Study Of Third Party Bone Marrow Mesenchymal Stem Cells On Graft Versus Tumour Effect After Mis-matched MHC Hematopoietic Stem Cells Transplantation

Objective:Allogeneic hematopoietic stem cells transplantation( allo-HSCT) can rebuild haematogenesis and immune function, which makes it one of the most effective methods to cure malignant hematological diseasees. The superiorly curable effect of allo-HSCT closely linked with GVT effect. GVHD and GVT are commonly simultaneous, which leads to that most approaches to lessen GVHD weaken GVT effect at the same time. Mesenchymal stem cells( MSC) have been observed the effects to advance haematopoietic reconstitution and relieve GVHD after being transplanted together with HSC.But whether it weakens GVT effect,is not clear. In this study, we used mouse inbred line C57BL/6H-2b→BALB/cH-2d to establish mis-matched MHC allo-HSCT of tumor-bearing mice, and transplanted BMMSC of closed populationed KM mice together with donor-deprived bone marrow cells and spleen cells after cultured in vitro. The aim of the study is to investigate the influence of third party MSC on GVT while enhanceed engraftment and induced immune tolerance. Methods:1 AnimalsSpecific pathogen-free (SPF) inbred mouse C57BL/6^H-2b (B6),BALB/c^H-2d at age of 8 to 12 weeks were used to form mismatched -MHC-transplantation models. KM mice at age of 4 to 6 weeks supplied BMMSC for experimental groups and Mouse B6 at age of 4 to 6 weeks supplied BMMSC for control groups. Both male and female were used in the experiment. All mice were supplied by Hebei Province Laboratory Animal Center. The certificate number is 803177.All the mice were bred in laminar flow animal tanks.2 Preparation of MSCB6 mice and KM mice who supplied BMMSC were executed by pulling off neck. Then both thighbones and shankbones were separated in germfree circumstance. Marrow cells were dashed out by 20%FBS DMEM/F-12(1:1)culture medium ,beaten upon gently and then planted in cultivable bottles in 10⁷/ml. The cells were exchanged semis new culture medium at the first and second 48th hour post-planting, then exchanged exclusive medium every 3 to 4 days. When cells inosculated to 90%, they were digested by 0.25% E and 0.02% EDTA, and existed from generation to generation in 1:2. The cells were used in the experiment after the third generation.3 Preparation of SPC and BMCExecuted B6 mice by pulling off neck and separated spleen ,thighbones and shankbones in germfree circumstance. Prepared suspension of SPC and BMC conventionly to be spared.4 Cell line of B16B16 were supplied by Hebei Province Laboratory AnimalCenter. After conventional culture and passage, prepared cell suspension of 1×10⁷/ml, and subcutaneously injected into the oxter of BALB/c,about 1×10⁶ cells per mouse.On the transplanting day , took out of the tumor in germfree circumstance and prepared the monoplast suspension. Then the cells were adjusted to the concentration of 1×10⁷/ml after taken count of in Typan Blue Staining (Cell Viability >95%).5 Establishment of MHC mismatched HSCT models with tumor bearing miceBoth donors and recipients drank acidification water before transplantation. B16 cells were subcutaneously injected into the oxter of BALB/c mice beforehand. Recipients were prepared for transplantation with 8.50 Gy sub-lethal irradiation (dose rate 0.63Gy/min) using 60Co source at day 0. Both BMC and SPC were injected intravenously into recipients in four hours after sub-lethally irradiation, ( BMC 5×10 6 + SPC1×10⁷ )/0.2ml/each mouse. KM mice deprived MSC in different dose were given to mice in each experimental group and B6 mice deprived MSC were given to mice in control group .6 Experimental animal groupsa. low dose third party MSCs group : B16+TBI+(BMC+SPC)_B6+MSCKM(5×10³)→BALB/c b. middle dose third party MSCs group: B16+TBI+(BMC+SPC)_B6+MSCKM(5×10⁴)→BALB/cc. high dose third party MSCs group: B16+TBI+(BMC+SPC)_B6+MSCKM (5×10⁵)→BALB/cd.high dose donor deprived MSCs group: B16+TBI+(BMC+SPC)_B6+ MSC_B6 (5×10⁵)→BALB/ce. with none MSCs transplanted group : B16+TBI +(BMC+SPC)_B6→BALB/cf. TBI group: TBI→BALB/cg. TBI+B16group: TBI+B16→BALB/ch. B16 group: B16→BALB/cNote: 4～5 animals in each group(not including the killed animals to obtain specimens in different time after HSCT). The experiment repeat twice.7 Result criterion standard7.1Transplant failure:Animal died with WBC≤1×10⁹/L, or died 15 day after HSCT with WBC≤1.5×10⁹/L.7.2 Judgment of GVHD:Animals with WBC>1.0×10⁹/L accompanied with more than three of the following symptoms to be settled for the GVHD: depressed, disarrayed of hair, depilating, denudative, hunched back, diarrhea, ophthalmia and loss of weight ,even died . Animals that appeared symptom in 10 days were settled for super acute GVHD(saGVHD)and that after 10 days before 30 days were settled for aGVHD .7.3 Judgment of tumor caused deth:Animal without obvious GVHD performance that died with WBC>1.5×10⁹/L,and the tumor obviously enlarged with necrosis .7.4 Judgment of long term survival:Animal of which survival time exceeded 30 days were settled for having long term survival.The survival time exceeded 60 days was recorded as 60 days.8 Content of observation after HSCTAfter HSCT the following conditions of the mice should be observed:food-taking condition,mental condition , aGVHD conditions and body weight everyday;the number of WBC in peripheric blood every two days;tumor size measuring with sliding caliper after the tumor could be touched every three days. Record the survival time and the reason of death of every mouse.9 Statistical analysisStatistical analysis was performed with SPSS 13.0 software package. (p<0.05) was considered significant for all statistical analyses, and Kaplan-Meier curve was protracted.Results:1 Influence of BMMSC on hematopoietic reconstruction The number of WBC in peripheric blood in all transplant groups and TBI group reached to the lowest level at 4th day after transplantation. The engraftment time from a to f by turns were: 6.0±0.76d,5.3±0.64d,4.9±0.6d,5.0±0.53d,6.7±0.74d,12.0±1.31d。The engraftment time in all transplant groups with third party MSCs and donor deprived MSCs was significantly sooner than f group in which the mice did not undergo a transplantation(p<0.05). The engraftment time of group a ,b,c and d ,in which third party MSCs or donor deprived MSCs were transplanted with HSC ,were sooner than that of without MSCs . The effect was observed to be dose-dependent. The engraftment time of third party MSCs group was not notablely compared with that of donor deprived MSCs group ,p>0.05.2 Influence on GVHD2.1Change of body weightBody weights of group a,b,c and d reached the lowest leval on d+7,and then appeared heterogeneity in changes in the contribution of engraftment ,GVHD and the tumor growth. Body weights of group f and g reached the lowest leval on d+10. Group f's recoverd slowly ,but group g's recoverd more quickly with the tumor growth and surpassed that of all other groups,untill died of prostration . group h's increased with the tumor growth untill died.2.2 Influence on General condition of aGVHDIncidence of aGVHD in group a,b,c,d and e was 100% (40/40) and the appear time of aGVHD by turns were 7.00±1.07d,12.6±1.06d,14.75±1.28d,14.63±0.92d,5.88±0.83d. Mice in group a and e were observed to develop aGVHD much sooner,and died quickly than that in group b,c and d . The time for mice in group b,c and d to appear aGVHD markedly later than that in group e(p<0.05) ,and aGVHD in the previous groups were more slightly than that in the post one .The effect presented dose dependent. The time for mice in group c to appear aGVHD was not notablely different compared with that in group d (p>0.05). But all the mice in the transplantation groups died of aGVHD eventually.2.3 Influence on Pathologic features of lung,liver and small intestine after aGVHDThe basic pathologic changes of all the transplant groups were similar.But the changes in the group a and e were more severe : lung was hyperemia, pulmonary alveolus cavity hemorrhage was obvious;liver cell dropsy accompanied with big piece stove necrosis, portal area , central vein and liver blood sinus were obviously expansion; small intestine gland and partial mucous epithelium necrosis, there were massive necrotic epithelial cell in the enteric cavity. The pathological changes of b,c and d group were obviously reduced , but there were no notable difference between each other .3 Influence on the tumor growthBecause of the severe aGVHD, the mice in group a and e died without notable tumors growing .The appear time of visible tumor of mice in group b,c ,d ,g and h by turns 9.00±0.53 d,8.50±0.93d,8.75±0.71d,7.63 d±1.06d,6.38 d±0.92d,and the tumor size on d+15 by turns were 0.235±0.070cm³ ,0.312±0.041cm³,0.299±0.038cm³,2.543±0.233cm³ ,2.132±0.239 cm³ . The tumor size on +15d tended slightly bigger with the increasing of MSCs dose,but there was no significantly statistics difference between each group(p>0.05). There was no significantly statistic difference between the tumor size of high dose third party MSCs group and high dose donor deprived MSCs group(p>0.05),which indicated that Both donor deprived and third party MSCs don't influence GVT effect .4 Influence on survival timeThe survival time of mice in group a--h by turns were 9.13±0.99d , 18.25±1.39d , 27.13±3.18d , 26.25±2.60d ,8.25±0.89d,39.00±2.73d,17.16±0.98d,36.88±2.90d.The mean survival periods of group b,c and d were longer than that in group e (p<0.05). There was no significantly statistic difference between the survival time of mice in group c and d(p>0.05).Conclusions:1 Both donor deprived and third party BMMSCs can promote haematopoietic reconstruction after mismatched MHC HSCT.The effect is dose dependent ,but not the source of MSC.2 Both donor deprived and third party MSCs can lessen severe aGVHD and prolong the survival periods of mice .The effect is dose dependent but not the source of MSCs.3 Both donor deprived and third party MSCs don't influence GVT effect in a certain range while lessen severe aGVHD.