The Study On The Effect Of Rabbit Bone Marrow Mesenchymal Stem Cells On Immunoregulation In Tumor Tissue

OBJECTIVE To investigate the immunoregulatory effect of rabbit bone marrow mesenchymal stem cells on T lymphocytes in vitro.METHODS MSCs were obtained by density gradient centrifugation with Percoll solution and sticking plastic bottle repeatedly;The surface markers CD29,CD34, CD44 and CD45 were tested by flow cytometry;The cultured MSCs were stained by ACP,PAS and NAP;T lymphocytes were harvested by using nylon column and T lymphocyte proliferation in the presence of PHA was evaluated by MTT;ELISA was used to detect the secretion of IL-2,IL-4,IL-10 and IFN-γ.RESULTS MSCs were homogenous population,the cell curve showed MSCs had a good ability of proliferation.The cultured MSCs were ACP(-),PAS(+),NAP(-), CD29(+),CD34(-),CD44(+),CD45(-).MSCs and the supernatant inhibited T lymphocytes proliferation and the inhibitory effect depended on the amount of MSCs.IL-2 and IFN-γsecretion of T lymphocytes were suppressed by MSCs or the supematant significantly,IL-4 and IL-10 secretion were promoted by MSCs at the same time.CONCLUSION MSCs obtained in this study was reliable.MSCs and the supernatant suppressed the proliferation of T lymphocytes.MSCs or the supernatant could suppress the secretion of IL-2 and IFN-γ,but MSCs had a different result on the secretion of IL-4 and IL-10.MSCs and the supematant might suppress T lymphocytes proliferation through alter cytokine variety and amount of T lymphocytes secretion.SIGNIFICANCE This experiment proved that MSCs could regulate the balance of Th1/Th2 by secretion of cytokines,induce the immunotolerance by inhibiting Th1 subset,which provided the theoretic support to treat autoimmune diseases. OBJECTIVE To study the effect of bone marrow mesenchymal stem cells on immunoregulation in tumor tissue in rabbit.METHODS 22 New Zealand white rabbits were randomly classified into the control group and the test group equally.Bone marrow mesenchymal stem cells were isolated from tibia of each animal.When the mesenchymal stem cells of each animal were cultured successfully,VX-2 tumor tissue was embedded in the thigh muscle of each animal.One week after the transplantation,the mesenchymal stem cells were self transplanted into tumor tissue in the test group while equal DMEM was injected in the control group one time every week for three weeks.F2 passage mesenchymal stem cells were marked by DAPI for locating the distribution in tumor tissue.The ultrasonography was performed for each animal 1,2,3,4 week(s)after the tumor mass was embedded in the thigh muscle.The maximum tumor diameter of each animal was recorded and the mean value of each group was calculated.One animal of each group was put to death in the third week and all the left animals were killed in the fourth week to observe the tumor development.All the tumor samples were made successive frozen section.Immunohistochemical stain and ELISA were performed to determine the content of CD4~+T,CD8~+T and cytokines.RESULTS The ultrasonography showed the tumor was growing slowly in the first and second week after the VX-2 tumor mass transplantation.The mean maximum tumor diameter of the control group and test group was no significant difference between them(P＞0.05).The tumor growth velocity of the test group increased gradually in the third and fourth week,and the difference of the mean maximum tumor diameter between the two groups increased gradually too,statistical significance appeared when comparing each other(P＜0.05).The mesenchymal stem cells were more likely to distribute in tumor stroma or the invasive border,only very few mesenchymal stem cells were seen in the tumor nest.The result of immunohistochemical stain showed that the contents of CD4~+T cells and CD8~+ T cells were decreased obviously,there was significant difference between the test group and the control group.The result of ELISA showed that the secretion of IL-2 and IFN-γwas decreased obviously in the test group while the secretion of IL-4 of test group was increased inversely,and there was significant difference between the test group and the control group.The secretion of IL-10 of the test group was also increase,but there was no significant difference between the two groups.CONCLUSIONS Rabbit bone marrow mesenchymal stem cells could make the balance of Th1/Th2 to drift to Th2 so that inhibit the anti-tumor immune response through altering the cytokines secretion.SIGNIFICANCE The results of our study show that rabbit bone marrow mesenchymal stem cells can inhibit the immune response in the tumor tissue,in addition,our topic prophase finding bone marrow mesenchymal stem cells may differentiate into myofibroblast under the induction of local tumor microenvironment, we thereby presume these cells may be related with tumor development.This hypothesis can explain the mechanism of tumor progression on another point of view. Because bone marrow mesenchymal stem cells possess the character of low immunogenicity and tumor have the ability to enrich mesenchymal stem cells,some researches have utilized mesenchymal stem cells as the drug carrier for tumor treatment.We have proved that mesenchymal stem cells can accelerate the tumor development in this study,so we think utilizing mesenchymal stem cells as the drug cartier for tumor treatment needs further researches to prove the biological safety.