采用99Tcm标记的新精氨酸-甘氨酸-天冬氨酸多肽序列显示胶原诱导性关节炎大鼠滑膜血管翳形成的研究

摘要

目的 观察放射性99Tcm标记的新精氨酸-甘氨酸-天冬氨酸多肽序列(99Tcm-3P4-RGD2)在胶原诱导性关节炎(CLA)大鼠体内的分布与显像,探索单光子放射型计算机断层扫描仪(SPECT)下99Tcm-3P4-RGD2成为RA治疗前后监测滑膜血管翳新生血管新标记物的可能性.方法 建立Ⅱ型胶原诱导性鼠关节炎模型,分别设贝伐单抗(bevacizumab,商品名Avastin)治疗组、重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白(rhTNFR:Fc)治疗组和空白对照组,观察治疗前后3组大鼠足垫厚度变化,评估关节炎指数积分和99cm-3P4-RGD2体内分布情况,计算四肢放射性核素平均计数率靶区摄取计数/纵隔摄取计数(T/NT).酶联免疫吸附试验(ELISA)同步检测不同治疗组CIA大鼠治疗前后血清血管内皮生长因子(VEGF)表达水平.治疗完毕后将大鼠处死行后足远端趾间关节病理切片,分别做苏木素-伊红(HE)、番红O染色并评价关节炎病理损伤评分.免疫组织化学法检测3组大鼠滑膜组织中VEGF表达水平,半定量积分法评价VEGF染色结果.统计方法采用配对样本t检验.结果 3组造模成功CIA大鼠四肢关节部位可出现异常放射性浓聚,其值分别为1.40±0.17,1.32±0.20,1.30±0.08,经过贝伐单抗和rhTNFR:Fc治疗后T/NT值均下降,分别为0.43±0.14,0.40±0.12差异有统计学意义(t=17.710,16.812,P＜0.05).随着贝伐单抗和rhTNFR:Fc治疗后,滑膜血管翳新生血管减少,大鼠足垫厚度变化、关节炎指数积分和血清VEGF表达水平均随之下降,其T/NT值与上述指标呈正相关(贝伐单抗治疗组r=0.753,0.800,0.892,P＜0.01；rhTNFR:Fc治疗组r=0.701,0.502,0.481,P=0.001,0.024,0.032).结论 CIA大鼠尾静脉注射99Tcm-3P4-RGD2后,可较好地显示大鼠四肢关节病变部位,99Tcm-3P4-RGD2有可能会成为新型的RA治疗前后监测滑膜血管翳新生血管的显像剂.%Objective To observe the distribution and imaging of radiolabelled arginine-glycineaspartic(RGD)peptide(99Tcm-3P4-RGD2)in collagen-induced arthritis(CIA),and to investigate the possibility of new markers for monitoring the angiogenesis of rheumatoid arthritis(RA)before and after treatment by single photon emission computed tomography(SPECT)labelled with 99Tcm-3P4-RGD2.Methods Type Ⅱ chicken collagen was injected into the tails of Wistar rats to establish arthritis models.The CIA-Wistar rats were randomly divided into three group:the Avastin group,the recombinant human tumor necrosis factor-α receptor Ⅱ group,and the blank control group.The changes of the arthritis index,joint pad thickness and radiological of T/NT before and after treatment were observed.The level of vascular endothelial growth factor(VEGF)in the serum was measured by enzyme-linked immuno sorbent assay(ELISA).The back metapedes pathological section was analyzed by Hematoxylin and Eosin stain(HE)and Safranin O stain as well as arthritic pathology index was also scored.The synovial expression level of VEGF was measured by immuno-histochemistry.The stain level of VEGF was assayed by semiquantitative approach.T test was applied for the comparison of two intergroup samples.Results Abnormal radioactive concentration appeared in the arthritis of the CIA-Wistar rats(1.40±0.17,1.32±0.20,1.30±0.08,P＜0.05).Through the treatment of Avastin and recombinant human tumor necrosis factor-α receptor Ⅱ,the radiological of T/NT decreased significantly(0.43±0.14,0.40±0.12,t=17.710,16.812,P＜0.05).After the treatment with Avastin and recombinant human tumor necrosis factor-α receptor Ⅱ,the severity of synovial pannus,joint pad thickness,arthritis index and the expression of VEGF were all decreased.The radiological of T/NT was positively correlated with the indices mentioned above(r=0.753,0.800,0.892,all P＜0.01 respectively with the the rhTNFR:Fc group)(r=0.701,P=0.001;r=0.502,P=0.024;r=0.481,P=0.032 individually with the rhTNFR:Fc group).Conclusion When 99Tcm-3P4-RGD2 is injected through the tail vein of Wistar rats,it can show the pathological changes of arthritis clearly.99Tcm-3P4-RGD2may be the new eikonogen to monitor the angiogenesis of RA before and after treatment.