Effect Of Mesenchymal Stem Cells For The Tropism And The Growth Of Colon Cancer

[Objective]1. To investigate pathological changes of subcutaneous transplantation tumor in nude miceby establishing animal models of colon cancer.2. To isolate, culture and purify bone marrow-derived mesenchymal stem cells(MSCs) ofnude mice, and identify MSCs according to suffice markers.3. To observe the effects of MSCs on subcutaneous transplantation tumor in nude mice bytransplanting MSCs to the nude mice of colon cancer.[Methods]1. The nude mice subcutaneously colon cancer, colon cancer cell lines Hct116method toestablish nude mice subcutaneous transplantation tumor model: Logarithmic phase Hct116cells,with0.25%of pancreatic enzyme digestion, PBS fluid percussion,1000r/min at roomtemperature centrifugal,10min, abandon the supernatant, Repeat the above steps3times, withserum-free culture medium heavy suspension cells into single cell suspension, adjust the cellconcentration to107/ml. Cell suspension liquid shake, with1ml sterile syringes from cellsuspension0.2ml (including2x106cells). On the right side of the waist skin disinfection nudemice, will be cells on the right side of the waist subcutaneous injection syringe. A total of20injections. Ten days after the injection, the continuous observation, minister of the right side ofthe waist to be nude mice out of subcutaneous tumor is greater than5mm in diameter, nudemouse model is established in colon cancer. Nude mouse model of colon cancer will only20nude mice according to random number table method is divided into two groups, experimentalgroup10, control group10.2. Whole bone marrow adherent culture was chosen to culture MSCs. Whole bone marrowwas collected from healthy adult rats in sterility condition. Both lower limbs of the rats wereremoved and femur medullary cavities were flushed with Dulbecco’s modification of Eagle’s medium(DMEM), low glucose until the diaphysis became white. The douche was centrifugedfor5min at1000rpm, then removed the supernatant, and resuspended the deposition withDMEM-low glucose,10%~15%fetal bovine serum(FBS) and antibiotics. The cells suspensionwas innoculated in75cm2cell culture flask and cultured in the incubator maintained at37℃in asaturated humidity atmosphere with5%CO2. Growth and adherence of MSCs was obsevedevery day. Medium was changed every3days and non-adherent cells were removedconcomitently. MSCs were subcultured at a ratio of1:2or1:3according to the density. Afterpassage3, surface markers were detected with flow cytometry analyses(FACS). The harvestedMSCs were resuspended with phosphate buffered saline (PBS) and than separated into7tubeswith each tube about1×106cells. CD34, CD45, CD90and CD29antigen on the surface ofMSCs were detected.3. MSCs were obtained from male SD rats and subcultured, proliferated and purified asdescribed above. The third generation of MSCs in a carbonyl CM-DiI fluorescent cyanine dye,dioctadecy tag, rate of fluorescent microscope. Then MSCs into cell suspension, theconcentration of1x106/ml, the first10days in Hct116cells injected into nude mice skin,colon cancer will nude mice model group2ml per of nude mouse tail vein injection containing1x106mark of MSCs cell suspension, the control group2ml per of nude mouse tailintravenous saline solution. Observation of transplanted tumor growth situation, every3to4days at the same time two groups of tumor transplanted tumor long and short diameter, calculatethe tumors had product, depicting tumor growth curve, transplant MSCs14days, euthanasianude mice, subcutaneous transplantation tumor, tumor weight, calculate. Tumor specimens forquick frozen pathological, fluorescence microscope frozen pathological tissue Dil positive cells;HE staining, observed the pathological changes.4.[Results]1. Line subcutaneous transplantation tumor tissue pathological dyeing can be observedthat HE transplanted tumor cells as an adenocarcinoma, cancer cell nuclear size, number,multinucleated tumor giant cells, visible pathological fission like, more diffuse plate shaped ornests, full of myxoid secretions in the cell.2. The original generation vaccination after24h in a microscope to see more cell wallstickers, appearance is spindle and polygon,3d after the cells began to accumulate, form a long fusiform fibroblasts samples, after training to9d, cell confluence of around90%, the cells arearranged polarity, colony spiral shaped. After in fluid and extend to the third generation, thecells tend to be purified, morphology, uniform membrane spread out. By the flow cytometryinstrument detection, cell surface markers CD29positive rate was98.6%, CD90were99.6%;And CD34, CD45were negative, positive rate of0.56%,0.89%respectively.3. Differences between subcutaneous transplantation tumor tissue volume had nostatistical significance (P>0.05);The difference between experimental tumor weight andweight control tumor has no statistical significance (P>0.05).Frozen pathological tissue inexperimental subcutaneous tumor fluorescent microscope visible CM-DiI hair redfluorescence positive cells.[Conclusion]1. Using nude mice subcutaneously colon cancer cell lines Hct116, can successful preparationof nude mouse model of colon cancer, this experiment method is stable and good repeatability.2. The whole bone marrow adherent method difference can obtain high purity MSCs,provides abundant source of MSCs for subsequent experiments.3. MSCs have the function of the targeted colon cancer tissue.4. MSCs had no significant effect on colon cancer tissue growth.