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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(P<0.05)。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(P>0.05),照射后3h组和24h组差异显著(P<0.05);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(P<0.05)。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(P>0.05)。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。
Objective To investigate the relationship between myocardial microvascular endothelial cell injury and endoplasmic reticulum stress induced by microwave irradiation. Methods The cultured myocardial microvascular endothelial cells of 3 to 4 generations were randomly divided into control group and irradiation group. 1. The myocardial microvascular endothelial cells were irradiated with 10mW / cm2, 30mW / cm2 and 50mW / cm2 microwave respectively for 6min. Cells were harvested 24h after irradiation. The cells were exposed to 30 mW / cm2 microwave for 6 min, and then cultured for 1 h, 3 h or 24 h, the endothelial cells were collected, and the control group ended the experiment at 24 h. Apoptosis was detected by annexin V-propidium iodide double staining. Phagocytosis staining was used to observe the changes of the microvascular endothelial cell skeleton, and the damage of microvascular endothelial cells was evaluated. The endoplasmic reticulum stress CRT, GRP78 and CCAAT / CHOP protein expression, to evaluate whether microwave irradiation induced endoplasmic reticulum stress in microvascular endothelial cells. Results The dose-response study of endothelial cell apoptosis showed that the apoptotic rate of the irradiated group was (2.34 ± 0.15)% and (2.72 ± 0.96)% respectively at 10mW / cm2, 30mW / cm2 and 50mW / cm2 after microwave irradiation , (2.62 ± 0.34)% respectively, which was significantly different from that of the control group (0.88 ± 0.32)% (P <0.05). Apoptosis rates were (1.12 ± 0.15)%, (1.49 ± 0.54)% and (1.85 ± 0.45)% at 1, 3 and 24 h after irradiation at 30 mW / cm2, respectively. Compared with the control group (1.10% ± 0.28)%, the difference was not significant at 1h after irradiation (P> 0.05), and at 3h and 24h after irradiation the difference was significant (P <0.05) The protein expression of CRT, GRP78 and CHOP in 30mW group was increased by 124%, 76% and 256% respectively compared with the control group. The protein expression of CHOP in the 50mW group increased by 52% and 189% respectively compared with the control group. Compared with the control group, the expressions of GRP78 and CHOP in 30mW CRT, GRP78, CHOP GRP78 and 50mW group were significantly different (P <0.05). There was no significant difference in CRT protein expression between CRT, GRP78, CHOP and 50mW group in 10mW group (P> 0.05). The control group endothelial cells showed little actin fibers. The dramatic increase of filamentous actin stress fibers caused by exposure of endothelial cells to microwaves. The formation of maximal stress fibers occurred 3 h after irradiation of endothelial cells or 30 mW of irradiation power. Conclusion Microwave irradiation can induce severe endoplasmic reticulum stress and cause myocardial microvascular endothelial cell injury in rats.