The Inhibiting Effect Of MSCs And Its Mechanism On Radiation Induced Thymic Lymphoma

With the development of radiotherapy techniques and equipment, the effects of cancertreatment are increased, and patients’ five-year survival rate is significantly increased.However, the induced second malignancies reports are also increased year by year, includingleukemia, colorectal cancer and bladder cancer. Thymus, as the main body immune organ, isalso sensitive to radiation, and radiation-induced thymic lymphoma is a highly invasive,even metastasizing tumor, which has poor prognosis. Therefore, it is necessary to pay moreattention to radiation-induced the second malignancies.Bone marrow mesenchymal stem cells (MSCs) are a class of pluripotent stem cells withmultipotent differentiation potential, immune regulation, targeting homing capability and soon. The studies have shown that MSCs had tissue repair function for brain injury, spinal cordinjury, kidney damage, pancreatic damage and skin damage.In this study, the method of bone marrow adherent was applied to isolate and cultivateMSCs in vitro. Cell cycle and cell surface markers of MSCs were detected by flowcytometry to identify MSCs. Selection of healthy childhood C57BL/6J mice were dividedinto normal group, the irradiation group and MSCs therapy group. Animal model wasprepared according to classical Kaplan thymic lymphoma methods, in which the mice ofMSCs therapy group were treated by0.2ml suspension with the concentration of2×106cells/ml by tail vein infusion after one-day and one-week irradiation. The generalconditions of the mice were observed every day, and the mice were killed to take thymusafter six months irradiation. Histological observation was detected by HE staining, theproliferation of thymocytes, cell cycle kinetics of each subpopulation cells, the expression ofCD3in each subsets and TCR Mf were discovered by flow cytometry, and RT-PCR wasused to test the expression of the related upstream and downstream genes of CyclinD1, such as P15(INK4B), P16(INK4A), P18(INK4C), P19(INK4D) and Rb.The results showed the growth of MSCs separated from whole bone marrow was in goodcondition. The results of flow cytometry showed the majority of cells are relatively inactiveand quiescent, and cell surface markers CD44expression was positive, whereas CD34was notexpressive, which accorded with the characteristics of MSCs. The mouse of radiation groupseemed gray and dull, and some of the mouse had poor mental symptoms after three-monthirradiation, while the conditions of MSCs therapy group were significantly improved.Histopathological observation results showed that the thymus cortex and medulla structureof mice at normal group was clear, the lymphocyte morphological was regular and the sizewas uniform, presenting circular or elliptic. However, the thymus structure of radiated-micewas completely destroyed, the lymphoid neoplastic cells was diffused distribution, the shapewas irregular, the size was differ, the nucleus was big with deep dyeing, and there appearedmirror cells and gypsophila phenomenon.There also existed nucleus fission, while thethymus structure of MSCs therapy group tended to be normal.In thymus T cells, the percentage of CD4-CD8-cell subsets in the irradiation group wassignificantly higher than in the normal group, while in the MSCs therapy group wasapproximate. The percentage of CD4+CD8+cell subsets in the irradiation group subsets wassignificantly lower than in the normal group and in the MSCs therapy group. the percentageof CD4+CD8-cell subsets in the MSCs therapy group was higher than in the in theirradiation group, which was approximate to the normal group. The percentage of CD4-CD8+cell subsets was low, which was approximate to0and had no obvious difference in thosethree groups.In whole thymocyte and CD4-CD8-cells subsets, the G1phase cells in irradiation groupwas significantly higher than in the normal group and MSCs group, whereas the number of Sphase cells in irradiation group was significantly higher than in the normal group and in theMSCs therapy group it was approximate to normal level. In CD4+CD8+and CD4+CD8+cellssubsets the number of S phase cells in irradiation group was significantly higher than in thenormal group and the MSCs therapy group.In whole thymus cells, CD4+CD8+and CD4+CD8-cell subsets, the number of parent, G2or G3, G4cells in the normal and MSCs therapy group were significantly higher thanthat in the model group, while G5, G6, or G8, G9, G10cells and proliferation index (PI)were significantly lower than in the model group. In thymus CD4-CD8-cell subsets, parent,G2and G3cells in the irradiation group were significantly higher than in the normal andMSCs therapy group, while the G4, G5, G6cells and PI in irradiation group weresignificantly lower than in the normal and MSCs group.For various CD4CD8cell subsets in normal group, the expression of CD3+cells wasobviously higher than that in the irradiation and MSCs therapy group. After MSCstransplantation the expression of CD3+cells is higher than in the radiation group, but stillsignificantly lower than in the normal group. The expression of CD3+cell was opposite. TheTCR mutation frequency (TCR Mf) in the irradiation and MSCs therapy group which wassignificantly higher than in the normal group.The result of RT-PCR showed that the mRNA expression of upstream geneP15(INK4B), P16(INK4A) and P18(INK4C) in the radiation group was higher than in thenormal group, whereas the expression declined after MSCs transplantation（p<0.05）.Compared with the control group, the relative mRNA expression of P19(INK4D) geneincreased significantly（p<0.05）. And compared with radiation group, the relative mRNAexpression of P19(INK4D) gene also increased significantly. However, the relativeexpression of Rb in irradiation group was significantly lower than in the normal and MSCstherapy group.Conclusion：MSCs transplantation can make thymic lymphoma cell cycle arrest remission, whichmay have inhibition function for radiation-induced thymic lymphoma. Its repair mechanismsmay be related to the MSCs for increasing expression of CyclinD1upstream genes，such asP15(INK4B), P16(INK4A), P18(INK4C), P19(INK4D)，and decreasing expression ofCyclinD1downstream gene Rb.