脂联素在免疫性血小板减少症患者外周血中的水平及其对巨核细胞系分化的作用

doi:10.3969/j.issn.1674-8115.2022.07.004

上海交通大学学报（医学版） ›› 2022, Vol. 42 ›› Issue (7): 866-874.doi: 10.3969/j.issn.1674-8115.2022.07.004

• 论著 · 基础研究 • 上一篇

脂联素在免疫性血小板减少症患者外周血中的水平及其对巨核细胞系分化的作用

李昕雨¹(), 左斌²(), 王文¹, 钮晓音³, 翁震², 何杨¹()

^1.苏州大学附属第一医院，江苏省血液研究所，苏州 215006
^2.苏州大学唐仲英血液学研究中心，苏州 215123
^3.上海交通大学医学院，上海市免疫学研究所，上海 200025

收稿日期:2022-02-24 接受日期:2022-07-12 出版日期:2022-07-28 发布日期:2022-09-04
通讯作者: 何杨 E-mail:lixinyu0417@163.com;binz@suda.edu.cn。;heyang1963@163.com
作者简介:李昕雨（1997—），女，硕士生；电子信箱：lixinyu0417@163.com
左斌（1985—），男，实验师，硕士；电子信箱： binz@suda.edu.cn。第一联系人：（李昕雨、左斌并列第一作者）
基金资助:
国家自然科学基金(82070123)

Levels of adiponectin in the peripheral blood of patients with immune thrombocytopenia and its effect on the differentiation of megakaryocyte cell line

LI Xinyu¹(), ZUO Bin²(), WANG Wen¹, NIU Xiaoyin³, WENG Zhen², HE Yang¹()

^1.The First Affiliated Hospital of Soochow University, Jiangsu Institute of Hematology, Suzhou 215006, China
^2.TANG Zhongying Hematology Research Center of Soochow University, Suzhou 215123, China
^3.Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China

Received:2022-02-24 Accepted:2022-07-12 Online:2022-07-28 Published:2022-09-04
Contact: HE Yang E-mail:lixinyu0417@163.com;binz@suda.edu.cn。;heyang1963@163.com
Supported by:
National Natural Science Foundation of China(82070123)

摘要/Abstract

摘要：

目的·探究脂联素在免疫性血小板减少症（immune thrombocytopenia，ITP）患者外周血血浆中的水平，以及其对巨核细胞分化、成熟的影响。方法·2021年1月至2022年2月，收集苏州大学附属第一医院血液科46例ITP患者及体检中心30名性别、年龄相对应的健康对照者外周血标本，使用人脂联素酶联免疫吸附测定（enzyme-linked immunosorbent assay，ELISA）试剂盒检测ITP患者与健康对照（HC）组的血浆脂联素水平，并分析ITP患者脂联素水平与其身体质量指数（body mass index，BMI）的相关性。在细胞实验方面，利用实时荧光定量PCR（RT-qPCR）、Western blotting分别在mRNA水平和蛋白水平上研究脂联素受体（ADIPOR1、ADIPOR2）在髓系细胞株K562，巨核细胞系细胞株MEG-01、Dami中的表达。用豆蔻酰佛波醇乙酯（phorbol 12-myristate 13-acetate，PMA）诱导K562细胞株分化，诱导MEG-01及Dami细胞株成熟，同时给予不同浓度的脂联素受体激动剂AdipoRon处理，采用流式细胞术检测CD41⁺细胞百分比、CD41平均荧光强度（mean fluoresence intensity，MFI）、多倍体细胞（≥4N）比例。结果·与HC组比较，ITP患者血浆脂联素水平显著升高（P=0.000），且与患者BMI无明显相关性（P=0.621）。RT-qPCR和Western blotting均发现K562、MEG-01及Dami细胞株表达ADIPOR1和ADIPOR2。10 μmol/L、20 μmol/L AdipoRon与PMA共同培养K562细胞株72 h后CD41⁺细胞比例显著低于PMA+DMSO组（均P=0.000）。20 μmol/L AdipoRon与PMA共同培养MEG-01细胞株72 h后CD41-MFI（P=0.047）和多倍体细胞比例（P=0.003）均显著高于PMA+DMSO组；但在Dami细胞株中，20 μmol/L AdipoRon与PMA处理后未发现两者显著升高。结论·ITP患者外周血血浆脂联素水平升高；人髓系细胞株K562、巨核细胞系细胞株MEG-01及Dami均表达脂联素受体ADIPOR1和ADIPOR2；脂联素受体激动剂可以在体外抑制巨核细胞分化，但其对巨核细胞成熟的影响尚不明确。

关键词: 免疫性血小板减少症, 脂联素, 脂联素受体, 巨核细胞, 脂肪细胞因子

Abstract:

Objective·To explore the level of adiponectin in the peripheral blood plasma of patients with immune thrombocytopenia (ITP) and its effect on the differentiation and maturation of megakaryocytes.

Methods·From January 2021 to February 2022, the peripheral blood samples were collected from 46 ITP patients in the Department of Hematology, The First Affiliated Hospital of Soochow University and 30 healthy controls with corresponding gender and age in the physical examination center. The plasma adiponectin levels in the ITP patients and healthy control (HC) group were detected by using human adiponectin enzyme-linked immunosorbent assay (ELISA) kits. And the correlation between adiponectin level and body mass index (BMI) in the ITP patients was analyzed. In terms of cell experiments, real-time quantitative PCR (RT-qPCR) and Western blotting were used to study the expression of adiponectin receptors (ADIPOR1 and ADIPOR2) in the myeloid cell line K562, and the megakaryocyte cell lines MEG-01 and Dami at mRNA and protein levels, respectively. The differentiation of K562 cell line and the maturation of MEG-01 and Dami cell lines were induced by phorbol 12-myristate 13-acetate (PMA). At the same time, the cells were treated with different concentrations of adiponectin receptor agonist AdipoRon. Flow cytometry was used to detect the percentage of CD41⁺ cells, the mean fluorescence intensity (MFI) of CD41, and the proportion of polyploid cells (≥4N).

Results·Compared with the HC group, the plasma adiponectin level of the ITP patients significantly increased (P=0.000), and there was no significant correlation between the adiponectin level and the BMI of the patients (P=0.621). RT-qPCR and Western blotting showed that ADIPOR1 and ADIPOR2 were expressed in K562, MEG-01 and Dami cell lines. The proportion of CD41⁺ cells in the K562 cell line co-cultured with 10 μmol/L or 20 μmol/L AdipoRon and PMA for 72 h were significantly lower than those in the PMA+DMSO group (P=0.000). The CD41-MFI (P=0.047) and the proportion of polyploid cells (P=0.003) in the MEG-01 cell line co-cultured with 20 μmol/L AdipoRon and PMA for 72 h were significantly higher than those in the PMA+DMSO group; however, in the Dami cell line, no significant increases of both were found after the treatment of 20 μmol/L AdipoRon and PMA.

Conclusion·The adiponectin level is elevated in the peripheral blood plasma of ITP patients. Human myeloid cell line K562, and megakaryocyte cell lines MEG-01 and Dami all express adiponectin receptors ADIPOR1 and ADIPOR2. Adiponectin receptor agonists can inhibit megakaryocyte differentiation in vitro, but its effect on megakaryocyte maturation is uncertain.

Key words: immune thrombocytopenia (ITP), adiponectin, adiponectin receptor (ADIPOR), megakaryocyte, adipocytokine

中图分类号:

R558⁺.2

李昕雨, 左斌, 王文, 钮晓音, 翁震, 何杨. 脂联素在免疫性血小板减少症患者外周血中的水平及其对巨核细胞系分化的作用[J]. 上海交通大学学报（医学版）, 2022, 42(7): 866-874.

LI Xinyu, ZUO Bin, WANG Wen, NIU Xiaoyin, WENG Zhen, HE Yang. Levels of adiponectin in the peripheral blood of patients with immune thrombocytopenia and its effect on the differentiation of megakaryocyte cell line[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(7): 866-874.

图/表 9

表1 RT-qPCR引物（5'→3'）

Tab 1 Primer sequences for RT-qPCR （5'→3'）

Gene	Forward primer	Reverse primer
ADIPOR1	CCAACTGGATGGTACACGAAG	TGGGAAGATAGAACAGATGCCTC
ADIPOR2	CGGTTTGCCACTCCTAAGC	CCACTCAAGCCAAGTCCCA
GAPDH	GTGAAGGTCGGAGTCAACGG	GTTCTCAGCCTTGACGGTGC

表2 ITP患者及HC组的临床资料

Tab 2 Clinical information of ITP patients and healthy controls

Item	ITP patient （n=46）	HC （n=30）	P value
Gender （female/male）/n	15/31	17/13	0.057
Age/year	48.5 （15‒81）	48 （25‒77）	0.510
BMI/（kg·m^-2）	24.05 （21.81， 25.79）	23.71 （22.44， 25.11）	0.761
Platelet count/（×10⁹ L^-1）	32.0 （13.0， 69.3）	220.5 （190.8， 260.5）	0.000
Disease subtype/n （%）			‒
Newly diagnosed	15 （32.6）	‒
Persistent	8 （17.4）	‒
Chronic	23 （50.0）	‒
rPLT/%^①	20.60 （8.80， 27.70）	‒	‒
PAIgG/%^②	37.45 （32.18， 47.45）	‒	‒
Platelet autoantibodies detected by MAIPA/n （%）^③			‒
Positive	9 （31.03）	‒
Negative	20 （68.97）	‒
TPO/（pg·mL^-1）^④	117.9 （48.88， 185.9）	‒	‒

图1 ITP患者和HC组血浆脂联素水平

Fig 1 Plasma adiponectin levels in the ITP patients and the healthy controls

图2 ITP患者血浆脂联素水平与BMI之间的关系Note：A. Correlation between plasma adiponectin level and BMI in the ITP patients. B. Plasma adiponectin levels in the ITP patients with different BMIs.

Fig 2 Relationship between plasma adiponectin level and BMI in the patients with ITP

图3 不同病程的ITP患者血浆脂联素水平Note： ndITP—newly-diagnosed ITP; pITP—persistent ITP; cITP—chronic ITP.

Fig 3 Plasma adiponectin levels in the ITP patients with different courses of disease

图4 脂联素受体在K562、MEG-01、Dami细胞株中的表达 (n=4)Note：A. Expression of ADIPOR1 and ADIPOR2 mRNA detected by RT-qPCR. B. Expression of ADIPOR1 protein detected by Western blotting. C. Expression of ADIPOR2 protein detected by Western blotting.

Fig 4 Expression of adiponectin receptors in the cell lines K562, MEG-01 and Dami (n=4)

图5 流式细胞术检测脂联素受体激动剂对K562细胞株分化的影响 (n=4)

Fig 5 Effect of AdipoRon on the differentiation of the cell line K562 detected by flow cytometry (n=4)

图6 流式细胞术检测脂联素受体激动剂对MEG-01细胞株成熟的影响 (n=3)Note： A. Percentage of CD41+cells. B. Expression level of CD41. C. Percentage of polyploid (≥4N) of megakaryocytes.

Fig 6 Effect of AdipoRon on maturation of the cell line MEG-01 detected by flow cytometry (n=3)

图7 流式细胞术检测脂联素受体激动剂对Dami细胞株成熟的影响 (n=3)Note： A. Percentage of CD41+ cells. B. Expression level of CD41. C. Percentage of polyploid (≥4N) of megakaryocytes.

Fig 7 Effect of AdipoRon on maturation of the cell line Dami detected by flow cytometry (n=3)

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脂联素在免疫性血小板减少症患者外周血中的水平及其对巨核细胞系分化的作用

Levels of adiponectin in the peripheral blood of patients with immune thrombocytopenia and its effect on the differentiation of megakaryocyte cell line

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