葡萄糖调节蛋白78靶向分子探针^(68)Ga-DOTA-VAP用于肿瘤特异性microPET/CT显像

^(68)Ga-DOTA-VAP molecular probe targeted glucose-regulated protein 78 for specific microPET/CT imaging of tumors

目的评价68Ga-1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸(DOTA)-丝氨酸-天冬酰胺-苏氨酸-精氨酸-缬氨酸-丙氨酸-脯氨酸(SNTRVAP,简称VAP)分子探针在体内外靶向葡萄糖调节蛋白78(GRP78)的特异性以及用于GRP78阳性肿瘤microPET/CT诊断的可行性。方法将多肽VAP与双功能螯合剂DOTA偶联后进行68Ga标记,制备68Ga-DOTA-VAP分子探针。用蛋白免疫印迹法验证人脑胶质瘤细胞株U87MG、人胰腺癌细胞株BxPC-3、人胚胎肾细胞株293T细胞中GRP78表达情况,并用阻断实验验证68Ga-DOTA-VAP与上述细胞的特异性结合。建立U87MG与BxPC-3荷瘤裸鼠模型,检测68Ga-DOTA-VAP在体内的生物学分布;用microPET/CT评价68Ga-DOTA-VAP在U87MG与BxPC-3荷瘤裸鼠模型的显像效果。采用两独立样本t检验、单因素方差分析和Dunnettt检验分析数据。结果68Ga-DOTA-VAP制备简便,标记率、放化纯均大于98%,体外稳定性好,2 h后放化纯仍为(98.27±0.22)%。GRP78在U87MG、BxPC-3和293T细胞中均有表达(分别为0.78±0.02、0.53±0.05和0.36±0.03),且在肿瘤细胞中的表达高于正常细胞(F=102.22,P<0.001;t值:0.43、0.18,均P<0.01)。U87MG细胞对68Ga-DOTA-VAP的摄取高于BxPC-3细胞(5154.00±216.70和4344.00±60.88;t=3.10,P=0.027),过量VAP多肽可显著降低U87MG、BxPC-3细胞对探针的摄取(3324.00±54.14、3270.00±131.10;t值:8.19、7.43,均P<0.01)。68Ga-DOTA-VAP经尾静脉注射后,在U87MG肿瘤组织的摄取高于BxPC-3肿瘤组织[(1.98±0.20)和(1.30±0.08)每克组织百分注射剂量率(%ID/g);t=5.48,P=0.005];注射过量VAP多肽后,肿瘤组织摄取显著降低[(0.99±0.02)和(0.62±0.05)%ID/g;t值:8.32、12.25,均P<0.05]。MicroPET/CT显像示U87MG、BxPC-3模型鼠肿瘤清晰可见,U87MG模型鼠显影效果更佳;注射过量VAP多肽后,2种模型鼠肿瘤显像均不明显。结论68Ga-DOTA-VAP分子探针可与GRP78特异性结合,其可反映体内GRP78表达水平并有希望用于GRP78阳性肿瘤的特异性显像诊断。

Objective To evaluate the specificity of^(68)Ga-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid(DOTA)-Ser-Asn-Thr-Arg-Val-Ala-Pro(SNTRVAP,VAP)molecular probe targeting glucose-regulated protein 78(GRP78)in vivo and in vitro and the feasibility of^(68)Ga-DOTA-VAP microPET/CT imaging in the diagnosis of GRP78-positive tumors.Methods^(68)Ga-DOTA-VAP was prepared by the combination of bifunctional chelating agent DOTA and VAP,followed by^(68)Ga labeling.Western blotting experiment was perfomed to detect the expression of GRP78 in U87MG,BxPC-3,and 293T cell lines,at the same time,cold polypeptide blocked experiments were conducted to verify the specific binding of^(68)Ga-DOTA-VAP to cells.U87MG and BxPC-3 subcutaneous transplantation tumor mouse models were established and the biodistribution of^(68)Ga-DOTA-VAP were explored in vivo.The imaging effect of^(68)Ga-DOTA-VAP in GRP78-positive tumor-bearing mouse models was evaluated by microPET/CT.Independent-sample t test,one-way analysis of variance and Dunnett t test were used for data analysis.Results^(68)Ga-DOTA-VAP was easily prepared with labeling yield and radiochemical purity>98%.It had good stability in vitro,and its radiochemical purity was still(98.27±0.22)%after 2 h.GRP78 was highly expressed in U87MG and BxPC-3 cells,but lowly expressed in 293T cells((0.78±0.02),(0.53±0.05)and(0.36±0.03),F=102.22,P<0.001;t values:0.43,0.18,both P<0.01).The uptake of^(68)Ga-DOTA-VAP in U87MG cells was higher than that in BxPC-3 cells(5154.00±216.70 vs 4344.00±60.88;t=3.10,P=0.027).Excessive unlabeled VAP polypeptide could significantly reduce the uptake of^(68)Ga-DOTA-VAP both in U87MG and BxPC-3 cells(3324.00±54.14,3270.00±131.10;t values:8.19,7.43,both P<0.01).The uptake of^(68)Ga-DOTA-VAP in U87MG tumor tissue was higher than that in BxPC-3 tumor tissue((1.98±0.20)vs(1.30±0.08)percentage activity of injection dose per gram of tissue(%ID/g);t=5.48,P=0.005),while co-injection of excessive unlabeled VAP polypeptide significantly reduced the uptake in U87MG and BxPC-3 tumors((0.99±0.02)and(0.62±0.05)%ID/g;t values:8.32,12.25,both P<0.05).MicroPET/CT imaging showed that^(68)Ga-DOTA-VAP could clearly display U87MG and BxPC-3 tumors,and U87MG had a better imaging effect.The tumors could not be clearly visualized after co-injection of excessive VAP polypeptide.Conclusion^(68)Ga-DOTA-VAP molecular probe binds with GRP78 specifically and can reflect the expression level of GRP78 in vivo,which may be a promising probe for the specific imaging diagnosis of GRP78-positive tumors.