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。

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.