Neurturin基因在体外培养的大鼠骨髓基质细胞中的表达

Expression of neurturin gene in the bone marrow stromal cells of rats in vitro

目的构建携带一种神经营养因子Neurturin(NTN)基因的腺病毒(Ad),使其在骨髓基质细胞(BMSCs)中表达。方法采用分子克隆技术,构建携带NTN基因的Ad载体,用脂质体法转染、包装HEK293细胞,制备具有感染活性的Ad-NTN病毒粒子;用病毒上清液感染原代培养的大鼠BMSCs,并用免疫细胞化学方法检测阳性细胞。用W estern B lot检测转染Ad-NTN的BMSCs细胞上清液。结果经酶切鉴定和DNA测序,得到重组Ad-NTN,对被感染的HEK293细胞进行病毒滴度测定,每毫升病毒贮存液可达1×108.5半数组织培养感染量(TC ID50);将病毒贮存液感染BMSCs,获得了表达NTN的BMSCs,阳性率为65%。W estern B lot检测证实BMSCs上清液中出现特异性NTN条带。结论重组Ad携带的NTN基因能够在BMSCs中表达。

Objective To obtain the gene engineering modified bone marrow stromal cells （BMSCs） expressing the Neurturin （ NTN ） gene. Methods An adenoviral vector encoding NTN was constructed with the technique of molecular cloning. High titer viral particles of adenovirus vector with NTN were obtained after transfection HEK 293 cells by liposome mediated transfection. Primary BMSCs were infected with viral stock and identified by immunocytochemistry, NTN in supernatant was tested by Western Bolt. Results Recombined adenoviral vector with NTN was verified by restriction endonuclease digestion and DNA sequencing. The titer of recombinant adenoviral stock could be reached up to 1 ×10^8.5 TCID50/ml viral stock. Primary cultured BMSCs from rat infected with adenoviral stock were proved to be the gene engineering modified BMSCs expressing the gene of NTN. The percentage of positive cells was approximately 65% by the method of immunocytochemistry. Western Blot showed that the supernatant of BMSCs transfected with Ad-NTN expressed a specific band combined with NTN antibody. Conclusion The gene engineering modified BMSCs can express the gene of NTN recombinant with adenoviral vector.