RUNX1c及GATA2表达质粒构建及其在肝细胞中表达的鉴定

Construction of recombinant plasmids containing RUNX1c or GATA2and identification of expressions of these genes in hepatocytes

目的构建含RUNX1c及GATA2基因的表达质粒,探讨水动力转染法能否实现其在小鼠肝中的表达。方法采用PCR方法分别扩增RUNX1c及GATA2基因序列,并分别连入T载体中,通过双酶切、连接反应将2个基因序列分别连入慢病毒载体pCDH-MCS-T2A-copGFP-MSCV中。将含RUNX1c及GATA2基因的慢病毒毒液转染至L-02细胞,通过qPCR方法检测目的基因表达。通过水动力高压转染方法将此两种质粒通过尾静脉注入小鼠体内,于转染24 h,取小鼠肝组织,对组织切片进行抗RUNX1及GATA2抗体的免疫荧光染色;于转染72 h,通过流式细胞术分选肝组织中GFP+细胞,实时定量PCR(qPCR)检测RUNX1c、GATA2及造血相关基因的表达。结果经双酶切及测序鉴定证明,pCDH-MCS-T2A-RUNX1c-copGFP-MSCV、pCDH-MCS-T2A-GATA2-copGFP-MSCV质粒构建成功。通过体外实验证实这两个载体携带的RUNX1c及GATA2基因能在人肝细胞系L-02中表达。水动力高压转染法可使含RUNX1c及GATA2的质粒进入肝组织,并在肝细胞中表达RUNX1及GATA2蛋白。通过分选肝组织中GFP+细胞,可发现这些细胞内过表达RUNX1及GATA2基因,Fli-1、Erg基因的表达也明显提高。结论成功构建了含有造血转录因子RUNX1c、GATA2基因的表达载体,并通过水动力转染法实现RUNX1c、GATA2的表达载体在小鼠肝中的表达,为进一步研究肝中过表达造血转录因子能否实现肝细胞转分化为造血细胞及观察其对造血系统修复的作用奠定基础。

Objective To construct plasmids containing RUNX1 c or GATA2 gene and investigate whether the hydrodynamics-based transfection method can help these plasmids into hepatocytes in vivo.Methods The full-length coding sequences of RUNX1 c and GATA2 were cloned by using PCR and ligated into pCDH-MCS-T2 A-copGFP-MSCV.The recombinant plasmids were packaged into lentiviruses and transduced into L-02 cells.Quantitative real-time PCR(qPCR)was used to detect the expressions of these genes in cells.The hydrodynamics-based transfection method was used to introduce the plasmids into the liver of mice.Immunostaining experiments for detection of the expressions of RUNX1 and GATA2 proteins were performed on the liver tissue sections from the mice after hydrodynamics-based transfection with the plasmids.GFP+cells from the liver tissue were sorted and analyzed for the expressions of these two genes and other hematopoietic genes.Results We constructed two plasmids pCDH-MCS-T2 A-RUNX1 c-copGFP-MSCV and pCDH-MCS-T2 A-GATA2-copGFP-MSCV.The expressions of RUNX1 and GATA2 genes could be detected in L-02 cells after infection with the lentiviruses with RUNX1 c and GATA2 gene.The expressions of RUNX1 and GATA2 proteins could be detected in hepatocytes from mice after hydrodynamics-based transfection with these plasmids.qPCR results showed that RUNX1 and GATA2 genes were highly expressed in GFP+cells sorted from the liver tissue of the mice after hydrodynamics-based transfection with these plasmids.It was also found that Fli1 and Erg genes were up-regulated in these GFP+cells from the liver.Conclusion Plasmids with RUNX1 c or GATA2 genes have been constructed,which could be transduced into the liver of the mice using hydrodynamics-based transfection method.The expressions of RUNX1 and GATA2 proteins can be detected in the hepatocytes of the mice after hydrodynamics-based transfection with the plasmids.This research can help us to find out more about the role of hematopoietic transcriptional factors in cell fate conversion of hepatocytes into hematopoietic cells in vivo and their effect on hematopoietic repair.