CRISPR/Cas9介导下敲除KNDC1的人脐静脉内皮细胞具有抗衰老能力

基础医学与临床 ›› 2020, Vol. 40 ›› Issue (9): 1175-1181.

CRISPR/Cas9介导下敲除KNDC1的人脐静脉内皮细胞具有抗衰老能力

孙靖玉, 姚赫, 胡刚, 魏洁, 郭君, 张鑫, 林雅军^*

北京医院国家老年医学中心国家卫生健康委员会老年医学重点实验室,北京 100730

收稿日期:2019-08-26 修回日期:2020-01-07 出版日期:2020-09-05 发布日期:2020-09-04
通讯作者: ^*linyajun2000@126.com
基金资助:
国家自然科学基金(81671391);中国医学科学院医学与健康科技创新工程(2018-I2M-1-002)

HUVECs with KNDC1 knockout mediated by CRISPR/Cas9 have anti-aging ability

SUN Jing-yu, YAO He, HU Gang, WEI Jie, GUO Jun, ZHANG Xin, LIN Ya-jun^*

Key Laboratory of Geriatrics of National Health Commission, National Center of Gerontology, Beijing Hospital, Beijing 100730, China

Received:2019-08-26 Revised:2020-01-07 Online:2020-09-05 Published:2020-09-04
Contact: ^*linyajun2000@126.com

摘要/Abstract

摘要： 目的利用CRISPR/Cas9基因编辑技术敲除人脐静脉内皮细胞(HUVECs)的KNDC1并观察其抗衰老能力。方法通过在线软件设计靶向于KNDC1外显子1、2、3的5个sgRNAs,并将其构建到CRISPR/Cas9 载体中;经测序确认序列正确后,将重组质粒转染HeLa细胞和HUVECs,48 h后用real-time PCR和Western blot分别检测KNDC1 mRNA和蛋白表达水平;用SA-β-gal染色检测细胞衰老情况;用2, 7-二氯二氢荧光素二乙酸酯(DCFH-DA)荧光探针检测细胞内活性氧水平。结果测序结果显示,设计的5个sgRNAs全部插入到了CRISPR/Cas9载体中,序列正确,构建成功。将5个重组质粒转染到HeLa细胞后发现第4和5号载体敲除效率较高,接下来将4和5号重组质粒转染到HUVECs中,发现与转染对照质粒相比,转染了4和5号重组质粒的HUVECs KNDC1 mRNA及蛋白表达水平显著降低(P＜0.05),衰老HUVECs数量和细胞内活性氧水平明显减少。结论利用CRISPR/Cas9技术能有效敲除HUVECs的KNDC1,敲除KNDC1的HUVECs具有抗衰老能力。

关键词: KNDC1, CRISPR/Cas9系统, 基因编辑, 脐静脉内皮细胞

Abstract: Objective To knock out the KNDC1 of human umbilical vein endothelial cells (HUVECs) by CRISPR/Cas9 gene editing technology and observe its anti-aging ability. Methods Five sgRNAs targeting KNDC1 Exon1, Exon2 and Exon3 were designed by online software and were constructed into CRISPR/Cas9 vector. After sequence confirmation, the recombinant plasmids were transfected into HeLa cells and HUVECs. After 48 h, KNDC1 mRNA levels and protein expression levels were detected by real-time PCR and Western blot; cell senescence was measured by SA-β-gal staining; intracellular reactive oxygen species were detected by 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe. Results The sequencing results showed that all the 5 sgRNAs were inserted into the CRISPR/Cas9 vector, the sequence was correct and these vectors were successfully constructed. After transfecting 5 recombinant vectors into HeLa cells, it was found that the knockout efficiency of vectors No. 4 and No. 5 was higher, and then the recombinant vectors of No. 4 and 5 were transfected into HUVECs. Compared with the control plasmid, the KNDC1 mRNA level and protein expression level in HUVECs transfected with recombinant plasmids 4 and 5 were significantly decreased (P＜0.05). The number of senescent HUVECs and the level of intracellular reactive oxygen species were significantly reduced. Conclusions The KNDC1 of HUVECs can be effectively knocked out by CRISPR/Cas9 technology, and HUVECs with KNDC1 knockout have anti-aging ability.

Key words: KNDC1, CRISPR/Cas9 system, gene editing, human umbilical vein endothelial cell

中图分类号:

Q782

孙靖玉, 姚赫, 胡刚, 魏洁, 郭君, 张鑫, 林雅军. CRISPR/Cas9介导下敲除KNDC1的人脐静脉内皮细胞具有抗衰老能力[J]. 基础医学与临床, 2020, 40(9): 1175-1181.

SUN Jing-yu, YAO He, HU Gang, WEI Jie, GUO Jun, ZHANG Xin, LIN Ya-jun. HUVECs with KNDC1 knockout mediated by CRISPR/Cas9 have anti-aging ability[J]. Basic & Clinical Medicine, 2020, 40(9): 1175-1181.

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