超抗原活化的耐受性淋巴细胞防治高危角膜移植免疫排斥反应的实验研究

The effect of tolerant lymphocytes activated by superantigen on inhibiting high-risk corneal transplantation immune rejection

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 investigate the immune tolerance of mice lymphocytes activated in vitro by superantigen staphylococcal enterotoxin B and the anti-immune rejection effects of these cells on high-risk corneal transplantation in mice. Design Experimental study. Participants BALB/c mice were used as the donor and C57BL/J mice were used as the receptor. Methods C57BL/J lymphocytes were isolated and collected from mice spleen in sterile condition, and then cultured with SEB and ConA separately. MTT method was used to measure cell proliferation. Flow cytometry was used to analyze the percentage of CD4＋CD25＋ cells and CD3＋NK1.1＋NKT cells on the first and sixth day of culture with SEB or ConA. BALB/c mice were used as the donor and C57BL/J mice were used as the receptor. The animals were injected subconjunctivally with either 0.05 ml NS （the control group） or 0.05 ml lymphocytes suspension （the treatment group） having cultured for 6 days with SEB （the concentration of lymphocytes was 1×10^6/ml）. Corneal grafts were observed for 35 days to examine the survival rate and histopathological and immunochemical changes. Main Outcome Measures The mean survival time （MST） of the allografts, histopathology and immunohistochemical of the graft. Results The cell proliferation rate measured by OD570nm was 0.15±0.01 （n=6） before cultured with SEB, ConA, increased to 0.25±0.07, 0.59±0.06 after cultured with SEB, ConA for 3 days, and change to 0.43±0.07 and 0.35±0.05 for 6 days. The percentage of CD3＋NK1.1＋NKT cells was increased from （1.21±0.19）% to （5.67±0.25）%, and CD4^＋CD25^ cells from （1.21±0.06）% to （0.98±0.12）%. The MST of the allografts was 28.60±3.75 d in the group injected with lymphocytes, and 22.13±4.91d in the NS group （P=0.006）. HE staining of the corneal grafts showed that the grafts in NS group were severe edema, and stromal fibrous boards were disordered, and there was copious inflammatory cells infiltration and neovascularization. In comparison, the grafts in treatment group were slight edema, and the stromal fibrous boards were ordered arrangement, and there was only limited inflammatory cell infiltration and no neovascularization. Immunohistochemieal staining showed that the amount of CD4＋ and CD8＋ lymphocytes in grafts was significantly lower in the treatment group than in the control group. Conclusions Both SEB and ConA could activate and stimulate the proliferation of mice lymphocytes. Mice lymphocytes could be activated by superantigen SEB and had immune tolerance. Recipient lymphocytes activated by SEB in vitro could significantly prolong the allografts survival.