAM的总体发病率为100％，且在第0天至第21天均无死亡。2．正常对照组与CD40siRNA治疗组大鼠心电图均正常，EAM组与siRNA组大鼠仅2例出现期前收缩。3．EAM组、CD40siRNA治疗组及siRNA对照组大鼠超声心动图均异常，表现为左心室扩大、左心室壁肥厚、心肌动度减低、心功能明显降低、心包积液。CD40siRNA治疗组大鼠左心室短轴收缩率（LVFS）和左心室射血分数（LVEF）显著高于EAM组[（46．70±8．25）％比（34．28±11．81）％，（80．92±11．76）％比（64．03±12．60）％]，差异均有统计学意义（F：0．652、0．234，P均〈0．05）。4．EAM组、CD40siRNA治疗组及siRNA对照组大鼠心肌组织病理检查显示心肌纤维断裂、心肌细胞坏死及炎性细胞浸润。CD40siRNA治疗组心肌组织病理学积分明显低于EAM组（2．10±1．07比3．40±0．72），差异有统计学意义（F=1．290，P〈0．05）。5．与正常对照组比较，EAM组大鼠外周血IFN-Y IL-4、IL-17及TGF-p水平均显著升高[（1245．55±244．56）ng／L比（501．53±18．93）rig／L，（78．03±10．47）ng／L比（30．77±1．74）ng／L，（80．82±13．33）ng／L比（27．98±3．01）ng／L，（156．27±12．11）ng／L比（4．33±0．79）ng／L]，差异均有统计学意义（t=7．408、10．764、9．250、30．608，P均〈0．05），CD40siRNA治疗组大鼠外周血IFN-1与IL-17水平较EAM组显著降低[（940．62±128．40）ng／L比（1245．55±244．56）ng／L，（60．42±12．40）ng／L比（80．82±13．33）ng／L]，差异均有统计学意义（t=2．704、2．745，P均〈0．05），IL．4与TGF-β水平显著升高[（97．91±13．62）ng／L．比（78．03±10．47）ng／L，（178．84±12．10）ng／L比（156．27±12．11）ng／L]，差异均有统计学意义（t=2．835、3．229，P均〈0．05）。结论CD40siRNA可明显减轻EAN大鼠心肌炎症，改善心功能，其机制可能与其降低Th1与Th17相关细胞因子水平，上调Th2与Treg相关细胞因子的水平，从而纠正Th1／Th2与Th17／Treg失衡有关。

Objective To investigate how gene silence of CD40 by small interfering RNA（siRNA） influencethe balance between Thl/Th2 and Th17/Treg in ratwith experimental autoimmune myocarditis（ EAM ）. MethodLewiratwere divided into normal - control group, EAM group, CD4o siRN- therapy group and siRN- control group by using random numbetable. EAM, CD4o siRN- therapy and siRN- control groupwere immunized on day 0 and day 7 with purified porcine cardiamyosin to establish EAM, and CD40 siRNwaadministered intravenously on day 7. The basidatof each group were observed,and the ratwere executed on day 21 to study the pathology of myo- cardial tissueand the myocardial histopathology scorewere calculated, and than the changein electrocardiogram（ECG） and echocardiogram were recorded. Enzyme -linked immunosorbenassay （ELISA） waused to determine the levelof interferon （IFN） - ~/, interleukin （IL） - 10, IL - 17 and transforming growth facto（TGF） - 13 in peripheral blood. Result（ 1 ） The overall incidence of EAM, CD4o siRN- therapy and siRN- control group wa100% , and no ratdied from day 0 to day 21. （2） There were only 2 premature ratin EAM and siRN-control groups,and the ECGof normal -control group and CD4osiRN-therapy group were normal. （3） The echoeardiogram of EAM, CD4o siRN- therapy and siRN- control group, displayed the lefventriculadilatation, hypertrophy of the lefventricle,the weakening of the lefventriculawall motion, and hearfailure, and pericardial effusion wadiscovered. The lefven- trieulafractional shortening（LVFS） and the lefventrieulaejection fraction （LVEF） of rattreated by CD40iRNwere highethan those of ratin the EAM group[ （46.70 ± 8.25 ）% v（34.28 ± 11.81 ）%, （80.92 ± 11.76）% v（64, 03 ±12.60 ） %, F = 0. 652,0. 234, all P 〈 0.05 ]. （ 4 ） Pathologiexamination of the EAM group, CD40 siRNtherapy group and siRNcontrol groups, showed myocardial fibefracture, myocardial tissue necrosiand inflammatory cell infiltration. Bumyocardial tissue pathological scoreof the cardiatissue of CD4o siRNtherapy group walowethan those of the EAM group（2.10 ± 1.07 v3.40 ±0.72 ,F = 1. 290,P 〈0.05）. （5） ELISmethod examination re- vealed thathe levelof IFN -Y, IL -4, IL - 17 and TGF - 13 in EAM group were increased compared with those of normal control group （1 245.55 ±244.56） ng/L v（501.53 ± 18.93） ng/L, （78.03 ± 10.47） ng/L v（30.77 ± 1.74） ng/L, （80.82 ± 13.33） ng/L v（27.98 ± 3.01 ） ng/L, （ 156.27 ± 12.11 ） ng/L v（4.33 ± 0.79） ng/L, = 7. 408,10. 764,9. 250,30.608, all P 〈 0.05 ]. However, the levelof IFN -Y/and IL - 17 in CD40 siRNtherapy group were lowethan those in the EAM group [ （940.62 ± 128.40 ） ng/L v（ 1 245.55 ± 244.56 ） ng/L, （60.42 ± 12.40 ） ng/L v（80.82 ± 13.33） ng/L,=2.704,2.745 ,all P 〈0.05] ,while the levelof IL -4 and TGF -Y were highe[ （97.91±13.62） ng/L v（78.03 ±10.47） ng/L,（178.84 ±12.10） ng/L v（156.27 ± 12.11） ng/L,=2. 835, 3. 229 ,all P 〈 0.05 1. CoelusiouThe dmiuistration of CD40 iRNmarkedly reducemyocardial inflammation of EAM ratand improvetheicardiafunction,which ean be explained by Thl - type and Thl7 - type cytokineinhibi- ted,Th2 -type and Treg- type eytokineincreased,and so then the imbalance of Thl/Th2 and Thl7/Treg corrected.