人脐动脉内皮细胞和平滑肌细胞体外培养鉴定及缝隙连接通讯功能测定

摘要

Objective:To use the two-photon laser scanning confocal microscope to identify umbilical artery endothelial cells (ECs) and smooth muscle cells (SMCs) that were cultured in vitro,determine the gap junctional intercellular communication (GJIC) between smooth muscle cells and ECs by using fluorescence redistribution after photobleaching (FRAP) of laser scanning confocal microscopy.Methods:Human umbilical artery ECs and SMCs were isolated and cultured,use the Ⅷ factor and SMα-actin antigen to identified ECs and of SMCs.The functions of GJIC in SMCs and ECs were detected by FRAP.Record real-time imaging results.Application of mobile fraction(M) to calculate the ratio of the mobile molecular labeled fluorescent molecules in the bleached region.Results:The first group of ECs and SMCs cultured alone,the bleached cells with the surrounding at least three allogenic cells connected,the average M value of SMCs after photobleaching is 31.79 ± 5.69,of ECs after photobleaching is 23.43 ± 2.11.The second group of ECs and SMCs mixed culture,ECs connect to SMCs independent,the average M value of SMCs after photobleaching is 14.47 ± 3.28,of ECs after photobleaching is 6.41 ± 0.80.Conclusion:FRAP real-time dynamic recovery curve can be directly observed fluorescence recovery of strength and speed.Reference to the FRAP recovery curve,the value of M can be a reliable indicator for relative quantification as a GJIC between the two groups.Between ECs and SMCs was confirmed by GJIC,and from ECs to SMCs direction is greater than from SMCs to the ECs direction.%目的:应用双光子激光扫描共聚焦显微镜鉴定体外培养的脐动脉内皮细胞(ECs)和平滑肌细胞(SMCs),应用荧光光漂白恢复技术(FRAP)测定血管ECs、SMCs之间的缝隙连接通讯(GJIC)功能.方法:人脐动脉ECs、SMCs分离培养,Ⅷ因子和SMα-actin相关抗原鉴定ECs和SMCs,应用FRAP技术测定血管内皮细胞、平滑肌细胞之间的GJIC功能,记录实时成像结果,应用动态比(M)计算漂白区域内标记荧光的分子中动态分子的比例.结果:第一组ECs和SMCs单独培养,选择漂白细胞与周围至少3个同种细胞相连接,SMCs被漂白后平均M值为31.79 ±5.69；ECs被漂白后平均M值为23.43 ±2.11；第二组ECs和SMCs混合培养,选择ECs和SMCs独立相连的2个细胞,SMCs被漂白后平均M值为14.47±3.28,ECs被漂白后平均M值为6.41±0.80.结论:FRAP实时动态恢复曲线可直接观察荧光恢复强度及速度,参照FRAP恢复曲线,M值可做为组间GJIC比较相对定量的可靠指标,通过检测证实ECs和SMCs之间存在GJIC,且荧光由ECs向SMCs方向的传递大于由SMCs向ECs方向的传递.