摘要
研究构建靶向抑制人Mbd3表达的sh RNA慢病毒颗粒,感染人脐带间充质干细胞并用嘌呤霉素筛选获得Mbd3稳定下调的细胞系,为进一步探讨Mbd3在诱导型多潜能干细胞形成中的作用提供实验基础。研究根据Gene Bank中公布的Mbd3基因序列设计特异性si RNA干扰序列并合成可产生sh RNA的双链DNA,经双酶切后克隆至p LVsh RNA-EGFP(2A)Puro载体上构建慢病毒重组质粒,转化DH5α大肠杆菌PCR检测筛选阳性质粒,利用HEK293Ta包装产生含有Mbd3sh RNA的慢病毒颗粒。通过RT-PCR和Western blot检测慢病毒感染人脐带间充质干细胞(h UC-MSC)后Mbd3的转录和蛋白表达情况,进一步检测重组慢病毒对Mbd3的沉默效果并筛选出最佳抑制效果的sh RNA慢病毒载体。结果显示成功构建并筛选出下调Mbd3的最佳慢病毒干扰载体,慢病毒感染人脐带间充质干细胞48h后于荧光显微镜下可见绿色荧光,经嘌呤霉素筛选9 d后,RT-PCR检测Mbd3表达显著降低,Western blot检测Mbd3蛋白表达明显减少。说明构建的sh RNA重组慢病毒包装成功,具有较高的感染活性,可有效抑制人脐带间充质干细胞Mbd3的表达,为后续研究脐带间充质干细胞跨胚层分化打下前期研究基础。
In this study, we constructed a shRNA lentivirus particle, it is able to targetedly suppress Mbd3 expression and infect human umbilical cord mesenchymal stem cells （hUC-MSCs） lines screened by puromycin in which Mbd3 is down regulated stably. Specific siRNA was designed and dsDNA that could generate shRNA was synthesized based on the gene sequence of Mbd3. After double digestion, pLVshRNA-EGFP（2A） Puro vector was used to construct recombination lentivirus plasmid and transformed into E. coli DH5αsubsequently. The positive plasmid was screened＆amp;nbsp;by PCR. Packaged lentivirus particle contained Mbd3 shRNA by HEK293Ta. Then hUC-MSCs were infected with this lentivirus and the effect of recombination lentivirus on the Mbd3 gene silence was detected through analysis of mRNA transcription level and protein level. The results showed that the lentivirus vector which could interfere inMbd3 gene expression efficaciously was constructed and screened. Green fluorescence was observed through fluorescence microscope 48 h after hUC-MSCs were infected with this lentivirus and the expressions of Mbd3 gene and Mbd3 protein were significantly decreased through the detection by RT-PCR and Western blot 9 days after screening by puromycin.
出处
《生物学杂志》
CAS
CSCD
2015年第3期15-20,共6页
Journal of Biology
基金
云南省重点新产品开发计划(2012AE001)
协和青年基金和中央高校基本科研业务费专项资金(33320140082)
