微波辐照诱导内质网应激致心肌微血管内皮细胞损伤

目的探讨微波辐照致心肌微血管内皮细胞损伤与内质网应激之间的关系。方法取培养3~4代心肌微血管内皮细胞随机分为对照组和辐照各组。1.分别采用10mW/cm2,30mW/cm2、50mW/cm2微波辐射心肌微血管内皮细胞,辐照时间均为6min。于照射后24h收集细胞。2.细胞被暴露于30mW/cm2微波6 min,继续培养1 h、3 h或24 h之后,内皮细胞被收集,对照组于24 h结束实验。以膜联蛋白V-碘化丙啶双染法检测细胞凋亡;鬼笔环肽染色法观察微血管内皮细胞骨架的变化,评价微血管内皮细胞的损伤情况;免疫印迹法检测内质网应激分子钙网蛋白(CRT)、葡萄糖调节蛋白78(GRP78)和CCAAT/增强子结合蛋白同源蛋白(CHOP)的蛋白表达,评价微波辐照是否引起微血管内皮细胞内质网应激。结果内皮细胞凋亡率的量效研究发现,微波辐照之后,10mW/cm2、30mW/cm2、50mW/cm2照射组的细胞凋亡率分别为(2.34±0.15)%、(2.72±0.96)%、(2.62±0.34)%,与对照组(0.88±0.32)%比较差异显著(P0.05)。时效研究则发现,30mW/cm2照射后1h、3h和24h细胞凋亡率分别为(1.12±0.15)%,(1.49±0.54)%和(1.85±0.45)%。与对照组(1.10%±0.28)%比较,照射后1h组差异不显著(P0.05),照射后3h组和24h组差异显著(P0.05);内质网应激分子的检测发现,30mW组CRT、GRP78、CHOP的蛋白表达分别较对照组升高124%,76%,256%。50mW组CHOP的蛋白表达分别较对照组升高52%,189%。与对照组相比,30mW组CRT、GRP78、CHOP GRP78、及50mW组GRP78、CHOP表达差异显著(P0.05)。10mW组CRT、GRP78、CHOP及50mW组CRT蛋白表达与对照组相比差异无显著性(P0.05)。对照组内皮细胞表现出很少的肌动蛋白纤维。内皮细胞暴露于微波引起的丝状肌动蛋白应力纤维数量的急剧增加。最大应力纤维的形成发生在内皮细胞受到照射后3h或照射功率为30mW。结论微波辐照可诱导严重内质网应激反应,造成大鼠心肌微血管内皮细胞损伤。

Cardiac microvascular endothelial cell injury induced by microwave irradiation and endoplasmic reticulum stress

Objective To study the relationship between the damage of cardiac microvascular endothelial cells by microwave radiation and endoplasmic reticulum stress. Methods Cardiac microvascular endothelial cells of the rat that were cultured 3-4 generation were divided into control and radiation groups. Of radiation groups, using 10mW/cm2,30mW/cm2,50mW/cm2 microwave radiated cardiac microvascular endothelial cells, respectively, and radiated for 6min. After radiated 24hours,cells were collected. The cells were exposed to 30mW/cm2 microwave for 6 minutes. After cultured for 1hour, 3hours or 24hours, endothelial cells were collected and the control group finished experiment at 24 hours. The annexin V-propidium iodide double staining method was used to detect apoptosis rate. The method of phalloidin staining to observe the changes of microvascular endothelial cytoskeleton was used. Westen blotting was used for detecting the protein expression of calreticulin, CHOP and GRP78. Results The dose effect study on the apoptosis showed that, after microwave irradiation, the apoptosis rates in 10mW/cm2, 30mW/cm2, 50mW/cm2 irradiation groups were (2.34±0.15)%, (2.72±0.96)%, (2.62±0.34)% and there were significant difference (P<0.05) comparing to the control group（0.88±0.32）%. Aging studies on the apoptosis found that, at 1hour, 3hours and 24hours of post exposure to 30mW/cm2,the apoptosis rate of cells were (1.12±0.15)%, (1.49±0.54)% and (1.85±0.45)%.The control group was (1.10±0.28)%.Post irradiated 1hour group had no significant difference (P>0.05), post irradiated 3hours group and 24hours group had significant difference (P<0.05). Molecular detection of endoplasmic reticulum stress showed that, comparing with the control group, the protein expressions of CRT, GRP78 and CHOP in the 30mW group increased by 124%, 76% and 256%, respectively. In the 50mW group,the protein expressions of GRP78 and CHOP increased by 52% and 189%, respectively. Comparing with the control group, the protein expressions of CRT, GRP78 and CHOP in the 30mW and GRP78 and CHOP in the 50mW group showed significant differences (P<0.05). The protein expressions of CRT, GRP78 and CHOP in the 10mW and CRT in the 50mW group showed no significant differences (P>0.05). Cytoskeleton staining showed that, under the control conditions, endothelial cells displayed a few actin stress fibers. Exposure of endothelial cells to microwave caused a dramatic increase in the number of F-actin stress fibers. Maximal stress fiber formation occurred when endothelial cells were challenged for 3h or with 30mW microwave. Conclusion Microwave radiation can induce serious endoplasmic reticulum stress, resulting in cardiac microvascular endothelial cell damage.