MR Tracing Of Bone Marrow Stem Cell Targeting Experimental Study Of Hepatocarcinoma In Rats

Part Ⅰ Bone Marrow Mesenchymal Stem Cell Culture, Purification andAmplificationObjective: To establish the separation, purification, cultivation,identification, amplification of bone marrow mesenchymal stem cells (BMSCs)method in Wistar rats.Methods: The adherence screening method for isolation and culture ofMSC from Wistar rat bone marrow and analysis of the basic biologicalcharacteristics. The morphological features and proliferation characteristicswere observed. The expression of CD34, CD45, CD29, and CD44wereanalyzed by flow cytometry1Electronmicroscopic features and multipledifferentiation potentials were confirmed by lineage-specific induceddifferentiation to osteoblasts and adipocytes.Result: Primary MSC were spindle-like, mainly to colony growth.Passaged for three generations, the heterogeneity of the cells significantlyincreased. Phenotype analysis of MSC did not express CD34and CD45, ahigh degree of expression for CD29, CD44. The negative control group, IgG2ain-FITC and IgG1-PE, obtain the negative experimental results.Conclusion: We successfully isolated, cultured and amplified the BMSCsof the rats, and were identified to achieve the purpose of the experiment, laidthe foundation for further study.Part Ⅱ Assessment of Charaterristics of Bone Marrow MesenchymalStem Cell Labeled with Superparamagnetic Iron Oxide Particles in VitroObjective: To explore magnetic labeling efficiency and cell growthactivity influence by superparamagnetic iron oxide particles (SPIO) andpoly-L-lysine (PLL) mark of rat bone marrow mesenchymal stem cells(BMSCs). To optimize the labeling ratio of SPIO and PLL.Methods: In this study, we established the seven study groups. The blank control group only contains unlabeled BMSCs (group A). In accordance withSPIO alone or with PLL concentration gradient (final concentration0.25,0.5,0.75and raised to1.0μg/ml) is divided into five experimental group (group Bto F). Each experimental group according to the different concentrations ofSPIO into four experimental tubes (the final concentration of USPIO were25,50,100and150μg/ml). Different concentrations of USPIO-PLL complexmixed media, adding that the BMSCs were incubated overnight. Experimentalgroup B to F cell markers after36h, take the number of labeled cells in1×105,and resuspended in0.5ml Ependoff tube. The labeling efficiency was assessedby iron content, Prussian blue staining, electron microscopy and in vitro MRimaging.24experimental tubes were put into the plastic model of the onecontaining agarose gel, using a1.5T MRI scans. Scan sequences includesT1WI, T2WI, and the SWI. Determination of the sequence signal strength, anddo statistical analysis with analysis of variance. The cell viability wasdetermined by trypan blue exclusion test. The observation of cell growth ismade by MTT assay. Using the electron microscope marks after the BMSCand intracellular SPIO particles. Using the flame method measure theintracellular iron content, and determine the iron content and the relationshipof the MR signal.Result: Group B to F of labeled cells remains its original form in thecytoplasm are visible Prussian blue staining particles. Cells with markedconcentration increase and the use of the PLL, the blue dye particlesincreased. Electron microscopy of SPIO-PLL labeled mixture of stem cellswithin the iron gathered into a group, most of particle sizes were in the10~20nm. Only SPIO-labeled stem cells within the small number of iron ions,sparse, a lot of iron there is no agglomeration of pellets. From group A to F,trypan blue staining cell viability were99.20%±1.11,97.41%±3.42,96.32%±3.67,97.24%±4.34%,95.43%±3.33and93.43%±5.33. Display morethan90%of the cells refused to dye trypan blue, we can see the mark of theiron ions of BMSCs to maintain good cell viability. Smaller iron ions withinthe cytoplasm of cells. According to the judgment of the growth curve of cells in each group simply use the iron ion concentration of200μg/ml cell growthinhibition and use1.0μg/ml PLL the growth of the cells of the same willproduce a certain extent. Flame measured separately pure SPIO labeled cellsand SPIO with PLL labeled cells compared with a significant difference (P<0.05). T1WI, T2WI and SWI each sequence scanning prompts are significantdifferences among the groups (P <0.05). T2WI and SWI image sequences ofgroup B to group F signal intensity gradually decreased, reflecting the changesof the iron.Conclusion: It showed that no more than160μg/ml for SPIO safeeffective concentration of marker, the amount of the PLL should be less thanraised to1.0μg/ml. In accordance with the experimental results, we believethat SPIO100μg/ml and PLL0.75μg/ml labeled cells without adverselyaffecting cell activity and growth, and the stronger the signalintensity of thelabeledcells.Part Ⅲ Sensitivity of superparamagnetic iron oxide-labeled bone marrowmesenchymal stem cells using Different MR SequencesObjective: To compare the two1.5T MR scaner sequence sensitivity forthe detection of magnetically labeled BMSCs.Methods: Use a20cm×15cm×12cm plastic model. The phantom modelfilled with a2%agarose gel. The experiment uses8EP tube, group A iscontrol group containing pure PBS solution, Group B to I was10,20,30,40,50,60,70μg/ml SPIO, respectively.Siemens and GE.1.5T two MR scaner,the scanning sequence, including T1WI, T2WI, T2~*, HASTE, SWI, ESWANand R2~*. MR signal values between the two groups were compared usinganalysis of variance.Result: The scanning sequence of different concentrations of SPIO has asignal strength differences (P <0.05). The best sequence of changes in thescanning sequence of signal values of the two machines and the results of thestatistical analysis of SPIO scans the Siemens MR machine SWI sequence andGE MR R2~*sequences. T2~*sequence of Siemens and GE ESWAN-sequencealso reflected to some extent, thetrend of the iron concentration, but the control group PBS signals are low, in the abovetwo sequences, resulting in itsidentification of the uncertainty of whether iron capacity.T2WI sequences canalso be reflected to some extent, the iron concentration trends, statisticsshowed limited ability to distinguish high concentrations of iron. T1WIsequence of signal changes did not reflect the changes of iron concentration.Conclusion: SPIO enhanced effect lasts for a long time, the scanacquisition time is more adequate, so more suitable for applications with goodsignal-to-noise ratio, non-breath-holdscanning sequence. The best sequence ofthe SPIO scans of the R2~*sequences and SWI sequence. Siemens T2~*sequence and GE's ESWAN-sequence to a certain extent reflect the trend ofthe iron concentration.Part Ⅳ Establishment of Implanted Hepatic Cancer Model in WistarRatsObjective: To establish a model of liver cancer in the liver of rat, andevaluate the pathological and MR imaging features.Methods: The rat CBRH7919tumor strain recovery, culture,amplification, passaged three generation. The third generation of tumor cellswere used as inoculum cells. Concentrated cancerous suspension ofCBRH-7919cell lines (concentration of about5×107/ml) were injectedsubcutaneouly into a Wistar rat liver. Two sites were selected for each rat weretransplanted.1week preoperatively, postoperative continuous2weeks foreach rat intramuscular dexamethasone2.0mg/day.2weeks after surgery withmagnetic resonance imaging (MRI) of tumor growth, screening modeling inrats, randomly selected from five rats were killed to remove the tumor in theliver pathology.Result: The success rate of implanted tumors was75%(15/20).15ratswere grown to23tumor nodules. The tumor size5mm-16mm. Thepathological types were hepatocellular carcinoma.Conclusion: Inoculated with the rat model of hepatocellular carcinoma inthe Wistar rat liver open to the immune suppression, is an easy to operate,simple, high success rate. Apply to diagnostic imaging and other aspects of the experimental study.PartⅤ In Vivo Tracing of SPIO Labeled BMSCs Transplanted forHepatic Cancer by MR Imaging in RatsObjective: At the animal model of the rat hepatoma-based, non-invasive invivo between the transplantation of bone marrow mesenchymal stem cells(BMSC) for sustainability, visualorientation and quantitative analysis. Toobserve the interaction and influence between HCC and BMSCs.Methods: Pre-transplant apply SPIO particles to tag. Mark the same wayasthe third part. HCC model rats were randomly divided into2groups, groupⅠ(9rats) magnetically labeled BMSCs injected group, the spleen injectedmagnetically labeledcells,1×107/0.5ml; group Ⅱ pure SPIO injection group,spleen to inject35μg/0.5ml/only (6rats). Experimental animals, respectively,one hour before surgery,postoperative1h,3h,6h,12h,24h,48h,1week,2weeks,4weeks of MR scanning. MR sequences includes T1WI, T2WI and T2~*and SWI. The experimental group, magnetically labeled before and aftercomparison of the liver tissue and carcinoma insignal strength and differentsequence of hepatic tumor CNR multiple time points using analysis ofvariance.Result: Control rats after injection of SPIO within the spleen, the liver ofrats one hour after injection, scanning MR signal decreased to the lowest.T1WI signal value decreased by18.12%, T2WI, the signal decreased by55.4%,the SWI signal decreased by71.55%, and T2~*signal value decreased by60.99%. To the second week after injection, the liversignal values basicallyback to normal. Experimental group in the6hours after the injection ofmagnetically labeled stem cells of the liver parenchyma and tumor of signalreduced the most significant. Liver T2WI signal decreased by16.44%, theSWI signaldecreased by38.84%, T2~*signal value decreased by12.97%.HCC T2WI signaldecreased by18.65%, the SWI signal decreased by26.76%,T2~*signal valuedecreased by34.51%.1week after injection of stem cells,liver tissue signal values recovered to before the injection level. Foci withintwo weeks after the injection tomaintain a mild signal to reduce, scan four weeks after the tumor signal is restored to thelevel before the injection.Contrast to noise ratio of the experimental group before and after injection ofliver cancer there are significant differences (P <0.05).