Nf1基因沉默促进人脑血管内皮细胞的增殖  

Nf1 gene silencing promotes proliferation of human brain vascular endothelial cells

作　　者：叶梦 梁晨思 霍丽蓉[1] Ye Meng;Liang Chensi;Huo Lirong(Department of Central Laboratory,the Fuxing Hospital,Capital Medical University,Beijing 100038,China)

机构地区：[1]首都医科大学附属复兴医院中心实验室,北京100038

出　　处：《脑与神经疾病杂志》2021年第2期115-120,共6页Journal of Brain and Nervous Diseases

基　　金：北京市自然科学基金(7182065);北京市西城区优秀人才项目(20160031);北京市西城区拔尖团队项目(201915)。

摘　　要：目的抑制1型神经纤维瘤病(NF1)发病基因Nf1在人脑血管内皮细胞(HBMECs)中的表达并检测HBMECs增殖改变情况,以研究NF1相关脑血管病发病机制。方法设计并合成3对Nf1基因小干扰RNA(siRNA)及其阴性对照;使用脂质体转染法将siRNA转染进HBMECs,通过实时荧光定量PCR(Real-time PCR)及Western blot法分别检测Nf1基因mRNA和蛋白质表达情况。将筛选出的具有最佳Nf1基因沉默效应的siRNA转染进HBMECs,并设置阴性对照,使用细胞计数试剂盒8(CCK8)法与细胞计数法检测细胞增殖情况。结果3对siRNA均具有一定的Nf1基因沉默效应,其中siRNA C基因沉默效应最佳,Nf1基因mRNA相对转录水平是对照组的(58.65±0.62)%,神经纤维瘤蛋白(neurofibromin)的相对表达量为对照组的(29.447.10)%,差异均有统计学意义(P<0.01)。CCK8法与细胞计数法结果均显示Nf1基因沉默后HBMECs 5d中有4d实验组相对细胞数多于对照组(P<0.05)。结论干扰HBMECs Nf1基因后,其增殖活性增强,这可能是NF1相关脑血管病的发病机制之一。Objective The expression in human brain microvascular endothelial cells(HBMECs)of Nf1 gene which causes neurofibromatosis type 1(NF1)was reduced,then detecting proliferation changes of HBMECs to contribute a better understanding of NF1 associated cerebrovascular disease.Methods Three pairs of double stranded small interfering RNA(siRNA)and the negative control were designed as well as synthesized.After transfected siRNAs into HBMECs by lipofection,Real-time PCR and Western blot were implemented to detect Nf1 gene silencing efficiency.The most effective siRNA or the negative control siRNA was transfected into HBMECs,then cell proliferation was detected by CCK8 assay and cell counting method.Results All three pairs of siRNAs had a certain gene silencing effect,among which the most effective siRNA was siRNA C,the relative level of mRNA was(58.65±0.62)%while the relative level of neurofibromin was 29.44%,7.10%compared to negative control(P<0.01).After Nf1 gene interference,the proliferation of the experimental group was stronger then the negative control no matter using CCK8 assay or cell counting method in 4 of 5 days(P<0.05).Conclusion The most effective Nf1-specific siRNA was successfully synthesized and screened.After interfering Nf1 gene of HBMECs,the proliferation was enhanced,which may be one pathogenesis of NF1 associated cerebrovascular disease.

关 键 词：1型神经纤维瘤病 脑血管病 人脑血管内皮细胞 

分 类 号：R743[医药卫生—神经病学与精神病学]