脑源性神经生长因子过表达慢病毒载体构建及转染脂肪干细胞的研究

Construction and identification of recombinant lentiviral vector expressing rat brain derived neurotrophic factor gene and Infection of adipose-derived stem cells

目的构建携带大鼠脑源性神经生长因子(BDNF)基因的重组慢病毒表达载体,并转染脂肪干细胞(ADSCs)来获得治疗的种子细胞。方法从已构建好的含BDNF的质粒克隆模板pEGFP-C-BDNF中,利用聚合酶链反应(PCR)方法提取目的基因BDNF,将该基因克隆到慢病毒载体表达质粒GV287(含Flag基因)中,得到重组的UBI-BDNF,通过PCR、酶切、测序和分析比对验证BDNF基因后,将UBI-BDNF质粒和包装质粒pHelper1.0、pHelper2.0共同转染人胚胎肾上皮细胞株293T细胞,经同源重组产生重组慢病毒UBI-BDNF。UBI-BDNF在293T细胞内大量扩增,应用实时定量聚合酶链反应(Real-time PCR)法鉴定和测定滴度,然后转染ADSCs,并通过免疫荧光法及酶联免疫吸附试验(ELISA)和蛋白质印迹法(Western blot)检测。结果克隆得到791 bp的目的BDNF全长基因,经过PCR扩增、酶切鉴定、序列测定证实,BDNF基因被成功克隆到慢病毒载体中,可实现BDNF基因的表达,且病毒滴度为2.0×109 TU/ml。免疫荧光检测BDNF在转染ADSCs中表达。ELISA检测转染ADSCs上清液中BDNF浓度为891.38~1 196.26 ng/L[(945.33±38.54)ng/L],而绿色荧光蛋白(GFP)转染ADSCs中未检测到。Western blot证实转染ADSCs表达BDNF蛋白。结论成功构建表达大鼠BDNF基因的慢病毒载体并能在ADSCs高表达。

Objective To construct a lentiviral brain derived neurotrophic factor(BDNF)expression vector and then infect the adipose derived mesenchymal stem cells(ADSCs)to produce therapeutic seed cells.Methods The BDNF gene was isolated and amplified by polymerase chain reaction(PCR)technique from pEGFP-C-BDNF plasmid which was constructed before.Then the gene was subcloned into the expression plasmid of lentiviral vector,GV287(containing Flag gene),to generate the lentiviral expression vector,UBI-BDNF.The correct BDNF gene was confirmed by PCR,endoenzyme digestion,sequencing,analysis and comparison.Recombinant lentivirnses containing BDNF gene and nag gene were produced by 293T cells following the co-transfection of UBI-BDNF and packaging plasmids-pHelper1.0 and pHelper2.0.The newly constructed recombinant lentivirus and the titer of virus were confirmed by real-time quantitative polymerase chain reaction(Real-time PCR),and then the ADSCs were transfected with the vectors after titer determination.Stable expression of BDNF in ADSCs was confirmed by immunofluorescence staining,enzyme linked immunosorbent assay(ELISA)and Western blotting.Results The evidence of endonuclease digestion,DNA sequencing and PCR analysis confirmed that BDNF gene was correctly inserted into the lentiviral vector,and the titer of virus was 2.0×109 TU/ml.BDNF could be expressed in the transfected cells confirmed by immunofluorescence staining.By ELISA,the density of BDNF was 891.38-1 196.26 ng/L[(945.33±38.54)ng/L]in the supernatant of BDNF-transfected cells but not detected in the GFP-transfected cells(P<0.05).By Western blotting also confirmed the BDNF expression in BDNF-transfected cells.Conclusion The recombinant lentivirus expressing BDNF was successfully constructed and over-expressed in ADSCs.