碳酸酐酶在红细胞分化发育过程中的功能研究

doi:10.19586/j.2095-2341.2022.0008

生物技术进展 ›› 2022, Vol. 12 ›› Issue (4): 584-590.DOI: 10.19586/j.2095-2341.2022.0008

碳酸酐酶在红细胞分化发育过程中的功能研究

吴静(), 张英楠, 徐长禄, 刘金花, 石莉红()

中国医学科学院血液病医院（中国医学科学院血液学研究所），北京协和医学院，实验血液学国家重点实验室；国家血液系统疾病临床医学研究中心；细胞生态海河实验室，天津 300020

收稿日期:2022-01-18 接受日期:2022-02-15 出版日期:2022-07-25 发布日期:2022-08-10
通讯作者: 石莉红
作者简介:吴静 E-mail：wj20142019@163.com；
基金资助:
国家自然科学基金青年科学基金项目(82100152);国家自然科学基金项目(81870089);中央高校基本科研业务费专项资金(3332020056);医科院医学与健康科技创新工程项目(2021-I2M-1-040);国家重点实验室课题(ZK21-09)

Research on the Function of Carbonic Anhydrase During Erythroid Differentiation

Jing WU(), Yingnan ZHANG, Changlu XU, Jinhua LIU, Lihong SHI()

State Key Laboratory of Experimental Hematology，National Clinical Research Center for Blood Diseases，Haihe Laboratory of Cell Ecosystem，Institute of Hematology & Blood Diseases Hospital，Chinese Academy of Medical Sciences & Peking Union Medical College，Tianjin 300020，China

Received:2022-01-18 Accepted:2022-02-15 Online:2022-07-25 Published:2022-08-10
Contact: Lihong SHI

摘要/Abstract

摘要：

目前红系分化调控相关的研究主要集中在细胞因子、转录因子、lncRNA及表观遗传方面，为了对红系分化调控机制进行更加深入的解析，研究了碳酸酐酶在红系分化中的功能。碳酸酐酶可以高效催化二氧化碳的水合，但它在红细胞发育过程中的功能尚不清楚。利用脐带血来源的CD34⁺细胞在体外进行红细胞诱导分化，在分化过程中通过慢病毒介导的基因敲降的方法能够降低碳酸酐酶1和碳酸酐酶2的表达，并使用流式细胞仪检测红细胞的生成和分化效率。研究结果表明，与对照组相比，碳酸酐酶1的表达缺陷使红细胞的晚期分化明显受阻，而碳酸酐酶2的表达缺陷则将红细胞的分化阻滞在早期阶段。研究结果表明，虽然作用窗口不同，但碳酸酐酶1和碳酸酐酶2在红系分化的过程中均发挥着重要的调控作用，这一发现对将来在体外红细胞生成具有指导意义。

关键词: 红细胞分化发育, 碳酸酐酶, 体外红细胞生成

Abstract:

The current research about the regulation of erythroid differentiation mainly focuses on cytokines， transcription factors， lncRNAs and epigenetics. To further dissect mechanisms about erythroid differentiation， the function of carbonic anhydrase during erythroid differentiation was investigated. Carbonic anhydrase can catalyze the hydration of carbon dioxide， but its function in erythroid development is unclear. Erythroid differentiation of CD34⁺ cells was induced in vitro. Lentivirus-mediated knockdown of carbonic anhydrase 1 and carbonic anhydrase 2 were performed in this study. The differentiation efficiency was detected by flow cytometry. Results showed that the knockdown of carbonic anhydrase 1 led to a significant decline in late erythroid differentiation. However， erythroid differentiation was blocked in the earlier stage after carbonic anhydrase 2 knockdown. Thus， our study demonstrated that carbonic anhydrase 1 and carbonic anhydrase 2 play important roles in different stages of erythroid differentiation and this finding will be instructive for erythroid generation in vitro in the future.

Key words: erythroid differentiation, carbonic anhydrase, erythropoiesis generation in vitro

中图分类号:

R331.1+41

吴静, 张英楠, 徐长禄, 刘金花, 石莉红. 碳酸酐酶在红细胞分化发育过程中的功能研究[J]. 生物技术进展, 2022, 12(4): 584-590.

Jing WU, Yingnan ZHANG, Changlu XU, Jinhua LIU, Lihong SHI. Research on the Function of Carbonic Anhydrase During Erythroid Differentiation[J]. Current Biotechnology, 2022, 12(4): 584-590.

图/表 5

表1 本研究shRNA序列

Table 1 shRNA sequences in this study

名称	引物序列（5′→3′)
ScrambledF	5′-GTGCGTTGTTAGTACTAATCCTATTT-3′;
ScrambledR	5′-AAATAGGATTAGTACTAACAACGCAC-3′;
CA1-sh1F	5′-GCTGATGGTTTGGCAGTTATT-3′;
CA1-sh1R	5′-AATAACTGCCAAACCATCAGC-3′;
CA1-sh2F	5′-GGGCAGTACAAATGAGCATGG-3′；
CA1-sh2R	5′-CCATGCTCATTTGTACTGCCC-3′;
CA1-sh3F	5′-GGGCATTCCTTCCATGTAAAT-3′;
CA1-sh3R	5′-ATTTACATGGAAGGAATGCCC-3′;
CA2-sh1F	5′-GGGCCTTCAGAAAGTTGTTGA-3′;
CA2sh1R	5′-TCAACAACTTTCTGAAGGCCC-3′;
CA2-sh2F	5′-GGAGTTTGATGACTCTCAGGA-3′;
CA2-sh2R	5′-TCCTGAGAGTCATCAAACTCC-3′

图1 CA家族基因在人类各造血细胞中的表达情况及CA1、CA2在红系分化过程中的动态变化过程A: 人骨髓各谱系血细胞单细胞转录组数据；B：脐带血和外周血来源的CD34+细胞RNA-seq数据。HSC—造血干细胞；MPP—多能祖细胞；CMP—常见髓系祖细胞；MEP—巨核细胞-红细胞祖细胞；LMPP—淋巴活化的多能祖细胞；MLP—多淋巴祖细胞；BNK—B淋巴细胞和NK细胞；GMP—粒细胞-巨噬细胞祖细胞；B—B淋巴细胞；CLP—共同淋巴祖细胞；NK—自然杀伤细胞；T—T淋巴细胞；hMDP—人单核-树突状细胞前体；cMOP—单核细胞前体；Mono/Mac—单核巨噬细胞；Neu—中性粒细胞；Ery—红细胞；CD34+—CD34+细胞；BFU-E—早期红系祖细胞集落形成单位；CFU-E—晚期红系祖细胞集落形成单位；ProE—原始红细胞；Early-BasoE—早期早幼红细胞；Late-BasoE—晚期早幼红细胞；PolyE—中幼红细胞；OrthE—晚幼红细胞。

Fig.1 The expression of CA family genes in each human hematopoietic lineage and the dynamic changes of CA1 and CA2 during erythroid differentiation

图2 HEL细胞中检测CA1和CA2的敲降A: 通过RT-qPCR检测HEL细胞中CA1的敲降；B：通过RT-qPCR检测HEL细胞中CA2的敲降

Fig.2 Detection of the CA1 and CA2 knockdown in HEL cells

图3 CA1的敲降对CD34+细胞分化的影响A：CD34+细胞体外诱导分化实验模式图；B：流式细胞仪分析CD34+细胞体外诱导分化Day 8、Day 11和Day 14时各个群体细胞的比例；C、D：统计学分析CD34+细胞体外诱导分化Day 8、Day 11和Day 14时CD71+CD235a+细胞和CD71-CD235a-细胞比例。*表示在P<0.05水平差异具有统计学意义，**表示在P<0.01水平差异具有统计学意义，***表示在P<0.001水平差异具有统计学意义，ns表示无统计学差异(P>0.05)。

Fig.3 The effect of CA1 knockdown on differentiation of CD34+ cells

图4 CA2的敲降对CD34+细胞分化的影响A：流式细胞仪分析CD34+细胞体外诱导分化Day 8、Day 11和Day 14时各个群体细胞的比例；B、C：统计学分析CD34+细胞体外诱导分化Day 8、Day 11和Day 14时CD71+CD235a+细胞和CD71-CD235a-细胞比例。*表示在P<0.05水平差异具有统计学意义，**表示在P<0.01水平差异具有统计学意义，***表示在P<0.001水平差异具有统计学意义。

Fig.4 The effect of CA2 knockdown on differentiation of CD34+ cells

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碳酸酐酶在红细胞分化发育过程中的功能研究

Research on the Function of Carbonic Anhydrase During Erythroid Differentiation

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