摘要
目的:评价不同密度低温缺氧复氧大鼠心脏成纤维细胞(RCF)对心肌细胞损伤和细胞间偶联的影响。方法:体外培养RCF,采用随机数字表方法分为3组(n=12):密度0.5×10^(5)个/ml组(T 0.5组)、密度为1.0×10^(5)个/ml组(T_(1.0)组)和密度为2.0×10^(5)个/ml组(T_(2.0)组)。3组置于缺氧装置中,以5 L/min的速度持续吹入95%N 2+5%CO_(2)15 min进行缺氧处理,然后置入4℃冰箱培养1 h进行低温处理;培养完成后,置于37℃、含95%空气+5%CO_(2)培养箱中复氧4 h。上述处理结束后,3组分别与相同密度(1.0×10^(5)个/ml)的心肌细胞采用Transwell小室间接共培养16 h,心肌细胞接种于Transwell小室下层,RCF接种于Transwell小室上层。共培养结束后,收集心肌细胞,采用CCK8法检测细胞活力,流式细胞术检测细胞凋亡率,实时荧光定量PCR法检测缝隙连接蛋白43(Cx43)mRNA的表达,Western blot法检测Cx43及磷酸化Cx43(p-Cx43)的表达。结果:与T 0.5组相比,T_(1.0)组和T_(2.0)组心肌细胞活力、细胞凋亡率、Cx43、p-Cx43及Cx43 mRNA表达水平降低(P<0.01);与T_(1.0)组相比,T_(2.0)组心肌细胞活力、细胞凋亡率、Cx43和p-Cx43表达水平降低(P<0.01),心肌细胞Cx43 mRNA表达差异无统计学意义(P>0.05)。结论:低温缺氧复氧RCF在一定范围内呈密度依赖性地诱导心肌细胞损伤,其机制可能与下调Cx43的表达,降低Cx43的活性有关。
Objective To evaluate the effects of different densities of rat cardiac fibroblasts(RCF)subjected to hypothermic hypoxia-reoxygenation on cardiomyocyte injury and intercellular coupling.Methods RCF was cultured in vitro and divided into 3 groups(n=12 each)using a random number table method:RCF density 0.5×10^(5) cells/ml group(T0.5 group),RCF density 1.0×10^(5) cells/ml group(T_(1.0) group),and RCF density 2.0×10^(5) cells/ml group(T_(2.0) group).The three groups were placed in an anoxic device,into which 95%N2+5%CO_(2) was continuously blown at the speed of 5 L/min for 15 min,and then placed in a 4℃refrigerator for 1 h for low temperature treatment.After completion of culture,cells were placed in a incubator containing 95%air+5%CO_(2) at 37℃for 4 h of reoxygenation.After the end of culture,RCF in three groups were indirectly co-cultured with cardiomyocytes of the same density(1.0×10^(5) cells/ml)in a Transwell chamber for 16 h,cardiomyocytes were seeded in the lower chamber of Transwell,and RCF were seeded in the upper chamber of Transwell.After the end of co-culture,cardiomyocytes were collected for determination of the cell viability(by CCK8 method),apoptosis rate(by flow cytometry),expression of connexin 43(Cx43)mRNA(by real-time fluorescence quantitative polymerase chain reaction),and expression of Cx43 and phosphorylated Cx43(p-Cx43)(by Western blot).Results Compared with T0.5 group,the cell viability,apoptosis rate and expression of Cx43,p-Cx43 and Cx43 mRNA were significantly decreased in T_(1.0) and T_(2.0) groups(P<0.01).Compared with T_(1.0) group,the cell viability,apoptosis rate and expression of Cx43 and p-Cx43 were significantly decreased(P<0.01),and no significant change was found in expression of Cx43 mRNA in cardiomyocytes in T_(2.0) group(P>0.05).Conclusions RCF subjected to hypothermic hypoxia-reoxygenation induces cardiomyocyte injury in a density-dependent manner in a certain range,and the mechanism may be related to down-regulation of the expression of Cx43 and reduction of the acti
作者
吴学艳
高鸿
蒙富雪
牛振英
冯玉蓉
王贵龙
曹莹
易菁
佟睿
安丽
Wu Xueyan;Gao Hong;Meng Fuxue;Niu Zhenying;Feng Yurong;Wang Guilong;Cao Ying;Yi Jing;Tong Rui;An Li(School of Anesthesiology,Guizhou Medical University,Guiyang 550004,China;Department of Anesthesiology,Affiliated Hospital of Guizhou Medical University,Guiyang 550004,China;The Third Affiliated Hospital of Guizhou Medical University,Duyun 558000,China;Department of Anesthesiology,People′s Hospital of Liuzhi Region,Guizhou Province,Liupanshui 553400,China;Department of Anesthesiology,Zhijin People′s Hospital of Bijie City,Guizhou Province,Bijie 552100,China;School of Clinical Medicine,Guizhou Medical University,Guiyang 550004,China)
出处
《中华麻醉学杂志》
CAS
CSCD
北大核心
2022年第2期176-180,共5页
Chinese Journal of Anesthesiology
