摘要
目的构建神经束蛋白155(neurofascin155,NF155)高表达的少突胶质细胞模型。方法 RT-PCR法提取NF155 cDNA,经T-A克隆及双酶切将其与pUM-T simple重组,构建NF155慢病毒载体质粒,NheⅠ、AgeⅠ双酶切后,片段大小及基因测序均显示NF155慢病毒载体质粒构建成功。取对数生长期少突胶质细胞分为空白对照组、阴性慢病毒感染组、NF155慢病毒感染组,空白对照组不做任何处理,阴性慢病毒感染组予10 μL空载体慢病毒+100 μL培养基,NF155慢病毒感染组予10 μL慢病毒载体质粒+100 μL培养基。培养12 h采用荧光定量PCR法检测NF155 mRNA,采用Western blotting法检测NF155蛋白。结果 NF155慢病毒感染组、空白对照组、阴性慢病毒感染组NF155 mRNA相对表达量分别为3.25±0.11、1.00±0.06、1.08±0.01,与空白对照组、阴性慢病毒感染组比较,NF155慢病毒感染组NF155 mRNA相对表达量升高( P 均<0.05)。NF155慢病毒感染组、空白对照组、阴性慢病毒感染组NF155 蛋白相对表达量分别为1.57±0.08、0.51±0.12、0.50±0.06,与空白对照组、阴性慢病毒感染组比较,NF155慢病毒感染组NF155蛋白相对表达量升高( P 均<0.05)。结论 成功构建NF155高表达的少突胶质细胞瞬转细胞模型。
Objective To constructthe oligodendrocytes model with high expression of neurofascin 155 (NF155). Methods NF155 cDNA was cloned by RT-PCR and inserted into pUM-T simple vector by T-A clone methodand double digestion to construct a lentivirus vector plasmid. Nhe I and Age I were used to cut off the target gene, the size of the fragment and gene sequencing showed that the lentivirus vector plasmid was successfully constructed. Oligodendrocytes in logarithmic growth phase were divided into the blank control group, negative lentivirus infection group, and NF155 lentivirus infection group. The blank control group was not treated,the negative lentivirus infection group was treated with 10 μL empty vector lentivirus + 100 μL medium, and the NF155 lentivirus infection group with 10 μL lentivirus vector plasmid + 100 μL medium. The mRNA and protein expression levels of NF155 were detected by real-time PCRand Western blotting. Results The relative expression levels of NF155 mRNA in the NF155 lentivirus infection group, blank control group and negative lentivirus infection group were 3.25±0.11, 1.00±0.06, and 1.08±0.01, respectively. The relative expression of NF155 mRNA in the NF155 lentivirus infection group was higher than that in the blank control group and negative lentivirus infection group( P <0.05).The relative expression levelsof NF155 protein in the lentivirus infection group, blank control group, and negative lentivirus infection group were 1.57±0.08, 0.51±0.12, and 0.50±0.06, respectively. The lentivirus infection group had higher relative expression of NF155 proteinthan the blank control group and negative lentivirus infection group( P <0.05). Conclusion The oligodendrocytes transient cell model with high expression of NF155 is successfully constructed.
作者
胡斌
王成举
陈鹏慧
张雨平
HU Bin;WANG Chengju;CHEN Penghui;ZHANG Yuping(TheSecond Affiliated Hospital of Army Medical University,Chongqing 400037,China)
出处
《山东医药》
CAS
2019年第18期28-31,共4页
Shandong Medical Journal
基金
国家自然科学基金面上项目(81671496)
