CAJ | 학술논문

目的采用99Tcm-3聚乙二醇4-RGD2(3P-RGD2)评估小细胞肺癌和肺腺癌细胞荷瘤鼠动物模型中整合素αvβ3表达水平.方法按试剂盒说明书制备99Tcm-3P-RGD2.选取H446人小细胞肺癌细胞进行受体竞争抑制实验,检测3P-RGD2与整合素αvβ3的特异亲和性.通过细胞摄取实验检测H446和A549人肺腺癌细胞对99Tcm-3P-RGD2的摄取情况,以流式细胞术和免疫荧光染色测定2种细胞中整合素αvβ3的表达.观察99Tcm-3P-RGD2在H446和A549肺癌荷裸鼠模型(各6只)的microSPECT/CT显像情况.显像后断颈处死裸鼠,取部分肿瘤组织制备单细胞悬液,以流式细胞术检测细胞整合素αvβ3表达;部分组织制成切片,以免疫组织化学法检测肿瘤组织整合素αvβ3的表达.采用配对t检验对实验数据进行统计学分析.结果 99Tcm-3P-RGD2标记率为(97.0±2.0)％,4h时后放化纯仍高达95％.3P-RGD2与整合素αvβ3特异性结合的半数抑制浓度(IC5o)为8.759 nmol/L.H446细胞对99Tcm-3P-RGD2的亲和性高于A549细胞,摄取率均于120 min达峰值,分别为(5.75±0.50)％和(3.35±0.28)％(t=9.324,P＜0.05).H446和A549肺癌细胞均表达整合素αvβ3,且H446高于A549[(18.01±2.83)％和(5.77±0.64)％,t=7.488,P＜0.05].免疫荧光染色示H446细胞整合素信号明显高于A549细胞.MicroSPECT/CT显像示注射99Tcm-3P-RGD2后3 h T/NT达最大值,H446荷瘤鼠T/NT比值为6.39±1.29,高于A549荷瘤鼠(3.62±0.33,t=6.869,P＜0.05).H446和A549肿瘤组织经流式细胞术检测,整合素αvβ3表达水平分别为(22.89±3.63)％和(10.23±1.94)％(t=13.967,P＜0.05).免疫组织化学检测结果示H446和A549肿瘤组织和新生血管内皮细胞均有整合素αvβ3表达.结论 99Tcm-3P-RGD2可用于整合素αvβ3阳性肺癌的显像,并可无创评估不同肺癌组织整合素αvβ3的表达水平.
목적 채용99Tcm-3취을이순4-RGD2(3P-RGD2)평고소세포폐암화폐선암세포하류서동물모형중정합소αvβ3표체수평.방법 안시제합설명서제비99Tcm-3P-RGD2.선취H446인소세포폐암세포진행수체경쟁억제실험,검측3P-RGD2여정합소αvβ3적특이친화성.통과세포섭취실험검측H446화A549인폐선암세포대99Tcm-3P-RGD2적섭취정황,이류식세포술화면역형광염색측정2충세포중정합소αvβ3적표체.관찰99Tcm-3P-RGD2재H446화A549폐암하라서모형(각6지)적microSPECT/CT현상정황.현상후단경처사라서,취부분종류조직제비단세포현액,이류식세포술검측세포정합소αvβ3표체;부분조직제성절편,이면역조직화학법검측종류조직정합소αvβ3적표체.채용배대t검험대실험수거진행통계학분석.결과 99Tcm-3P-RGD2표기솔위(97.0±2.0)％,4h시후방화순잉고체95％.3P-RGD2여정합소αvβ3특이성결합적반수억제농도(IC5o)위8.759 nmol/L.H446세포대99Tcm-3P-RGD2적친화성고우A549세포,섭취솔균우120 min체봉치,분별위(5.75±0.50)％화(3.35±0.28)％(t=9.324,P＜0.05).H446화A549폐암세포균표체정합소αvβ3,차H446고우A549[(18.01±2.83)％화(5.77±0.64)％,t=7.488,P＜0.05].면역형광염색시H446세포정합소신호명현고우A549세포.MicroSPECT/CT현상시주사99Tcm-3P-RGD2후3 h T/NT체최대치,H446하류서T/NT비치위6.39±1.29,고우A549하류서(3.62±0.33,t=6.869,P＜0.05).H446화A549종류조직경류식세포술검측,정합소αvβ3표체수평분별위(22.89±3.63)％화(10.23±1.94)％(t=13.967,P＜0.05).면역조직화학검측결과시H446화A549종류조직화신생혈관내피세포균유정합소αvβ3표체.결론 99Tcm-3P-RGD2가용우정합소αvβ3양성폐암적현상,병가무창평고불동폐암조직정합소αvβ3적표체수평.
Objective To evaluate expression levels of integrin αvβ3 in H446 human small cell lung cancer cells and A549 human adenocarcinoma cells and their lung cancer xenografts with 99Tcm-3(poly(ethylene glycol),PEG)4-RGD2(99Tcm-3P-RGD2).Methods 99Tcm-3P-RGD2 was prepared according to the instruction of the kit-formulation.The half-inhibition concentrations (IC50) for 125 Ⅰ-cyclo (Arg-Gly-AspD-Tyr-Lyx) (1~Ⅰ-c(RGDyK))of 3P-RGD2 binding to integrin αv β3 were measured.In vitro cellular uptake of 99Tcm-3P-RGD2 with H446 cells and A549 cells was measured.Flow cytometry and immunofluorescence staining were performed for detecting the expression of integrin αv β3.Molecular imaging with microSPECT/ CT was performed in nude mice bearing H446 (n =6) and A549 tumor (n =6) xenografts,respectively.Tumor tissues were prepared for the flow cytometry and immunohistochemisty analysis to measure the expression of integrin αvβ3.Paired t test was used for data analysis.Results The labeling rate of 99Tcm-3P-RGD2 was (97.0±2.0)％,and the radiochemical purity was about 95％ after 4 h.The IC50 value was 8.759 nmol/L.The affinity of 99Tcm-3P-RGD2 to H446 cells was higher than that of A549 cells ((5.75± 0.50) ％ vs (3.35±0.28) ％; t =9.324,P＜0.05),both reached the peak at 120 min.The expression level of integrin αvβ3 on H446 cells was also higher than that on A549 cells ((18.01± 2.83)％ vs (5.77±0.64) ％; t=7.488,P＜ 0.05),which was further confirmed by immunofluorescence staining.From microSPECT/CT images,T/NT ratios in H446 and A549 xenografts were 6.39± 1.29 and 3.62±0.33 at 3 h postinjection,respectively(t =6.869,P＜0.05).The levels of integrin αvβ3 in H446 and A549 tumor tissue were (22.89±3.63)％ and (10.23± 1.94) ％,respectively (t =13.967,P＜0.05).Immunohistochemistry results showed that the integrin αvβ3 was expressed both in tumor cells and neo-vasculature.Conclusion 99Tcm-3P-RGD2 may be a potential tracer for molecular imaging targeting to integrin αv β3 in lung cancers,and it may contribute to quantitating the levels of integrin αvβ3 non-invasively.