NF1～180mut表达载体构建及其在脑血管细胞中的表达

Construction of NF1-180mut Expression Vector and Its Expression in Cerebral Vascular Cells

目的前期研究中发现一合并脑血管狭窄的Ⅰ型神经纤维瘤病家系,为探索血管狭窄的病因,本研究构建该家系患者NF1基因共同突变产物NF1~180mut的过表达质粒,观察该质粒在脑血管细胞中的表达。方法设计NF1~180mut全长序列的相应引物,采用p EGFP-N1作为载体框架,构建p EGFP-N1/NF1~180mut表达载体,通过Western blot法及共聚焦检测其蛋白表达;之后采用脂质体转染重组质粒于脑血管平滑肌细胞或内皮细胞中观察NF1~180mut在两种脑血管细胞中的表达。结果(1)成功构建了NF1~180mut表达载体;(2)该表达载体可在脑血管内皮细胞及平滑肌细胞内表达;(3)初步通过细胞计数研究及增殖实验显示NF1~180mut对脑血管平滑肌细胞和血管内皮细胞的生长及增殖有一定的作用。结论本研究中构建了NF1-Q181X突变后的产物NF1~180mut表达载体,为进一步深入研究NF1~180mut导致脑血管狭窄发生的通路提供了良好的体外研究工具。

Objective One NF1 family combined with cerebrovascular stenosis was found by us. The common feature in the NF1 patients was Q181X mutation in NF1 gene. Truncated protein （180 amino acids, NF1-180mut） was produced. To further determine the role of NF1-180mut in cerebrovascular stenosis, we constructed the recombinated pEGFP-N1/NF1-180mut plasmids and transfected cerebrovascular cells with the plasmids. Methods The primers of total NF1-180mut were devised. Using pEGFP-N1 as the carrier frame, we constructed the pEGFP-N1/NF1-180mut expression vector and the protein expression was detected by western-blot and confocal. Then we observed the expression of NF1-180mut in cerebrovascular smooth muscle cells or endothelial cells by liposome mediated transfection of the recombinant plasmid. Results （1） NF1-180mut expression vector was constructed successfully. （2） The expression vector can be expressed in brain vascular endothelial cells and smooth muscle cells. （3） The preliminary study of cell count and proliferation test showed that NF1-180mut had a certain effect on cerebrovascular smooth muscle cells and endothelial cells. Conlusion In this study, we constructed the expression vector of NF1-180mut from NF1-Q181X mutation, which provides a good in vitro research tool for further study on the pathway of NF1-180mut induced cerebral vascular stenosis.