Zeste同源物2增强子在桥本甲状腺炎B淋巴细胞亚群中的表达及其抑制剂的治疗机制及效果研究

doi:10.12114/j.issn.1007-9572.2023.0623

中国全科医学 ›› 2024, Vol. 27 ›› Issue (21): 2639-2645.DOI: 10.12114/j.issn.1007-9572.2023.0623

• 论著·甲状腺疾病专题研究 • 上一篇下一篇

Zeste同源物2增强子在桥本甲状腺炎B淋巴细胞亚群中的表达及其抑制剂的治疗机制及效果研究

易圣果¹, 曹业迪¹, 赵雪¹, 卢桂芝¹, 张杨¹, 丛铁川², 张澜波³, 张继新⁴, 梁振威⁵, 屈晨雪⁶, 张俊清¹, 高莹¹^,^*()

1．100034　北京市，北京大学第一医院内分泌内科
2．100034　北京市，北京大学第一医院耳鼻咽喉-头颈外科
3．100034　北京市，北京大学第一医院甲状腺乳腺外科
4．100034　北京市，北京大学第一医院病理科
5．100034　北京市，北京大学第一医院超声医学科
6．100034　北京市，北京大学第一医院检验科

收稿日期:2023-08-20 修回日期:2023-12-10 出版日期:2024-07-20 发布日期:2024-04-18
通讯作者: 高莹
作者贡献：
易圣果提出研究思路，设计研究方案，研究命题提出，部分实验方案实施，论文起草；曹业迪、赵雪负责样本采集，进行实验，研究过程的实施；卢桂芝、张杨负责数据收集、采集、清洗等；丛铁川、张澜波负责患者筛选、甲状腺样本收集；张继新负责甲状腺病理诊断；梁振威负责甲状腺B超及影像学选取、数据留存；屈晨雪负责临床检验指标化验与检测；张俊清负责文章的质量控制与审查，监督管理；高莹负责研究命题的设计、方案修正，最终版本修订，文章的审查，对文章整体负责。
基金资助:
国家自然科学基金面上项目(8217030553)

EZH2 Expression in B Cell Lymphocyte Subsets of Hashimoto's Thyroiditis and the Therapeutic Mechanism and Effect of Its Inhibitors

YI Shengguo¹, CAO Yedi¹, ZHAO Xue¹, LU Guizhi¹, ZHANG Yang¹, CONG Tiechuan², ZHANG Lanbo³, ZHANG Jixin⁴, LIANG Zhenwei⁵, QU Chenxue⁶, ZHANG Junqing¹, GAO Ying¹^,^*()

1 Department of Endocrinology, Peking University First Hospital, Beijing 100034, China
2 Department of Otorhinolaryngology-Head and Neck Surgery, Peking University First Hospital, Beijing 100034, China
3 Department of Thyroid and Breast Surgery, Peking University First Hospital, Beijing 100034, China
4 Department of Pathology, Peking University First Hospital, Beijing 100034, China
5 Department of Ultrasound Medicine, Peking University First Hospital, Beijing 100034, China
6 Department of Clinical Laboratory, Peking University First Hospital, Beijing 100034, China

Received:2023-08-20 Revised:2023-12-10 Published:2024-07-20 Online:2024-04-18
Contact: GAO Ying

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摘要/Abstract

摘要： 背景甲状腺自身抗体是诊断桥本甲状腺炎（HT）的标志，B淋巴细胞在HT的发病机制中发挥重要作用。Zeste同源物2增强子（EZH2）是一种表观遗传学蛋白，在淋巴细胞的发育与功能调控中扮演重要角色。目的本研究探讨EZH2在HT甲状腺组浆母细胞及浆细胞中的表达，进一步探讨EZH2抑制剂在实验性自身免疫甲状腺炎（EAT）模型中的治疗作用。方法收集北京大学第一医院2010—2020年6例行甲状腺手术的患者，取肿瘤对侧的甲状腺组织（HT及正常甲状腺组织各3例），通过RNA-seq筛选B淋巴细胞相关基因的表达情况；收集16例HT患者的甲状腺组织和8例健康对照（HD）甲状腺组织，分别利用免疫组化及免疫荧光验证EZH2在HT甲状腺组织中B淋巴细胞中的表达；收集25例HT甲状腺细针穿刺液（FNA）、19例HT外周血以及12例健康人外周血样本应用流式细胞分析检测EZH2在浆母细胞及浆细胞中的表达改变。将15只7周龄NOD.H-2h4小鼠EAT模型分为对照组（n=5）、EAT无注射（n=5）或注射EZH2抑制剂GSK126处理组（10 mg/kg，腹腔注射3次/周，n=5），8周后观察甲状腺炎症程度及甲状腺球蛋白抗体（TgAb）水平的改变。结果 RNA-seq结果显示，相较于正常甲状腺组织，HT甲状腺组织中EZH2水平上调，一些与B淋巴细胞表型相关的基因例如CD19、CD27、CD38、CD52相应增加。免疫组化结果显示，16例HT甲状腺组织标本中EZH2免疫组化染色均可在生发中心（GC）见到阳性细胞，呈强阳性，8例正常甲状腺组织染色中未观察到阳性细胞。HT甲状腺组织中EZH2染色高表达在GC区，EZH2特异性地表达在CD19+B淋巴细胞中。流式细胞术检测结果显示HT FNA样本中CD19+B淋巴细胞、浆母细胞及浆细胞比例均高于HD外周血、HT外周血样本（P<0.01），HT FNA样本中EZH2在CD19+B淋巴细胞、浆母细胞中的阳性比例高于HT外周血（P<0.005）。小鼠实验中，EAT组甲状腺的淋巴细胞浸润较对照组增加。GSK126处理组炎症程度评分和TgAb水平高于对照组，低于EAT组（P<0.001）。结论 EZH2在HT甲状腺组织CD19+B淋巴细胞中表达异常升高，可能促进了B淋巴细胞分化成浆细胞进而促进自身抗体生成破坏甲状腺，EZH2抑制剂可以减缓EAT模型甲状腺炎症程度。浆母细胞中EZH2表达增加可能参与了HT的发病机制，EZH2可能成为治疗HT的新靶点，相关机制需要进一步深入研究。

关键词: 桥本甲状腺炎, B淋巴细胞亚群, Zeste同源蛋白2增强子, 甲状腺功能减退症, 靶向治疗

Abstract:

Background

Thyroid autoantibody is a marker for the diagnosis of Hashimoto's thyroiditis (HT), and B cells are essential in the pathogenesis of HT. Enhancer of Zeste homolog 2 (EZH2), which is an important epigenetic regulator, plays an important role in the regulation of lymphocytes development and function.

Objective

To investigate EZH2 expression in plasmoblasts and plasma cells in HT, and further explore the therapeutic effect of EZH2 inhibitors in experimental autoimmune thyroiditis (EAT) model.

Methods

The thyroid tissues from 6 patients who underwent thyroidectomy (3 HT patients with PTC, 3 patients with PTC alone) in Peking University First Hospital between 2010 and 2020 were obtained from the contralateral lobe with thyroid cancer, and screened for the expression of B-lymphocyte-related genes by RNA-seq; thyroid tissues from 16 HT patients and 8 normal thyroid tissues were collected and verified for the the expression of EZH2 in B cells in HT thyroid tissues by immunohistochemistry or immunofluorescence, respectively. Fine-needle aspiration (FNA) samples from patients with HT (n=25), and peripheral blood from patients with HT (n=19) or healthy donors (n=12) were analyzed by flow cytometry to define altered EZH2 expression in plasmablasts and plasma cells. Fifteen seven-week-old NOD.H-2^h4 mice were randomly divided into the control (n=5), EAT without injection group (n=5), and EZH2 inhibitor+ GSK126 injection group (10 mg/kg, intraperitoneal injection 3 times /week, n=5). The degree of thyroid inflammation and changes in TgAb levels were observed after 8 weeks.

Results

RNA sequencing analysis showed that EZH2 and genes associated with the B-cell phenotype such as CD19, CD27, CD38, CD52 were higher expressed in HT hyroid tissues compared with normal thyroid tissues. Immunohistochemical results showed that immunohistochemical staining for EZH2 in 16 HT thyroid tissue specimens was strongly positive with positive cells observed in the GC region, and no positive cells were observed in the staining of 8 normal thyroid tissues. EZH2 staining in HT thyroid tissue was highly expressed in the GC region, and EZH2 was specifically expressed in CD₁₉⁺ B cells. The results of flow cytometry assay showed that the proportion of CD₁₉⁺ B cells, plasmablasts and plasma cells in HT FNA samples was higher than that of HD peripheral blood and HT peripheral blood samples (P<0.01), and the proportion of EZH2 positivity in CD₁₉⁺ B cells and plasma cells was higher in HT FNA samples than that of HT peripheral blood (P<0.005). In the mouse experiments, lymphocytic infiltration of the thyroid tissues was increased in the EAT group compared to the control group. In the GSK126 treatment groups, the thyroid inflammatory score and serum TgAb titer were significantly higher than the control group and lower than the EAT group.

Conclusion

EZH2 over-expression in CD₁₉⁺ B cells of HT hyroid tissues may promote the differentiation of B cells into plasma cells and auto-antibody production, which leads to the destruction of thyroid tissues. EZH2 inhibitors can slow down the degree of thyroid inflammation in the EAT model. Increased EZH2 expression in plasmablasts may be involved in the pathogenesis of HT. EZH2 may serve as a therapeutic target for HT, although further studies are needed.

Key words: Hashimoto thyroiditides, B-lymphocyte subsets, Enhancer of Zeste homolog 2 protein, Hypothyroidism, Targeted therapy

中图分类号:

R 581.4

易圣果,曹业迪,赵雪,等. Zeste同源物2增强子在桥本甲状腺炎B淋巴细胞亚群中的表达及其抑制剂的治疗机制及效果研究[J]. 中国全科医学, 2024, 27(21): 2639-2645. DOI: 10.12114/j.issn.1007-9572.2023.0623.
YI Shengguo,CAO Yedi,ZHAO Xue, et al. EZH2 Expression in B Cell Lymphocyte Subsets of Hashimoto's Thyroiditis and the Therapeutic Mechanism and Effect of Its Inhibitors[J]. Chinese General Practice, 2024, 27(21): 2639-2645. DOI: 10.12114/j.issn.1007-9572.2023.0623.

图/表 8

图1 HT甲状腺组织和正常甲状腺组织中差异基因火山图

Figure 1 Differential gene volcano plot in HT thyroid tissues and normal thyroid tissues

图2 HT及正常甲状腺组织的免疫组化染色结果（×40）注：EZH2=Zeste同源物2增强子，HT=桥本甲状腺炎；HT1~HT3来源于不同的患者，N为正常甲状腺组织。

Figure 2 Immunohistochemical staining results of HT and normal thyroid tissues

图3 HT甲状腺组织的免疫荧光染色结果（×40）注：红色荧光为EZH2，绿色荧光为CD19+细胞，蓝色荧光为DAPI。

Figure 3 Immunofluorescence staining of HT thyroid tissue

图4 HT FNA样本、外周血样本流式细胞分析圈门注：A为淋巴细胞圈门，B为CD19+B淋巴细胞圈门，C为浆母细胞（CD27+CD38+细胞），D为浆细胞（CD27+CD138+细胞）。

Figure 4 Flow cytometric analysis circle gates of samples from FNA in HT thyroid tissue and peripheral blood

表1 HD外周血、HT外周血及HT FNA样本中CD19+B淋巴细胞、浆母细胞、浆细胞比例比较（%）

Table 1 Comparison of percentages of CD19+B cells，plasmablasts and plasma cells in HD peripheral blood，HT peripheral blood and HT FNA samples

样本	例数	CD₁₉⁺B淋巴细胞	浆母细胞（CD₂₇⁺CD₃₈⁺B淋巴细胞）[M（P₂₅，P₇₅）]	浆细胞（CD₂₇⁺CD₁₃₈⁺B淋巴细胞）[M（P₂₅，P₇₅）]
HD外周血	12	10.91±1.90	4.21（2.35，8.90）	0.39（0.20，0.62）
HT外周血	19	8.05±0.83	6.81（4.14，9.05）	0.30（0.18，0.63）
HT FNA	25	21.70±2.21^ab	9.69（5.54，18.75）^ab	2.89（1.54，5.67）^ab
H（F）值		16.26^c	9.48	37.33
P值		<0.001	0.009	< 0.001

表2 HT外周血、FNA样本中EZH2在CD19+B淋巴细胞、浆母细胞、浆细胞中的阳性比例比较（%）

Table 2 Comparison of the proportion of HT peripheral blood and FNA samples positive for EZH2 in CD19+ B cells，plasmablasts and plasma cells

样本	例数	CD₁₉⁺B淋巴细胞	浆母细胞（CD₂₇⁺CD₃₈⁺B淋巴细胞）[M（P₂₅，P₇₅）]	浆细胞（CD₂₇⁺CD₁₃₈⁺B淋巴细胞）
HT外周血	19	6.56±0.77	17.9（9.82，30.1）	67.32±3.52
HT FNA	25	17.32±2.84	63.5（37.6，79.1）	66.98±4.80
Z（t）值		3.223^a	57.000	0.055^a
P值		<0.005	<0.001	0.957

图5 对照组、EAT组以及GSK126处理组小鼠甲状腺HE染色

Figure 5 HE staining of thyroid in control group，EAT group and GSK126 treatment group

表3 3组小鼠甲状腺炎症程度评分、外周血TgAb水平比较

Table 3 Comparison of the degree of thyroid inflammation scores and peripheral blood TgAb levels in 3 groups of mice

组别	只数	炎症程度评分[M（P₂₅，P₇₅），分]	TgAb水平（OD值）
对照组	5	0（0，0）	0.06±0.01
EAT组	5	4（3，4）^a	0.85±0.11^a
GSK126处理组	5	2（2，3）^ab	0.38±0.09^ab
F（H）值		84.00^c	22.97
P值		<0.001	<0.001

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Zeste同源物2增强子在桥本甲状腺炎B淋巴细胞亚群中的表达及其抑制剂的治疗机制及效果研究

EZH2 Expression in B Cell Lymphocyte Subsets of Hashimoto's Thyroiditis and the Therapeutic Mechanism and Effect of Its Inhibitors

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