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目的探讨自然杀伤T细胞(NKT)对大鼠角膜移植免疫排斥反应的防治作用。设计实验性研究。研究对象8只Fisher344大鼠为供体,16只Lewis大鼠为受体。方法无菌取Lewis大鼠脾脏中淋巴细胞,RPMI1640培养基体外培养三周后,流式细胞仪分选出NKT细胞(浓度5×10~4/ml)。取8只Fisher344大鼠为供体,16只Lewis大鼠为受体行穿透性角膜移植术。将受体大鼠随机分为治疗组和对照组,每组各8只。治疗组在手术结束时结膜下注射0.1ml上述浓度的NKT细胞,对照组注射相同体积生理盐水。术后观察记录植片的存活情况,术后第10天,每组2只大鼠取材,行组织病理学、免疫组织化学和流式细胞仪检测。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。结果对照组角膜植片平均存活时间为(8.00±1.58)天,NKT细胞治疗组为(26.00±1.34)天。组织病理学检查可见对照组大鼠角膜植片重度水肿,角膜基质纤维板层排列紊乱,大量炎性细胞浸润,新生血管长入植片。而治疗组植片仅表现为轻度水肿,少量炎性细胞浸润。免疫组织化学染色可见对照组植片中大量CD4+和CD8+淋巴细胞浸润,治疗组中仅见少量CD4+和CD8+淋巴细胞浸润。流式细胞仪检查结果表明在治疗组脾脏中NKT细胞为(3.90±0.32)%,明显高于对照组(1.85±0.21)%;外周血中治疗组NKT细胞为(1.34±0.12)%,对照组为(3.59±0.23)%。结论NKT细胞结膜下注射可延长角膜移植片存活时间,为防治角膜移植免疫排斥反应提供新的思路。
Objective To investigate the preventive and therapeutic effects of natural killer T cells (NKT) on immune rejection in rat corneal allografts. Design experimental research. Study object 8 Fisher344 rats as donors, 16 Lewis rats as the receptor. Methods Lymphocytes from spleens of Lewis rats were collected aseptically. After cultured in RPMI1640 medium for three weeks in vitro, NKT cells were sorted by flow cytometry (concentration 5 × 10 ~ 4 / ml). Eight Fisher344 rats were used as donors and 16 Lewis rats as the recipients for penetrating keratoplasty. The recipient rats were randomly divided into treatment group and control group, each group of eight. At the end of surgery, the treatment group received subconjunctival injection of 0.1 ml NKT cells in the above concentrations, while the control group received the same volume of normal saline. Postoperatively, the survival of the implants was recorded. On the 10th postoperative day, 2 rats in each group were drawn for histopathology, immunohistochemistry and flow cytometry. The main indicators of corneal graft survival time, histopathology and immunohistochemical staining of lymphocyte infiltration. Results The average corneal graft survival time was (8.00 ± 1.58) days in control group and (26.00 ± 1.34) days in NKT cell treatment group. Histopathological examination showed that corneal graft in rats in the control group had severe edema, disordered corneal stromal fibrin layer, a large number of inflammatory cell infiltration and neovascularization. The treatment group showed only mild edema, a small amount of inflammatory cell infiltration. Immunohistochemical staining showed a large number of CD4 + and CD8 + lymphocytes infiltration in the control group, only a small amount of CD4 + and CD8 + lymphocytes infiltration in the treatment group. The results of flow cytometry showed that the percentage of NKT cells in the spleen of the treated group was (3.90 ± 0.32)%, which was significantly higher than that of the control group (1.85 ± 0.21)%. The NKT cells in the peripheral blood were (1.34 ± 0.12)%, Group was (3.59 ± 0.23)%. Conclusion Subconjunctival injection of NKT cells can prolong the survival time of corneal graft and provide a new idea for preventing and treating corneal graft rejection.