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
원기구단백-4항체파향표기합성형혈관평활기세포MR체외성상적실험연구
MR imaging of tropomyosin-4 antibody targeting synthetic phenotype vascular smooth muscle cells in vitro
  • 万方数据
    中华放射学杂志 중화방사학잡지
    Chinese Journal of Radiology
    2013年 12期 1132-1138 ,共7页

    尚松安%马占龙%陈相汛%陈宇辰%安艳丽%滕皋军

    상송안%마점룡%진상신%진우신%안염려%등고군

    肌,平滑,血管%磁共振成像%细胞 肌,平滑,血管%磁共振成像%細胞
    기,평활,혈관%자공진성상%세포
    Muscle,smooth,vascular%Magnetic resonance imaging%Cells
    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.
    목적 분리、배양병감정합성형혈관평활기세포(VSMC),검측파향표기원기구단백-4(TPM-4),병채용면역분자성상기술구건TPM-4항체파향형MRI탐침,탐토파향합성형VSMC체외MR성상적가행성.방법 체외분리배양합성형VSMC여내피세포(EC),채용SMA、Ⅷ인자면역세포화학염색방법분별행세포감정;면역형광쌍중염색험증합성형VSMC고표체TPM-4단백;응용화학교련법합성파향TPM-4 MRI분자탐침,TPM-4표기탐침(TPM4-USPIO)위실험탐침조、IgG표기탐침(IgG-USPIO)위음성탐침조、미표기탐침(USPIO)위대조탐침조,PBS위공백조,실험조탐침분별여합성형VSMC급EC공부육,음성조、대조조탐침여VSMC EC공부육.채용보로사람염색분석탐침특이파향성,새서람(MTT)비색법분석불동철농도조건하(함철농도제도분별위0、5、10、20、40 μg/ml)대세포체외생물학활성적영향;채용7.0TMR소묘의검측탐침자학특성,병대불동탐침조표기적합성형VSMC(n=3)이급1×103、5×103、1 × 104、5 ×104、1×105개/관(n=3)불동농도제도TPM4-USPIO표기세포행체외세포T2WI성상분석.T2신호수거이급MTT결과수거조간비교선행단인소방차분석(ANOVA),약차이유통계학의의,재용LSD법행조간량량비교.결과 성공분리병배양료합성형VSMC,합성형VSMC중TPM-4정고표체;성공구건TPM-4、IgG항체표기파향형탐침,TPM4-USPIO、IgG-USPIO탐침이예솔분별위0.0350×106、0.0316×106 mol/s,동USPIO(0.0292×106 mol/s)자학특성상사,은정성고;보로사람염색결과현시실험조탐침여합성형VSMC정특이성결합,여EC무특이성결합,MTT결과표명철농도≤40 μg/ml범위내대합성형VSMC증식활성무영향;실험탐침조표기세포T2WI정저신호,교음성탐침조、대조탐침조급공백조신호변화명현,T2이예시간위(116.67 ±2.08) ms,명현단우이상3조탐침[(217.67±2.52)、(219.33±2.08)급(205.33±1.53) ms],차이구유통계학의의(F=1670.43,,＜0.01);1×103、1 ×104、1×105 3충농도세포지간T2이예시간분별위(184.33±2.08)、(169.67±1.15)、(116.67±2.08) ms,차이구유통계학의의(F=684.35,P＜0.01),T2 WI신호제도감저,동일수량급세포지간T2치변화차이무통계학의의(P치균＞0.05).결론 혈소판연화생장인자(PDGF)-BB처리후가획득합성형VSMC,TPM4-USPIO탐침능구고효、파향성결합합성형VSMC,고장강MRI T2WI신호변화현저,위혈관성질병재체파향합성형VSMC적상관분자영상학연구제공료신적절입점.
    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.
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