| 苏 宏,李 钊,刘婷婷,陈超飞,王 敏.Etk敲除及过表达细胞株构建并探讨Etk对细胞增殖的影响[J].,2020,(5):801-807 |
| Etk敲除及过表达细胞株构建并探讨Etk对细胞增殖的影响 |
| Construction of Etk Knockout and Overexpression Cell Strains and Investigation for the Effect of Etk on HUVECs Proliferation |
| 投稿时间：2019-12-19 修订日期：2020-01-12 |
| DOI：10.13241/j.cnki.pmb.2020.05.001 |
| 中文关键词: Etk CRISPR-Cas9敲除 慢病毒过表达 细胞增殖 |
| 英文关键词: Etk CRISPR-Cas9 Knockdown Lentivirus overexpression Cell proliferation |
| 基金项目:国家自然科学基金重大研究计划培育项目（91539110） |
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| 中文摘要: |
| 摘要 目的：利用CRISPR-Cas9技术在人脐静脉内皮细胞（Human umbilical vein endothelial cells，HUVECs）中构建Etk（Epithelial and endothelial tyrosine kinase）敲除稳定细胞株以及利用慢病毒构建Etk过表达稳定细胞株，并初步探讨Etk基因对HUVECs细胞增殖的影响。方法：利用CRISPR-Cas9技术，使用在线工具设计针对Etk的sgRNA (https://chopchop.rc.fas.harvard.edu/)。将sgRNA利用连接酶整合到病毒载体中，包被病毒并感染HUVECs敲除HUVECs中的内源性Etk。利用嘌呤霉素筛选得到Etk敲除的稳定细胞株。PCR扩增Etk基因序列，将其整合到pLEX-MCS慢病毒过表达载体中构建Etk过表达重组质粒。包被病毒并感染HUVECs，在HUVECs中过表达Etk，利用嘌呤霉素筛选得到Etk过表达的稳定细胞株。利用qRT-PCR和Western-Blotting检测Etk的敲除和过表达情况。利用CCK-8盒子检测两种稳定细胞株细胞增殖情况。结果：利用CRISPR-Cas9技术有效的敲除HUVECs中内源性Etk，同对照相比，Etk的mRNA和蛋白水平显著地降低（P＜0.01）。同时利用慢病毒在HUVECs中过表达Etk，同对照相比，在过表达Etk稳定细胞株中Etk的mRNA和蛋白表达显著上调（P＜0.01）。CCK-8检测发现，Etk敲除降低细胞增殖能力；而Etk过表达增加细胞增殖能力。结论：通过CRISPR-Cas9技术成功在HUVECs中敲除Etk，利用慢病毒过表达系统成功的在HUVECs中过表达Etk，并且初步验证了Etk促进HUVECs细胞增殖。 |
| 英文摘要: |
| ABSTRACT Objective: In order to study the function of Etk in HUVECs proliferation, we created Etk knockdown cell strain by CRISPR-Cas9 and build Etk overexpression cell strain by lentivirus transfection. Methods: According to CRISPR-Cas9 method, sgRNAs of Etk were designed by the online tool (https://chopchop.rc.fas.harvard.edu/). After cloned into a lentivirus vector and packaged into lentivirus, sgRNA were delivered into HUVECs by lentivirus transfection. Etk knockdown cell strain was selected by puromycin. The CDS sequence of Etk was amplified by PCR and cloned into LentiORF pLEX-MCS vector. After packaged into lentivirus and transfection into HUVECs, Etk overexpression cell strain was selected by puromycin. qRT-PCR and Western Blotting were used to analyze the mRNA and protein level of Etk in Etk knockdown and overexpression cell strains. Meanwhile, CCK-8 kit was used to examined cell proliferation in these two cell strains. Results: CRISPR-Cas9 knockdown significantly decreased Etk expression in HUVECs. The mRNA and protein levels of Etk were markedly decreased (P＜0.01) in Etk knockdown cell strain compared to its control cell strain. The mRNA and protein levels of Etk were significantly increased in Etk overexpression cell strain compared to its control cell stain (P＜0.01). Regarding CCK-8 analysis, Etk knockdown decreased cell proliferation while Etk overexpression increased cell proliferation. Conclusion: We used CRISPR-Cas9 to create Etk knockdown cell strain and lentivirus system to creat Etk overexpression cell strain in HUVECs. In addition, we proved that Etk promotes HUVECs proliferation. |
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