利用Crispr/Cas9基因编辑系统在人胚胎干细胞中探索ELABELA的潜在新受体

doi:10.3969/j.issn.1674-8115.2022.09.012

上海交通大学学报（医学版） ›› 2022, Vol. 42 ›› Issue (9): 1258-1264.doi: 10.3969/j.issn.1674-8115.2022.09.012

• 论著 · 基础研究 • 上一篇

利用Crispr/Cas9基因编辑系统在人胚胎干细胞中探索ELABELA的潜在新受体

周月¹(), 程晨², 郑恩霖², 孟卓¹, 王鉴¹, 王青洁¹, 何勇宁², 孙锟¹()

^1.上海交通大学医学院附属新华医院小儿心血管科，上海 200092
^2.上海交通大学医学院附属仁济医院上海肿瘤研究所，上海 200127

收稿日期:2022-07-08 接受日期:2022-09-06 出版日期:2022-09-28 发布日期:2022-09-28
通讯作者: 孙锟 E-mail:we.crazy@sjtu.edu.cn;sunkun@xinhuamed.com.cn
作者简介:周　月（1995—），女，回族，博士生；电子信箱：we.crazy@sjtu.edu.cn。
基金资助:
国家自然科学基金(82130015);上海市卫生健康委员会协同创新集群项目(2020CXJQ01)

Exploring potential new receptors for ELABELA in human embryonic stem cells by Crispr/Cas9-mediated gene editing system

ZHOU Yue¹(), CHENG Chen², ZHENG Enlin², MENG Zhuo¹, WANG Jian¹, WANG Qingjie¹, HE Yongning², SUN Kun¹()

^1.Department of Pediatric Cardiovascular, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China
^2.Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China

Received:2022-07-08 Accepted:2022-09-06 Online:2022-09-28 Published:2022-09-28
Contact: SUN Kun E-mail:we.crazy@sjtu.edu.cn;sunkun@xinhuamed.com.cn
Supported by:
National Natural Science Foundation of China(82130015);Science and Research Fund of Shanghai Municipal Commission of Health and Family Planning(2020CXJQ01)

摘要/Abstract

摘要：

目的·利用Crispr/Cas9基因编辑技术在人胚胎干细胞（human embryonic stem cell，hESC）中探索小分子多肽ELABELA（ELA）是否存在潜在的新受体。方法·收集干细胞向心肌细胞定向分化过程中不同天数（0~13 d）的细胞，检测其ELA及其受体APJ的动态表达水平。在干细胞向心肌细胞定向分化过程中加入APJ抑制剂ML221，观察心肌细胞标志物MYH6、TnnT2及NKX2.5的mRNA表达水平是否发生变化。利用人胚肾悬浮细胞（HEK-293F）表达重组ELA绿色荧光蛋白（green fluorescent protein，GFP），研究ELA发挥结合作用的区域在其C末端还是N末端，构建GFP序列分别插入ELA-32序列的C末端及N末端的2种重组质粒。利用Crispr/Cas 9基因编辑技术构建ELA唯一已知受体APJ敲除的hESC并进行验证。在构建成功的敲除细胞系及正常细胞系中外源性加入2种GFP-ELA重组蛋白，通过荧光显微镜观察ELA是否能够进入细胞。结果·在干细胞向心肌细胞定向分化过程中，在干细胞时期，ELA高表达而APJ低表达；ELA表达高峰在分化第6天而APJ表达高峰出现在分化第3天。外源性加入APJ抑制剂ML221后，MYH6、TnnT2及NKX2.5的mRNA表达水平无显著变化（P>0.05）。经过基因DNA测序及Western blotting验证，成功构建敲除APJ干细胞系。敲除APJ后，外源性加入ELA N端荧光蛋白，仍能够进入细胞内；而ELA C端荧光蛋白未出现荧光，表示ELA C端荧光蛋白不能进入细胞。结论·hESC中存在非APJ的ELA其他受体， ELA与受体发生结合的部位主要位于C末端。

关键词: 人胚胎干细胞, ELABELA, APJ, 基因编辑

Abstract:

Objective To verify the existence of potential new receptor of ELABELA (ELA) in human embryonic stem cells (hESC) by Crispr/Cas9-mediated gene editing system. Methods The dynamic expression levels of ELA and its receptor APJ were examined by collecting cells at different days (0?13 d) during the directed differentiation of stem cells into cardiomyocytes. The mRNA expression levels of cardiomyocyte markers MYH6, TnnT2 and NKX2.5 were observed by adding the APJ inhibitor ML221 during the directed differentiation of stem cells to cardiomyocytes. Human embryonic kidney suspension cells (HEK-293F) were used to express recombinant ELA green fluorescent protein (GFP). To verify whether ELA binds through its C-terminus or N-terminus, two recombinant plasmids with GFP sequences which were inserted into the C-terminus or the N-terminus of ELA-32 sequence were constructed. The only known receptor for ELA, APJ-knockout hESC, was constructed and validated by using Crispr/Cas 9 gene editing technology. Two GFP-ELA recombinant proteins were added exogenously to the successfully constructed knockout cell lines and normal cell lines, and the ability of ELA to enter the cells was observed by fluorescence microscopy. Results During the stem cell period, ELA was highly expressed while APJ was basically not expressed; the peak of ELA expression was at the 6th day of differentiation while the peak of APJ expression appeared at the 3rd day of differentiation. The efficiency of stem cell directed differentiation to cardiomyocytes was not affected by exogenous addition of the APJ inhibitor ML221 (P>0.05). After DNA sequencing and Western blotting validation, the APJ-knockdown stem cell line was successfully constructed. When APJ was knocked down, exogenous addition of ELA N-terminal fluorescent protein was still able to enter the cells, while the absence of fluorescence of ELA C-terminal fluorescent protein indicated that ELA C-terminal fluorescent protein could not enter the cells. Conclusion There are other ELA receptors in hESCs that are not APJ. Otherwise, the binding sites of ELA and its receptor are mainly located at the C-terminal of ELA.

Key words: human embryonic stem cell (hESC), ELABELA, APJ, gene editing

中图分类号:

R394.2

周月, 程晨, 郑恩霖, 孟卓, 王鉴, 王青洁, 何勇宁, 孙锟. 利用Crispr/Cas9基因编辑系统在人胚胎干细胞中探索ELABELA的潜在新受体[J]. 上海交通大学学报（医学版）, 2022, 42(9): 1258-1264.

ZHOU Yue, CHENG Chen, ZHENG Enlin, MENG Zhuo, WANG Jian, WANG Qingjie, HE Yongning, SUN Kun. Exploring potential new receptors for ELABELA in human embryonic stem cells by Crispr/Cas9-mediated gene editing system[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(9): 1258-1264.

图/表 4

图1 ELA 、 APJ 的表达及APJ抑制剂对心肌标志物表达的影响Note： A/B. During the differentiation of hESCs to cardiomyocytes, ELA (A) was relatively highly expressed at D0, while APJ (B) was largely absent. ELA expression peaked at D6, while APJ expression peaked at D3. C. Addition of APJ inhibitor (ML221) did not affect the differentiation efficiency of hESCs to cardiomyocytes.

Fig 1 Expression of ELA and APJ and the effect of APJ inhibitor on the expression of myocardial markers

图2 GFP-ELA重组蛋白的构建及荧光表达情况Note：A. N-terminal and C-terminal GFP-ELA plasmids construction strategy. B. Exogenous addition of GFP-ELA-purified protein in hESCs revealed that exogenous ELA could still enter the cells during the period of low APJ expression.

Fig 2 Construction of GFP-ELA recombinant protein and observation of fluorescent expression

图3 APJ 敲除干细胞系的构建Note：A. Gene-level construction strategy for APJ-knockdown cell lines with gRNA in red. B. Sanger sequencing verified that part of the APJ gene sequence on exon 1 was sheared. C. Western blotting showed that APJ was successfully knocked down from the protein level.

Fig 3 Construction of APJ knockout hESC lines

图4 荧光显微镜观察GFP-ELA重组蛋白的表达Note： The addition of exogenous GFP-ELA-purified protein to the APJ-knockout hESCs revealed that exogenous ELA was still able to enter the cells.

Fig 4 GFP-ELA recombinant protein expression observed by fluorescence microscopy

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利用Crispr/Cas9基因编辑系统在人胚胎干细胞中探索ELABELA的潜在新受体

Exploring potential new receptors for ELABELA in human embryonic stem cells by Crispr/Cas9-mediated gene editing system

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