The Clinical And Experimental Research On The Regulatory T Cell And Stomach Cancer

Part â… : The clinical research on the CD4~+CD25~+ regulatory T cell in the tissue of stomach cancer and it's relationship with clinicopathologic character.Purpose: To analyze the component of T cell in the microenviroment around stomach cancer cell,especially CD4~+CD25~+ regulatory T cell,and observe the relationship between CD4~+CD25~+ regulatory T cell and the clinicopathologic characters. Method: From July2003 to June 2004 ,there are 22 patients of stomach cancer and 16 patients of benign diseases of stomach with different clinical symptoms came into our research.AU the patients came to the No.2 affiliated hospital of Suzhou University and accepted the gastroscopy and pathology examination ,stomach cancer patients underwent the operation to resect the tumor. The specimens we got from the stomach resected by operation or from gastroscopy,send to the laboratory immediately and digested by the mixed enzymes to single cells, separated by density gradient centrifugation, flowcytometry to check CD3 , CD4, CD8 and CD25 expressed on the surface of the cell ,thenanalyze the component of the subpopulation of T cell, especially the cell coexpressed CD4 and CD25 molecular. Collect the clinical and pathologic characters of cancer patient, use statistic software to analyze the relationship between CD4~+CD25~+ regulatoryT cell and the clinicopathologic characters . Result: It is higher that the CD4~+CD25~+regulatory T cell subset in T lymphocyte in the stomach cancer than that of benign diseases(p<0.01) ,30.95% vs 19.05%,while CD4~+CD25~+Treg/CD8~+T is higher in cancer than that in benign disease( p<0.05),0.70 vs 0.43 .Analyze the relationgship between CD4~+CD25~+Treg and the clinicopathologic characters with SPSS ,result shows that thereare no significant relationship between them(p>0.05). Conclusion: CD4+CD25+ regulatory T cell is prevalent in stomach cancer microenviroment. The prevalence of CD4+CD25+ regulatory T cell around cancer cells maybe happened in the early stage of stomach cancer. Part II :The purification of CD4+CD25+ regulatory T cell and CD8+ T cell from peripheral blood in vitro.Purpose:To purify CD4+CD25+ regulatory T cell and CD8+T cell from peripheral blood in vitro and observe the possibility to expand CD4+CD25+ regulatory T cell and CD8+ T cell in vitro. Method: Blood was gotted from a healthy volunteer. Cells were purified by negetive or positive seletion utilizing magnetic cell sorting using MACS microbeads (Miltenyi biotec).CD4+CD25+ regulatory T cell were sorted by two steps .-first negetive selection of CD4+ T cells.second positive selection of CD25+ T cells. CD8+T cell were sorted by positive selection .To check the purity of the cells utilizing flow cytometric analysis .The cell then cultured in RPMI1640 median which contain certain cell factors. Result: CD4+CD25+ regulatory T cell and CD8+ T cell's purity are 86.55% with 91.78% respectively . CD8+T cell expanded significantly by high concentration of IL-2's stimulation.But it was poor for CD4+CD25+ regulatory T cell's to expand. Conclusion: we can obtain CD4+CD25+ regulatory T cell and CD8+ T cell with high purity by utilizing magnetic cell sorting using MACS microbeads .It is easy to get great number of CD8+T cell by culture with IL-2.While it is difficult to expand CD4+CD25+ regulatory T cell by culture with IL-2 AND anti-CD3 antibody .Part IIIrThe experiment on the inhibition of CD4+CD25+ regulatory Tcell to CD8+T cell.Purpose:To study the inhibition of CD4+CD25+ regulatory T cell to CD8+ T cell in vitro,including the CD8+ T cell's proliferation and cytotoxity against tumor cell .Method :The cell sorted with the method introduced in part II ,were used in thefollowing experiment. 1) The experiment of CD8+ T cell's cytotoxity to SGC-7901 stomach cancer cell. 2) The experiment of CD4+CD25+ regulatory T cell inhibit CD8+ T cell's proliferation. 3) The experiment of CD4+CD25+ regulatory T cell inhibit CD8+ T celPcytotoxity to SGC-7901 stomach cancer cell.Result: 1) CD8+ T cell cultured with high concentration of IL-2 had the ability to kill SGC-7901 stomach cancer cell,it's quantity-dependant.When effector-target ratio is 10:1, the ratio that cancer cell be killed is near to 50%, when effector-target ratio is 50:1,it is 78.08%.2) CD4+CD25+ regulatory T cell inhibit proliferation of CD8+ T cell, the degree of inhibition is parallel to the quantity of CD4+CD25+ regulatory T cell. 3) Exogenous IL-2 can reduce the degree of inhibition (49.1 % vs 31.4%). 4) CD4+CD25+ regulatory T cell inhibit CD8+ T cell's cytotoxity to kill SGC-7901, When ratio of CD4+CD25+ regulatory T cell/CD8+T cell greater than l:4,the ratio that SGC-7901 cell be killed was decreased. Conclusion: CD4+CD25+ regulatory T cell can inhibit CD8+ T cell's proliferation and cytotoxity to SGC-7901 stimach cancer cell, it's quantity-dependant. CD4+CD25+ regulatory T cell which increased in the tissue of stomach cancer ,showed a suppressive effect in the cancer microenviroment,and played a role in cancer progression and may present a barrier to immunotherapy.