摘要
目的:培养人真皮微血管内皮细胞,建立人真皮微血管内皮细胞低氧/复氧模型。方法:采用酶消化及联合磁珠分选法,得到人真皮微血管内皮细胞(HDMECs),体外培养传至P5代,采用Ⅷ因子免疫荧光及CD309、CD34流式细胞学进行鉴定。用缺氧Buffer加低氧舱的方法低氧处理P5代HDMECS。实验分为四组,Con组不做任何处理、H8组低氧8小时组,H8R12组低氧8小时复氧12小时组、H8R24组低氧8小时复氧24小时组。TUNEL(脱氧核糖核苷酸末端转移酶介导的缺口末端标记法)染色法检测细胞凋亡,蛋白质印迹法(Western Blot)检测半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)蛋白的表达。结果:联合法分离的细胞,经过2次CD31磁珠分选后,可见细胞短梭形,呈铺路石样。细胞传至P5,Ⅷ因子免疫荧光染色阳性率为(95±2.61)%。流式细胞仪检测CD309阳性率为(95.4±1.34)%;CD34阳性率为(42.5±3.23)%。TUNEL染色、Western Blot结果显示:H8组的细胞凋亡率和活化的Caspase-3表达高于Con组(P<0.05),H8R12组、H8R24组细胞凋亡率和活化的Caspase-3表达高于H8组(P<0.05)。结论:本研究采用磁珠分选的办法成功培养了HDMECs,并且建立了HDMECs低氧/复氧模型,为临床研究游离皮瓣缺血再灌注损伤机制提供实验基础。
Objective：Using enzymatic digestion and magnetic bead separation combined method to extract the flap microvascular endothelial cells, create an endothelialcell model of hypoia/reoxygenation model in vitro. Methods： using enzyme digestion and combined magnetic bead separation method to get people flap microvascular endothelial cells culture spread to the P5 In vitro, used immunofluorescence and fluid cytology to identiflcation it. Adopt the method of oxygen Buffer with low oxygen tank processing P5 HDMECs, Experiment is divided into four groups, Con not do any processing, H8 group hypoxia 8h group, H8R12 reoxygenation 12h af- ter hypoxia of 8h , H8R24 reoxygenation 24h after hypoxia of 8h. Percentage of apoptosis cells was detected by TUNEL staining method （TdT-mediated dUTP Nick-End Labeling）. Protein imprinting method （Western Blot） detection protein expression of Caspase-3. Results ： the combined method to separate cells, after two CD31 magnetic bead separation, and cultured in vitro, cells were spindle shaped, a paving stone. To P5, immunofluorescence staining positive rate of factor VIII was （95 ±2. 61 ） %. Flow cytometry was used to detect the positive rate of CD309 was （95.4 ± 1.34） % respectively; the positive rate of CD34 was （42. 5 ±3.23 ） %. Four groups of cell TUNEL apoptotic dyeing and Western Blot results show that the H8 group of apoptosis rate and the activation of Caspase-3 expression above Con group （P 〈 0. 05 ）, H8R12 group, H8R24 apoptosis rate and the activation of Caspase-3 expression above H8 group （P 〈 0.05 ）. Conclusion：This research adopts magnetic bead separation method and had successed training the people flap microvascular endothelial cells （HDMECs） , and established the hypoxia reoxygenation model of flap microvascular endothelial cell, provides the basis for clinical research the mechanism of free flap ischemia-reperfusion injury.
出处
《心肺血管病杂志》
CAS
2015年第2期132-136,共5页
Journal of Cardiovascular and Pulmonary Diseases
基金
国家自然基金资助项目(81272130)
北京市卫生高层次人才
关键词
人真皮微血管内皮细胞
低氧/复氧
细胞凋亡
Human dermal microvascular endothelial cells
Hypoxia/reoxygenation
Cell apoptosis
