低温暴露对真皮微血管内皮细胞功能的影响

Effects of low temperature exposure on derminal microvascular endothelial cells function

目的:探讨低温暴露对真皮微血管内皮细胞损伤效应及血管内皮生长因子(VEGF)表达的影响。方法:利用非连续密度梯度Percoll分离液原代分离培养大鼠真皮微血管内皮细胞(DMVECs),免疫荧光检测CD31抗原的表达并结合形态学鉴定细胞。将DMVECs暴露于不同低温(28℃、12℃及0℃)条件下24 h,建立低温细胞暴露模型,通过倒置显微镜观察细胞形态,测定细胞培养液中乳酸脱氢酶(LDH)活性评价细胞膜完整性,应用半定量反转录-聚合酶链式反应(RT-PCR)检测血管内皮生长因子(VEGF)的表达。结果:培养的DMVECs呈典型的鹅卵石样排列,CD31抗原表达呈阳性。大鼠DMVECs于不同低温暴露24 h后,与37℃组比较,28℃组及12℃细胞形态趋于"成纤维样",而0℃组细胞出现明显的皱缩;28℃,12℃及0℃组细胞培养液中LDH活性(U/L)分别为54.17±3.02,64.66±3.03,82.13±10.91,与37℃组(12.23±3.03)比较统计学差异显著(P<0.01);28℃组及12℃组VEGFmRNA表达上调(P<0.05),但0℃组与37℃无统计学差异。结论:随温度降低,大鼠DMVECs损伤程度加重,其中VEGF可能参与低温致皮下血管通透性的增加。

Objective： To explore the damage effects and expression of vascular endothelial growth factor（VEGF）exposed with different low-temperatures on rat derminal microvascular endothelial cells（DMVECs）.Methods： Primary DMVECs were obtained by discontinuous Percoll gradient centrifugation.The DMVECs were identified by phase contrast microscope and immunofluorescence studies for CD31 antigen.Applied 28℃,12℃and 0℃to interfere with rat DMVECs as cold-exposure model.The changes of cells morphology were observed under invert microscope.The membrane integrity was determined by lactate dehydrogenase（LDH） activity.RT-PCR was used to examine the expression of vascular endothelial growth factor mRNA in cells.Results： The monolayer of cultured PMVECs displayed the shape of pavingstone.CD31 antigen and binding BSI results by fluorescence microscope identified the cultured cells were DMVECs.After 24 h cold exposure,the cell morphology of 0℃ group was shrunken,the other groups were ＂Fibroblast-like＂.The LDH activity（U/L） in the medium of 28℃,12℃and 0℃ groups was 54.17±3.02,64.66±3.03,82.13±10.91 respectively,which was significantly higher than that of 37℃ group（12.23±3.0,P＆lt;0.01）.The VEGF mRNA expression level was up-regulated in 28℃ group and 12℃ group versus control group（P＆lt;0.05）,but unchanged in 0℃ group.Conclusion： The rat DMVECs injury severity are deteriorated with temperature decreasing,and VEGF might be involved in the regulation of membrane permeability in this period.