造血干细胞逃逸细胞毒性淋巴细胞杀伤的机制的研究

The Study of the Possible Mechanism of the Hematopoietic Stem Cells Escaping from the Attacking of Cytotoxic T Lymphocytes

目的:研究造血干细胞(HSC)逃逸细胞毒性淋巴细胞(CTL)杀伤的机制,并比较外周血造血干细胞(PB-HSC)、骨髓造血干细胞(BM-HSC)和脐带血造血干细胞(UCB-HSC)3种常用造血干细胞逃逸CTL杀伤的能力,以期发现更适用于移植的HST类型。方法:以CTL为效应细胞,并以PB-HSC、BM-HSC及UCB-HSC为靶细胞,以去除了造血干细胞的外周血单个核细胞(PB-MNC)为对照组细胞,利用乳酸脱氢酶释放法分析效应细胞对靶细胞的杀伤率,并用克隆增殖试验分析效应细胞对靶细胞增殖能力的抑制率;用流式细胞技术分析PB-HSC、BM-HSC、UCB-HSC及PB-MNC表面HLA-Ⅰ类分子、HLA-Ⅱ类分子的表达情况。结果:CTL对3种HSC的杀伤率明显低于PB-MNC,有统计学差异;而CTL对3种HSC的杀伤率和增殖能力的抑制率之间差别均无统计学差异。PB-HSC、BM-HSC、UCB-HSC及PB-MNC表面均高表达HLA-Ⅰ类分子,相互之间比较均无统计学差异。PB-HSC、BM-HSC及PB-MNC表面均高表达HLA-Ⅱ类分子,相互之间比较也无统计学差异;只有UCB-HSC表面低表达HLA-Ⅱ类分子,与以上3种细胞比较有统计学差异。结论:3种HSC对CTL都具有一定程度的免疫逃逸能力,但其逃逸能力差异无统计学意义。UCB-HSC表面低表达HLA-Ⅱ类分子,可能在它逃逸特异性免疫杀伤的机制中发挥一定作用。

Objective：To study the possible mechanism of hematopoietic stem cells＇ immune escaping from cytotoxic T lym phocyte （CTL） and compare the ability for immune escaping of 3 kinds of common hematopoietic stem cells （peripheral blood hematopoietic stem cell（PB-HSC）, bone marrow hematopoietic stem cell（BM-HSC）and umbilical cord blood hematopoietic stem cell（UCB-HSC）. Methods： The cytolytic activity of CTLs against the hematopoietic stem cells were assessed in 4-h LDH release assays using CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit. The inhibition of proliferation of hematopoietic stem cells was detected by Colony Forming Cell Assays using Human Methylcellulose Complete Media. Detection of MHC-Ⅰ, MHC-Ⅱ proteins on the PB-HSC, BM-HSC and UCB-HSC were performed by Flow cytometry analysis. Results：Three kinds of hematopoietie stem cells all had the characteristic of immune escaping from CTLs. Concerned with the ability of immune escaping from CTLs, three kinds of hematopoietic stem cells had no significant difference. High levels of HLA-Ⅰ expression were detected in all of these four groups cells. High levels of HLA-Ⅱ expression were recorded in PB-HSC, BM-HSC and PB-MNC as compared with low levels expression in UCPrHSC. Conclusion： Three kinds of hematopoietic stem cells all have the characteristic of immune escaping from CTI.s. Their immune escaping from CTL may be due to the T cells in innate non special immunity and low level of HLA-Ⅱ molecule may be the possible mechanisms of UCB-HSC＇s immune escaping from T cell special immune cytolysis.