中国神经再生研究(英文版) ›› 2014, Vol. 9 ›› Issue (5): 504-512.doi: 10.4103/1673-5374.130074

• 原著:脑损伤修复保护与再生 • 上一篇    下一篇

重组腺病毒介导24-脱氢胆固醇还原酶神经元的过表达

  

  • 收稿日期:2014-01-10 出版日期:2014-03-12 发布日期:2014-03-12
  • 基金资助:

    国家自然科学基金面上项目(No. 31271494)和辽宁省优秀人才支持计划(No. LJQ2011004)

Recombinant adenovirus-mediated overexpression of 3β-hydroxysteroid-Δ24 reductase

Xiuli Lu 1, Dan Jia 1, Chenguang Zhao 1, Weiqi Wang 1, Ting Liu 1, Shuchao Chen 1, Xiaoping Quan 1, Deliang Sun 1, Bing Gao 2   

  1. 1 Department of Biochemistry and Cell Biology, School of Life Science, Liaoning University, Shenyang, Liaoning Province, China
    2 Department of Cell Biology and Genetics, School of Basic Medical Sciences, Shenyang Medical College, Shenyang, Liaoning Province, China
  • Received:2014-01-10 Online:2014-03-12 Published:2014-03-12
  • Contact: Bing Gao, M.D., Ph.D., Department of Cell Biology and Genetics, School of Basic Medical Sciences, Shenyang Medical College, Shenyang 110034, Liaoning Province, China, gaobing_cn@yahoo.com.cn. Xiuli Lu, Ph.D., Department of Biochemistry and Cell Biology, School of Life Science, Liaoning University, Shenyang 110036, Liaoning Province, China, luxiuli@yahoo.com.cn.
  • Supported by:

    This study was financially supported by the National Natural Science Foundation of China (General Program), No. 31271494; Excellent Talent Support Program of Liaoning Province, No. LJQ2011004.

摘要:

24-脱氢胆固醇还原酶是一种具有胆固醇合成作用和抗神经细胞凋亡作用的多功能蛋白,其过表达能够保护神经元免于Aβ沉积引起的细胞凋亡。实验以分别构建人源以及鼠源SYN1启动子驱动24-脱氢胆固醇还原酶神经元特异性表达的重组腺病毒,用Ad-r(h)SYN1-DHCR24分别感染293细胞、小鼠神经母细胞瘤细胞株N2A、以及小鼠胰腺癌细胞株MIN6细胞。Western bolt结果显示24-脱氢胆固醇还原酶在293细胞和N2A神经元中特异性表达,而MIN6组未出现明显特异性条带,证明构建的腺病毒成功实现了神经特异性表达靶蛋白24-脱氢胆固醇还原酶,而在非神经元中不表达。TUNEL结果说明此腺病毒转染的神经元凋亡减少。活性氧检测和免疫荧光结果说明此腺病毒转染的神经元是通过清除过量的活性氧实现抑制细胞凋亡的。结果提示构建的重组腺病毒24-脱氢胆固醇还原酶能够实现24-脱氢胆固醇还原酶的神经元内特异性表达,为进行以24-脱氢胆固醇还原酶为阿尔茨海默病基因治疗靶点的研究奠定了基础。

关键词: 神经再生, 神经退行性变, 24-脱氢胆固醇还原酶, 重组腺病毒, 神经元, 氧化应激, SYN1, 基因治疗, 神经保护, NSFC

Abstract:

3β-Hydroxysteroid-Δ24 reductase (DHCR24) is a multifunctional enzyme that localizes to the endoplasmic reticulum and has neuroprotective and cholesterol-synthesizing activities. DHCR24 overexpression confers neuroprotection against apoptosis caused by amyloid β deposition. The present study aimed to construct two recombinant adenoviruses driving DHCR24 expression specifically in neurons. Two SYN1 promoter DNA fragments were obtained from human (h) and rat (r). Recombinant Ad-r(h)SYN1-DHCR24 was transfected into AD-293, N2A (mouse neuroblastoma), and MIN6 (mouse pancreatic carcinoma) cells. Western blot analysis showed DHCR24 was specially expressed in 293 and N2A cells, but no specific band was found in MIN6 cells. This demonstrates that the recombinant adenoviruses successfully express DHCR24, and no expression is observed in non-neuronal cells. TUNEL assay results showed apoptosis was inhibited in adenovirus-transfected neurons. Detecting reactive oxygen species by immunofluorescence, we found that adenovirus transfection inhibits apoptosis through scavenging excess reactive oxygen species. Our findings show that the recombinant DHCR24 adenoviruses induce neuron-specific DHCR24 expression, and thereby lay the foundation for further studies on DHCR24 gene therapy for Alzheimer’s disease.

Key words: nerve degeneration, 3β-hydroxysteroid-Δ24 reductase, recombinant adenovirus, neuron, oxidative stress, synapsin-1, gene therapy, neuroprotection, NSFC grant, neural regeneration