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目的探讨Ferumoxide-PLL标记Flk1+CD31-CD34-人骨髓间充质干细胞(hBMSC)的方法及其在食蟹猴脑实质内移植活体示踪的可行性。方法采用Ferumoxide-PLL标记hBMSC,台盼蓝染色、普鲁士蓝染色和透射电镜扫描鉴定标记效率及细胞活力。体外磁共振成像(MRI)分别扫描标记和未标记细胞,计算T2*的弛豫时间和弛豫率(R2*)变化。通过立体定向手术将标记的hBMSC移植入食蟹猴右侧基底节区,采用MRI扫描活体示踪细胞。采用免疫组织化学、普鲁士蓝和HE染色对脑组织切片进行干细胞存活、分化及病理学研究。结果Ferumoxide-PLL标记hBMSC效率为96%,普鲁士蓝染色、电镜可显示标记hBMSC细胞质内铁颗粒。1×106和5×105两组Ferumoxide-PLL标记细胞的T2*的弛豫时间分别为68.86和79.88ms,而未标记细胞分别为12.71和15.24ms。标记细胞的R2*分别为78.68和65.61/s,分别是未标记细胞(14.52和12.52/s)的5.4和5.2倍。移植后3周MRI扫描T2WI仍可发现hBMSC呈明显的低信号。病理及免疫荧光结果显示hBMSCs在移植区大量存活,移植区有大量新生血管,但未见hBMSC向神经细胞分化。结论Ferumoxide-PLL可高效标记hBMSC,能显著增加其MRI图像对比度。MRI可活体示踪干细胞。移植入食蟹猴脑内的hBMSC可大量存活并促进新生血管形成。
Objective To investigate the method of Ferumoxide-PLL labeling Flk1 + CD31-CD34-human bone marrow mesenchymal stem cells (hBMSC) and its feasibility in living cadaver transplanted in cynomolgus monkey. Methods The labeling efficiency and cell viability were identified by Ferumoxide-PLL labeled hBMSC, trypan blue staining, Prussian blue staining and transmission electron microscopy. Marked and unlabeled cells were scanned by magnetic resonance imaging (MRI) separately, and the relaxation time and relaxation rate (R2 *) of T2 * were calculated. The labeled hBMSCs were transplanted into the right basal ganglia of cynomolgus monkey by stereotactic surgery. The cells were screened by MRI. Immunohistochemistry, Prussian blue and HE staining were used to study the survival, differentiation and pathology of stem cells. Results Ferumoxide-PLL labeled hBMSC had an efficiency of 96%. Prussian blue staining and electron microscopy showed that the hBMSCs were labeled with cytoplasmic iron particles. The T2 * relaxation times of 1 × 106 and 5 × 105 Fermoxide-PLL labeled cells were 68.86 and 79.88 ms, respectively, while the unlabeled cells were 12.71 and 15.24 ms respectively. R2 * of the labeled cells were 78.68 and 65.61 / s, respectively 5.4 and 5.2 times higher than that of unlabeled cells (14.52 and 12.52 / s). Three weeks after the transplantation MRI scan T2WI still showed hBMSC showed a significant low signal. The results of pathology and immunofluorescence showed that hBMSCs survived in the graft area with a large number of neovascularization, but hBMSC did not differentiate into neurons. Conclusion Ferumoxide-PLL can effectively label hBMSC, which can significantly increase the contrast of MRI images. MRI can be used to track the stem cells. HBMSC transplanted into the brain of cynomolgus monkeys can largely survive and promote neovascularization.