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目的检测超抗原金黄色葡萄球菌肠毒素B(SEB)体外活化的小鼠淋巴细胞的免疫耐受功能,探讨该细胞对小鼠高危角膜移植免疫排斥反应的防治作用。设计实验研究。研究对象14只BALB/C小鼠作为供体,28只C57BL/J小鼠作为受体。方法无菌取C_(57)BL/J小鼠脾脏淋巴细胞,制备成5×106/ml的混悬液,分别与SEB和刀豆蛋白A(ConA)体外共培养,MTT法测定细胞增殖。用流式细胞仪测定SEB和ConA体外活化的淋巴细胞在第0、6天时的CD4~+CD25~+调节性T细胞和CD3~+NK1.1~+NKT细胞百分比。以BALB/c小鼠为供体,C_(57)BL/J小鼠为受体,建立小鼠高危角膜移植动物模型,术后分为实验组、对照组,并分别结膜下注射0.05 ml培养6天的SEB活化的淋巴细胞悬液(浓度1×10~6个细胞/ml)及相同体积的生理盐水,术后观察记录植片的存活状况,并进行组织病理学检查和免疫组织化学检测。主要指标角膜植片平均存活时间,组织病理学及免疫组织化学染色检查淋巴细胞浸润情况。结果SEB、ConA与淋巴细胞共培养前OD_(570nm)值均为0.15±0.01(n=6),共培养后第3天为0.25±0.07和0.59±0.06,第6天为0.43±0.07和0.35±0.05。SEB组培养后的淋巴细胞中CD3~+NK1.1~+NKT细胞由0天时的(1.21±0.19)%升高到(5.67±0.25)%,CD4~+CD25~+调节性T细胞由(0.37±0.06)%升高到(0.98±0.12)%。活化淋巴细胞结膜下注射后小鼠角膜植片平均存活(28.60±3.75)天,而生理盐水组为(22.13±4.91)天,两者比较差异有统计学意义(P=0.006)。HE染色显示对照组植片中度水肿,角膜基质纤维板层结构排列紊乱,有炎性细胞浸润,植片中可见新生血管。而实验组植片仅轻度增厚,基质板层纤维排列规则,未见炎性细胞浸润及新生血管长入。免疫组织化学染色显示治疗组小鼠角膜植片中CD4~+和CD8~+淋巴细胞数量较对照组明显减少。结论SEB和ConA均能活化小鼠淋巴细胞并使其增殖。SEB组培养后的淋巴细胞中CD3~+NK1.1~+NKT细胞和CD4~+CD25~+调节性T细胞含量比培养前升高。SEB活化的小鼠淋巴细胞具有免疫耐受功能,能够延长小鼠高危角膜移植植片的存活时间。
Objective To detect the immune tolerance of lymphocytes activated by superantigen Staphylococcus aureus enterotoxin B (SEB) in vitro and to explore the preventive and therapeutic effects of this cell on the rejection of high-risk corneal allograft in mice. Design experiment research. Fourteen BALB / C mice served as donors and 28 C57BL / J mice as recipients. Methods The spleen lymphocytes of C57BL / J mice were aseptically harvested to prepare 5 × 106 / ml suspension. The cells were co-cultured with SEA and ConA respectively. The cell proliferation was measured by MTT assay. The percentages of CD4 ~ + CD25 ~ + regulatory T cells and CD3 ~ + NK1.1 ~ + NKT cells on days 0 and 6 of SEB and ConA-activated lymphocytes were measured by flow cytometry. The BALB / c mice were used as donors, and C_ (57) BL / J mice were used as recipients to establish a mouse model of high-risk corneal transplantation. The rats were divided into experimental group and control group, 6 days of SEB activated lymphocyte suspension (concentration of 1 × 10 ~ 6 cells / ml) and the same volume of saline, postoperative observation of graft survival and histopathological examination and immunohistochemistry . The main indicators of corneal graft survival time, histopathology and immunohistochemical staining of lymphocyte infiltration. Results The values of OD_ (570nm) before co-culture of SEB and lymphocytes were 0.15 ± 0.01 (n = 6), 0.25 ± 0.07 and 0.59 ± 0.06 on the third day after co-culture and 0.43 ± 0.07 and 0.35 ± 0.05. The percentage of CD3 ~ + NK1.1 ~ + NKT cells in the SEB group increased from (1.21 ± 0.19)% to (5.67 ± 0.25)% at day 0, and CD4 ~ + CD25 ~ 0.37 ± 0.06)% to (0.98 ± 0.12)%. The corneal graft survived an average of (28.60 ± 3.75) days after subconjunctival injection of activated lymphocytes and (22.13 ± 4.91) days in saline group, with significant difference (P = 0.006). HE staining showed moderate edema in the control group, disordered corneal stromal fibrin layer structure, infiltration of inflammatory cells, implants visible neovascularization. In the experimental group, the implants were only slightly thickened, and the fibers in the stroma lamellae arranged regularly with no infiltration of inflammatory cells and angiogenesis. Immunohistochemical staining showed that the number of CD4 ~ + and CD8 ~ + lymphocytes in the corneal grafts of the treatment group was significantly decreased compared with the control group. Conclusion Both SEB and ConA can activate mouse lymphocytes and proliferate them. The levels of CD3 ~ + NK1.1 ~ + NKT cells and CD4 ~ + CD25 ~ + regulatory T cells in the SEB group were higher than those in the pre-culture group. SEB-activated mouse lymphocytes have immune tolerance function, which can prolong the survival time of high-risk corneal graft in mice.