| PartⅠ：Experimental study of the SPIO-labeled Tibetan miniaturepig bone marrow mesenchymal stem cells in vitroObjective To learn the morphology and ultrastructural changes of Tibetan miniaturepig bone marrow mesenchymal stem cells(BMSCs) after SPIO-labeled, to explore thesafety、feasibility of this labeling method, and provide certain foundation for cell trac-er in vivo. Methods1. Bone marrow was extracted from adult Tibetan miniature pig,BMSCs were routinely isolated, purified and cultured, The cell growth characteristicswere observed in the light microscope,and its growth curves were recorded. Adipoge-nic, osteogenic induce and detection of surface antigens (CD45, CD90and CD10~5)were used to identify the obtained cells.2. Prussian blue staining was used to observeiron ions in the cells after labeled by different concentrations of SPIO, cell viabilitywas detected by Placenta blue exclusion experiments and MTT assay was applied toobserve cell growth before and after labeled.3. We had used SPIO (25ug/ml) tolabel different concentrations of cells （1×10~7/ml、1×10~6/ml、1×10~5/ml、1×10~4/ml、1×10~3/ml）and blank controlled group,used different concentrations (50/ml,25/ml,12.5ug/ml, agarose) of SPIO to labeled the same dose of cells(1×10~5/ml), MRI imag-ing results were compared after labeled.4. The morphology changes of the BMSCsafter labeled by different concentrations of SPIO were observed under transmissionelectron microscopy.Results1.24hours later some bone marrow stem cells startedadherence after inoculation. Division and prolif-eration of adherent cells was visible after48h when completely changing the fluid and it was more obviously5-7days later,then we can find a uniform cell prolifer ation colony and becomes long spindle grad-ually. BMSCs can maintain a good state of growth and the growth curve is basicallyconcordance at the first12genera tions. BMSCs can be successfully induced intobone cells and fat cells，and we can use Alizarin Red and Oil Red O to detected cal-cium nodules and fat particles for certificating it respectively. The positive rate ofCD90, CD10~5and CD45, which are surface markers of BMSCs, is96.9%,95.7%and1.1%.2. Labeling rate of SPIO-labeled BMSCs Blue iron particles can be visibled byinverted microscope in the intracytoplasm of cells of each tag group with differentconcentrations of SPIO incubation overnight, and the labeling rate is close to10~0%.MTT assay show that three different SPIO concentration have no effect on BMSCs’growth and proliferation within24h, and the concentration of50ug/ml have influenceon its growth and proliferation over time.3.MRI scan With the same SPIO-labeledconcentration of25ug/ml and BMSCs on the order of magnitude followed by1×10~7/ml,1×10~6/ml,1×10~5/ml,1×10~4/ml,1×10~3/ml, we can find the signalgradually increased as the density decreases.On the other hand, with the same cellsconcentration of1×10~5/ml, the higher of the concentration of iron, the more obvioussignal changes. The T2*WI signal changes is more obvious in MR sequence.4.Transmission electron microscopy reveal the high density of iron particles could befound in the cytoplasm of labeled BMSCs, which exist in the form of small vesicularand other structures are no significant differences. Conclusions The method whichmarked BMSCs is simple and feasible, and has no significant effect to biologicalactivity of MSCs in appropriate concentrations. MRI imaging show a negative changewhich is positively correlated with the cell density and SPIO concentration. The highdensity of iron particles which exist in the vesicles can be observed by electronmicroscope. The labeling method can be applied to in vivo tracer experiments. PartⅡ：Experimental study on the influence of the distribution ofSPIO-labeled autologous BMSCs by different infusion pathwayin Tibetan miniature pig grafted kidneysObjective Learn the distribution、survival and migration of SPIO-labeled Tibetanminiature pig autologous BMSCs by two pathways of transplant renal artery and iliacvein infusion in vivo of renal transplant recipients and explore effective infusionpathway. Methods We selected Tibetan miniature pigs who were8-12months withweighs about15-20Kg. Using the matching method of human organ transplant：selecting a total of12Tibetan miniature pigs with the same ABO bloodgroup,cross-matching negative,complement dependent cytotoxicity(CDC) also knownas micro-lymphocyte toxicity experiments negative（dead cells <10~%）,one-waymixed lymphocyte culture（one-way MLR）low reactivity （receptors lymphocytesvalue-added rate <0.60）. Adopted a form of one supply for two, according to thematching results we selected2as receptors with"CTX+Pred" associated inductionprogram and method of donor kidneys entire cut as cadaver human donor, and renalgraft were planted in the lateral retroperitoneal.The pig recipients was divided intotwo groups as transplant renal artery infusion group and iliac vein infusion group, n=4cases. By the transplant renal artery infusion group:it was infused by the transplantrenal artery that the concentration of the25ug/ml of SPIO-labeled autologous BMSCs(approximately2×10~6/kg) after open blood flow in surgery.By the common iliac veininfusion group:it was infused by the common iliac vein that the concentration of25ug/ml of SPIO-labeled autologous BMSCs (about2×10~6/kg) after open blood flowin surgery.The two groups of receptors pigs were sacrificed after the postoperative24h,and respectively took renal transplantation、pronephros、lung、heart、liver、spleen、pancreas and other organs,and then Prussian blue staining, HE staining andtransmission electron microscopy were performed. Results Above simulate thematching method of human organ transplant established the renal transplantationmodel in addition to the one died of anesthetic accident by the external iliac veininfusion, the rest are successful.Postoperative Prussian blue staining of biopsy revealed:by the transplant renal artery infusion group SPIO-labeled autologousBMSCs are mainly distributed in the transplanted kidney and lung,find no apparentpositive cells in other organs;by the external iliac vein infusion group as a result ofthese observations indicate that the cells are mainly distributed in the lungs, a smallamount in the transplanted kidney, other organs only to find1-2suspicious-positivecells in the liver and heart.Transmission electron microscopy find only in thetransplanted kidney of the renal artery infusion group, others are not found.Conclusions It is more conducive to the colonization、differentiation of Tibetanminiature pig autologous BMSCs in the allograft that by the transplant renal arteryinfusion than by the common iliac vein infusion. It can reduce the "capture" effect bylung and other organs... |
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