| Objective: The superiorly curable effect of allogeneic hematopoietic stem cells transplantation (allo-HSCT) is closely linked with graft versus tumor (GVT) effect. And the preventive measures of GVHD such as the application of TCD and immunosuppressant can weaken GVT effect and effective engraftment.Now both GVHD and GVT are thought to be immunoreaction in which T cells act mostly. Though they relate with each other, they can separate from each other. Nowadays CD8+Tc2 cells have been proved to be correlative with GVT effect.Bone marrow mesenchymal stem cells (BM-MSC) have the characteristics of self-renewal and the ability to differentiate along multiple lineages. Because MSCs exhibit low immunogenicity and demonstrate significant suppressive activity in cell cultures containing alloreactive T cells, it can expand the range of clinic application in allogeneic organ and cells transplantation. The further research in functionary mechanism of allo-HSCT has indicated that BMMSC could restrain the happening of aGVHD. However, there has no verdict for GVT in malignant disease. The aim of this study is to investigate that MSCs induce immune tolerance and influence on GVT based on mouse-transplanted model with tumor.Methods:1 Animals and cell line1.1 AnimalsSpecific pathogen-free (SPF) inbred mouse C57BL/6H-2b (B6), CB6F1 (BALB/cH-2d×C57BL/6F1, CB6F1H-2d×b) at age of 8 to 12 weeks were used to form haploidentical transplantation models. Mouse B6 at age of 4 to 6 weeks supplied BMMSCs. Both male and female were used and randomized in the experiment. All mice were supplied by He bei Province Laboratory Animal Center.1.2 Cell line of B16B16 cells, a melanoma cell line, could form tumor when subcutaneously injected into the left axilla of CB6F1 mice. The cells were cultivated in 10%FBS H-DMEM culture medium, and they were cultivated in a fully humidified atmosphere of 5% CO2 at 37℃. The cells growing in logarithmic phase were chosen in the experiment.2 Preparation of MSCsMouse B6 who supplied BMMSCs was executed by cervical dislocation. Then both femora and shankbones were separated in germfree circumstance. Marrow cells were dashed out by 10%FBS F12/DMEM culture medium and beaten upon gently and planted in cultivable flasks at the concentration of 107 cells/ml. The cells were first exchanged new culture medium at 72th hour post-planting, then exchanged medium every 3 to 4 days. When cells inoculated to 90%, they were digested by 0.25% E and 0.02% EDTA, and passaged from 1 to 2. The cells were used in the experiment after the third generation.3 Establishment of mouse models with tumorB16 cells were subcutaneously injected into the axilla of CB6F1 mice. When the tumor grew up to 1×1×1cm3, it was exenterated and thrown out necrotic tissue and divided into small pieces. Then they were whetted on 300-nylon net and suspend again to obtain MNC. Then the cells were adjusted to fit concentration after taken count of in Trypan Blue Staining (Cell Viability >95%).4 Establishment of haploidentical mouse-transplanted models with tumorBoth donors and recipients drank acidified water before transplantation. For the construction of haploidentical transplantation models for tumor-burden mice, B16 cells were subcutaneously injected into the axilla of CB6F1 mice beforehand. Recipients were prepared for transplant with 950 radsγsub-lethal irradiation (dose rate 0.63Gy/min) using 60Co source at day 0. Both bone marrow cells and spleen cells were injected intravenously into recipients in two hours after sub-lethally irradiation(,BM cells 2.3×106+ splenocytes 7×106)/each mouse. For syngeneic transplantation models, B16 cells were subcutaneously injected into the axilla of CB6F1 mice. After given lethally irradiation, these mice were transplanted donor cells from CB6F1 mice. MSCs in different dosage were given to each group mice to see their effect on aGVHD and graft anti-tumor effect.5 Animal groupsa syngenic transplantation group: CB6F1→TBI+B16+CB6F1;b haploidentical transplantation group without MSC(GVHD group): B6→TBI+B16+CB6F1;c haploidentical transplantation group with low dosage MSC : B6→CB6F1+TBI+B16+MSC(5×103/each mouse);d haploidentical transplantation group with middle dosage MSC : B6→CB6F1+TBI+B16+MSC(5×104/ each mouse);e haploidentical transplantation group with high dosage MSC : B6→CB6F1+TBI+B16+MSC(5×105/ each mouse);f TBI without transplantation group;g TBI+B16 without transplantation group;h B16 group to form tumor.6 Influence on immunocytesThe survival mice were killed at +21d, and the peripheral blood of each group were collected as well as the pre-transplant mice. All samples were analyzed on a FACSCallibar immediately for the content of CD3+CD8+ cells and CD3+CD4+cells and CD3-CD56+cells and ratio of CD4+ /CD8+ calculated.7 Content of observationPsychosis of each animal group after HSCT was observed during the experiment, and the survival time recorded, and the mean survival time calculated. The survival curves of tumor-burden mice were protracted. Recipients were weighed every two days, and WBC of periphery blood counted from d-1 to d+60, and tumor measured and compared.8 Statistical analysisStatistical analysis was performed using SPSS 13.0 software package. (P<0.05) was considered significant for all statistical analyses, and Kaplan-Meier curve was protracted.Results:1 Changes of animal body weight: The weight of mice reached to the lowest level at +6d～+7d post-transplant in all transplant groups. And allogeneic BMT group came back sooner than others. TBI group reached to the lowest level at +10d～+11d, then rose slowly. TBI+B16 group was at +18d～+19d, but it increased faster than other groups following the increase of tumor mass.2 Recovery of peripheral WBC: WBC in all transplant groups treated with TBI reached to the lowest level at 4th day after transplant. The time of engraftment in a, b, c, d, e, f and g groups was 9±2.1d, 8.4±1.2d, 6.2±1.7d, 4.3±1.1d, 5.8±2.3d, 20±2.0d, 20±1.51d respectively. And the time of hematopoietic reconstitution in all transplant groups with MSC was significantly sooner than that in a group and b group. However, the time of hematopoietic reconstitution in f and g group was slower than that in other groups, and there were significant differences. 3 Incidence of aGVHD: In b group incidence of aGVHD was 100% (17/17), beginning at 10±2.3d, while in e group that was decreased to 60% (6/10), beginning at 27±3.0d. The signs of aGVHD in MSC groups were slight, even there had no signs. In the experiment it was found that the size of tumor in mouse with severe aGVHD was smaller than that in mild one.4 Effect of MSCs on tumor mass after transplant: The size of tumor in c group at +30d was 1.22±0.41cm3, which was the smallest in all groups. However, the size of tumor in B16 group was significantly bigger than that in others. Though the size of tumor in TBI+B16 group was not significantly different from that in auto-HSCT, both of them were significantly bigger than that in haploidentical HSCT and cotransplantation of bone marrow cells with BMMSC. It indicated that there was no GVT effect in auto-HSCT group. The tumor size of cotransplantation of BM cells with BMMSC was increscent gradually along with the increase of MSCs, but there was no significantly statistic difference among them. The tumor mass of mice with severe aGVHD was smaller than that with slight aGVHD.5 Survival time and rate: The mean survival time of a, b, c, d and e group was 58.7±3.3d, 54.4±7.6d, 59.6±1.1d, 59.4±1.7d, 58.0±4.0d respectively. The survival time in f, g and h group shortened significantly comparing with a, b, c, d and e group. And the survival rate of b group at +50 day was significantly lower than that in c, d and e group. 6 Effect of MSC on immunocytes: CD3+ in syngenic transplantation group (a group) at 21d was significantly lower than haploidentical transplantation group. Following with MSC increased, CD3+ T cells in c, d and e group decreased significantly after transplant, but there were no difference between groups. It indicated that MSC could weaken aGVHD, and the effect was direct correlated with MSC dose.CD8+ T cells in b, c, d, e group were significantly higher than that before transplant. However, the content of CD8+ T cells in cotransplantation groups declined following the increase of MSCs infused.CD4+/CD8+ in a group was significantly higher than other groups. And the ratio of CD4+/CD8+ in b, c, d, e group lower significantly after transplant, which indicated that immunity recovered slowly in haploidentical transplantation group.No significant difference was observed in the content of CD4+ T cells in all groups between pre-transplant and post-transplant. The content of CD3-CD56+ cells increased following the increase of MSCs infused, but no significant difference was observed in the content of CD56+ cells in all groups.Conclusions:1 The tumor size of semi-transplanted mice was obviously smaller than that of auto-transplanted ones. It indicated that semi-transplanted mice had considerable GVT effect. 2 While lessening severity of aGVHD, MSCs could promote hematopoietic cells engraftment and prolong survival time.3 MSCs could postpone the happening of aGVHD, which was direct correlation with MSC dose. MSC could weaken GVT effect, though there was no statistic significance in our experiment.4 In the experiment we found that the size of tumor in mouse with severe aGVHD was smaller than that in mild ones. It could explain the relation between GVHD and GVT effect.5 CD8+T cells recovered faster than CD4+T cells in vivo, which indicated that BMMSC could change T cells sub-group after transplant, then influenced GVT and GVHD. CD56 in each group was not considered significant in the experiment. We cound not eliminate the reason of hCD56.6 CD3+CD4+, CD3+CD8+, CD3-CD56+ and CD4+/CD8+ in haploidentical transplantation groups resumed slowly comparing with auto-BMT.
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