干细胞在心肌组织中增殖及胞吐对磁共振在体示踪信号影响规律及其机制的实验研究

To develop effective mesencymal stem cells (MSCs)-based therapies for myocardial repair, the location, distribution, and long-term viability of the cells must be determined in a noninvasive manner. Magnetic resonance imaging (MRI) of cells labeled with magnetically visible contrast agents has the potential to fulfill this aim. The most sensitive existing markers for cell labeling using MRI are superparamagnetic iron oxide (SPIO) particles. But the accuracy of quantification of transplanted stem cells by MRI was unreliable because the signal of SPIO will underestimate the quantification of live stem cells in myocardium. It is supposed that the proliferation and exocytosis should be the main mechanism that MSCs exclude SPIO. In the culture medium, the proliferation of MSCs will be assessed by the EdU incorporation assay. The flow cytometry and the exocytosis of MSCs will be explored by the fluorescence of FM4-64 displaying in the cell membrane and by patch clamp detecting membrane capacitance. Furthermore, the animal study will be executed to explore the proliferation, exocytosis and apoptosis in vivo. The animal models of myocardial infarction are prepared with rats. Accordingly the rats suffered from sham operation will be control groups. All the animal models are divided into six experimental groups and the corresponding control groups according to the detection time. MSCs coming from transgenic rats expressing the enhanced green fluorescent protein (eGFP) are labeled by superparamagnetic iron oxide particles (SPIO) and are injected into the borderline area of myocardial infarction zone in the experimental groups and the same zone in the control group. MRI and bioluminescent imaging will be combined to evaluate quantitatively the dynamic relationship between the number of surviving MSCs and SPIO. The dynamic tendency of the quantity, proliferation, apoptosis and exocytosis of MSCs will be examined by histopathological and molecular methods. With intracellular contrast agent SPIO as the tracer, the quantitative relationship between magnetic resonance signal and the number of MSCs can be inferred. Electron microscopy can prove the distribution of the caveolae foam including SPIO. Furthermore, the expression of GFP is detected by Western-Blot and is used as the gold standard of quantification of live MSCs in myocardium. Finally, the GFP-positive MSCs will be extracted by immunomagnetic beads with GFP antibody and the proliferation, exocytosis will be detedcted. The objective of the study was to investigate how the proliferation, exocytosis and apoptosis of MSCs influence the SPIO labling rate and the SPIO content in cytoplasm of MSCs.In addition, the influence of ischemia and hypoxia to the proliferation, exocytosis and apoptosis will be explored. As a result, the accuracy of MRI quantifying MSCs in vivo will be re-estimated according the trend of proliferation, exocytosis and apoptosis.

目前对于骨髓间充质干细胞移植于心肌组织后的增殖和胞吐规律尚不明确。干细胞分裂增殖及胞吐可能是干细胞被顺磁性氧化铁颗粒（SPIO）标记后排除标记物的主要机制，也是造成磁共振在体示踪信号不能客观反映实际存活干细胞数量的主要原因。本项目以大鼠为研究对象，将磁共振成像及生物发光成像相结合, 以病理组织学、分子生物学及细胞学实验为佐证，动态量化分析干细胞移植于心肌后增殖、凋亡、胞吐等生物学行为演变规律。借助细胞内对比剂SPIO明确磁共振在体示踪信号衰减规律与干细胞生物学行为之间的联系。结合细胞学实验、通过膜片钳技术检测膜电容变化探索干细胞在体外及在心肌组织中的胞吐规律，以EdU掺入法、流式细胞仪及增殖细胞核抗原免疫组化检测干细胞在体内外的增殖规律。在此基础上，探索干细胞在心肌组织中的分裂增殖及胞吐对磁共振在体量化移植干细胞的影响规律；并进一步探索缺血缺氧对干细胞增殖及胞吐等生物学行为的影响。

目前对骨髓间充质干细胞移植于心肌组织后的增殖规律尚不明确。借助超顺磁性氧化铁颗粒（SPIO），可以在一定程度上提示骨髓间充质干细胞移植至缺血心肌后的生存情况，且通过磁共振成像，可实现在体示踪移植入心肌中的干细胞，从而进一步探索干细胞存活量与磁共振信号之间的关系。本研究首先从细胞学实验的角度探讨在间充质干细胞分裂增殖过程中干细胞胞浆中的超顺磁性氧化铁颗粒标记率的演变。将两组骨髓间充质细胞分别以15％胎牛血清的高糖DMEM以及含5％胎牛血清的低糖DMEM培养。每隔24小时估测细胞氧化铁颗粒标记率。SPIO标记干细胞48小时后，细胞爬片普鲁士蓝染色可见所有细胞中均含有蓝染颗粒，位于细胞核周围。将SPIO标记的细胞以不同条件的培养基继续培养，两组细胞SPIO标记率均逐渐减低。在两组细胞，均在标记以后第四次传代后24小时，SPIO的标记率下降至10％以下，在高糖DMEM组历时9天，而在低糖DMEM组历时16天。在标记后第四次传代后24小时，两组细胞SPIO标记率无统计学差异。提示SPIO标记的间充质干细胞排除SPIO的机制之一为细胞的分裂增殖。细胞分裂增殖越快，细胞清除SPIO的速率也越快。本项目尝试以大鼠完成心机梗死模型制备，但动物死亡率高，难以耐受二次开胸移植干细胞。故以中华小型猪制备心肌梗死模型1例并二次开胸完成自体骨髓间充质干细胞注射获得成功。干细胞移植后24 h及21 d，快速梯度回波序列T2*像检测干细胞移植点低信号区的面积及信号强度。细胞移植后24 h，经SPIO标记的MSCs注射点于MRI显示为边界清晰的卵圆形T2＊低信号区，移植3周后，T2＊低信号区与正常心肌区的对比程度较前下降。移植3周后，处死动物行病理学检查，可见氧化铁颗粒存在于部分移植干细胞核周围。MRI可在一定时间内间接反映MSCs在心肌局部的数量变化，其准确性有待进一步研究。