毛囊干细胞体外诱导成血管内皮细胞的实验研究

Induction of hair follicle stem cells into endothelial cells in vitro

目的建立简单、可靠的毛囊干细胞体外定向分化为血管内皮细胞的方法。方法无菌条件下切取1周龄SD大鼠触须部皮肤,Dispase酶和Ⅳ型胶原酶混合液消化,显微镜下分离毛囊隆突部,改良的组织块贴壁法培养rHFSCs,差速贴壁法纯化,流式细胞仪检测及细胞免疫荧光染色联合鉴定。用含10 ng/ml和20 ng/ml VEGF165作为主要诱导因子将细胞分为两种浓度组别,不传代的情况下分别在1、2、3周内观察诱导后细胞形态;通过流式细胞和细胞免疫荧光染色检测分化效率;选取最佳诱导因子浓度和工作时间,对诱导后的细胞进行体外官腔形成实验,并在透射电镜下观察W-P小体。流式细胞术检测分化效率实验数据的比较采用单因素方差分析及独立t检验。结果分离、培养、纯化的rHFSCs克隆能力好,活力强,生长曲线呈S型。流式细胞及免疫荧光染色联合检测β1(98.9%)、α6整合素(97.9%),CK15(68.1%)、P63(98.5%)呈高表达,阴性表达CD31(13.6%)、VE-cadherin(17.9%)。在诱导液的作用下,两组细胞从1周到3周后,均由扁平铺路石样改变到完全被长梭形样细胞占据。流式细胞及免疫荧光染色检测CD31和VE-cadherin显示,CD31在诱导1周时表达最强,两种诱导因子比较无差异[(65.27±0.57%)vs(66.13±0.60)%,t=1.812,P=0.145]。而VE-cadherin在诱导1周时表达也最强,10ng/ml要优于20ng/ml VEGF165的诱导作用[(95.57±0.85)%vs(78.10±1.25)%,t=19.977,P=0.001]。10 ng/ml VEGF165诱导1周后的细胞体外管腔形成实验阳性,在透射电镜下可以观察到内皮细胞特有结构W-P小体。结论毛囊干细胞体外在10ng/ml VEGF165的诱导下,1周后可成功得到性能良好的血管内皮细胞。可为组织工程血管、细胞移植治疗缺血性疾病等提供理想的种子细胞。

Objective To establish a simple, reliable method of hair follicle stem cells directional differentiation into vascular endothelial cells. Methods The tentacles of skin of 1-week SD rat were removed in aseptic condition and digested with Dispase enzyme and type IV collagenase mixture. The hair follicle bulges were picked out under a microscope. After culture, HFSCs were identified by flow cytometry and immunofluorescence staining. Cells were cultured in the presence of 10 ng/ml and 20 ng/ml VEGF165 and cell morphology were inspected at 1, 2, and 3 weeks. Differentiation efficiency was evaluated by flow cytometry and immunofluorescence staining; Formation of newspeak and W-P corpuscle was observed under transmission electron microscope. The experimental data were compared with one factor analysis of variance and t test. Results rHFSCs formed clone and positive for integrin 131 （98.9%）, integrin α6 （97.9%）, CK15 （68.1% ）, and P63 （98.5 % ）. Expression levels of CD31 （13.6 % ） and VE-cadherin （17.9 % ） were low. Under induction, spindle-shaped cells appeared after 1 week. Cells expressed high-level CD31 in the presence of both 10 ng/ml and 20 ng/ml VEGF 165 after 1 week [（65.27 ±0.57） % vs （66.13 4-0.60）%, t = -1.812, P = 0.145]. VE-cadherin was also expressed after 1 week （95.57 ± 0.85 vs 78.10± 1.25, t = 19.977, P = 0.000）. At the presence of 10 ng/ml VEGF165, induced cells had newspeak and W-P corpuscles specific for endothelial cells. Conclusion With induced factor of 10 ng/mlVEGF165 concentration, rHFSCs can get good performance of vascular endothelial cells successfully after 1 week.which provide vascular tissue engineering, cell transplantation treatment of ischemic diseases ideal seed cells.