损伤抑制基因Dsup对人胚胎干细胞生物学性质的影响

Biological effects of damage suppressor gene(Dsup)expression on human embryonic stem cells

目的通过CRISPR/Cas9技术将水熊虫Dsup基因定点敲入胚胎干细胞(hESC-H9)的AAVS1位点,构建Dsup基因过表达的稳定细胞株,研究Dsup表达对hESC-H9生物学性质的影响。方法构建CRISPR/Cas9 AAVS1位点敲入系统,采用PCR技术扩增Dsup序列,并将其插入pAAVS1-Donor-GFP-Puro,将构建的pAAVS1-Dsup-GFP-Puro与pAAVS1-CRISPR-Cas9载体共同转染hESC-H9,通过CRISPR/Cas9技术介导的同源重组使Dsup基因插入hESC-H9的AAVS1位点,经嘌呤霉素筛选后利用流式分选技术分选GFP、TRA-1-60、SSEA-4等3种荧光标记阳性的细胞群,获得稳定敲入Dsup基因并保持干性的细胞。将获得的hESC-H9-Dsup传至第9代,对Dsup mRNA表达水平、GFP、细胞表面干性标志物(TRA-1-60、SSEA-4)以及干性基因(OCT4、SOX2、NANOG)进行检测,并研究hESC-H9-Dsup的增殖、凋亡以及辐射耐受情况,以探索Dsup基因对hESC-H9生物学活性的影响。结果成功构建pAAVS1-Dsup-GFPPuro重组供体质粒,与pAAVS1-CRISPR-Cas9质粒共同转染hESC-H9后通过药物筛选获得了在AAVS1位点敲入的hESC-H9-Dsup。经实时定量PCR检测,Dsup mRNA在hESC-H9-Dsup中成功表达,且其表达对hESC-H9表面干性标志物TRA-1-60、SSEA-4以及干性基因OCT4、SOX2、NANOG表达未见明显影响。hESC-H9-Dsup增殖速度稍高于hES-H9-Control/WT。hESC-H9-Dsup与hESC-H9-Control/WT凋亡无显著差异(P>0.05),但hESC-H9-Dsup的辐射耐受能力显著高于hESC-H9-Control(P<0.001)。结论成功构建了hESC-H9-Dsup,Dsup基因表达未对其生物学性质产生明显影响,且表现出明显的辐射耐受能力,为后续研究Dsup基因对人类细胞的影响及探讨可能的应用价值奠定了基础。

Objective To investigate the effects of damage suppressor gene(Dsup)expression on the biological characteristics of human embryonic stem cells(hESC-H9)by using CRISPR/Cas9 technique to construct stable cell lines with Dsup overexpression.Methods The CRISPR/Cas9 AAVS1 insertion system was constructed.The Dsup sequence was amplified by PCR and inserted into pAAVS1-Donor-GFP-Puro.The constructed pAAVS1-Dsup-GFP-puromycin(Puro)vector and pAAVS1-CRISPR-Cas9 vector were co-transfected into the hESC-H9,and Dsup was inserted into the hESC-H9 AAVS1 site through CRISPR/Cas9-mediated homologous recombination.Puromycin was used for screening while flow cytometry was used to sort GFP,TRA-1-60 and SSEA-4 positive cells.The stable hESC-H9-damage suppressor(Dsup)was obtained and passaged to the ninth generation before Dsup mRNA expression,GFP,cell surface markers(TRA-1-60,SSEA-4)and genes expression(OCT4,SOX2,NANOG)were detected.Furthermore,the proliferation,apoptosis and radiation tolerance of the hESC-H9-Dsup were detected to explore the effects of Dsup onhESC-H9.Results There combinant plasmid pAAVS1-Dsup-GFP-Puro was successfully constructed.After co-transfection of hESC-H9 with pAAVS1-CRISPRCas9 vector,the hESC-H9-Dsup was obtained by drug screening.The Dsup expression did not affect the expressions of TRA-1-60,SSEA-4 or OCT4,SOX2 or NANOG.The proliferation rate of hESC-H9-Dsup was slightly higher than that of hESC-H9-Control/WT.There was no significant difference in apoptosis between hESC-H9-Dsup and hESC-H9-Control/WT.The radiation tolerance ability of hESC-H9-Dsup was markedly higher than that of hESC-H9-Control.Conclusion The hESC-H9-Dsup is successfully constructed,and the Dsup expression has no significant effects on its basic biological characteristics and manifests good radiation tolerance ability,which can contribute to subsequent studies on the influence and the possible application of Dsup to human cells.