CAJ | 학술논문

目的构建人转铁蛋白(Tf)介导的磁性-近红外荧光(NIRF)分子探针,探讨其主动靶向标记人骨髓间充质干细胞(hMSCs)并用于多模态成像的可行性.方法 Tf与cy5.5、超微超顺磁性氧化铁(IO)连接,构建分子探针Tf-cy5.5-IO,确定连接效率并表征.利用人血清白蛋白(HSA)构建对照粒子HSA-cy5.5-IO.将hMSCs分为未标记组(A)、靶向探针组(B)、HSA对照组(C)和竞争实验组(D)4组,并以表达人转铁蛋白受体(hTfR)的肿瘤细胞(HeLa)作为阳性对照,进行激光共聚焦、铁含量、流式细胞仪及透射电镜检测.每组2×105个细胞行磁共振(MRI)和NIRF成像,定量检测R2值和平均荧光强度值(AI)变化率.结果 IO、Tf、cy5.5成功连接,摩尔比1∶2.89∶7.89,整体粒径(23.4 ±2.4)nm.激光共聚焦扫描示Tf-cy5.5-IO快速进入hMSCs和HeLa细胞内,其分布与人转铁蛋白受体(hTfR)表达位置一致.B组细胞铁含量和AI均明显高于其他组(均P＜0.01),探针对hTfR具主动靶向性.MRI和NIRF成像显示Tf-cy5.5-IO使hMSCs T2WI信号减低,NIRF成像荧光强度增加,R2和AI值的增高均较其他组明显(P＜0.05).结论 Tf-cy5.5-IO特异性识别hTfR,通过MRI和NIRF可实现hMSCs多模态主动靶向成像.
목적 구건인전철단백(Tf)개도적자성-근홍외형광(NIRF)분자탐침,탐토기주동파향표기인골수간충질간세포(hMSCs)병용우다모태성상적가행성.방법 Tf여cy5.5、초미초순자성양화철(IO)련접,구건분자탐침Tf-cy5.5-IO,학정련접효솔병표정.이용인혈청백단백(HSA)구건대조입자HSA-cy5.5-IO.장hMSCs분위미표기조(A)、파향탐침조(B)、HSA대조조(C)화경쟁실험조(D)4조,병이표체인전철단백수체(hTfR)적종류세포(HeLa)작위양성대조,진행격광공취초、철함량、류식세포의급투사전경검측.매조2×105개세포행자공진(MRI)화NIRF성상,정량검측R2치화평균형광강도치(AI)변화솔.결과 IO、Tf、cy5.5성공련접,마이비1∶2.89∶7.89,정체립경(23.4 ±2.4)nm.격광공취초소묘시Tf-cy5.5-IO쾌속진입hMSCs화HeLa세포내,기분포여인전철단백수체(hTfR)표체위치일치.B조세포철함량화AI균명현고우기타조(균P＜0.01),탐침대hTfR구주동파향성.MRI화NIRF성상현시Tf-cy5.5-IO사hMSCs T2WI신호감저,NIRF성상형광강도증가,R2화AI치적증고균교기타조명현(P＜0.05).결론 Tf-cy5.5-IO특이성식별hTfR,통과MRI화NIRF가실현hMSCs다모태주동파향성상.
Objective To prepare the magnetic near infrared fluorescent (NIRF) bifunctional molecular probe with human holo-transferrin (Tf) as a targeted ligand and detect human transferrin receptor (hTfR) actively.Methods Molecular probe Tf-cyS.5-IO was prepared and purified by conjugating Tf,superparamagnetic iron oxide (IO) and near infrared fluorescent dye (cy5.5).The particle size and morphology was determined by transmission electron microscopy (TEM),zeta potential and particle sizing analyzer.Human serum albumin (HSA) was used for conjugating with cy5.5 and IO as control.hMSCs and HeLa (as a positive control) were divided into 4 groups:A non-labeled,B Tf-cy5.5-IO,C HSA-cy5.5-IO and D competition assay to confirm the targeted connection.The fluorescent signals from intracellular probe were detected with laser scanning confocal microscope (LSCM) and flow cytometry.Intracellular iron was detected with iron concentration assay and TEM.MRI and NIRF imaging of 2 × 105 cells were performed respectively.Enhancements of R2 value and average intensity (AI) were analyzed qualitatively.Results The conjugation between IO,Tf and cy5.5 was confirmed with a molar ratio of 1 ∶ 2.89 ∶ 7.89.The hyperdense aqueous diameter of probe was 23.39-± 2.42 nm.LSCM showed the fluorescence from Tf-cy5.5-IO and cy3-1abeled monoclonal antibody against hTfR in cells and two markers were localized in intracellular compartments of similar appearance.After co-incubating with Tf-cy5.5-IO,the intracellular iron and average intensity were significantly higher than cells of other groups (P ＜ 0.01).MRI and NIRF images showed that,after incubation,intracellular Tf-cy5.5-IO decreased the T2WI signal of human mesenchymal stem cells (hMSCs) and AI on NIRF image increased.Enhancements of R2 value and AI were higher in B group than those in other groups (P ＜ 0.05).Conclusion Tf-cy5.5-IO probe can recognize and conjugate with hTfR specifically.And targeted imaging in vitro of hTfR expressed in hMSCs may be performed by MRI and NIRF multimodal imaging.