热打击对体外培养人血管内皮细胞骨架及细胞周期的影响

The effect of heat stress on the cytoskeleton and cell cycle of human umbilical vein endothelial cell in vitro

目的：探讨热打击对体外培养人脐静脉血管内皮细胞（HUVEC）细胞骨架及细胞周期的影响。方法体外培养HUVEC，并分别置于37℃、5%CO2培养箱（对照组）或43℃细胞培养箱（热打击组）培养1 h。采用考马斯亮蓝R-250染色法，高倍镜下观察热打击对HUVEC细胞骨架的影响。热打击组细胞培养1 h后继续置于37℃、5%CO2培养箱孵育，分别于0、6、12、18、24 h采用流式细胞仪检测热打击对HUVEC细胞周期的影响。结果镜下观察对照组HUVEC细胞骨架形态正常；热打击组随热打击时间的延长，细胞骨架逐渐增粗、变短，且出现明显的应力纤维。对照组细胞G0/G1期DNA含量为48.57%，S期为44.62%，G2/M期为6.81%。在热打击后HUVEC细胞各时间点G0/G1期DNA含量为38.07%～55.19%，热打击后0、6、12、18、24 h组分别为54.06%、55.19%、48.23%、38.07%和41.03%；S期DNA含量为35.33%～48.18%，热打击后0、6、12、18、24 h组分别为35.33%、39.50%、42.50%、48.18%和47.99%；G2/M期DNA含量为5.31%～13.75%，热打击后0、6、12、18、24 h组分别为10.61%、5.31%、9.27%、13.75%和10.98%。说明当HUVEC细胞离开热打击环境后，G0/G1期DNA含量在6 h内明显高于对照组，至12 h时才恢复至对照组水平。结论热打击可改变HUVEC细胞骨架结构，且细胞周期出现阻滞，细胞被阻滞在G0/G1期。

ObjectiveTo study the effect of heat stress on the cytoskeleton and cell cycle of human umbilical vein endothelial cell （HUVEC） in vitro.Methods HUVEC was cultured in vitro in 5%CO2 medium at 37℃ （control group） or 43℃ （heat stress group） for 1 hour. Coomassie brilliant blue R-250 staining was used to determine the effect of heat stress on the cytoskeleton. The cells in heat stress group were subsequently cultured at 37℃in 5%CO2 medium after heat stress for 1 hour, and cell cycle of HUVEC was determined at 0, 6, 12, 18 and 24 hours with flow cytometry.Results Under light microscopy normal cytoskeleton was observed in control group, but thicker and shorter cytoskeleton was found after a rise of temperature, and stress fibers were found in heat stress group. The DNA content of HUVEC at all time points in G0/G1 stage was 38.07%-55.19% after heat stress. The DNA content in control group was 48.57%, and it was 54.06%, 55.19%, 48.23%, 38.07%, and 41.03% at 0, 6, 12, 18, 24 hours in G0/G1 stage in heat stress group. DNA content in S phase was 35.33%-48.18%. The DNA content in control group was 44.62%, and it was 35.33%, 39.50%, 42.50%, 48.18%, and 47.99% at 0, 6, 12, 18, 24 hours in S stage in heat stress group. DNA content in G2/M phase was 5.31%-13.75%. The DNA content in control group was 6.81, and it was 10.61%, 5.31%, 9.27%,13.75%, and 10.98% at 0, 6, 12, 18, 24 hours in G2/M stage in heat stress group. It was demonstrated that compared with control group, the DNA content in G0/G1 stage was significantly increased when the HUVEC were separated from heat stress within 6 hours, and it recovered at a similar level as control group at 12 hours.Conclusion Heat stress can change the cytoskeleton of HUVEC, and cause stagnation at G0/G1 stage in cell cycle.