CAJ | 학술논문

目的探讨树突状细胞(DC)表面特异的胞间黏附分子3捕获非整合素(DC-SIGN)在免疫介导肾毒血清件肾炎(NTN)肾小管间质损伤中的作用,以及抗P选择素功能域单抗(PsL-EGFmAb)的干预调节.方法 WKY大鼠随机分为正常对照组、模型组及PsL-EGFmAb干预组.模型组注射预制的兔抗大鼠肾毒血清1 nd/kg;PsL-EGFmAb组在注射肾毒血清同时及注射后2 h,注入PsL-EGFmAb 2 mg/kg;正常对照组则注射等量生理盐水.随后于实验第4、7、14天,分别观察大鼠肾功能及肾组织病理变化,并采用免疫荧光法检测肾组织DC-SIGN+DC分布;实时定量PCR检测P选择素、T细胞表达和分泌因子(RANTES)、肿瘤坏死因子α(TNF-α)、白细胞介素(IL)-10、干扰素(IFN)-γ、IL-4的mRNA表达;流式细胞仪检测肾脏分离DC表面主要组织相容性复合体Ⅱ(MHC Ⅱ)、DC-SIGN、CD80表达;细胞迁移试验及混合淋巴细胞反应(MLR)检测DC迁移与刺激T细胞的能力;ELISA法测定MLR上清中IFN-γ、IL-4含量.结果 NTH大鼠第4天起,未成熟DC-SIGN+DC即以肾间质为主浸润并于14 d成熟,且迁移及刺激T细胞增殖能力增强,其肾内分布与新月体形成、肾小管间质损伤程度及肾功能改变呈正相关.此外,大鼠第4天起肾内趋化因子及促炎因子RANTES、TNF-α mRNA表达持续上调,而抗炎因子IL-10 mRNA于第4天明显增强随后呈下调趋势;至14 d时IFN-γ/IL-4 mRNA比值增高,与DC成熟状况呈正相关.经PsL-EGFmAb干预,伴随Dc表面DC-SIGN及相应共刺激分子CD80表达下降,DC成熟、迁移及刺激T细胞增殖能力受抑,肾内促炎因子下降而抗炎因子上调,Th1/Th2偏移受到抑制.同时大鼠肾内新月体形成减少,肾小管间质损伤程度减轻,且肾功能改善.结论 DC-SIGN介导了DC肾间质浸润,并可能是局部免疫反应失衡以及肾小管间质病变的重要调控因素.PsL-EGFmAb在抑制DC迁移的同时可通过靶向DC-SIGN调抑DC成熟及功能,进而发挥防治效应. 目的探討樹突狀細胞(DC)錶麵特異的胞間黏附分子3捕穫非整閤素(DC-SIGN)在免疫介導腎毒血清件腎炎(NTN)腎小管間質損傷中的作用,以及抗P選擇素功能域單抗(PsL-EGFmAb)的榦預調節.方法 WKY大鼠隨機分為正常對照組、模型組及PsL-EGFmAb榦預組.模型組註射預製的兔抗大鼠腎毒血清1 nd/kg;PsL-EGFmAb組在註射腎毒血清同時及註射後2 h,註入PsL-EGFmAb 2 mg/kg;正常對照組則註射等量生理鹽水.隨後于實驗第4、7、14天,分彆觀察大鼠腎功能及腎組織病理變化,併採用免疫熒光法檢測腎組織DC-SIGN+DC分佈;實時定量PCR檢測P選擇素、T細胞錶達和分泌因子(RANTES)、腫瘤壞死因子α(TNF-α)、白細胞介素(IL)-10、榦擾素(IFN)-γ、IL-4的mRNA錶達;流式細胞儀檢測腎髒分離DC錶麵主要組織相容性複閤體Ⅱ(MHC Ⅱ)、DC-SIGN、CD80錶達;細胞遷移試驗及混閤淋巴細胞反應(MLR)檢測DC遷移與刺激T細胞的能力;ELISA法測定MLR上清中IFN-γ、IL-4含量.結果 NTH大鼠第4天起,未成熟DC-SIGN+DC即以腎間質為主浸潤併于14 d成熟,且遷移及刺激T細胞增殖能力增彊,其腎內分佈與新月體形成、腎小管間質損傷程度及腎功能改變呈正相關.此外,大鼠第4天起腎內趨化因子及促炎因子RANTES、TNF-α mRNA錶達持續上調,而抗炎因子IL-10 mRNA于第4天明顯增彊隨後呈下調趨勢;至14 d時IFN-γ/IL-4 mRNA比值增高,與DC成熟狀況呈正相關.經PsL-EGFmAb榦預,伴隨Dc錶麵DC-SIGN及相應共刺激分子CD80錶達下降,DC成熟、遷移及刺激T細胞增殖能力受抑,腎內促炎因子下降而抗炎因子上調,Th1/Th2偏移受到抑製.同時大鼠腎內新月體形成減少,腎小管間質損傷程度減輕,且腎功能改善.結論 DC-SIGN介導瞭DC腎間質浸潤,併可能是跼部免疫反應失衡以及腎小管間質病變的重要調控因素.PsL-EGFmAb在抑製DC遷移的同時可通過靶嚮DC-SIGN調抑DC成熟及功能,進而髮揮防治效應.
목적 탐토수돌상세포(DC)표면특이적포간점부분자3포획비정합소(DC-SIGN)재면역개도신독혈청건신염(NTN)신소관간질손상중적작용,이급항P선택소공능역단항(PsL-EGFmAb)적간예조절.방법 WKY대서수궤분위정상대조조、모형조급PsL-EGFmAb간예조.모형조주사예제적토항대서신독혈청1 nd/kg;PsL-EGFmAb조재주사신독혈청동시급주사후2 h,주입PsL-EGFmAb 2 mg/kg;정상대조조칙주사등량생리염수.수후우실험제4、7、14천,분별관찰대서신공능급신조직병리변화,병채용면역형광법검측신조직DC-SIGN+DC분포;실시정량PCR검측P선택소、T세포표체화분비인자(RANTES)、종류배사인자α(TNF-α)、백세포개소(IL)-10、간우소(IFN)-γ、IL-4적mRNA표체;류식세포의검측신장분리DC표면주요조직상용성복합체Ⅱ(MHC Ⅱ)、DC-SIGN、CD80표체;세포천이시험급혼합림파세포반응(MLR)검측DC천이여자격T세포적능력;ELISA법측정MLR상청중IFN-γ、IL-4함량.결과 NTH대서제4천기,미성숙DC-SIGN+DC즉이신간질위주침윤병우14 d성숙,차천이급자격T세포증식능력증강,기신내분포여신월체형성、신소관간질손상정도급신공능개변정정상관.차외,대서제4천기신내추화인자급촉염인자RANTES、TNF-α mRNA표체지속상조,이항염인자IL-10 mRNA우제4천명현증강수후정하조추세;지14 d시IFN-γ/IL-4 mRNA비치증고,여DC성숙상황정정상관.경PsL-EGFmAb간예,반수Dc표면DC-SIGN급상응공자격분자CD80표체하강,DC성숙、천이급자격T세포증식능력수억,신내촉염인자하강이항염인자상조,Th1/Th2편이수도억제.동시대서신내신월체형성감소,신소관간질손상정도감경,차신공능개선.결론 DC-SIGN개도료DC신간질침윤,병가능시국부면역반응실형이급신소관간질병변적중요조공인소.PsL-EGFmAb재억제DC천이적동시가통과파향DC-SIGN조억DC성숙급공능,진이발휘방치효응.
Objective To explore the role of dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) in the tubulointerstitial lesions of immune-mediated nephrotoxic nephritis (NTN) and the intervention regulation by anti-P-selectin lectin-EGF domain monoclonal antibody (PsL-EGFmAb). Methods WKY rats were randomly divided into control,NTN and PsL-EGFmAb-treated groups. The mrs in NTN group were injected with 1 ml nephrotoxic rabbit serum per kilogram of rat body weight; the ones in PsL-EGFmAb-treated group were injected with 2 mg PsL-EGFmAb per kilogram of rat body weight simultaneously and 2 h later after nephrotoxic rabbit serum injection; and those in control group were injected with equal volume of 0.9% saline. Renal function and pathology were observed at day 4, 7 and 14 after the induction of NTN. Distribution of DC-SIGN + dendritic cells (DCs) in renal tissues was measured by immunofluorescence. Real-time PCR was performed to examine the expression of P-selectin,RANTES, TNF-α, IL-10, IFN-γ and IL-4. Expression of MHC Ⅱ , CD80 and DC-SIGN on dendritic cells was analyzed by flow cytometry. Transendothelial migration was used to detect the ability of DCs migration. DCs ability to activate T cells was determined by mixed lymphocyte reaction (MLR). ELISA was used to detect the concentration of IFN-γ and IL-4 in the supernatant of MLR. Results At day 4, immature DC-SIGN+ DCs infiltrated the rat renal tubulointerstium of NTN group, matured at day 14, and enhanced the ability to migrate and activate T cells. The distribution of DC-SIGN + DCs was significantly related to the form of crescent, tubulointerstial lesions and renal function. In addition, expression of chemokine RANTES and proinflammatory cytokine TNF-α continuously augmented since day 4, while anti-inflammatory eytokine IL-10 decreased after markedly increased at day 4. At day 14, IFN-γ/IL-4 mRNA increased, which was obviously related to DCs maturation. The intervention of PsL-EGFmAb supressed the expression of DC-SIGN and CD80 on DCs, depressed DCs maturation, migration and ability to activate T cells,down-regulated proinflammatory cytokines and up-regulated anti-inflammatory cytokines in kidney,and thus regulated Th1/Th2 bias. At the same time, kidneys showed the decrease of crescents,improvement of tnbulointerstium damage and renal function. Conclusions DC-SIGN may mediate DCs tubulointerstitial infiltration. It may be also a potent regulator of local immune reaction imbalance and pathology of tubulointerstium. PsL-EGFmAb may depress DCs migration and downregulate DCs maturation and function through DC-SIGN, and thus having a role in prevention and treatment.