利用CRISPR/Cas9技术构建永生化脐带间充质干细胞系及MYC-AS1转基因研究

Construction of immortalized umbilical cord mesenchymal stem cell line using CRISPR/Cas9 system and MYC-AS1 transgenic research

人脐带间充质干细胞(hMSCs)是一种具有多向分化潜能的多能干细胞,在组织修复和某些疾病治疗的临床应用上发挥着重要作用。利用CRISPR/Cas9技术在腺相关病毒AAVS1位点插入具有磷酸甘油酸激酶PGK启动子和SV40-polyA的SV40 LT基因,使hMSCs达到永生化。并利用慢病毒构建具有抑癌作用的反义长链非编码RNA MYC-AS1转基因的hMSCs,从该细胞系的培养液中分离提取表达MYC-AS1的外泌体。通过处理肝癌HepG2细胞,研究外泌体对HepG2表型的影响。结果表明:与野生型MSCs相比,永生化细胞MSCs-SV40增殖显著加快,但其定向分化特性并未改变,可在体外稳定传代超过40代,且未发现成瘤性。MYC-AS1转基因hMSCs的外泌体能表达MYC-AS1,且显著抑制HepG2肿瘤细胞的增殖和迁移。这一研究通过CRISPR/Cas9技术敲入SV40 LT基因成功构建了hMSCs,其MYC-AS1转基因细胞的外泌体对HepG2肿瘤细胞具有明显的抑制作用,这对hMSCs及其外泌体的基础研究和临床应用研究具有重要意义。

Human umbilical cord mesenchymal stem cells(hMSCs)are multipotent stem cells with multiple differentiation potential,which play an important role in tissue repair and clinical application of some diseases.It is of great significance to construct immortalized umbilical cord mesenchymal stem cell line for its function research,gene editing and clinical application.In this study,CRISPR/Cas9 system was used to insert the SV40 LT gene with phosphoglycerate kinase PGK promoter and SV40-polyA into the adeno-associated virus site AAVS1 to immortalize hMSCs.Then,we used lentivirus to construct an antisense long non-coding RNA MYC-AS1 transgenic mesenchymal stem cell line,and the exosomes expressing MYC-AS1 were isolated from the culture medium of the cell line.Objective was to study the effect of the transgenic exosomes on the phenotype of HepG2 cells.The results showed that the proliferation of immortalized MSCs-SV40 LT was significantly faster than that of wild-type MSCs,but its directional differentiation characteristics did not change.The immortalized MSCs-SV40 LT can be stably passaged for more than 40 generations in vitro,and no tumorigenicity was found.The exosomes of MYC-AS1 transgenic mesenchymal stem cells can express MYC-AS1 and significantly inhibit the proliferation and migration of HepG2 tumor cells.In conclusion,hMSC cell lines were successfully constructed by using CRISPR/Cas9 system to knock in the SV40 LTgene.The exosomes of MYC-AS1 transgenic cells had obvious inhibitory effect on HepG2 tumor cells.The results of this study are of great significance for the basic research and clinical application of hMSCs and its exosomes.