应用要点：广州市政府科技计划项目“遗传上HLA半相合骨髓细胞优化移植技术平台建设”(200523-E0551)。前期的小鼠体内实验研究表明，TJU103可以明显降低异基因造血干细胞移植小鼠移植物抗宿主病的发生率和程度，并且不增加感染率，不影响小鼠的移植物抗白血病效应，从而明显延长小鼠移植后的生存期。

同行评价：移植物抗宿主病是影响异基因造血干细胞移植疗效的关键因素。目前主要是应用免疫抑制剂减轻急性移植物抗宿主病，但同时使增加了患者白血病复发和感染的机率；而慢性移植物抗宿主病的治疗尚没有特效药物。本实验通过TJU103特异性阻断CD4+ T细胞活化的抗原信号通路，打破CD4+T细胞与CD8+T细胞间的协同作用，降低移植物抗宿主病的发生率，诱导免疫耐受，结果显示，TJU103降低T淋巴细胞增殖反应的同时并不影响其抗白血病效应。

相关链接：①单向混合淋巴细胞培养的样本数量还不够大。②由于本实验是细胞水平的体外实验研究，无法对TJU103是否影响造血干细胞移植后的感染率进行研究。

摘要
目的：前期研究表明，吲哚亚甲基异烟腙(简称TJU103)可以明显降低异基因造血干细胞移植小鼠移植物抗宿主病的发生率和程度，明显延长小鼠移植后的生存期。实验拟观察TJU103诱导造血干细胞移植免疫耐受和对移植物抗白血病的影响，探索一条既能降低移植物抗宿主病又能保留移植物抗白血病的移植途径。
方法：实验于2007-07/10在南方医科大学珠江医院血液科实验室完成。①实验材料：人急性髓系白血病细胞株KG1a由中国医学科学院血液学研究所宋增璇教授惠赠。10份自愿捐献的健康人外周血白细胞由广州市中心血站提供，等分为供者与受者。②实验方法：采用梯度密度沉淀法常规分离人外周血单个核细胞。以供者外周血单个核细胞作为反应细胞，受者正常外周血单个核细胞作为刺激细胞，体外建立异基因造血干细胞移植中供、受者间的单向混合淋巴细胞反应体系，应用TJU103阻断CD4+T细胞激活的抗原信号通路，设单独刺激细胞和反应细胞对照以及1640完全培养基空白对照。③实验评估：于培养第1、3、5天应用MTT检测TJU103对供者淋巴细胞的增殖活性的影响，于第5天用乳酸脱氢酶释放法分别检测10∶1， 20∶1，40∶1效靶比时供者淋巴细胞对受者单个核细胞和急性髓系白血病细胞KG1a的细胞毒活性影响，同时采用流式细胞仪检测CD4+CD25+T细胞的百分比。
结果：第3天和第5天实验组A值低于对照组 (P < 0.05)。单向混合淋巴细胞5 d后实验组各效靶比供者T细胞杀伤受者外周血单个核细胞的活性明显低于对照孔 (P < 0.05)；二者对KG1a细胞的杀伤活性差异不显著 (P > 0.05)。TJU103对供者CD4+CD25+T细胞无影响。
结论：TJU103降低T淋巴细胞增殖反应的同时并不影响其抗白血病效应，有望用于临床异基因造血干细胞移植中移植物抗宿主病和移植物抗白血病的免疫调节。
关键词：干细胞移植；TJU103；T淋巴细胞增殖；细胞毒活性；移植物抗宿主病；移植物抗白血病；免疫耐受

周健，涂三芳，郭坤元，孙明，胡亮杉，吴远彬，卢晓珣，王杨.TJU103体外诱导造血干细胞移植的免疫耐受[J].中国组织工程研究与临床康复，2008，12(16):3049-3052 [www.zglckf.com/zglckf/ejournal/upfiles/08-16/16k-3049(ps).pdf]

Immunotolerance to haemopoietic stem cell transplantation induced by TJU103 in vitro

Abstract

AIM： Pristine investigation demonstrated that TJU103 could markedly decrease graft versus host disease (GVHD) incidence and intensity, and obviously prolonge life span after allogenetic haemopoietic stem cell transplantation (allo-HSCT) in mice. This experiment is designed to explore the effects of TJU103 on tolerance and graft versus leukemia (GVL) in allo-HSCT in vitro in order to seek an effective access to transplantation remaining GVL and with less GVHD.
METHODS: Experiments were performed at the Department of Hematology of Zhujiang Hospital of Southern Medical University from July to October 2007. ①Human acute myelogenous leukemia lines KG1a was presented by professor Song in Hematology Institute of Chinese Academy of Medical Sciences. All 10 peripheral blood samples were collected from healthy volunteers at Guangzhou Blood Center, and were divided equally into donors and recipients. ② Peripheral blood mononuclear cells (PBMCs) were obtained by traditional precipitation method discriminated by gradient density. Donor PBMCs and recipient PBMCs were took as responsive cell and antigenic stimulus cell，respectively. One-way mixture lymphocyte culture (MLC) response system between donor and recipient was modeled in vitro. The antigen activated pathway of CD4 positive T cell was blocked by TJU103. The blank control group of 1640 complete medium and control group of alone responsive cell and antigenic stimulus cell were set. ③Effects of TJU103 on donor T lymphocytes proliferation was estimated by MTT assay at MLC 1, 3, 5 days. Cytotoxicity of donor T lymphocytes blocked by TJU103 against recipient T lymphocytes and KG1a cell were measured by LDH releasing assay at different effector-to-target cell ratios (E:T, 10:1, 20:1 40:1) at MLC 5 days. Meanwhile, the ratio of donor CD4+CD25+ T lymphocytes was assayed by flow cytometry (FCM).
RESULTS: The A numerical values of experiment group were lower than control group at MLC 3 and 5 days (P < 0.05). Cytotoxicities of experiment group donor T cells against recipient PBMCs were significantly lower than control group at same E:T ratios at MLC 5 days (P < 0.05), but there was no difference in the ability of cytotoxicity against KG1a cell between them (P > 0.05). The ratio of donor CD4+CD25+ T lymphocyte was not affected by TJU103.
CONCLUSION: TJU103 may prohibit the T-cell proliferation significantly, but the ability of anti-leukemia is not influenced. TJU103 might be used to regulate the immune response of GVHD and GVL in clinical transplantation.

Zhou J, Tu SF, Guo KY, Sun M, Hu LS, Wu YB, Lu XX, Wang Y.Immunotolerance to haemopoietic stem cell transplantation induced by TJU103 in vitro.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(16):3049-3052(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-16/16k-3049(ps).pdf]