外磁场诱导下SPIO、hNIS、EGFP共标记人脐带间充质干细胞的靶向迁移及活体示踪

发布时间：2018-08-04 15:03

【摘要】：目的：采用纳米磁性靶向技术和细胞生物学等技术，将人钠/碘同向转运体（hNIS）报告基因、增强型绿色荧光蛋白（EGFP）报告基因和超顺磁氧化铁颗粒（SPIO）对人脐带间充质干细胞共标记，建立间充质干细胞活体单光子发射计算机断层成像(SPECT)、核磁共振成像（MRI）、活体小动物荧光拟合成像活体示踪系统，利用多模式显像机理，精确区分局部器官干细胞聚集量；外加磁场使干细胞在获得靶向移动能力的同时，用MRI及SPECT99mTc显像、活体小动物荧光拟合成像进行示踪，探讨建立干细胞靶向迁移能力与示踪的多模式分子影像技术的可行性。方法： 1.共标记干细胞系的建立：分离鉴定人脐带间充质干细胞，选择EGFP报告基因来间接反应目的基因hNIS的表达，PCR扩增后将目的基因hNIS克隆到pcDNA3.1中，再将EGFP连接到hNIS的下游，，然后与入门载体通过BP反应同源重组得到入门克隆,最后将入门克隆与表达载体同源重组产生表达克隆,经酶切、PCR及测序证实成功构建重组质粒pCMV-NIS-EF1-GFP-PGK-puro,再感染人脐带间充质干细胞。获得稳定表达hNIS及EGFP的干细胞系后，进行125I内流及125I外流实验鉴定hNIS蛋白功能。将适当浓度的SPIO与细胞系孵育获得hNIS及EGFP、SPIO共同标记的干细胞，并验证其活性及干性。 2.不同外磁场暴露时间筛选的体外研究：对照组为未标记的hUCMSCs，实验组为SPIO-hNIS-EGFP-hUCMSCs细胞并在培养板下方放置磁场（200mT）连续3天，按每天接受的磁场暴露时间分为1小时组、6小时组、12小时组、24小时组。台盼兰染色检测细胞活力，MTT法进行细胞增殖能力测试，Transwell实验测磁场干预下的迁移能力、成脂成骨诱导分化实验测分化能力，Western blot测BCL-1、Bax、Caspase-3、Caspase-9等凋亡因子的表达的影响，筛选合适的暴露时间为活体体内研究提供依据。 3.外磁场作用下干细胞的靶向迁移及活体示踪：建立全层皮肤缺损裸鼠模型，在细胞移植后0小时、24小时、48小时及7天进行核磁共振显像外磁场MRI、SPECT、活体小动物荧光成像示踪。分别测量MRI图像参数原位干细胞团的面积减少率、信噪比、载噪比、细胞团位移。普鲁士蓝染色及冰冻荧光染色鉴定皮肤创面干细胞数量。比较两组间皮肤创面愈合时间。结果： 1.成功分离培养人脐带间充质干细胞，并鉴定其分化增殖能力及干性。第三代慢病毒包装系统介导下完成构建包含目的基因hNIS的真核质粒并获得稳定表达的hNIS-EGFP-hUCMSCs细胞系，经Western blot鉴定hNIS表达良好。hNIS-EGFP-hUCMSCs实验组细胞摄取125I活性125I内流实验及外流实验证实人脐带间充质干细胞表达hNIS后的功能良好。成功建立SPIO、hNIS EGFP共标记的人脐带间充质干细胞系命名为SPIO-hNIS-EGFP-hUCMSCs，普鲁士蓝染色证实SPIO进入细胞内,细胞标记率达98%。SPIO-hNIS-EGFP-hUCMSCs在生物活性、细胞干性、增殖活性、分化能力与原始hUCMSCs无显著差别，可以作为动物模型的治疗细胞，并为下一步SPECT、MRI、活体小动物荧光显像示踪干细胞做好准备。 2.表面磁力200mT的永磁铁作用下，连续三天每天磁场暴露时间6小时在细胞活性，增殖能力，分化能力方面与空白对照组无差异，磁场暴露时间达12小时以上细胞生物活性及分化能力得到全面抑制。Transwell细胞迁移实验证明磁场暴露时间达到6小时后，SPIO-hNIS-EGFP-hUCMSCs细胞迁移能力不会随着暴露时间发生进一步增加。外磁场暴露时间不超过6小时/天，不会诱发过多的促凋亡基因表达。磁场暴露时间达12小时以上可能会诱发SPIO-hNIS-EGFP-hUCMSCs程序性死亡。 3.MRI示踪显像获得较高质量的图像，SPIO-hNIS-EGFP-hUCMSCs成功受到植入磁体产生磁场梯度的影响，由磁场引导干细胞在皮下区域向皮肤缺损部位快速迁移，此效应在外磁场作用后的第一个24小时最为明显。SPECT显像示标记干细胞在在0小时、24小时、48小时、7天的检测与MRI显像示踪移植干细胞趋势相符，但空间分辨率较MRI低。活体小动物荧光成像在0小时，24小时和48小时的外部磁场促进SPIO-hNIS-EGFP-hUCMSCs与MRI显示一致的趋势，但第7天的活体荧光成像细胞移植的示踪呈阴性，考虑单位细胞浓度过小。普鲁士蓝与冰冻荧光切片显示外磁场组具有较多的干细胞进入皮肤创面附近。结论：首次运用慢病毒转染系统成功建立SPIO-hNIS-EGFP-hUCMSCs细胞系，并在生物活性、细胞干性、增殖活性、分化能力与原始hUCMSCs无显著差别；外磁场暴露时间对干细胞生物特性造成较大影响，每天暴露时间6小时是最佳时间；首次将外磁场应用于皮下干细胞移植治疗以增加干细胞的归巢，MRI与基于hNIS报告基因SPECT显像相结合，可以更为精确的示踪干细胞，提高敏感度，减少假阳性。
[Abstract]:Objective:
Using nanotechnology of magnetic targeting and cell biology, the human Na / iodide transporter (hNIS) reporter gene, enhanced green fluorescent protein (EGFP) reporter gene and superparamagnetic iron oxide particle (SPIO) were used to mark human umbilical cord mesenchymal stem cells, and the single photon emission computed tomography (SPECT) of mesenchymal stem cells was established. Nuclear magnetic resonance imaging (MRI), living small animal fluorescence fitting imaging system, using multi-mode imaging mechanism to accurately distinguish the aggregation of local organ stem cells; while the magnetic field makes the stem cells to get the ability to target movement, MRI and SPECT99mTc imaging, the fluorescent pseudo synthetic images of living small animals are traced to explore the establishment of dry cells. The feasibility of cell targeting migration and tracer multimodal molecular imaging technology.
Method:
1. the establishment of a co labeled stem cell line: isolation and identification of human umbilical cord mesenchymal stem cells, selected EGFP reporter gene to indirectly respond to the expression of the target gene hNIS, PCR amplification of the target gene hNIS to pcDNA3.1, then EGFP connected to the downstream of hNIS, and then the entry vector through BP reaction homologous recombination to get the introductory clone, finally will be PCR and sequencing confirmed that the recombinant plasmid pCMV-NIS-EF1-GFP-PGK-puro was successfully constructed, and human umbilical cord mesenchymal stem cells were successfully constructed by enzyme digestion, PCR and sequencing. After obtaining the stable expression of hNIS and EGFP stem cell lines, the hNIS protein function was identified by 125I flow and 125I flow test. A proper concentration of SP was used. IO was incubated with cell lines to obtain hNIS and EGFP, SPIO co labeled stem cells, and to verify their activity and dry matter.
2. in vitro study of different external magnetic field exposure time: the control group was unmarked hUCMSCs, the experimental group was SPIO-hNIS-EGFP-hUCMSCs cells and the magnetic field was placed under the culture plate (200mT) for 3 days. The exposure time was divided into 1 hours group, 6 hour group, 12 hour group, 24 hour group. Trypan blue staining was used to detect cell vitality, M TT assay was used to test the cell proliferation ability, Transwell test was used to measure the migration ability of magnetic field intervention, the differentiation ability of fat induced osteogenesis induced differentiation, and the effect of Western blot on the expression of BCL-1, Bax, Caspase-3, Caspase-9 and so on, and to screen the appropriate exposure time to provide the basis for the study in vivo.
3. the target migration of stem cells and tracing in vivo under the action of 3. external magnetic field: a nude mouse model of full layer skin defect was established. The magnetic resonance imaging external magnetic field MRI, SPECT, and living small animals were traced at 0 hours, 24 hours, 48 hours and 7 days after the cell transplantation. The area reduction rate and the signal to noise ratio of the MRI map in situ stem cell mass were measured respectively. Prussian blue staining and frozen fluorescence staining were used to identify the number of stem cells in skin wounds. The healing time of skin wounds was compared between the two groups.
Result:
1. the human umbilical cord mesenchymal stem cells were successfully isolated and cultured, and their differentiation and proliferation ability and their dry character were identified. Under the third generation lentivirus packaging system, the true nuclear particles containing the target gene hNIS were constructed and the stable expression of hNIS-EGFP-hUCMSCs cell lines were obtained. The hNIS expression good.HNIS-EGFP-hUCMSCs experimental group cells were identified by Western blot The 125I active 125I flow test and the Exodus experiment proved that human umbilical cord mesenchymal stem cells have good function after hNIS expression. The human umbilical cord mesenchymal stem cell line marked by hNIS EGFP was named SPIO-hNIS-EGFP-hUCMSCs, and Prussian blue staining confirmed that SPIO entered the cell and the cell labeling rate was 98%.SPIO-hNIS-EGFP-hUCMSCs to 98%.SPIO-hNIS-EGFP-hUCMSCs. There is no significant difference in bioactivity, cell dry, proliferative activity and differentiation ability from the original hUCMSCs, which can be used as a therapeutic cell for animal models and prepare for the next SPECT, MRI, and living small animal fluorescence imaging to trace the stem cells.
Under the action of permanent magnet of 2. surface magnetic 200mT, the exposure time of magnetic field was 6 hours a day for three days. There was no difference between the blank control group and the cell activity, proliferation ability and differentiation ability. The exposure time of the magnetic field was over 12 hours and the cell biological activity and differentiation ability were completely inhibited by the.Transwell cell migration experiment to prove the time of magnetic field exposure. After 6 hours, the migration ability of SPIO-hNIS-EGFP-hUCMSCs cells does not increase with the exposure time. The exposure time of the external magnetic field does not exceed 6 hours / day and does not induce excessive expression of the apoptotic gene. The SPIO-hNIS-EGFP-hUCMSCs programmed death may be induced by the exposure time of the magnetic field for more than 12 hours.
3.MRI tracer imaging obtained high quality images. SPIO-hNIS-EGFP-hUCMSCs was successfully affected by magnetic gradient of implanted magnets. The magnetic field guided stem cells in the subcutaneous region to rapidly migrate to the skin defect. The effect of the first 24 hours after the external magnetic field was the most obvious.SPECT imaging labeled stem cells in 0 hours, 2 4 hours, 48 hours, 7 days and MRI imaging traced the trend of transplanted stem cells, but the spatial resolution was lower than that of MRI. The fluorescent imaging of living small animals in 0 hours, 24 hours and 48 hours promoted the consistent trend of SPIO-hNIS-EGFP-hUCMSCs and MRI, but the tracing of living fluorescent imaging cells for seventh days was negative. Prussian blue and frozen fluorescence sections showed that more stem cells entered the skin near the wound in the external magnetic field group.
Conclusion:
The SPIO-hNIS-EGFP-hUCMSCs cell line was successfully established by the lentivirus transfection system for the first time, and there was no significant difference in biological activity, cell dry, proliferation activity and differentiation ability with the original hUCMSCs; the exposure time of external magnetic field had a great influence on the biological characteristics of stem cells, and the time of 6 hours a day was the best time; the application of the external magnetic field for the first time was applied. In the treatment of subcutaneous stem cell transplantation to increase the homing of stem cells, the combination of MRI and hNIS based SPECT imaging can more accurately trace the stem cells, raise sensitivity and reduce false positive.
【学位授予单位】：暨南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R329;R814.42

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