RNA干扰神经干细胞中NgR基因的表达修复大鼠面神经缺损的体外实验研究

施勇; 周梁; 田洁; 汪洋

doi:10.13201/j.issn.1001-1781.2014.10.017

RNA干扰神经干细胞中NgR基因的表达修复大鼠面神经缺损的体外实验研究

- 复旦大学附属眼耳鼻喉科医院耳鼻咽喉科(上海, 200031)
基金项目:
上海市卫生局青年基金项目（No：2009Y013）

详细信息

通讯作者: 周梁,E-mail:zhoulent@126.com

中图分类号: R338

收稿日期: 2013-12-09

Silenced NgR gene expression by RNA interference to promote rats facial nerve regeneration in vitro

- Department of Otolaryngology, Eye and ENT Hospital, Fudan University, Shanghai, 200031, China

More Information

Corresponding author: ZHOU Liang, E-mail: zhoulent@126.com

Received Date: 09 December 2013

摘要

摘要: 目的：应用RNA干扰技术抑制神经干细胞中NgR基因表达，观察神经干细胞的体外分化，为体内面神经修复提供营养支持。方法：采用PCR扩增，限制性内切酶酶切，T4DNA连接酶连接，将NgR与pGCsi载体连接，构建重组载体NgR shRNA，通过测序鉴定。NgR shRNA通过Lipofectamine 2000感染神经干细胞，荧光显微镜下观察EGFP的表达，Western Blot检测NgR的表达，免疫细胞化学鉴定转染前后神经干细胞向神经元分化的比例，转染后的神经干细胞种植于PLGA管，扫描电镜观察。结果：成功构建NgR shRNA质粒，成功感染神经干细胞，Western Blot结果表明干扰后的神经干细胞中的NgR表达减弱、免疫细胞化学结果示干扰后的神经干细胞向神经元分化比例明显高于对照组（P<0.01），差异有统计学意义。结论：成功构建NgR shRNA质粒，感染神经干细胞，使神经干细胞向神经元进一步分化，能更有效促进神经轴突的再生，为面神经损伤修复提供高效稳定的基因平台。
- 神经再生 /
- 神经干细胞 /
- RNA干扰 /
- Nogo受体 /
- 短发夹RNA
Abstract: Objective: To suppress NgR gene expression in neural stem cells and observe differentiation of neural stem cells in vitro after interfered which provide nutritional support for the facial nerve repair in vivo. Method: PCR amplification, restriction endonuclease digestion, T4DNA ligase connections were used to connected NgR with rector pGCsi, and constructed recombinant vector(NgR shRNA).Lipofectamine 2000 were used to transfect the NSC. The expression of NgR was examined by Western Blot. The proportion of neural stem cells transformed into neurons after transfection was tested by Immunocytochemistry. Neural stem cells were planted in PLGA tubes after transfected, and were scanned by electron microscopy. Result: NgR shRNA plasmid was constructed and infected neural stem cells successfully.Western Blot showed that the expression of NgR decreased in neural stem cells after interference. Immunocytochemistry showed that the rate of the neural stem cells transformed into neurons after interfered was significantly higher(P<0.01). Conclusion: Neural stem cells were transformed into neurons after NgR shRNA plasmid infected neural stem cells, which promoted axonal regeneration more effectively and provided a efficient and stable gene platform for facial nerve repair.
- nerve regeneration /
- neural stem cells /
- RNA interference /
- NgR /
- shRNA

HTML全文

参考文献(10)

[1]	施勇,周梁,田洁,等.神经干细胞移植到神经导管修复大鼠面神经缺损的研究[J].临床耳鼻咽喉头颈外科杂志,2012,26(22):1040-1042.
[2]	GRANDPRE T, NAKAMURA F, VARTANIAN T, et al. Identification of the Nogo inhibitor of axon regeneration as a Reticulon protein[J].Nature,2000,403:43-44.
[3]	王朝鲁,孙红辉,杨有庚.Nogo受体基因干扰RNA表达载体的构建[J].中国实验诊断学,2006,10(11):1253-1254.
[4]	WANG Y T, LU X M, ZHU F, et al. The use of a gold nanoparticle based adjuvant to improve the therapeutic efficacy of hNgR-Fc protein immunization in spinal cord-injured rats[J].Biomaterials,2011,32:7988-7998.
[5]	SARAH C, BORRIE BASTIAN E, BAEUMER CH-RISTINE E, et al. The Nogo-66 receptor family in the intact and diseased CNS[J].Cell Tissue Res,2012,349:105-117.
[6]	GITLIN L, KARELSKY S. Short interfering RNA confers intracellular antiviral immunity in human cells[J].Nature,2002,418:430-434.
[7]	MCDONALD C L, BANDTLOW C, REIND M. Targeting the nogo receptor complex in diseases of the central nervous system[J].Current Medicinal Chemistry, 2011,18:234-244.
[8]	LI W, WALUS L, RABACCHI SA, et al. A neutralizing anti-Nogo66 receptor monoclonal antibody reverses inhibition of neurite outgrowth by central nervous system myelin[J].J Biol Chem,2004,279:43780-43788.
[9]	KIM J E, LIU B P, PARK J H, et al. Nogo-66 receptor prevents raphe spinal and rubro spinal axon regeneration and limits functional recovery from spinal cord injury[J].Neuron, 2004, 44:439-451.
[10]	KOICHI T, TATEKI K, KEN M, et al. Myelin-associated glycoprotein reduces axonal branching and enhances functional recovery after sciatic nerve transection in rats[J].Glia, 2007, 55:1498-507.