PKA基因过表达与敲除对人诱导多能干细胞体外巨核细胞分化的影响*

doi:10.3969/j.issn.1671-2587.2026.01.005

临床输血与检验 ›› 2026, Vol. 28 ›› Issue (1): 29-35.DOI: 10.3969/j.issn.1671-2587.2026.01.005

PKA基因过表达与敲除对人诱导多能干细胞体外巨核细胞分化的影响^*

杨玥¹, 岳伟², 黄韦华¹, 李津杞¹, 李彦欣³, 顾海慧¹

¹中国人民解放军海军军医大学第一附属医院输血科，上海 200433;
²盐城市第一人民医院输血科，江苏盐城 224005;
³上海交通大学医学院附属上海儿童医学中心转化所，国家卫健委儿童血液肿瘤重点实验室，上海 200000

收稿日期:2025-07-24 发布日期:2026-02-13
通讯作者: 顾海慧，主要从事临床输血与细胞治疗研究，（E-mail）haihuigu@126.com。
作者简介:杨玥，主要从事临床输血与患者血液管理，（E-mail）978357160@qq.com。
基金资助:
*本课题受国家自然科学基金面上项目（No.81970165）资助

Regulatory Role of Protein Kinase A in Megakaryocyte Differentiation from Human Induced Pluripotent Stem Cells

YANG Yue¹, YUE Wei², HUANG Weihua¹, LI Jinqi¹, LI Yanxin³, GU Haihui¹

¹Department of Transfusion Medicine, The First Affiliated Hospital of Naval Medical University, Shanghai 200433;
²Department of Transfusion Medicine, The First People's Hospital of Yancheng, Yancheng, Jiangsu 224005;
³Pediatric Translational Medicine Institute, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Key Laboratory of Pediatric Hematology & Oncology of National Health Commission, Shanghai 200000

Received:2025-07-24 Published:2026-02-13

摘要/Abstract

摘要： 目的研究蛋白激酶A（PKA）在人诱导多能干细胞（iPSCs）向巨核细胞（MKs）分化过程中的调控作用。方法构建PKA过表达质粒，利用慢病毒转染系统将其导入人脐血来源的iPSCs中建立PKA过表达细胞系;采用CRISPR/Cas9基因编辑技术构建PKA基因敲除iPS细胞系。通过蛋白水平检测验证两种细胞系中PKA的表达情况。在无血清、无饲养层的“旋转-拟胚体（Spin-EB）”培养体系中，将细胞分为对照组（untreated组）、PKA过表达组（OV组）和PKA敲除组（KO组），在培养至第21天时，收集各组细胞，采用流式细胞术检测巨核系标志物（CD41⁺）和成熟巨核标志物（CD41⁺CD42a⁺）的表达水平。结果构建PKA过表达和PKA敲除的人iPS细胞系：PKA OV iPSCs 和PKA KO iPSCs，并经western-blot验证。在“Spin-EB”分化体系中培养至第21天，PKA OV iPSCs分化生成的CD41⁺细胞比例及细胞数量无显著差异（P>0.05），CD41⁺CD42a⁺细胞比例和数量明显减少（P<0.01;P<0.05）。在PKA KO iPSCs中，分化生成的CD41⁺细胞比例及数量、CD41⁺CD42a⁺细胞比例及数量均显著增加（P<0.001;P<0.001;P<0.01;P<0.01）。结论本研究成功构建了PKA基因修饰iPSCs，并验证PKA在iPSCs分化为MKs中的调控作用，为优化人诱导多能干细胞源血小板生成体系提供了新靶点。PKA过表达抑制了iPSCs向成熟MKs分化，而PKA敲除则显著促进了iPSCs向MKs的分化。

关键词: 人诱导多能干细胞, 体外分化, 巨核细胞, 蛋白激酶A

Abstract: Objective To investigate the regulatory role of protein kinase A (PKA) in the differentiation of human induced pluripotent stem cells (iPSCs) into megakaryocytes (MKs). Methods A PKA overexpression plasmid was constructed and introduced into iPSCs derived from human umbilical cord blood via a lentiviral transduction system to establish a PKA overexpression cell line. A PKA gene knockout iPSC line was generated using CRISPR/Cas9 gene-editing technology. The expression of PKA in both cell lines was verified at the protein level. In a serum-free, feeder-free "Spin-EB" culture system, three groups were established: control group (untreated group), PKA overexpression group (OV group), and PKA knockout group (KO group). On day 21 of culture, cells from each group were collected, and the expression levels of megakaryocytic lineage markers (CD41⁺) and mature megakaryocytic markers (CD41⁺CD42a⁺) were detected by flow cytometry. Results No significant difference was observed in CD41⁺ cell proportion or count between PKA OV and control groups (P>0.05); PKA OV significantly reduced the proportion and count of CD41⁺CD42a⁺ mature MKs (P<0.01; P<0.05); PKA KO markedly increased both CD41⁺ and CD41⁺CD42a⁺ cell proportions and counts (P<0.001; P<0.001; P<0.01; P<0.01). Conclusion This study successfully established PKA gene-modified iPSC lines and demonstrated the regulatory role of PKA in the differentiation of iPSCs into MKs, providing a new target for optimizing the generation of platelets from human iPSCs. PKA overexpression inhibited the differentiation of iPSCs into MKs, whereas PKA knockout significantly promoted this differentiation.

Key words: Human induced pluripotent stem cells, In vitro differentiation, Megakaryocytes, Protein kinase A

中图分类号:

R394.2R446.1

杨玥, 岳伟, 黄韦华, 李津杞, 李彦欣, 顾海慧. PKA基因过表达与敲除对人诱导多能干细胞体外巨核细胞分化的影响^*[J]. 临床输血与检验, 2026, 28(1): 29-35.

YANG Yue, YUE Wei, HUANG Weihua, LI Jinqi, LI Yanxin, GU Haihui. Regulatory Role of Protein Kinase A in Megakaryocyte Differentiation from Human Induced Pluripotent Stem Cells[J]. JOURNAL OF CLINICAL TRANSFUSION AND LABORATORY MEDICINE, 2026, 28(1): 29-35.

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PKA基因过表达与敲除对人诱导多能干细胞体外巨核细胞分化的影响^*

Regulatory Role of Protein Kinase A in Megakaryocyte Differentiation from Human Induced Pluripotent Stem Cells

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