| Objective:We integrated molecular imaging, magnetic targeting nano-tech, cryobiology andmany other technologies to mark hNIS reporter gene, EGFP reporter gene and SPIOUmbilical cord mesenchymal-derived stem cells to set up the in-vivo tracking system ofSPECT, mesenchymal stem cells, MRI, and the fluorescent fitting imaging of livinganimals, and to use different imaging mechanisms to precisely distinguish the regionalconvergence of mesenchymal stem cells, the number of differentiated stem cells and theratio of dead stem cells; The additional magnetic field enables the stem cell to be tracedwhen it obtained the ability of target remove by using MRI and SPECT99mTc and livelittle animal fluorescent fitting image so as to have a complete and direct understanding ofthe progress of stem cell from injection to differentiation, and from a new perspectivemake deep exploration into the targeting migration ability of stem cell and tracingtechnology.Method:(1) The establishment of totally marked stem cell lines: separation andidentification of the umbilical cord mesenchymal stem cells, then we select EGFP reportergene to indirectly reflect the expression of the target gene hNIS. After the expanding ofPCR, we cloning the target gene hNIS into pDAN3.1and connect the EGFP with thedownstream of hNIS, then get started cloning vector by homologous recombinationtogether with entry carrier via BP reation, the fianl step is to produce the express cloneby the homologous recombination with entry clone and expression carrier. After theverification of digestion, PCR and sequence tesing, the recombinant plasmidpCMV-NIS-EF1-GFP-PGK-puro was successfully built up, then to infect the humanumbilical cord mesenchymal stem cells. After the acquisition of stem cell lines that steadily express hNIS and EGFP,125I inflow and125I outflow experiments should beconducted to identify the protein function of hNIS. Incubate spio at a proper drgree ofconcentration with the cell line to acquire stem cells marked by hNIS, EGFP and SPIO tovindicate their activity and dryness.(2) The in-vitro study conducted is according to different exposure duration inthe external magnetic field: the blank control groups will have unmarked hUCMSCs,and the experimental groups will have SPIO-hNIS-EGFP-hUSMSCs cells and be put anexternal magnetic field under the culture plate for3consecutive days. They will bedivided to1-hour group,6-hour group,12-hour group, and24-hour group in accordancewith the length of duration in which they are exposed to magnetic field every day. Cellviability was determined by trypan ble, cell proliferation was determined by MTT assy,the dispersal ability in the magnetic field was determined by the Transwell experiment,cell differentiation was determined by the osteoinductive differentiation, the expression ofapoptosis factor such as BCL-1, Bax, Caspase-3and Caspase-9was determined byWestern blot. Optimal exposure time was screening for the in-vivo experiment andclinical study.(3) The targeted migration of stem cells under the influence of the externalmagnetic field and in-vivo tracking: set up a denuded mouse model with its overallskin compromised, and conduct magnetic resonance imaging and MRI, SPECT, andin-vivo fluorescence imaging track in0hour,24hours,48hours and7days after the cellsare transplanted. Respectively measure the reduction rate in size, signal to noise Ratio,carrier to noise ratio and transfer of cell clusters of the image parameter of MRI which isoriginally situated in stem cell clusters. The number of stem cells in the skin wound wasdetermined by the prssian blue staining and frozen fluorescent staining. Compare thehealing time of the skin of the two groups.Results:(1) Mesnchymal stem cells derived from human umbilical were isolated and cultured,and the proliferation as well as stem was determined. With mediation of the third slowvirus packaging system, the research finishes constructing eukaryotic plasmid whichcontains target gene hNIS and obtains stable expression hNIS-EGFP-hUCMSCs cell line. The Western blot identifies that the expression of hNIS is fine. the experiment group ofhNIS-EGFP-hUCMSCs took in the activity of125I, the internal flow experiment and theexternal flow experiment of125I all proved the good result that after the hNIS express ofmesenchymal stem cells in human umbilical cord. Mesnchymal stem cells derived fromhuman umbilical were co-labeled by SPIO and hNIS EGFP, the stem cells were named bySPIO-hNIS-EGFP-hUCMSCs. The cell uptake of SPIO was proved by prussian bluestaining with a mark rate of98%. There’s no obvious difference betweenSPIO-hNIS-EGFP-hUCMSCs and original hUCMSCs in biological activity, cellstemming, proliferation as well as differentiation, whick makesSPIO-hNIS-EGFP-hUCMSCs therapeutic cells in animal model, and Fluorescent imagingcell in the SPECT, MRI and in-vivo animal model.(2) In the magnetic field of200mT, cells exposured6h has no difference with theblank control in cell viability, proliferation and differentiation. While the cell viability anddifferentiation of cells exposured12h were completely inhibited. According to theTranswell cell migration experiment, the migration ability ofSPIO-hNIS-EGFP-hUCMSCs won’t increase with a longer exposure time after exposuredto the magnetic field for6h. No excessive expression of proapoptosis gene will beinduced with magnetic exposure time less than6h per day. Apoptosis of SPIO-hNIS-EGFP-hUCMSCs may be induced with exposure time more than12h.(3) MRI tracer imaging obtains relatively high-quality image, the hUCMSCs markedby SPIO, hNIS and EGFP is successfully influenced by implanted into magnetic fieldgenerating magnetic field gradient, the magnetic field guides stem cells quickly moves inthe subcutaneous area to the defect part of skin. This effect is most obvious during thefirst24hours after external magnetic field begin coming into play. The detections ofSPECT imaging position indicating stem cells in0h,24h,48h and7days agree with thetrend of MRI imaging tracing transplanted stem cells, but its spatial resolution is lowerthan that of MRI. The fluorescent image of SPIO-hNIS-EGFP-hUCMSCs in animal at0h,24h, and48h agreed with MRI, while the tracing appeared negative in day7due to thelow cell concentration. Prussian blue and fluorescence seciton showed that there were agreat deal of stem cells near the skin wound. Conclusion:SPIO-hNIS-EGFP-hUCMSCs cell line was established by the Lentivirustransfection system for the first time, and it showed no obvious difference with originalhUCMSCs in biological activity, cell stemming as well as proliferation. The biologicalcharacteristics of stem cells are greatly affected by the exposure time to the magnetic field,and the optimal time is6h per day; for the first time, external magnetic field was used toincrease homing of stem cells in the stem cell transplant therapy. With MRI and SPECTimaging based on reporter gene hNIS, the trace of stem cells can be more precise,sensetive,and with less false positive. |
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