IL-10基因修饰骨髓间充质干细胞对角膜移植排斥的作用和机制研究

Although corneal allografts enjoy a remarkable degree of immune privilege, immune rejection remains the leading cause of keratoplasty failure. Mesenchymal stem cells (MSCs) have been proved to have immunosuppressive properties and have been successfully used as cellular therapy in organ transplantation. Immunosuppression activity of Interleukin 10 (IL-10) can enhance the body's immune tolerance. IL-10 has close relationship with cells, cytokine and other immune regulatory factors in the organ transplant rejection. Herein we investigated the immunomodulatory effects of lentivirus-mediated IL-10 gene transfected MSCs on rat corneal allograft rejection model. Recipient corneas were examined following transplant, and rejection was defined according to Larkin. T cell proliferation tests by BrdU to measure the direct suppressive effect. Using ELISA kit to measure the concentrations of cytokine productions related to T helper cells, such as IL-2, IL-4, IL-10, IFN-γ and IL-17. The ratio and expression of CD4+CD25+Foxp3+Tregs, which closely related to Anterior Chamber-Associated Immune Deviation (ACAID), are determined and analyzed by flow cytometry and PCR, respectively..In the present study, we investigated the effect of MSCs on corneal allograft rejection across the full MHC barrier and the therapeutic efficacy of MSCs. We also studied the effects of MSCs on T cell proliferation, on the Th1/Th2 cytokine profiles of T cells, and on the Treg population. We observed that MSCs could inhibit corneal allograft rejection and prolong corneal allograft survival on rat model. This effect might be due to an inhibition of the pro-inflammatory Th1 response and to upregulation of Tregs. These data support the potential use of MSCs as a treatment for corneal allograft rejection. We also gave systemic treat with MSCs alone or MSCs combined with different doses of Cyclosporin A (CsA) in penetrating keratoplasty. Postoperative MSCs injection reduced Th1 pro-inflammatory cytokines and elevated IL-4 cytokine secretion from T lymphocytes derived from cornea-transplanted rats. Tregs were upregulated by MSCs treatment. When 2 mg/kg CsA was given together with MSCs, graft survival was significantly prolonged. In addition, subconjunctival injections of MSCs also prolonged the survival time of cornea allograft, and the MSCs of injected upregulated the expression of IL-10 in the local cornea. Nevertheless, the mechanism of MSCs immunosuppressive properties is not clear, the role of local application is less than the systemic, and CsA's therapeutic dosages are limited by the potentially life-threaten complications associated with immune suppression system and by drug toxicity. Thus, less toxic and more efficient methods of immunomodulation are needed to reduce corneal allograft rejection.

免疫排斥反应是角膜移植手术失败的最主要原因。间充质干细胞（mesenchymal stem cells，MSCs）具有免疫抑制功能，它在其它组织器官移植排斥方面作用的研究已经开展。白介素-10（interleukin-10，IL-10）具有免疫抑制活性，能够增强机体免疫耐受。慢病毒介导IL-10基因转染MSCs治疗角膜移植排斥反应研究尚未见报道。本研究采用大鼠异体角膜移植排斥模型，观察IL-10基因修饰后的MSCs对角膜移植排斥反应的影响，并通过T细胞增殖实验来检测实验中各组大鼠免疫应答水平，检测因子分泌水平以及CD4+CD25+Foxp3+调节性T细胞的变化，观察其在异体角膜移植大鼠的免疫调节作用，为其在未来临床应用提供基础实验参考依据，进一步明确其免疫调节的关键机理，为角膜移植排斥反应机制的深入研究提供基础，为基因联合细胞治疗角膜移植排斥提供思路和可能。

角膜移植是治疗角膜失明的有效方法。然而，免疫排斥反应是角膜移植手术失败的最主要原因。间充质干细胞（mesenchymal stem cells，MSCs）具有免疫抑制功能，它在其它组织器官移植排斥方面作用的研究已经开展。白介素-10（interleukin-10，IL-10）具有免疫抑制活性，能够增强机体免疫耐受。慢病毒介导IL-10基因修饰的骨髓来源MSCs（IL-10-BM-MSCs）治疗角膜移植排斥反应研究此前未见报道。本研究采用大鼠异体角膜移植排斥模型，观察IL-10-BM-MSCs对角膜移植排斥反应的影响，并检测角膜植片中免疫细胞浸润情况，检测引流淋巴结中包括调节性T细胞在内的淋巴细胞的变化，观察其在异体角膜移植大鼠的免疫调节作用。结果显示，与IL-10蛋白或普通BM-MSCs相比，结膜下注射IL-10-BM-MSCs可减少角膜植片中的免疫细胞浸润，并使植片存活时间增加一倍（18±0.707 天）。与此同时，为进一步揭示其抗角膜移植排斥的机制，本研究通过对植片进行RNA测序检测长链非编码RNA表达谱、RNAscope原位杂交和体内外的功能缺失/获得实验发现，IL-10-BM-MSCs主要通过在植片浸润的CD68+巨噬细胞中上调一种新型lncRNA来促进角膜移植物的生存。以上结果为IL-10基因修饰后的MSCs在未来临床应用提供基础实验参考依据，为角膜移植排斥反应机制的深入研究提供基础，为基因联合细胞治疗角膜移植排斥提供思路和可能。