| 梁丽荣,芦 睿,王晓霞,马瑞雪,齐 鑫,刘 冰,张永权,张 惠.HIPK2对七氟醚暴露后原代海马神经元凋亡的影响[J].现代生物医学进展英文版,2018,(15):2814-2818. |
| HIPK2对七氟醚暴露后原代海马神经元凋亡的影响 |
| Effect of HIPK2 on the Apoptosis of Primary Neuron after Sevoflurane Exposure |
| Received:March 19, 2018 Revised:April 12, 2018 |
| DOI：10.13241/j.cnki.pmb.2018.15.003 |
| 中文关键词: 全麻，七氟醚 HIPK2慢病毒载体 凋亡 |
| 英文关键词: General anesthesia Sevoflurane HIPK2 lentivirus vector Apoptosis |
| 基金项目:国家自然科学基金项目（81371265） |
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| 中文摘要: |
| 摘要 目的：构建shHipk2慢病毒表达载体，并探讨同源结构域相互作用蛋白激酶2(homeodomain-interacting protein 2，HIPK2)在七氟醚诱导的原代海马神经元凋亡中的作用。方法：设计并构建针对HIPK2靶基因的慢病毒，转染至原代海马神经元，通过免疫荧光检测转染效率，应用RT-PCR验证干扰效果。病毒转染3 d后，处理组经过4.1%的七氟醚暴露6 h后，进行相应的检测。采用CCK8比色法检测各组细胞活性，LDH检测细胞毒性，FITC/PI流式细胞仪及Western Blot检测细胞凋亡。结果：RT-PCR显示较Scr组与Con组相比，shHIPK2组HIPK2 mRNA水平明显下降(P<0.0001)，干扰效率为75.29 %，提示靶向沉默HIPK2的慢病毒shRNA表达载体构建成功。Scr+Sev组细胞活力较Scr组相比显著下降而细胞毒性明显增加(P<0.05)，shHIPK2+Sev组与shHIPK2组相比细胞活力与细胞毒性均无统计学差异(P>0.05)。流式凋亡细胞检测结果显示Scr+Sev组凋亡细胞数明显高于Scr组(P<0.05)，而shHIPK2+Sev组与ShHIPK2组相比无统计学差异(P>0.05)。Western Blot结果显示Scr+Sev组Cl-caspase 3蛋白水平较Scr组相比明显升高，shHIPK2+Sev组与shHIPK2组相比无统计学差异(P>0.05)。结论：HIPK2可促进七氟醚引起的原代海马神经元的凋亡。 |
| 英文摘要: |
| ABSTRACT Objective: To construct shHipk2 lentiviral expression vector and explore the role of HIPK2 (homologous domain inter- acting protein kinase 2) in the apoptosis of primary neurons after Sevoflurane exposure. Methods: The lentivirus against HIPK2 target gene was designed, constructed, and then infected to primary hippocampal neurons. The infection efficiency was detected by immunoflu- orescence, and the interference effect was verified by RT-PCR. Cell activity was detected by CCK8 colorimetric assay, cytotoxicity was detected by LDH, and apoptosis was detected by FITC/PI flow cytometry and Western blot. Results: RT-PCR showed that the level of HIPK2 mRNA in the shHIPK2group decreased by 75.29 %, which suggested that the lentivirus shRNA expression vector of target silent HIPK2 was successfully constructed. Cell activity decreased and cytotoxicity increased in the Scr+Sev group, but there was no significant difference in the cell viability and cytotoxicity between shHIPK2+Sev group and shHIPK2 group. The proportion of apoptotic cells in the Scr+Sev group was significantly higher than that of Scr group(P<0.05), but there was no significant difference between shHIPK2+Sev group and shHIPK2 group(P>0.05). The level of Cl-caspase3 protein in the Scr+Sev group was significantly higher than that in the Scr group(P<0.05), and there was no statistical difference between the group of shHIPK2+Sev and the shHIPK2(P>0.05). Conclusion: HIPK2 could promote the apoptosis of primary hippocampal neurons induced by sevoflurane. |
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