CD107α表达对NK细胞与细胞毒性T细胞细胞毒功能缺陷性疾病筛查价值探讨

Screening for cytotoxic defects with flow cytometric detection of CD107α on natural killer cells andcytotoxic T lymphocyte cells

目的建立自然杀伤性细胞及细胞毒性T细胞表面溶酶体相关膜蛋白质1（LAMP-1，CD107α）表达流式细胞术检测方法，探讨其对自然杀伤性（NK）细胞与细胞毒性T淋巴细胞（CTL）细胞毒功能及相关性疾病的筛查价值。方法收集疑诊Chediak-Higashi综合征（Chediak-HigashiSyndrome，CHS）患儿3例、噬血淋巴组织细胞增生症（HLH）患儿3例及10例健康对照儿童外周血，提取外周血DNA及RNA，PCR或RT-PCR扩增目的基因，测序分析基因背景。外周血单个核细胞（PBMC）以重组人白介素受体2（IL-2）刺激过夜，再以植物凝集素（PHA）或T细胞刺激剂Anti-CD3活化刺激细胞2h后，流式细胞术分析NK细胞及CTLCD107α表达频率及强度。结果10例健康儿童PBMC刺激前后NK细胞[（0．27±0．07）％伽．（5．80-4-2．83）％，P〈0．05]及CTL[（0．18±0．07）％郴．（4．47±2．36）％，P〈0．05]表面CD107α表达频率均显著升高，其平均荧光强度（MFI）亦显著升高。与10例正常儿童比较，3例疑似CHS患儿外周血PBMC经Anti-CD3活化后CTL表面CD107α表达无明显上升（0．3％、0．9％、0．2％），对照组为（4．47±2．36）％；PHA刺激后两例患儿NK细胞表面CD107α表达频率亦无明显变化（0．5％、0．6％），对照组为（5．80±2．83）％，其MFI变化趋势与CD107α表达率类似。3例HLH患儿NK细胞（3．3％、4．1％、2．7％），对照组为（5．80±2．83）％；CTL（2．8％、1．7％、1．9％），对照组为（4．47±2．36）％；表面CD107α刺激前后其表达频率及MFI与正常对照组差异无统计学意义。3例疑似CHS患儿中两例患儿LYST基因突变分别为（c．5411-5414del TTTC，L1741fsX1758和C．7975C〉T，R2596X）和（c．4863G〉A，R1563H和c．5392-5393delAA．E1739fsX1756），确诊为CHS综合征，1例基因诊断尚未明确。3例HLH患儿PRF、MUNC13-4及STXll基因检查结果均未发现突变。结论成功建立CD107α表达流式细胞检测方法，并在CHS中验证，该方法可能用作细胞毒功能的筛查方法，为后续基因确诊及发现新的致病基因奠定基础。

Objective To establish a novel flow cytometry-based assay for measuring the expression of lysosomal-associated membrane protein 1 （LAMP-1,CD107α） on the cell surface of natural killer （NK） cells and cytotoxic T lymphocyte （CTL） and evaluate the screening value of this assay for cytotoxic defects- related diseases such as familial hemophagocytie lymphopro-liferative （FHL） syndrome. Method Three suspected Chediak-Higashi Syndrome （CHS） patients, three suspected FHL patients and 10 healthy children were enrolled in the study from October 2010 to June 2011. Their PBMCs were separated and activated overnight with IL-2. After the granule release of NK cells activated by phytohemagglutinin （PHA） and CD8 ＋ T cells by anti-CD3, the CD107a expression were analyzed by flow cytometry. The peripheral blood DNA and RNA of the patients were extracted to analyze the oathogenie genes via DNA-PCR/RT-PCR anddirect sequencing. Result The CD107α expression on CTL in the ten healthy children significantly increased after activation by anti-CD3 [ （0. 18 ± 0. 07 ） % vs. （4. 47 ± 2. 36 ） %, P 〈 0. 05 ] and NK cells after activation by PHA [ （0. 27±0.07 ） % vs. （ 5.80 ＋ 2. 83 ） %, P 〈 0. 05 ]. The frequency of CD107α- expression NK cells in three suspected CHS after activation was significantly elevated when compared with the healthy control [0. 5%, 0. 6% vs. （5.80 ±2. 83）% ] except patient 2. After the anti-CD3 activation, the frequency of CD107α expression on CTL ceils also showed no significant difference [0. 3%, 0. 9%, 0. 2% vs. （4. 47 ＋2. 36）% ] in three patients. All of their mean fluorescence intensity （MFI） showed the same trend. Patient 1 and 3 were identified to have LYST mutations （ Patient 1 ： c. 5411-5414 del TTTC, L1741fsX1758 and c. 7975 C 〉 T, R2596X; Patient 3： c. 4863G 〉 A, R1563H and c. 5392-5393delAA, E1739fsX1756）. There was no mutation identified in the LYST gene for patient 2. CD107α expression of NK cells and CTL in the suspected FHL patients and in mirror of these findings, no underlying gene variation of PRF, MUNC13-4 and STXll were identified. Conclusion We developed a method to quantitatively assess cytotoxicity of the NK cells and CTL by measuring the expression of CD107α on the cell membrane, which appeared to be an effective and rapid screening test for cytotoxic defects-related diseases such as FHL and other HLH secondary to primary immunodeficiency.