Abstract
目的
检测免疫抑制剂治疗中系统性硬化症(systemic sclerosis, SSc) 患者外周血T淋巴细胞、B淋巴细胞及自然杀伤(natural killer，NK) 细胞的表达水平，分析其与临床实验室指标之间的相关性，进而探讨外周血淋巴细胞亚群检测在SSc治疗中的意义。
方法
采用流式细胞术检测使用免疫抑制剂的32例SSc患者(SSc组)和30例健康对照(healthy control，HC)组外周血T、CD4+T、CD8+T、B、NK细胞数量及比例，比较SSc组与HC组外周血淋巴细胞亚群的差异，分析外周血淋巴细胞亚群与SSc其他实验室及临床指标之间的相关性。
结果
与HC组相比，SSc组中T、CD4+T、CD8+T、B、NK细胞数量均明显减少(P＜0.05)，同时，NK细胞占淋巴细胞的百分比也明显降低(P=0.004)；此外，使用免疫抑制剂的SSc患者中65%以上外周血存在各淋巴细胞亚群细胞数量减少。CD4+T淋巴细胞数量降低组与正常组相比，其出现雷诺现象的比例明显升高(P=0.024)，红细胞沉降率和C-反应蛋白也明显升高(P＜0.001，P=0.018)；CD8+T淋巴细胞数量降低组与正常组相比，红细胞沉降率明显升高(P=0.022)；B淋巴细胞数量降低组与正常组相比，发生指尖溃疡的风险明显增高(P=0.019)；NK细胞数量降低组与正常组相比，发生指尖溃疡的风险明显增高(P=0.033)，而体内免疫球蛋白(immunoglobulin，Ig)M水平明显降低(P=0.049)。相关性分析可见，红细胞沉降率与总T淋巴细胞(r=-0.455，P=0.009)、CD4+T淋巴细胞(r=-0.416，P=0.018)、CD8+T淋巴细胞(r=-0.430，P=0.014)、B细胞(r=-0.366，P=0.039)数量呈负相关。
结论
使用免疫抑制剂的SSc患者外周血中T、CD4+T、CD8+T、B及NK细胞数量明显减少，某些淋巴细胞亚群的变化可能与雷诺现象、指尖溃疡的发生有关，与红细胞沉降率、C-反应蛋白呈明显负相关，使用免疫抑制剂治疗SSc中应定期检测外周血淋巴细胞亚群的细胞数量。
Keywords: 系统性硬化症, T淋巴细胞, B淋巴细胞, 自然杀伤细胞
Abstract
Objective
To explore the significance of lymphocytes in systemic sclerosis (SSc), by detecting the levels of T lymphocytes, B lymphocytes and natural killer (NK) cells, and analyzing the correlation between the lymphocytes and clinical laboratory indexes.
Methods
The numbers and proportion of T, CD4+T, CD8+T, B, and NK cells were detected by flow cytometry in peripheral blood of 32 SSc patients who had taken immunosuppressive drugs and 30 healthy controls (HC). The comparison of the lymphocyte subsets in SSc with them in the HC groups, and the correlation between the lymphocytes and other clinical and laboratory indicators were analyzed by the relevant statistical analysis.
Results
Compared with the HC group, the numbers of T, CD4+T, CD8+T, and NK cells in peripheral blood of SSc group, who had taken immunosuppressive drugs, were significantly decreased (P < 0.05). More-over, the proportion of NK cells in peripheral blood of the SSc group was also significantly lower than that in the HC group (P=0.004). In addition, all the lymphocyte subsets were decreased in peripheral blood of more than 65% of the SSc patients who had taken immunosuppressive drugs. Compared with CD4+T normal group, the positivity of Raynaud's phenomenon, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) was significantly increased in CD4+T reduction group, respectively (P＝0.024, P < 0.001, P=0.018). ESR was higher in CD8+T reduction group than CD8+T normal group (P＝0.022). The prevalence of fingertip ulcer was significantly increased in B cell decrease group (P=0.019). Compared with NK cell normal group, the prevalence of fingertip ulcer was significantly increased in NK cell lower group (P=0.033), IgM was remarkablely decreased yet (P=0.049). The correlation analysis showed that ESR was negatively correlated with the counts of T lymphocytes (r＝－0.455, P＝0.009), CD4+T lymphocytes (r＝－0.416, P＝0.018), CD8+T lymphocytes (r＝－0.430, P=0.014), B cells (r＝－0.366, P＝0.039).
Conclusion
The number of CD4+T, CD8+T, B, and NK cells significantly decreased in peripheral blood of SSc patients who had used immunosuppressive drugs, some lymphocyte subsets might be related with Raynaud's phenomenon and fingertip ulcer, and reflected the disease activity by negatively correlated with ESR and CRP; the numbers of lymphocyte subsets in peripheral blood should be detected regularly in SSc patients who had taken immunosuppressive drugs.
Keywords: Systemic sclerosis, T lymphocytes, B lymphocytes, Natural killer cells
系统性硬化症(systemic sclerosis，SSc)是一种病因不明、发病机制复杂的慢性自身免疫性疾病，主要病理改变为血管病变，早期出现雷诺现象，之后皮肤或内脏器官逐步纤维化。临床上根据纤维化的部位是否局限分为局限型和弥漫型，全球SSc患病率为1/10 000 [1]，虽然近年来SSc患者生存率有所改善，但其死亡率仍然很高, 尤其是弥漫型，高于其他风湿病[2]。该病虽无特效治疗药物，但使用免疫抑制剂如硫唑嘌呤、甲氨蝶呤、环磷酰胺或环孢素A以及吗替麦考酚酯等药物进行早期干预，可提升患者生活质量，改善肺间质纤维化及肺动脉高压，并可大幅度降低患者的死亡率。SSc患者体内免疫细胞存在过度活化，使用免疫抑制剂可抑制其活化，然而，淋巴细胞被过度抑制后可能产生其他不良结果。本研究拟分析应用激素和免疫抑制剂治疗中SSc患者外周血淋巴细胞的变化，探索淋巴细胞亚群检测在SSc治疗中的意义。
1. 资料与方法
1.1. 临床资料
选取2018年1月至2019年8月在北京大学人民医院风湿免疫科门诊和住院就诊的SSc患者32例(SSc组)，其中女性31例，男性1例，平均年龄(50.56±13.60)岁，病程为5(0.5~28)年，32例患者均符合2013年《美国风湿病协会/欧洲风湿病防治联合会系统性硬化症分类标准》[3]，流式细胞仪进行检测前长期或大于1个月持续使用免疫抑制剂，排除患有其他结缔组织病、感染性疾病或肿瘤等相关性疾病。年龄、性别与之匹配的30例北京大学人民医院健康体检者作为健康对照(healthy control，HC)组，其中女性26例，男性4例，平均年龄(49.07±14.26)岁，HC组与SSc组在性别(P=0.189)和年龄(P＝0.27)上差异无统计学意义。本研究获得北京大学人民医院医学伦理委员会批准(批准号：2016PHB168-01)，所有研究对象均签署知情同意书。
1.2. 研究方法
淋巴细胞亚群的检测：采集SSc组及HC组静脉血4 mL，用乙二胺四乙酸(ethylene diamine tetraacetic acid，EDTA)抗凝，各取50 μL全血加入2个流式管内，向流式管内分别加入20 μL CD3-FITC/CD8-PE/CD45-PerCP/CD4-APC和20 μL CD3-FITC/CD16+CD56-PE/CD45-PerCP/CD19-APC荧光标记抗体(购自深圳迈瑞公司)，混匀，室温避光孵育15 min，加入500 μL溶血素(购自美国BD公司)，室温避光孵育15 min，上机检测(BriCyte E6流式仪，深圳迈瑞公司)，收集细胞数量及比例。按照SSc患者外周血淋巴细胞数量是否降低分为降低组和正常组，比较两组间临床实验室指标的差异。
其他实验室指标的检测：采集SSc组及HC组静脉血4 mL，离心收集血清，分别采用免疫印迹法、免疫比浊法进行以下检测：硬皮病抗体谱，红细胞沉降率(erythrocyte sedimentation rate，ESR)，C-反应蛋白(C-reactive protein, CRP)，免疫球蛋白(immunoglobulin，Ig)A/G/M/，补体(complement, C) 3、C4。
1.3. 统计学分析
采用统计软件SPSS 24.0对数据进行分析，计量资料如符合正态分布，以均数±标准差表示，两组间比较采用独立样本t检验，相关性分析采用Pearson分析；非正态分布的计量资料，以中位数(四分位数)表示，两组间比较采用Mann-Whitney U检验，相关性分析采用Spearman‘s分析。计数资料以百分率表示，采用Fisher精确概率法检验，P < 0.05为差异有统计学意义。用百分位数法(95%CI)定义淋巴细胞亚群正常参考值范围。
2. 结果
2.1. 一般资料
32例SSc患者中均不同程度地使用激素和免疫抑制剂, 如吗替麦考酚酯、硫唑嘌呤、他克莫司、青霉胺、环孢素A，伴有一种或同时伴有多种临床表现，如雷诺现象、局限性皮肤改变、弥漫性皮肤改变、指尖溃疡、皮肤肿胀或硬化、肺间质纤维化、肺动脉高压、关节炎、抗核抗体(antinuclear antibody，ANA)阳性、抗硬皮病抗体-70(anti-Scl-70)阳性。
2.2. 外周血淋巴细胞亚群计数
SSc患者外周血总T淋巴细胞(CD3+)、CD4+T淋巴细胞(CD3+CD4+)、CD8+T淋巴细胞(CD3+CD8+)、B淋巴细胞(CD3-CD19+)、NK细胞(CD3-CD16+CD56+)数量与HC组相比，均明显降低(P < 0.05，表 1)。根据HC组计算95%CI，得到各淋巴细胞亚群数量正常参考值范围分别为(×106/L)：总T淋巴细胞1 410~1 615, CD4+T淋巴细胞752~897, CD8+T淋巴细胞530~662, B淋巴细胞206~274, NK细胞255~360。SSc组中总T淋巴细胞及各淋巴细胞亚群数量均出现明显降低，其降低率最低为65.63%，而淋巴细胞数量正常占比最高为34.37%。
表 1.
SSc患者与正常对照组外周血T淋巴细胞亚群、B细胞及NK细胞的比较
Comparison of T lymphocyte subsets, B cells and NK cells in SSc patients and HC group
| Group | Number of lymphocytes/(×106/L) | ||||
| T cells | CD4+T cells | CD8+T cells | B cells | NK cells | |
| HC, healthy control；SSc, systemic sclerosis; T cells, T lymphocytes cells; B cells, B lymphocytes cells; NK cells, natural killer cells. | |||||
| HC | 1 539(1 296-1704) | 835(678-926) | 566(477-721) | 228(169-293) | 262(216-336) |
| SSc | 1 010(554-1368) | 639(355-803) | 347(205-529) | 141(73-262) | 143(59-179) |
| P | < 0.001 | 0.017 | 0.001 | 0.022 | < 0.001 |
2.3. 外周血淋巴细胞亚群百分比
SSc组患者外周血CD4+T细胞占淋巴细胞百分比明显高于HC组(P=0.017)，而NK细胞的百分比则明显低于HC组(P=0.004, 表 2)。
表 2.
SSc患者与HC组外周血T淋巴细胞亚群、B细胞及NK细胞的百分比
Percentage of T lymphocyte subsets, B cells and NK cells of peripheral blood between SSc group and HCgroup
| Group | Percentage of cells/% | ||||
| T cells | CD4+T cells | CD8+T cells | B cells | NK cells | |
| HC, healthy control; SSc, systemic sclerosis; T cells, T lymphocytes cells; B cells, B lymphocytes cells; NK cells, natural killer cells. | |||||
| HC | 73.61±6.36 | 41.41±7.28 | 29.65±6.28 | 11.29±3.83 | 14.27±4.79 |
| SSc | 76.62±10.14 | 48.05±11.35 | 28.80±9.60 | 12.16±6.88 | 10.02±5.43 |
| P | 0.099 | 0.017 | 0.260 | 0.573 | 0.004 |
2.4. T淋巴细胞亚群、B细胞及NK细胞数量减少与临床实验室指标的关系
CD4+T降低组较正常组出现雷诺现象的比例明显增高(100% vs.66.67%，P=0.024)，ESR(25 vs.9，P < 0.001)和CRP (7.93 vs. 2.21，P=0.018) 均明显升高(表 3)；CD8+T降低组较正常组的红细胞沉降率明显增高(23 vs. 9，P=0.022, 表 4)；B细胞降低组较正常组发生指尖溃疡的比例明显升高(50% vs. 9.09%，P=0.019，表 5)；NK细胞降低组均较正常组发生指尖溃疡的比例明显升高(40% vs. 5.88%，P=0.033)，而IgM水平却明显降低(0.77 vs. 0.93, P=0.049，表 6)。
表 3.
CD4+T淋巴细胞与临床实验室指标之间的关系
Relationship between CD4+T lymphocyte cells reduction and clinical laboratory index
| Items | Reduction group (n=14) | Normal group (n=18) | P |
| ANA, antinuclear antibody; Scl-70, anti-Scl-70; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein; IgG, immunoglobulin G; IgM, immunoglobulin M; IgA, immunoglobulin A; C3, complement 3; C4, complement 4. | |||
| Age/years，x±s | 51.64±14.56 | 49.72±13.17 | 0.699 |
| Women，n(%) | 13 (92.86) | 18 (100) | 1.000 |
| Disease duration/years，M(P25, P75) | 8.50 (1，28) | 3.75 (0.5，20) | 0.085 |
| Raynaud‘s phenomenon，n(%) | 14 (100) | 12 (66.67) | 0.024 |
| Diffuse cutaneous changes，n(%) | 13 (92.86) | 12 (66.67) | 0.104 |
| Fingertip ulcer，n(%) | 3 (21.43) | 4 (22.22) | 1.000 |
| Pulmonary interstitial fibrosis，n(%) | 12 (83.71) | 11 (61.11) | 0.235 |
| ANA，n(%) | 13 (92.86) | 16 (88.89) | 1.000 |
| Scl-70, n(%) | 5 (35.71) | 6 (33.33) | 1.000 |
| ESR/(mm/h), M(P25, P75) | 25 (18.00，41.50) | 9.00 (7.50，14.50) | ＜0.001 |
| CRP/(mg/L), M(P25, P75) | 7.93 (1.76，14.72) | 2.21 (0.47，3.14) | 0.018 |
| IgG/(g/L), M(P25, P75) | 14.25 (10.20，19.70) | 11.05 (9.65，15.40) | 0.180 |
| IgM/(g/L), M(P25, P75) | 0.81 (0.64，1.05) | 0.85 (0.66，1.04) | 0.955 |
| IgA/(g/L), x±s | 2.11±1.28 | 2.24±1.49 | 0.798 |
| C3/(g/L), x±s | 0.85±0.24 | 0.89±0.17 | 0.583 |
| C4/(g/L), x±s | 0.19±0.08 | 0.19±0.07 | 0.979 |
表 4.
CD8+T淋巴细胞与临床实验室指标之间的关系
Relationship between CD8+T lymphocyte cells reduction and clinical laboratory index
| Reduction group (n=15) | Normal group (n=17) | P | |
| Abbreviations as in Table 3. | |||
| Age/years，x±s | 47.76±13.79 | 53.73±13.10 | 0.221 |
| Women，n(%) | 15 (100) | 16 (94.12) | 0.531 |
| Disease duration/years，M(P25, P75) | 4 (0.5，20) | 6.5 (1，28) | 0.189 |
| Raynaud‘s phenomenon，n(%) | 12 (70.59) | 14 (93.33) | 0.178 |
| Diffuse cutaneous changes，n(%) | 13 (76.47) | 12 (80) | 0.355 |
| fingertip ulcer，n(%) | 3 (17.65) | 4 (26.66) | 0.678 |
| Pulmonary interstitial fibrosis，n(%) | 12 (70.59) | 11 (73.33) | 1.000 |
| ANA，n(%) | 14 (93.33) | 15 (88.24) | 1.000 |
| Scl-70, n(%) | 5 (33.33) | 6 (35.29) | 1.000 |
| ESR/(mm/h), M(P25, P75) | 23.00 (11.50，27.50) | 9.00 (7.50，17.00) | 0.022 |
| CRP/(mg/L), M(P25, P75) | 7.15 (0.71，12.83) | 2.43 (0.55，3.14) | 0.142 |
| IgG/(g/L), M(P25, P75) | 13.50 (9.30，16.40) | 11.20 (9.50，15.70) | 0.551 |
| IgM/(g/L), M(P25, P75) | 0.77 (0.52，0.94) | 0.91 (0.72，1.18) | 0.370 |
| IgA/(g/L), x±s | 1.96±1.27 | 2.39±1.48 | 0.387 |
| C3/(g/L), x±s | 0.87±0.20 | 0.88±0.21 | 0.940 |
| C4/(g/L), x±s | 0.20±0.07 | 0.18±0.08 | 0.520 |
表 5.
B细胞与临床实验室指标之间的关系
Relationship between B cells reduction and clinical laboratory index
| Reduction group (n=10) | Normal group (n=22) | P | |
| Abbreviations as in Table 3. | |||
| Age/years，x±s | 56.00±12.61 | 48.09±13.57 | 0.129 |
| Women，n(%) | 10 (100) | 21 (95.45) | 1.000 |
| Disease duration/years，M(P25, P75) | 10.5 (1, 28) | 4 (0.5, 20) | 0.052 |
| Raynaud‘s phenomenon，n(%) | 10 (100) | 16 (72.73) | 0.142 |
| Diffuse cutaneous changes，n(%) | 10 (100) | 15 (68.18) | 0.069 |
| fingertip ulcer，n(%) | 5 (50) | 2 (9.09) | 0.019 |
| Pulmonary interstitial fibrosis，n(%) | 8 (80) | 15 (68.18) | 0.681 |
| ANA，n(%) | 8 (80.00) | 21 (95.45) | 0.224 |
| Scl-70, n(%) | 4 (40.00) | 7 (31.82) | 0.703 |
| ESR/(mm/h), M(P25, P75) | 21.00 (10.00, 26.00) | 10.50 (7.50, 20.00) | 0.092 |
| CRP/(mg/L), M(P25, P75) | 6.78 (0.97, 9.9) | 2.50 (0.55, 5.19) | 0.325 |
| IgG/(g/L), M(P25, P75) | 12.25 (10.15, 14.4) | 11.70 (9.50, 15.70) | 0.889 |
| IgM/(g/L), M(P25, P75) | 0.81 (0.52, 0.94) | 0.87 (0.72, 1.18) | 0.562 |
| IgA/(g/L), x±s | 2.79±1.74 | 1.91±1.12 | 0.092 |
| C3/(g/L), x±s | 0.90±0.21 | 0.86±0.20 | 0.639 |
| C4/(g/L), x±s | 0.21±0.07 | 0.19±0.08 | 0.491 |
表 6.
NK细胞与临床实验室指标之间的关系
Relationship between NK cells reduction and clinical laboratory index
| Reduction group (n=15) | Normal group (n=17) | P | |
| Abbreviations as in Table 3. | |||
| Age/years，x±s | 53.00±14.05 | 48.41±12.23 | 0.349 |
| Women，n(%) | 15 (100) | 16 (94.12) | 1.000 |
| Disease duration/years，M(P25, P75) | 6.5 (1, 28) | 5 (0.5, 20) | 0.455 |
| Raynaud‘s phenomenon，n(%) | 13 (86.67) | 13 (82.35) | 0.659 |
| Diffuse cutaneous changes，n(%) | 12 (80) | 13 (82.35) | 1.000 |
| fingertip ulcer，n(%) | 6 (40) | 1 (5.88) | 0.033 |
| Pulmonary interstitial fibrosis，n(%) | 11 (73.33) | 12 (70.59) | 1.000 |
| ANA，n(%) | 14 (93.33) | 15 (88.24) | 1.000 |
| Scl-70, n(%) | 5 (33.33) | 6 (35.29) | 1.000 |
| ESR/(mm/h), M(P25, P75) | 23.00 (10.5, 40.00) | 10.00 (7.50, 18.00) | 0.069 |
| CRP/(mg/L), M(P25, P75) | 5.83 (0.97, 12.12) | 2.57 (0.55, 3.86) | 0.242 |
| IgG/(g/L), M(P25, P75) | 11.70 (9.25, 15.60) | 11.70 (9.80, 15.70) | 0.655 |
| IgM/(g/L), M(P25, P75) | 0.77 (0.52, 0.94) | 0.93 (0.76, 1.39) | 0.049 |
| IgA/(g/L), x±s | 2.43±1.53 | 1.97±1.23 | 0.348 |
| C3/(g/L), x±s | 0.85±0.24 | 0.90±0.16 | 0.548 |
| C4/(g/L), x±s | 0.21±0.09 | 0.18±0.06 | 0.365 |
2.5. SSc外周血淋巴细胞数量与实验室指标之间的相关性
通过相关性分析可得出，ESR与总T淋巴细胞(r=-0.455，P=0.009)、CD4+T淋巴细胞(r=-0.416, P=0.018)、CD8+T淋巴细胞(r=-0.430，P=0.014)、B淋巴细胞(r=-0.366，P=0.039)数量之间存在明显负相关。然而，T淋巴细胞亚群、B淋巴细胞及NK细胞数量与CRP、IgA、IgG、IgM、C3、C4之间无明显相关性(P＞0.05, 表 7)。
表 7.
T淋巴细胞亚群、B细胞及NK细胞数量与ESR、CRP、IgA、IgG、IgM、C3、C4之间的相关性
Correlation between the number of T lymphocyte subsets, B cells and NK cells and ESR, CRP, IgA, IgG, IgM, C3, C4
| Items | ESR | CRP | IgA | IgG | IgM | C3 | C4 | |||||||||||||
| r | P | r | P | r | P | r | P | r | P | r | P | r | P | |||||||
| ESR, erythrocyte sedimentation rate; CRP, C-reactive protein; IgA, immunoglobulin A; IgG, immunoglobulin G; Ig M, immunoglobulin M; C3, complement 3;C4, complement 4;T cells, T lymphocytes cells; B cells, B lymphocytes cells; NK cells, natural killer cells. | ||||||||||||||||||||
| T cells | -0.455 | 0.009 | -0.292 | 0.104 | 0.112 | 0.541 | -0.079 | 0.669 | -0.024 | 0.898 | 0.317 | 0.077 | -0.022 | 0.903 | ||||||
| CD4+T cells | -0.416 | 0.018 | -0.235 | 0.195 | 0.112 | 0.541 | -0.182 | 0.381 | -0.142 | 0.437 | 0.301 | 0.095 | -0.008 | 0.964 | ||||||
| CD8+T cells | -0.430 | 0.014 | -0.325 | 0.070 | 0.021 | 0.909 | 0.030 | 0.869 | 0.148 | 0.418 | 0.290 | 0.107 | -0.021 | 0.911 | ||||||
| B cells | -0.366 | 0.039 | -0.218 | 0.231 | 0.051 | 0.782 | 0.209 | 0.251 | 0.281 | 0.120 | 0.138 | 0.452 | -0.289 | 0.108 | ||||||
| NK cells | -0.196 | 0.283 | -0.093 | 0.363 | 0.043 | 0.817 | 0.161 | 0.378 | 0.338 | 0.059 | 0.162 | 0.376 | -0.069 | 0.708 | ||||||
3. 讨论
SSc的发病机制尚不明确，但学者普遍认为, 在遗传易感基础上，受环境因素诱导免疫系统被激活，进而针对自身组织器官产生过度免疫反应，早期出现血管内皮损伤及炎症反应，进而造成成纤维细胞活化和基质积聚，最终导致慢性和进行性多器官组织纤维化[4-7]。免疫系统在SSc发病中至关重要，其过度活化是血管病变和纤维化的启动因素[8-9]。免疫抑制剂可有效抑制淋巴细胞过度活化，缓解SSc患者病情，改善临床症状，是治疗SSc的常用药物。然而，免疫抑制剂的使用可能导致淋巴细胞受到过度抑制，从而影响淋巴细胞的正常免疫功能。本研究发现，使用免疫抑制剂的SSc组外周血中T、CD4+T、CD8+T、B及NK细胞数量明显低于HC组，且65%以上的SSc患者出现了T、CD4+T、CD8+T、B及NK细胞数量异常，这表明SSc患者中存在淋巴细胞过度被抑制。淋巴细胞亚群百分比的对比结果显示，SSc组NK细胞百分比明显低于HC组，而CD4+T细胞百分比却明显高于HC组，其他亚群百分比差异无统计学意义，这一结果提示免疫抑制剂的使用导致淋巴细胞被过度抑制，尤其是NK细胞。
雷诺现象是SSc最常见的临床表现，有报道称约96%的SSc患者会出现雷诺现象。同时，雷诺现象也是SSc患者最早期出现的典型症状，可以在其他器官出现病变前很多年就出现[10-11]。雷诺现象的发生与CD4+T细胞密切相关。CD4+T细胞在内源性或外源性因素刺激下可通过分泌多种炎性细胞因子，如白介素(interleukin，IL)-2，干扰素(inter-feron，IFN)-γ，IL-4, IL-17等，作用于血管内皮细胞和平滑肌细胞，产生炎性损伤，最终导致血管通透性增加，出现雷诺现象[12-14]。本研究发现，CD4+T细胞数量降低组出现雷诺现象的概率明显高于CD4+T细胞正常组，但其原因及机制尚不明确，有待进一步研究。
指尖溃疡是紧随雷诺现象后常见的SSc的临床表现，如果治疗不当，指尖溃疡的终生携带率高达71.4%，最终可发展为指端坏疽，疼痛和手功能的丧失将严重影响SSc患者的生存质量[15]。以往的研究发现，SSc患者外周血中B细胞活化状态增强，患者皮肤活检中存在B细胞及其基因表达异常，与皮肤纤维化相关的细胞因子IL-6水平升高[16-17]。此外，活化B细胞分泌的IL-6可与转化生长因子-β共同诱导基质合成，抑制胶原降解，最终导致组织纤维化[18-19]。本研究证实使用免疫抑制剂的SSc组存在B细胞数量明显减少，而且在B细胞降低组，指尖溃疡的发病率明显升高，这表明B细胞与指尖溃疡发生发展密切相关，在免疫抑制剂的使用中应定期检测B细胞数量，以防B细胞被过度抑制，影响B细胞发挥正常免疫功能。
NK细胞是机体固有免疫系统的重要组成成分，其通过释放多种细胞因子调节免疫系统中T、B细胞的发育和活化，使后者进入耐受状态[18]。NK细胞可通过抑制商陆丝裂原PWM诱导的B细胞分化及抗体应答，直接抑制B细胞或抑制B细胞的抗原提呈[20]。本文结果显示使用免疫抑制剂的SSc组NK细胞数量和百分比均明显降低，这导致NK细胞抑制B细胞产生自身抗体的作用减弱，加重自身抗体的产生。有文献报道SSc患者的皮肤纤维化可能与NK细胞的减少有关[21-22]。我们的结果显示，NK细胞降低组较正常组发生指尖溃疡的概率明显升高，进一步证实NK细胞与皮肤纤维化的形成有关。
ESR、CRP是反映SSc疾病活动状态的常用指标，本研究发现，CD4+T降低组存在ESR和CRP明显升高，CD8+T降低组也存在ESR明显升高。此外，相关性分析结果显示，T、CD4+T、CD8+T及B细胞与ESR呈明显负相关。这表明，淋巴细胞亚群被过度抑制后SSc的病情可能不但没有缓解，反而处于活动状态，也可能因为患者病情重，尽管淋巴细胞亚群被抑制，但疾病仍处于活动状态，其原因及机制有待进一步研究。
综上所述，SSc患者治疗中免疫抑制剂的使用导致绝大多数患者外周血T、CD4+T、CD8+T、B及NK细胞数量明显减少，某些淋巴细胞数量的减少可能会增加雷诺现象和指尖溃疡的发生，加重SSc疾病活动度，因此，在使用免疫抑制剂进行临床治疗SSc中应定期检测外周血淋巴细胞亚群细胞数量及比例，及时调整免疫抑制剂的使用。
Funding Statement
国家自然科学基金(81701614)和北京大学人民医院研究与发展基金(RDC2019-01)
Supported by the National Natural Science Foundation of China (81701614)and Peking Univercity People's Hospital Research and Development Funds (RDC2019-01)
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