An Experimental Study In Pre-treated Bone Marrow Mesenchymal Stem Cells With Vegf In Treatment Of Acute Radiation-induced Intestine Injury In Rats

Part One:Isolation,culture,purification and identification biologicalcharacterization of bone bone mesenchymal stem cellsObjective： BMSCs of the SD rats were isolated, cultured，purified andbiological characterization,to lay the foundation of the the entire issue.Method: Combine by density gradient centrifugation and bone marrowadherent culture, to culture and purify of BMSCs.And cell growth wasobserved under a microscope. Identify cell surface markers with flowcytometry.Identify its osteogenic, adipogenic differentiation potential.Results: The isolated and cultured BMSCs were fusiform, uniformsize, similar to fibroblasts. The flow cytometry results showed thatBMSCs surface express CD44, CD90, and CD45negative. P3-generation BMSCs can be osteogenic, adipogenic differentiation.Conclusion: In this part of the experiment, the BMSCs successfullyisolated and cultured is high purity, uniform morphology, which can beused in later experimental studies. Part Two: The experimental study of the CXCR4expresstion ofBMSCs stimulated by VEGFObjective: To evaluate the effect on VEGF-stimulated BMSCs homingMethods: The first part of the isolated and cultured of BMSCs wererandomly divided into three groups, A groups: control group stimulatedby complete culture medium; Group B: adding VEGF in F12DMEM tomake the final VEGF concentration of10ng/ml. Group C: adding VEGFin F12DMEM to make the final VEGF concentration of50ng/ml. Themembrane and inner membrane expression of CXCR4each group after48h stimulation were measured by flow cytometry.The results:The membrane and inner membrane of BMSCs each groupcan express CXCR4, and the expression of the membrane is higher themembrane one,and the highest is C group, P <0.05Conclusion:1. The membrane and inner membrane of BMSCs and BMSCsstimulated by VEGF can express CXCR4.2. After VEGF stimulation, the membrane and inner membraneexpression increased; and the highest expression is under50ng/mlconcentration; Part Two: SDF1-expression in acute radiation intestine injuryObjective:1. To establish a model of acute radiation intestine injury;2, To further explore the effects of VEGF on BMSCs homing.Methods:90female SD rats were randomly divided into3groups (n=30), the control group (A), BMSCs treatment group (group B), andBMSCs stimulated by VEGF treatment group (group C). Groups B andC were injected of1×106/ml BMSCs suspension1ml and BMSCs whichis stimulated by50ng/ml VEGF suspension1ml for48h，within4hoursafter irradiation by tail vein; A group were intravenously infusion ofsaline1ml within4hours after irradiation.1,3,7,14,21days after modelingsix rats of each group were randomly sacrificed to get whole bloodspecimens and two terminal ileum. Intestinal tissue structure wasobserved under a microscope, measuring villus height and crypt depth;Measurement serum concentrations of citrulline and VEGF by Elisa. TheSDF-1expression of intestinal tissue was measured byImmunohistochemistry SP after modeling three days.Results:1. Established intestinal radiation injury model successfully;3,7,14days after modeling, mental status, physical activity, food intake andexcretion of rats in group C were better than the situation in group A,group B, but1,21days after modeling no significant difference betweenthe three groups of rats. HE staining showed that3,7and14days aftermodeling, compared with group A and group B group A have more complete intestinal structure,less necrosis of small intestinal epithelialcells,fewer inflammatory cells, and rich glandular structure. Bymeasuring the intestinal villus height and crypt depth assess of bowelfunction7,14days after modeling.In the same period, the result is C>B> A, P <0.05statistically significant.1,21days after the modeling,villus height and crypt depth, had no significant differencen each groupin the same period, P>0.05.2. The SDF-1expression of intestinal tissue was measured byImmunohistochemistry SP.It is found that SDF-1expression levels ofgroup C were significantly higher than A, group B, and group B higherthan that in group A.Conclusions:1, The dose of the establishment of intestinal radiationinjury model is12Gy by linear accelerator;2, Radiation bowel injury can self-repair cycle in about2to3weeks;3, BMSCs stimulated by VEGF can promote intestinal repair of acuteradiation injury, can improve the efficiency of its repair, but there is nosignificant difference in long-term effects;4, BMSCs stimulated by VEGF can promote intestinal damage repairthrough increaseing BMSCs homing.Its mechanisms is related toSDF-1/CXCR4.