| Objective: Acute graft versus host disease (aGVHD) has been consider which was the major obstruct of allogeneic hematopoietic stem cells transplantation(allo-HSCT). Bone marrow mesenchymal stem ce11s(BMMSCs) are another stem cells in bone marrow which different with hematopoietic stem cells, it could be promote the hematopoiesis reconstruction , reduces aGVHD, and induce immunity tolerance in allo-HSCT. Since the low immunogenic in BMMSCs, it was considered to take as a new therapeutics for aGVHD treatment, and donor's or third-party MSCs might be have the same founction. To investigate the effect of third-party BMMSCs on acute GVHD after MHC mismatch HSCT in mice, different quantity of third-party BMMSCs were used. The influence of engraftment and immune restitution by third-party BMMSCs were observed simultaneouslyMethods1 AnimalsDonor mice: Specific pathogen-free (SPF) mouse C57BL/6H-2b (B6), aged 6~8 weeks; recipient mice: Specific pathogen-free (SPF) mouse BALB/cH-2d , aged 6~8 weeks; third-party MSCs donor mice: Specific pathogen-free (SPF) mouse KM, aged 6~8 weeks; The animals provided by experiment animal center of HeBei province. All of them were breeded in hecto-grade asepsis laminar air flow bench.2 Isolation and Cultivation of BMMSCsMice were killed through dislocating cervical vertebra, their femur and tibia were exposed on super-clean bench. Medullary canal was syringed with complete medium (CCM), which was F12-DMEM culture fluid and contained penicillin (100μg/ml), streptomycin (100μg/ml), HEPES (0.03mol/L) and 10%FBS. After prepared as unicell suspension through blowing softly, Rinse solution was inoculated directly into aseptic 25cm2 plastic culture flask . The culture was performed with 5%CO2 at 37℃and saturated humidity. First half moulting fluid was after 48 hours and population parameter moulting fluid was after 4 days. Hereafter, moult fluid per 3-4days. When the cells got confluence, they were dissociated by using 0.25% diastase vera. Mesenchymal stem ce11s that undergone at least three times dissociation were used for transplantation.3 Preparation mouse spleen and bone marrow cellsAfter executing donor mice by breakdowning vertebra, abstract spleen, femoral and shin bone of double side. Prepare cells of bone marrow and spleen in routine method. Count cells number by congo blue. More than 95% cells should refuse to dye. 4 Establishment of MHC non-syngeneic transplantation model All of the animals drink acidified water three days before transplantation. The recipients received whole body irradiation by a linear accelerator(total dose is 10 Gy, rate of dose is 0.63Gy/min).The recipients were injected with cells according to groups four hours after irradiation. The cell quantity is: bone marrow cells 5×106+spleen 1×107/0.2ml/mice. The experimental group needs at the same time infusion different origin different dosage BMMSCs/0.2ml/mice, and control group infusion physiological saline /0.2ml/ mice.5 Animal groupsa TBI without transplantation group BALB/c +TBIb allogeneic transplantation group BALB/c +TBI + (BMC+SPC)B6c allogeneic transplantation+donor BMMSCs group BALB/c +TBI + (BMC+SPC)B6 + MSC B6 (5×105)d allogeneic transplantation+low dose third-party BMMSCs group BALB/c +TBI + (BMC+SPC)B6 + MSCKM(5×103)e allogeneic transplantation+middle dose third-party BMMSCs group BALB/c +TBI + (BMC+SPC) B6 + MSCKM (5×104)f allogeneic transplantation+high dose third-party BMMSCs groupBALB/c +TBI + (BMC+SPC) B6 + MSCKM (5×105) Note: 4~5 animals in one group(not including killed animals at different times after transplantation in order to obtain serum specimens). The experiment duplicate twice.6 Result determinationWBC≤1×109/L when animal died or WBC≤1×109/L 15 days after transplantation was due to failure of transplantation. WBC > 1.5×109/L accompanying three symptoms of the following: depressed, disarrayed of hair, depilating, denudative, hunched back, diarrhea, ophthalmia and loss of weight.Super-acute GVHD was diagnosed when above-mentioned symptoms occurrenced within 10 days after transplantation. Acute GVHD was diagnosed when above-mentioned symptoms occurrenced from 10 to 30 days after transplantation. The time of engraftment: Continual two times WBC>1×109/L. Selects when takes the lung, the liver, the small intestine to carry on the pathology inspection.7 Content of observationPsychosis and aGVHD performance of each animal group after HSCT was observed during the experiment, and the survival time recorded. Recipients were weighed every two days, and WBC of periphery blood counted from d-1 to d+30.Blood of survival each group after engraftmeng the 7th day was prepared, and all samples were analyzed on a FACSCallibar immediately for the content of CD8+ cells, CD4+cells and ratio of CD4+ /CD8+ calculated. Before taking the transplant, and transplants the latter 7 days of each group of mouse, the methylcellulose semi-solid raise examines the CFU-GM production rate.8 Statistical treatmentAnalyze data by using SPSS13.0. Mean士standard deviation expresses grouped data. T-test was used between two groups. Draw Kaplan-meier survival curves.Results1 Influence of third-party BMMSCs to hematopoietic reconstruction1.1 Engraftment timeAll transplant group's WBC of periphery blood reaches the lowest level in +3d, and each group succeeds transplants. Engraftment time of a,b,c,d,e,f each group is: 0d,6.7±1.3d,5.0±0.7d,5.7±0.8d,4.8±1.2d,4.6±1.0d. The c,d,e,f group compare with b group, the engraftment time reduces obviously(P<0.01). The infusion compares the engraftment time for c group and f group the time not to have the difference (P>0.05). d, e, f during each group carries on the comparison: d group compares with f group, the engraftment time has the obvious difference (P<0.01); d,e group and e,f group is quite even not obvious difference (P>0.05).1.2 CFU-GM colony countingThe number of CFU-GM in a,b,c,d,e,f each group is: 0,23.0±2.0,60.9±2.2,23.4±3.1,49.6±2.3,60.1±3.0. The c,e,f each group compares with b group, the number of CFU-GM increases obviously (P<0.01);d group compares not obvious difference with the number of CFU-GM for b group (P>0.05); d,e,f during each group carries on the comparison, during each group has the obvious difference (P<0.01).2 Third-party BMMSCs the influence which occurs to aGVHD2.1 aGVHD occurrence time and suevival timeCarries on mouse's body weight which the different allogeneic transplantation group along with the GVHD appearance to assume the declining trend. The b and d group present Super-acute GVHD in +3~+5 day; c group and f group present Super-acute GVHD in +9~+12 day; e group present Super-acute GVHD in +7~+9 day .The survival time in a,b,c,d,e,f each group is : 8.5±1.0d,7.8±0.8d,22.3±4.1d,8.9±1.0d,14.9±1.6d,22.5±2.3d. The c,d,e,f each group compares the survival time with the b group to lengthen obviously(P<0.05). The infusion compares the survival time for the c group and f group not to have the difference(P>0.05). Carries on the comparison d,e,f during each group: during each group has the obvious difference(P<0.01).2.2 Histopathology observationThe histopathology observes each group of GVHD to have the time pathology change basically consistent. The b group pathological change degree is most remarkable : the lung hyperemia, the pulmonary alveolus cavity hemorrhage is obvious;liver cell dropsy and is accompanied by the big piece stove necrosis, collects the district and the central vein and the liver blood sinus obvious expansion, the extravasated blood; the small intestine gland and partial mucous epithelium necrosis, in the enteric cavity has necrosis epithelial cell which falls off massively. The c,d,e,f each group of pathological change degree obviously reduces, during its each group compares not obvious difference.3 The restoration of the mouse immune cell after transplantation In transplantes latter 7 days, the survival mouse for c,e,f groups. The number of CD4+ respectively is: 5.59%, 7.85%, 6.74%; The number of CD8+ respectively is: 14.37%, 13.16%, 15.19%。With normal mouse's CD4+, CD8+ (44.40%, 10.26%) the value compares, these three group of CD4+ value obviously reduces, but CD8+ value obvious markup, and the rate of CD4+/CD8+ is inversion, respectively is 0.39, 0.60, 0.44,4.33(normal:4.33).Conclusions1 Both the donor BMMSCs group and the third-party BMMSCs group can delay the occurrence time of aGVHD in non-syngeneic transplantation model and accelerate the time of engraftment.2 the function of the third-party BMMSCs reduceing GVHD and promoting engraftment to become with its dosage is being related.
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