Adenovirus-mediated short hairpin RNA interference against p75 neurotrophin receptor in pheochromocytoma cells

作者：Feng Dongxu; Li Haopeng^*; Xu Siyue; Liu Yu; Hou Xiaofei

来源：Neural Regeneration Research, 2011, 6(7): 517-522.

DOI：10.3969/j.issn.1673-5374.2011.07.006

• p75 neurotrophin receptor
• RNA interference
• adenovirus
• rat pheochromocytoma cells
• human embryonic kidney 293 cells
• apoptosis
• cauda equina syndrome

Previous studies have confirmed that motor neuron apoptosis in the anterior horn of the lumbosacral spinal cord is positively correlated with p75 neurotrophin receptor (p75NTR) expression in rat models of cauda equina syndrome. This study used adenovirus to carry a short hairpin RNA (shRNA) for p75NTR gene silencing, to reduce p75NTR expression in the damaged phase and to decrease motor neuron apoptosis. Three p75 siRNA template oligonucleotide segments (shRNA) were designed, and cloned into the 1.0 CMV shuttle vector. HEK293 cells were cotransfected with shuttle vector (carrying shRNA) and an adenovirus vector framework expressing enhanced green fluorescent protein. Thus, this study successfully obtained adenovirus carrying p75shRNA. The obtained viruses were named Ad.shRNA1, Ad.shRNA2, and Ad.shRNA3. The recombinant adenoviruses were separately used to infect cultured pheochromocytoma cells (PC12). Forty-eight hours later, p75NTR mRNA and total protein were analyzed from the PC12 cells. Compared with the negative controls, RNA interference rates were separately 98.49 +/- 0.68%, 95.08 +/- 1.79% and 96.60 +/- 1.14% at the mRNA level, and 72.89 +/- 2.17%, 58.83 +/- 1.15% and 59.88 +/- 0.44% at the protein level in the Ad.shRNA1, Ad.shRNA2, and Ad.shRNA3 groups, respectively. Thus, recombinant adenovirus shRNA-mediated gene silencing successfully suppressed p75NTR expression.

• 出版日期2011-3-5
• 单位西安交通大学; 内蒙古科技大学包头医学院