低剂量地西他滨对免疫性血小板减少症患者来源的骨髓间充质干细胞生物学行为的影响

doi:10.3969/j.issn.1674-8115.2022.06.010

上海交通大学学报（医学版） ›› 2022, Vol. 42 ›› Issue (6): 758-767.doi: 10.3969/j.issn.1674-8115.2022.06.010

• 论著 · 基础研究 • 上一篇

低剂量地西他滨对免疫性血小板减少症患者来源的骨髓间充质干细胞生物学行为的影响

王昕芃¹(), 王君颖¹, 蔡佳翌², 付婉彬¹, 钟华¹()

^1.上海交通大学医学院附属仁济医院血液科，上海 200127
^2.上海交通大学医学院附属仁济医院中心实验室，上海 200127

收稿日期:2022-02-11 接受日期:2022-05-11 出版日期:2022-06-28 发布日期:2022-08-19
通讯作者: 钟华 E-mail:wxpyoyo@126.com;zhh_lj@163.com
作者简介:王昕芃（1996—），女，硕士生；电子信箱：wxpyoyo@126.com。
基金资助:
上海交通大学医学院附属仁济医院南院临床多学科合作共建项目(2014MDT01-07)

Effect of low-dose decitabine on the biological behavior of bone marrow mesenchymal stem cells derived from patients with immune thrombocytopenia

WANG Xinpeng¹(), WANG Junying¹, CAI Jiayi², FU Wanbing¹, ZHONG Hua¹()

^1.Department of Hematology, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China
^2.Central Laboratory, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China

Received:2022-02-11 Accepted:2022-05-11 Online:2022-06-28 Published:2022-08-19
Contact: ZHONG Hua E-mail:wxpyoyo@126.com;zhh_lj@163.com
Supported by:
Program for Clinical Multi-Disciplinary Teamwork of Renji Hospital (South Campus), Shanghai Jiao Tong University School of Medicine(2014MDT01-07)

摘要/Abstract

摘要：

目的·研究低剂量地西他滨对原发免疫性血小板减少症（immune thrombocytopenia，ITP）患者来源的骨髓间充质干细胞（mesenchymal stem cell，MSC）增殖、凋亡等生物学行为的影响。方法·纳入2020年4月—2021年10月上海交通大学医学院附属仁济医院血液科门诊或住院的ITP发病期患者10例，以及10例无骨髓异常的非ITP患者作为对照（normal control，NC），收集骨髓穿刺标本，分离MSC，流式细胞术检测其表面抗原标志物。使用CCK-8试剂盒和EdU细胞增殖检测试剂盒检测及比较ITP患者来源（MSC-ITP）和对照组来源（MSC-NC）的MSC增殖水平差异，并使用微管荧光探针检测细胞形态的变化。使用Hoechst 33258染色观察细胞凋亡情况，采用Annexin V-FITC/PI细胞凋亡双染试剂盒检测细胞凋亡水平。通过不同浓度的地西他滨以不同的时长处理MSC-ITP，探索地西他滨促其增殖的最佳浓度及处理时间。以最佳浓度的地西他滨处理MSC-ITP，观察其对细胞凋亡水平的影响。Western blotting检测细胞凋亡相关通路蛋白的表达，以及地西他滨的影响。结果·与MSC-NC相比，MSC-ITP细胞形态异常，细胞核碎裂、皱缩较多，体外增殖水平减弱，基础凋亡率增加。地西他滨刺激MSC-ITP增殖的最佳工作浓度为2.5 μmol/L，最佳处理时间为24 h。2.5 μmol/L地西他滨处理MSC-ITP 24 h后细胞核形态得到改善，细胞核的碎裂和凝聚减少，凋亡率显著降低（P<0.05）。Western blotting结果表明，地西他滨处理后，MSC-ITP中线粒体凋亡通路相关蛋白BAX降低，胱天蛋白酶（caspase3）和cleaved-caspase3减少（均P<0.05）。结论·ITP患者骨髓来源的MSC形态异常，体外增殖水平减弱，基础凋亡率增加；低剂量地西他滨可以促进该细胞增殖，抑制其凋亡；该作用可能是通过线粒体凋亡途径介导的。

关键词: 免疫性血小板减少症, 骨髓间充质干细胞, 地西他滨, DNA甲基转移酶抑制剂, 细胞凋亡

Abstract:

Objective·To investigate the effects of low-dose decitabine on the biological behaviors of proliferation and apoptosis of bone marrow mesenchymal stem cells (MSC) derived from the patients with primary immune thrombocytopenia (ITP).

Methods·Ten patients with ITP onset who were outpatients or inpatients in the Department of Hematology, Renji Hospital, Shanghai Jiao Tong University School of Medicine, from April 2020 to October 2021 were included, and 10 non-ITP patients without bone marrow abnormalities were included as controls (normal control, NC). The bone marrow aspiration specimens were collected, MSCs were isolated, and flow cytometry was used to detect their surface antigenic markers. Differences in MSC proliferation levels between the ITP patients (MSC-ITP) and the controls (MSC-NC) were detected by using the CCK-8 kit and the EdU cell proliferation assay kit, and the changes in cell morphology were detected by using microtubule fluorescent probes. Apoptosis was observed by using Hoechst 33258 staining, and the level of apoptosis was detected by using Annexin V-FITC/PI apoptosis double-staining kit. The optimal concentration and treatment time of decitabine to promote the proliferation of MSC-ITP were explored by treatment with different concentrations and different durations. Western blotting was performed to detect the expression of apoptosis-related proteins and the effect of decitabine.

Results·Compared with MSC-NC, MSC-ITP showed abnormal morphology, more nuclear fragmentation and crinkling, reduced proliferation level in vitro, and increased basal apoptosis rate. The optimal working concentration of decitabine to stimulate MSC-ITP proliferation was 2.5 μmol/L, and the optimal treatment time was 24 h. After 2.5 μmol/L decitabine treating MSC-ITP for 24 h, cell nuclear morphology was improved, nuclei fragmentation and coalescence were reduced, and apoptosis rate was significantly decreased (P<0.05). Western blotting results showed that BAX, a mitochondrial apoptosis pathway-related protein, cystatin protease 3 (caspase3) and cleaved-caspase3 were reduced in MSC-ITP after treatment with decitabine (P<0.05).

Conclusion·The MSCs originating from ITP patients' bone marrow have abnormal morphology, reduced proliferation level and increased basal apoptosis rate in vitro; low-dose decitabine can promote the proliferation and inhibit the apoptosis of the cells, which may be mediated through the mitochondrial apoptosis pathway.

Key words: immune thrombocytopenia (ITP), bone marrow mesenchymal stem cell, decitabine, DNA methyltransferase inhibitor, apoptosis

中图分类号:

R558⁺.2

王昕芃, 王君颖, 蔡佳翌, 付婉彬, 钟华. 低剂量地西他滨对免疫性血小板减少症患者来源的骨髓间充质干细胞生物学行为的影响[J]. 上海交通大学学报（医学版）, 2022, 42(6): 758-767.

WANG Xinpeng, WANG Junying, CAI Jiayi, FU Wanbing, ZHONG Hua. Effect of low-dose decitabine on the biological behavior of bone marrow mesenchymal stem cells derived from patients with immune thrombocytopenia[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(6): 758-767.

图/表 7

表1 纳入研究的ITP患者和对照组的临床信息

Tab 1 Clinical information of the ITP patients and the normal controls enrolled in the study

ITP patient	Gender	Age/year	Platelet count/（×10⁹ L^－1）	NC subject	Gender	Age/year	Platelet count/（×10⁹ L^－1）
P1	Female	48	44	N1	Female	60	137
P2	Female	44	34	N2	Female	74	206
P3	Female	70	69	N3	Female	65	169
P4	Female	71	102	N4	Male	31	145
P5	Male	72	61	N5	Female	66	172
P6	Female	84	38	N6	Male	39	151
P7	Male	77	20	N7	Female	51	183
P8	Female	45	48	N8	Male	68	201
P9	Female	85	34	N9	Male	45	195
P10	Male	61	7	N10	Female	75	141

图1 流式细胞术鉴定体外培养MSC表型Note：n=5. Neg CKTL—negative cocktail.

Fig 1 Phenotype of MSCs cultured in vitro identified by flow cytometry

图2 ITP患者来源MSC增殖水平和细胞形态检测Note：A. Morphology of MSC-NC and MSC-ITP under light microscope (×40). B. CCK-8 method showing growth curves of MSC-NC (n=5) and MSC-ITP (n=5). C. Observation under fluorescent microscope (n=5, ×200).

Fig 2 Detection of proliferation and morphology of MSCs derived from ITP patients

图3 ITP患者来源MSC基础凋亡水平检测Note：A. Hoechst 33258 staining under fluorescence microscopy showed MSC-ITP and MSC-NC nuclei (n=6, ×200). White arrows indicate fragments of apoptotic cells and condensed nuclei. B. Apoptotic rates of MSC-ITP and MSC-NC determined by using annexin V-FITC/PI method and flow cytometry (n=10). ①P=0.004, ②P=0.001.

Fig 3 Detection of basal apoptosis in MSCs derived from ITP patients

图4 CCK-8法测定不同剂量、不同处理时间下地西他滨对MSC-ITP增殖的影响Note：A. 0 h. B. 12 h. C. 24 h. D. 48 h. n=5. ①P=0.011, ②P=0.007.

Fig 4 Effect of decitabine on MSC-ITP proliferation under different doses and working hours detected by CCK-8 assay

图5 2.5 μmol/L地西他滨处理24 h后MSC-ITP的凋亡检测Note：A. Hoechst 33258 staining under fluorescence microscopy showing nuclear morphology of cells (n=6, ×200). B. Apoptotic rates determined by using annexin V-FITC/PI method and flow cytometry (n=10). ①P=0.027, ②P=0.015.

Fig 5 Apoptosis detection of MSC-ITP after 2.5 μmol/L decitabine treatment for 24 h

图6 Western blotting检测凋亡相关蛋白BAX、caspase-3和cleaved-caspase3的表达量Note：A. The bands in Western blotting. B. Quantitative analysis of caspase-3. C. Quantitative analysis of cleaved-caspase3. D. Quantitative analysis of BAX. ①P=0.000, ②P=0.000, ③P=0.005, ④P=0.038.

Fig 6 Expression levels of apoptosis-related proteins BAX, caspase-3 and cleaved-caspase3 detected by Western blotting

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低剂量地西他滨对免疫性血小板减少症患者来源的骨髓间充质干细胞生物学行为的影响

Effect of low-dose decitabine on the biological behavior of bone marrow mesenchymal stem cells derived from patients with immune thrombocytopenia

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