腺病毒和慢病毒载体感染骨髓间质干细胞的比较

Transduction of recombinant marrow stem cells using lentiviral vector and adenoviral vector:a comparative study

目的比较腺病毒和慢病毒载体感染骨髓间质干细胞(rMSCs)的荧光病毒基因表达的稳定性及持续时间。方法将含有绿色荧光蛋白(GFP)基因的慢病毒载体质粒同辅助质粒一起共感染293T细胞,重组包装;将慢病毒和腺病毒载体分别感染rMSCs,培养5周,分别在1、3、5周通过流式细胞仪检测GFP的表达情况。从GenBank中调出人骨形态发生蛋白2(hBMP2)基因序列,设计带有NheI、AgeI双酶切位点引物。从人肝脏组织中抽提总RNA,再逆转录成cDNA,扩增出hBMP2基因的全长序列。测序验证后克隆到慢病毒载体pHIV-CS-CDF-CG-PRE上,通过PCR扩增、酶切、测序等方法鉴定重组慢病毒载体。将该重组慢病毒载体质粒同辅助质粒一起共感染293T细胞,通过重组而包装成能表达hBMP2基因的有活性的慢病毒。结果两种病毒载体感染后1周,绿色荧光蛋白表达分别是90%和71%,到5周时,慢病毒载体的绿色荧光蛋白表达明显高于腺病毒载体,分别是79%和0.05%。扩增出1.2kb左右的hBMP2全长基因与GeneBank中的hBMP2阅读框架序列完全一致;成功克隆和构建了含hBMP2基因的慢病毒载体pHIV-hBMP2;对慢病毒载体进行了成功包装和分析。结论含hBMP2基因的慢病毒载体可成功构建。慢病毒载体对大鼠骨髓间质干细胞的转染效率明显优于腺病毒载体。

Objective To compare the efficiency of transfection of marrow stem cells （MSCs） by using lentiviral vector and adenoviral vector. Methods MSCs were obtained from the femur bone marrow of rat and cultured. Recombinant plasmid pHIV-CS-CG-PRE containing green fluorescent protein （GFP） gene, and package plasmids pRSV-Rev, pMDLg/pPRE, and pMD. G were infected into the human embryonic kidney cells of the line 293T. The rat MSCs were transfected with the DFP recombinant lentiviral particles, and flow cytometry was used to detect the expression of GFP 1, 3, and 5 weeks later. Full-length human bone morphogenetic protein =2 （hBMP2） gene was searched out from the GenBank and isolated from human liver cDNA from a sample of liver tissue resected from a patient with liver rupture and then cloned. Thus the lentivirul vector with BMP-2 gene, pHIV-CS-CDF-CG-PRE-BMP-2 was constructed and transfected into 293 cells. Indirect immunofluorescence assay, ELISA, and Western blotting were used to detect the expression of hBMP2 gene. Results After infected by the viral vectors the GFP expression was significantly better in the lentiviral vector-infected rMSCs than in the adenoviral vector-infected ones. FCM showed that the GFP expression rates of the lentiviral vector-infected rMSCs 1, 3, and 5 weeks after the infection were 90%, 83. 2%, and 79% respectively, and he GFP expression rates of the adenoviral vector-infected rMSCs 1, 3, and 5 weeks after the infection were 71%, 0. 13%, and 0. 05% respectively, all significantly lower than on the former group. Indirect immunofluorescence assay, ELISA, and Western blotting showed that recombinant lentivirus successfully expressed the target protein in the transfected 293T cells. Conclusion Lentiviral vector with hBMP2 gene can be constructed successfully. The transfection efficiency of BMP2 gene by lentivirus is significantly higher than that of adonovirus.