原肌球蛋白-4抗体靶向标记合成型血管平滑肌细胞MR体外成像的实验研究

MR imaging of tropomyosin-4 antibody targeting synthetic phenotype vascular smooth muscle cells in vitro

目的 分离、培养并鉴定合成型血管平滑肌细胞（VSMC）,检测靶向标记原肌球蛋白-4（TPM-4）,并采用免疫分子成像技术构建TPM-4抗体靶向型MRI探针,探讨靶向合成型VSMC体外MR成像的可行性.方法 体外分离培养合成型VSMC与内皮细胞（EC）,采用SMA、Ⅷ因子免疫细胞化学染色方法分别行细胞鉴定;免疫荧光双重染色验证合成型VSMC高表达TPM-4蛋白;应用化学交联法合成靶向TPM-4 MRI分子探针,TPM-4标记探针（TPM4-USPIO）为实验探针组、IgG标记探针（IgG-USPIO）为阴性探针组、未标记探针（USPIO）为对照探针组,PBS为空白组,实验组探针分别与合成型VSMC及EC共孵育,阴性组、对照组探针与VSMC EC共孵育.采用普鲁士蓝染色分析探针特异靶向性,噻唑蓝（MTT）比色法分析不同铁浓度条件下（含铁浓度梯度分别为0、5、10、20、40 μg/ml）对细胞体外生物学活性的影响;采用7.0TMR扫描仪检测探针磁学特性,并对不同探针组标记的合成型VSMC（n=3）以及1×103、5×103、1 × 104、5 ×104、1×105个/管（n=3）不同浓度梯度TPM4-USPIO标记细胞行体外细胞T2WI成像分析.T2信号数据以及MTT结果数据组间比较先行单因素方差分析（ANOVA）,若差异有统计学意义,再用LSD法行组间两两比较.结果 成功分离并培养了合成型VSMC,合成型VSMC中TPM-4呈高表达;成功构建TPM-4、IgG抗体标记靶向型探针,TPM4-USPIO、IgG-USPIO探针弛豫率分别为0.0350×106、0.0316×106 mol/s,同USPIO（0.0292×106 mol/s）磁学特性相似,稳定性高;普鲁士蓝染色结果显示实验组探针与合成型VSMC呈特异性结合,与EC无特异性结合,MTT结果表明铁浓度≤40 μg/ml范围内对合成型VSMC增殖活性无影响;实验探针组标记细胞T2WI呈低信号,较阴性探针组、对照探针组及空白组信号变化明显,T2弛豫时间为（116.67 ±2.08） ms,明显短于以上3组探针[（217.67±2.52）、（219.33±2.08）及（205.33±1.53） ms],差异具有统计学意义（F=1670.43,,＜0.01）;1×103、1 ×104、1×105 3种浓度细胞之间T2弛豫时间分别为（184.33±2.08）、（169.67±1.15）、（116.67±2.08） ms,差异具有统计学意义（F=684.35,P＜0.01）,T2 WI信号梯度减低,同一数量级细胞之间T2值变化差异无统计学意义（P值均＞0.05）.结论 血小板衍化生长因子（PDGF）-BB处理后可获得合成型VSMC,TPM4-USPIO探针能够高效、靶向性结合合成型VSMC,高场强MRI T2WI信号变化显著,为血管性疾病在体靶向合成型VSMC的相关分子影像学研究提供了新的切入点.

Objective To isolate,culture,and identify the synthetic phenotype vascular smooth muscle cells （VSMC） and identify the specific marker protein （tropomyosin-4,TPM-4） of synthetic phenotype.To employ the immune molecular imaging technique to develop MRI of probe targeted with TPM-4 antibody VSMC in vitro.Methods The synthetic phenotype VSMC and endothelial cells （EC） were isolated and cultured in vitro,respectively.Immunocytochemistry （ICC） staining for α-smooth muscle actin （SMA） and Ⅷ factor was performed for cell identification,respectively.The high expression level of TPM-4 protein was tested by immunofluorescence double staining.The MRI molecular probe was built by chemical cross-linking,TPM-4 conjuncted probe （TPM4-USPIO） as the experimental group,IgG conjuncted probe （IgG-USPIO） as the negative group,unconjuncted probe （USPIO） as the control group,and PBS as the blank group.The synthetic VSMC were incubated with probes within experimental group,negative group,control group,respectively,and EC were incubated with experimental group as another control group.Prussian blue staining was employed to analyze the specific-targeting and MTT assay was used to test bioactivity of the probe under different concentrations （0,5,10,20,40 μg/ml） in vitro.7.0 T MRI scanner was used to detect the magnetic properties.With 7.0 T MRI scanner,the T2WI images of different probes labeled synthetic VSMC and different concentration gradient （1 × 103,5 × 103,1 × 104,5 × 104）TPM4-USPIO labeled cells were obtained and analyzed.T2 signal and MTT data among groups were compared using single factor analysis of variance （ANOVA） and LSD test.Results The synthetic phenotype of VSMC were isolated and cultured successfully,and the VSMC could express the TPM-4 protein.The synthetic phenotype VSMC had a high level of the protein expression.The probe was made successfully.The T2 relaxivity of TPM4-USPIO and IgG-USPIO were 0.0350 × 106,0.0316 × 106 mol/s,respectively,with high stability as USPIO （0.0292 × 106 mol/s）.Prussian blue staining results showed that the experimental group probe could specifically bind to the synthetic VSMC.MTT results showed that iron concentration within 40 μg/ml or less had no effect on VSMC proliferation activity.The T2 WI of experimental group showed lower signal than the control group.The T2 relaxivity was （116.67 ± 2.08） ms,which was less than the control group [（217.67 ±2.52）,（219.33 ±2.08）ms,respectively] and the blank group [（205.33 ± 1.53）ms]（F =1670.43,P 〈 0.01）.The T2 relaxivity of the different concentration gradient labeled cells （1 × 103 、1 × 104 、1 × 105） were （184.33 ± 2.08）,（169.67 ± 1.15）,（116.67 ± 2.08） ms,respectively （F =684.35,P 〈0.01）.No significant difference of the T2WI gradual signal dim was found between cells with the same order concentration（P 〉 0.05）.Conclusions The synthetic phenotype of VSMC can be obtained by PDGF-BB treatment.TPM4-USPIO probe is efficient,specific and targeted at combination with synthetic VSMC.The T2WI signal changed obviously under high field MRI scanner,which provides a new way for molecular imaging research.