摘要
目的构建稳定沉默SUMO特异性蛋白酶1(SENP1)基因的人Ⅱ型肺泡上皮细胞系HEPApiC。方法构建沉默SENP1基因的LV3-SENP1-RNAi慢病毒载体。将HEPApiC细胞分为实验组、对照组、空载组,实验组感染LV3-SENP1-RNAi,空载组感染空载体,对照组不做特殊处理;感染72 h后观察绿色荧光强度,采用qRT-PCR检测HEPApiC细胞中的SENP1 mRNA,采用Western blotting法检测SENP1蛋白。结果经酶切鉴定及测序分析,成功构建LV3-SENP1-RNAi慢病毒载体质粒,包装后得到高滴度的病毒颗粒。感染LV3-SENP1-RNAi的HEPApiC细胞中GFP表达随时间延长逐渐增强,说明LV3-SENP1-RNAi成功感染HEPApiC细胞。实验组、对照组、空载组细胞中SENP1 mRNA相对表达量分别为0.026、0.050、0.057,SENP1蛋白相对表达量分别为0.161±0.015、0.781±0.046、0.811±0.008;实验组SENP1 mRNA及蛋白相对表达量降低,与对照组及空载组相比,P均<0.05。结论成功构建了稳定沉默SENP1基因的HEPApiC细胞系。
Objective To establish human type II alveolar epithelial cells（HEPApiC） that can stably silence SUMO specific protease 1（SENP 1）. Methods We constructed the LV3-SENP1-RNAi lentiviral vector silencing SENP1 gene.HEPApiC cells were divided into the experimental group,control group and empty vector group. The experimental group was infected with LV3-SENP1-RNAi,the empty vector group with empty vector and the control group was not treated. After72-hour infection,the green fluorescence intensity was observed,the SENP1 mRNA in HEPApiC cells was detected by qRT-PCR,and SENP1 protein was detected by Western blotting. Results After restriction enzyme digestion and sequencing,the LV3-SENP 1-RNAi lentiviral vector plasmid was successfully constructed and we got a virion with a high titer.The expression of GFP in HEPApiC cells infected with LV3-SENP1-RNAi was gradually increased with time,indicating that LV3-SENP1-RNAi successfully infected HEPApiC cells. The relative expression of SENP1 mRNA in the experimental group,the control group and the empty vector group was 0. 026,0. 050,and 0. 057; and the relative expression of SENP1 protein was 0. 161 ± 0. 015,0. 781 ± 0. 046 and 0. 811 ± 0. 008,respectively. The expression of SENP1 mRNA and protein in the experimental group was lower than that in the control group and empty vector group,all P 〈0. 05. Conclusion HEPApiCs cell line stably silencing SENP1 is successfully established.
作者
赵许
董文斌
雷小平
李清平
康兰
赵帅
张婵
ZHAO Xu DONG Wenbin LEI Xiaoping LI Qingping KANG Lan ZHAO Shuai ZHANG Chan(The Affiliated Hospital of Southwest Medical University, Luzhou 646000, Chin)
出处
《山东医药》
CAS
北大核心
2017年第3期16-19,共4页
Shandong Medical Journal
基金
国家自然科学基金资助项目(81571480)
四川省科技厅科研项目(2014NZ0014)
泸州市政府-泸州医学院联合专项资金资助项目(2013LZLY-J08)
关键词
小泛素相关修饰蛋白质类
人Ⅱ型肺泡上皮细胞
基因沉默
RNA干扰
small ubiquitin-related modifier proteins
human type Ⅱ alveolar epithelial cells
gene silencing
RNA interference
