摘要
目的探讨自噬对放射性碘125标记抗Toll样受体5(^(125)I-anti-TLR5)靶向同种移植排斥显像的影响及特点。方法建立小鼠皮肤同种移植模型,分为对照组(无药物处理)、雷帕霉素组、3-甲基腺嘌呤(3MA)组和联合组(雷帕霉素+3MA处理)。采用Iodogen法常规制备^(125)I-anti-TLR5,测定其在生理盐水和血清中的稳定性;体外根据浓度不同将细胞分为对照组、雷帕霉素组(10 ng/mL)、3MA组(10 mmol/L)和联合组(10 ng/mL雷帕霉素+10 mmol/L3MA),研究^(125)I-anti-TLR5与处理细胞的结合与解离;在移植后9 d,在小鼠同种皮肤移植模型尾静脉注射^(125)I-anti-TLR5,72 h后进行生物学分布研究,24、48、72 h进行全身磷屏自显影显像,分析T/NT比值(靶与非靶比值);采用病理及免疫组化染色法分析移植皮片Beclin1/TLR5的表达。结果^(125)I-anti-TLR5标记率高,稳定性好。与雷帕霉素组相比,联合组细胞与^(125)I-anti-TLR5结合率与解离率无明显改变。注射^(125)I-anti-TLR5后72 h,标记物主要经肝肾代谢。移植皮片放射性浓聚,雷帕霉素组和联合组T/NT比值显著高于对照组(P<0.001,P<0.001)。全身动态磷屏放射自显影显像显示,各组48 h移植皮片即可显像,其中雷帕霉素组移植皮片放射性浓聚最明显(P<0.001);雷帕霉素组和联合组72 h显像明显,放射性活度比均显著高于对照组(P<0.001)。移植后12 d对照组移植皮片炎症细胞浸润明显,而雷帕霉素组与联合组炎性浸润减少;对照组及雷帕霉素组自噬分子Beclin1表达增高,而联合组无明显降低;雷帕霉素组及联合组TLR5表达无显著变化。结论自噬抑制对雷帕霉素作用下TLR5表达及^(125)I-anti-TLR5靶向同种移植排斥显像无明显影响,^(125)I-anti-TLR5可用于免疫耐受状态下同种移植排斥显像。
Objective To evaluate the effect of autophagy on targeting imaging of allotransplanted skin graft by ^（125）I-antiTLR5.Methods Mouse alloskin transplantation models were established and divided into 4 groups：control group,rapamycin group,3-Methyladenine（3MA） group,and rapamycin ＋ 3MA group.Anti-TLR5 was labeled with ^（125）I by Iodogen method,and stability in normal saline and serum was determined respectively.Spleen cells were divided into control group,rapamycin group（10 ng/mL）,3MA group（10 mmol/L） and rapamycin ＋ 3MA group（10 ng/mL rapamycin ＋ 10 mmol/L 3MA）.Then the uptake and dissociation of ^（125）I-anti-TLR5 were studied.^（125）I-anti-TLR5 was injected to transplantation models through tail veins on day 9 after allotransplantation.Biodistribution at 72 h after ^（125）I-anti-TLR5 injection and whole-body phosphor-autoradiography at 24 h,48 h,and 72 h were performed.T/NT ratio（target to non-target） was analyzed.Beclin1/TLR5 expression was detected with histological and immunohistochemical staining.Results ^（125）I-anti-TLR5 was successfully obtained with good labeling rate and stability.The uptake and dissociation of tracer were not apparently changed in rapamycin ＋ 3MA group compared with rapamycin group.Biodistribution studies showed that ^（125）I-anti-TLR5 was mainly metabolized through the liver and kidney,and high radioactivity in grafted skin was detected.The T/NT ratio in rapamycin group and rapamycin ＋ 3MA group was significantly higher than that in control group（P〈0.001,P〈0.001）.Whole body phosphor-screen autoradioimaging showed clear skin grafts radioactivity-imaging in rapamycin group at 48 h compared with the control group（P〈0.001）.Radioactivity activity ratio of rapamycin group and rapamycin ＋ 3MA group remarkably increased（P〈0.001） at 72 h.Allograft histology assay showed that acute inflammatory infiltration was decreased in rapamycin group and rapamycin ＋ 3MA group.The expression of Beclin1 was not significantly decre
出处
《山东大学学报（医学版）》
CAS
北大核心
2017年第9期46-52,共7页
Journal of Shandong University:Health Sciences
基金
国家自然科学基金(81371601)
