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

Part One: Isolation, culture and identification bone mesenchymalstem cells of SD ratsObjective： Isolation, culture and identification bone mesenchymal stemcells of rats， to obtain BMSCs of high purity, and stability of thebiological characteristics,lay the foundation for the research.Methods：1.Isolation、culture bone mesenchymal stem cells from maleSD rat femur and tibia by combined used the methods of density gradientcentrifugation and cells adherent,and cell morphology was observed.2.The surface antigens identification: The expression of CD90, CD45,CD25and CD34of the third generation cells were analyzed by flowcytometry.3.Von Cusa staining (Von Kossa) staining, oil red O stainingwere used to identification bone mesenchymal stem cells thedifferentiation potential. of the osteogenic, adipogenicResults:1.Mesenchymal stem cells of isolated and cultured by combinedused the methods of density gradient centrifugation and cells adherentgrowth by clone type,spindle, Uniform size. The P3generation BMSCspurity up to98.5%.2.Cultured BMSCs measured by flow cytometryshow expression,CD44and CD90surface antigen, do not express the CD34surface antigen.3.The cultured BMSCs by induced candifferentiate into bone cells and adipocytes.Conclusions: Combined used the methods of density gradientcentrifugation and cells adherent can successfully isolate and culture ratBMSCs. And size uniformly, power proliferation, high purity, stablebiological characteristics, can be used for the experimental study of thesubject of BMSCs. Part Two: An experimental study in pre-treated bone marrowmesenchymal stem cells with TGF-differentiation toepithelial cell.Objective：Explore the capacity of BMSCs stimulated by TGF-todifferentiation epithelial cell and find the optimum inductionconcentration. lay the foundation for the next experimental studyMethods：BMSCs cultured in3plates with12-well cell culture plate.Each plate were randomly divided into four groups, group A: controlgroup. Group B: add200ng/ml TGF-. Group C: add250ng/ml TGF-.Group D: add300ng/ml TGF-. VEGF concentration of1,3and7daysof cell culture medium was detected by ELISA, BMSCs were detected byimmunofluorescence technique whether have CK protein in the day of1、3、7. Results:1.Culture medium of each group were detected VEGF, group Chave the highest concentration of the same period, P〈0.05.2.CK proteindetected in the first seventh days in the group which added by TGF-,group C have the highest concentration of the same period.Conclusions:1.Cultured BMSCs can secrete VEGF itself.2.TGF-canstimulate BMSCs secrete more VEGF, and the most were250ng/ml.3.TGF-can induce BMSCs differentiation to epithelial cells,and the most were250ng/ml Part three: An experimental study in pre-treated bone marrowmesenchymal stem cells with TGF-in treatment ofacute radiation-induced intestine injury in ratsObjective：1.Establish the model of acute intestinal injury by irradiationin rats.2.Observation the effect of infusion BMSCs、 BMSCs ofpre-treated with TGF-to treatment acute radiation-induced intestinaldamage.And to investigate the efficacy mechanisms.Methods：1.SD rats irradiatied by X-ray using a linear accelerator at ourhospital Department of Radiation Oncology:. The total dose of12Gy.2.Irradiated180female SD rats were randomly divided into threegroups (60/group), group A (control group), B group (BMSCs treatmentgroup) and group C (BMSCs treatment group stimulated by TGF-). Agroup in the4hours after irradiation tail vein infusion of1ml of normal saline; B group in the4hours after irradiation tail vein infusion of1×106/ml BMSCs suspension of SD rats; C group in the tail vein within fourhours after irradiation infusion of1×106/ml by250ng/mlTGF-stimulated BMSCs suspension of SD rats1ml. After irradiation1day,3days,7days,14days,21days.Each group skill six rats randomly and getspecimens from whole blood and terminal ileum. Light microscopestructure of intestinal tissue observed villus height and crypt depth; serumand ileal tissue citrulline and IL-6, TNF-, VEGF concentration wasmeasured by ELISA.Results:1.SD rat acute intestinal injury radioactive modeling: successfulestablishment of a model of acute radiation-induced intestinal injury inSD rats.2.BMSCs stimulated by TGF-and BMSCs of acute radiationthe intestinal injury treatment effect（:1）compared with the same period ingroup A:B group and C group SD rats show less intestinal epithelial cellnecrosis and inflammatory cell infiltration,more villi and glands; villusheight and crypt depth C> B> A, P <0.05.（2）compared with the sameperiod in group A: B group and C group levels of IL-6, TGF-concentration of3,7, and14days is lower, P <0.05, and group Cconcentration is lower than in group B (P <0.05), the first1day,21daysno significant difference, P>0.05.（3）compared with the same period ingroup A: B group and C group VEGF, citrulline concentration of3,7, and14days is high, P <0.05. And concentration of group C than in group B (P <0.05),1day,21days, no significant difference (P>0.05).（4）organization citrulline and IL-6, TGF-and VEGF concentrations werehigher than the same period serum concentrations.Conclusions:1.Use linear accelerator can successfully established acuteradiation-induced intestinal damage.2.BMSCs stimulated by TGF-andBMSCs can promote intestinal structure and function recover of acuteradiation intestinal injury3.BMSCs stimulated by TGF-and BMSCsproliferation of acute radiation-induced intestinal damage repair andanti-inflammatory related.4.BMSCs stimulated by TGF-promote theefficacy of bowel function recover of acute radiation intestinal injurymore significant than BMSCs...