| Nogo受体慢病毒干扰载体在大鼠神经干细胞中的表达及其干扰效率鉴定 |
| 投稿时间：2015-04-02 修订日期：2015-04-15 点此下载全文 |
| 引用本文：刘欣春,朱悦.Nogo受体慢病毒干扰载体在大鼠神经干细胞中的表达及其干扰效率鉴定[J].医学研究杂志,2015,44(11):60-63 |
| DOI：
10.11969/j.issn.1673-548X.2015.11.017 |
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| 基金项目:辽宁省自然科学基金资助项目(2014021062) |
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| 中文摘要:目的 在体外分离培养的大鼠神经干细胞内,通过慢病毒介导的shRNA实现持续稳定的NgR沉默。方法 设计并合成表达NgR shRNA的慢病毒载体,通过转染293T细胞系收集包装好的病毒颗粒。分离培养初生大鼠脊髓神经干细胞,以不同MOI值感染慢病毒,流式细胞术确定感染所需的最佳MOI值。感染慢病毒的神经干细胞经潮霉素筛选后进行单克隆培养,Western blot法检测NgR的沉默效率。取感染后不同天数的神经干细胞,实时定量PCR检测NgR沉默的稳定性。结果 神经干细胞分离培养6天后可见神经球形成;流式细胞术测定慢病毒感染的最佳MOI=10;慢病毒感染后NgR的沉默效率可达80%,并可在7~28天内维持在70%~80%。结论 慢病毒介导的shRNA可以在体外分离培养的大鼠神经干细胞内实现较为稳定的NgR基因沉默,为开展神经干细胞移植治疗脊髓损伤奠定了基础。 |
| 中文关键词:Nogo受体 神经干细胞 慢病毒 |
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| Expression and Interference Efficiency Evaluation of Lentiviral shRNA Targeting at Nogo-receptor in Neural Stem Cell of Rat. |
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| Abstract:Objective To realize a stable knockdown of NgR in primary cultured rat neural stem cells by lentivirus mediated shRNA. Methods Lentiviral vector expressing shRNA targeting at NgR was designed and sythesized. After transfection in 293T cells, the virus granules was packeged and collected. Rat spinal neural stem cell was isolated and cultured, followed by lentivirus transfection at different MOIs. The best MOI was determined by flow cytometry detecting the expression of GFP. After hygromycin screening and monoclonal culture, the knockdown effeciency on NgR was determined by Western blot. The stability of the knockdown effect on NgR was detected by quantitative real-time PCR by using NSC on different culture days after transfection. Results Neural spheres were observed under light microscope 6 days after isolation. The best MOI was 10 as detected by flow cytometry. The expression of NgR was knocked down by 80% by the lentiviral shRNA, which is stable and can be maitained at 70%-80% in 7-28 days after infection. Conclusion The shRNA mediated by lentivirus can stably interfere the expression of NgR in primary cultured rat neural stem cells and is hopefully to be used in future study on NSC transplantation after spinal cord injury. |
| keywords:NgR Neural stem cell Lentivirus |
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