γ-分泌酶抑制剂对毛囊干细胞诱导分化为血管内皮细胞效率的影响

γ- secretase inhibitors on the efficiency of the hair follicle stem cell differentiation into vascular endothelial cells

目的观察Notch信号通路的特异性阻断剂——γ-分泌酶抑制剂（DAPT）对毛囊干细胞（rHFSCs）诱导分化为血管内皮细胞（ECs）效率的影响。方法运用改良的rHFSCs分离培养、纯化鉴定的方法得到种子细胞，并用透射电镜观察其内部结构。用10恤晷／L血管内皮生长因子165（VEGF165）作为诱导因子进行诱导，流式细胞仪检测分化效率。用不同浓度DAPT（0．5、1．0μmol／L）进行抑制，将他们分为诱导组和抑制组。两组细胞均处理1周，用实时荧光定量聚合酶链反应（FQ—PCR）和Western blot法分别检测两组CD31、血管内皮钙黏蛋白（VE—cadherin）mRNA和蛋白表达，联合DiL标记的乙酰化低密度脂蛋白（Dil—gc—LDL）吞噬功能检测和体外成血管实验形成实验进行对比，综合评价DAPT在抑制Notch信号通路后对rHFSCs诱导分化为ECs的影响。结果改良分离、培养、纯化的rHFSCs克隆能力好，活力强。流式细胞仪检测β1、α6整合素、细胞角蛋白15（CK15）、p63呈高表达，分别为98．9％、97．9％、68．1％和98．5％；低弱表达CD31、VE—cadherin，分别为13．6％和17．9％；透射电镜显示细胞处于原始状态。诱导1周后，流式细胞仪检测高表达内皮细胞标志物CD31和VE—cadherin，分别为61．5％和95．9％。抑制Notch信号通路后两组FQ-PCR结果显示，CD31变化差异无统计学意义（P=0．136）、VE-cadherinmRNA变化差异有统计学意义（P=0．003），Western blot法得到CD31、VE-cadherin蛋白表达明显降低（P=0．000），体外Dil—ac-LDL吞噬功能，成血管形成能力明显减弱。结论DAPT通过阻断Notch信号通路，虽没有显著影响CD31、VE—cadherinmRNA水平，但能较好抑制CD31、VE-cadherin蛋白表达，体外吞噬和成血管的能力，从而减低rHFSCs诱导分化为ECs的效率。

Objective To study the specificity of the Notch signal pathway blockers - γ- - secre- tase inhibitors （DAPT） on the efficiency of the hair follicle stem cell （rHFSCs） differentiation into vascu- lar endothelial cells （ECs）. Methods Applying the modified method of rHFSCs separation, culture, pur- ification, identification for seed cells, and using transmission electron microscope to observe the internal structure. With 10 μg/Lvascular endothelial growth factor 165 （VEGF165） as inducing factor to carry on the inductio, and flow cytometry instrument to detect differentiation efficiency. Then divided them into induction and inhibition group. With different concentrations of DAPT （0. 5, 1.0 μmol/L） to suppress, Two group of cells are both handled for 1 week, with the method of real - time fluorescent quantitative polymerase chain reaction （ FQ - PCR） and Western blotting, respectively detecting the expression of mRNA and protein CD31, vascular endothelial cadherin （VE -cadherin） in the two groups, then conbined with Dil -labeled acetylated low density lipoprotein （ Dil - ac - LDL） phagocytosis test and Lumen formation experi- ment test, finally comparing the result of two group. To comprehensive evaluate DAPT on the influence of rHFSCs differentiation ECs after it inhibited Notch signaling pathway. Results The rHFSCs with modified of isolation, culture, purification has strong cloning ability and vitality. Flow cytometry instrument testing shows β1, α6 integrin and CK15 with a high expression, respectively, 98. 9%, 97. 9%, 68.1% and 98.5% ; and Low weakly expressed CD31, VE - cadherin, respectively, 13.6% and 17.9% , Transmission electron microscopy （sem） show that cells in original condition. Flow cytometry instrument testing shows CD31, VE -cadherin with high expression afer induced Cells for a week, respectively, 61.5% and 95.9%. After inhibition of Notch signaling pathway, according to the results of FQ -PCR： there is no ob- vious difference of variation of mRNA of CD31 （ P = 0. 136） and VE - cadherin （P = 0. 003 ） between two groups, the protein expression of CD31 and VE - cadherin significantly reduced through Western blotting method （P =0. 000）, Dil -ac -LDL phagocytosis and lumen formation ability in vitro decreased significantly. Conclusion After interdicting the Nntch signal pathway, although DAPT has no obvious influence on the mRNA level of CD31 and VE - cadherin, but can inhibit protein expression of CD31 and VE - cad- herin and ability of phagocytosis and lumen formation in vitro, thereby reducing efficiency of rHFSCs differentiation for ECs. It lays a experimental basis on further research of the mechanism of Notch signaling pathway in inducing vascular endothelial cells.