CRISPR/Cas9介导下敲除KNDC1的人脐静脉内皮细胞具有抗衰老能力  

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

作　　者：孙靖玉 姚赫 胡刚[1] 魏洁[1] 郭君 张鑫 林雅军[1] 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)

机构地区：[1]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

出　　处：《基础医学与临床》2020年第9期1175-1181,共7页Basic and Clinical Medicine

基　　金：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)。

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。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 HUVECstransfected 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.

关 键 词：KNDC1 CRISPR/Cas9系统 基因编辑 脐静脉内皮细胞 

分 类 号：Q782[生物学—分子生物学]