The Study Of Treatment With Bone Marrow Mesenchymal Stem Cells In Irradiation-induced Acute Skin Injury

Objective:Isolation, culture, amplification bone mesenchymal stem cells of rat and its antigens and the potency of multi-directional differentiation were identified. To establish a steady culture of BMSCs. Establishing the rats local acute skin irradiation injury model, observe the effect of treatment with BMSCs and study of the mechanism.Methods:1. Isolation , culture of BMSCs: Take SD rats, aseptic conditions separation rats double lower limbs tibiofibulas, PBS flush the marrow cavity. With Ficoll separation medium isolation individual nucleus cell, DMEM-LG cultivation, observation its morphology.2. Identification of BMSCs:(1) The surface antigens identification: The expression of CD90 , CD45, CD25 and CD34 of the third generation cells were analyzed by flow cytometry.(2) The differentiation ability identification: The third-passage cells were respectively cultured in osteogenic, adipogenic, confirmed using silver nitrate and oil red O.3. Establishing the model of local acute skin irradiation injury: select 60 randomly in 62 male SD rats and anesthesia with 1% sodium pentobarbital. The model of acute skin irradiation injury in rats replicated by 45 Gy x-ray to right buttock skin. (area,2cm×2cm) .4. Experiment group and interference model: The rats are divided into 3 groups randomly as follow after irradiation:the control group (A group, n=20). The caudal vein group (B group, n=20). The local multipoint injections group (C group, n=20). NS(1ml) and BMSCs(2×106,1ml) were directly transplanted into A and B group respectively as soon as the irradiation process was finished. BMSCs(2×106,1ml) were local multipoint injected into C group when ulcerating sore were finded after the irradiation process on 14th.5. Observation index:The rats were observed every other 3 day, and take wound score and area measurement. Parts of the skin ulcer tissue ( 3 rats every time) were taken respectively on 14th, 28th, 42th, 56th day when the irradiation process was finished. Observe the pathological changes of injured skin used HE stain. The expression of TGF-β₁, SDF-1, PGE2 were studied by Immunohistochemisty and parts of the skin ulcer tissue were taken from unirradiation rats.Results:1. Isolation,culture of BMSCs results : Using Ficoll liquid gradient centrifugation combining with attachment metho can get spindle BMSCs . The original generation training 14 days or so can get multiple cells cloned cooker.2. Identification restlts of BMSCs(1) The surface antigens identification:BMSCs surface antigen profiles were positive for CD29,CD90 and negative for CD34, CD45 after analyzed by flow cytometry.(2) The differentiation ability identification:BMSCs have appeared the phenotypic character istics of osteogenic and adipogenic respectively.By silver nitrate dyeing osteoblast were black, into fat cells by oil red O dyeing visible cell has red oil droplets.3. Establishing the model : The model of acute skin irradiation injury in rats were established successfully on SD rat skin.4. Therapeutic effect: Compared with the control group BMSCs treatment groups were ulcer lighter and wound area smaller ,the healing time shorter, But the caudal vein group and local multipoint injected group has no diffidence in principle of statistics.5. Mechanism discussed:TGF-β₁ factor of BMSCs treatment group was higher at 42th and slight expression at 56 th day compared with contorl group. SDF-1 factor of BMSCs treatment group was higher at 42th day compared with contorl group. PGE2 factor of BMSCs treatment group was lower at 14th an 28th compared with contorl group.Conclusions :1. BMSCs isolated, cultured, proliferated applying density gradient centrifugation, attachment method combining with monoclone cultivation have high autoploidy.2. The model of acute skin irradiation injury in rats were established successfully on rats skin.3. BMSCs can promote effectively acute skin irradiation injury healing.4. BMSCs in acute skin irradiation injury repair may be related to regulating the microenvironment of wound healing fibrosis of value-added factor TGF-β₁, the chemokine SDF-1, inhibit inflammation has relationship...