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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 n＝12)：密度0.5×10n 5个/ml组(Tn 0.5组)、密度为1.0×10n 5个/ml组(Tn 1.0组)和密度为2.0×10n 5个/ml组(Tn 2.0组)。3组置于缺氧装置中，以5 L/min的速度持续吹入95%Nn 2+5%COn 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%COn 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(1.0×10n 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。n 结果:与Tn 0.5组相比，Tn 1.0组和Tn 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(n P<0.01)；与Tn 1.0组相比，Tn 2.0组心肌细胞活力、细胞凋亡率、Cx43和p-Cx43表达水平降低(n P0.05)。n 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 n in vitro and divided into 3 groups (n n=12 each) using a random number table method: RCF density 0.5×10n 5 cells/ml group (Tn 0.5 group), RCF density 1.0×10n 5 cells/ml group (Tn 1.0 group), and RCF density 2.0×10n 5 cells/ml group (Tn 2.0 group). The three groups were placed in an anoxic device, into which 95% Nn 2 + 5% COn 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% COn 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 (1.0×10n 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).n Results:Compared with Tn 0.5 group, the cell viability, apoptosis rate and expression of Cx43, p-Cx43 and Cx43 mRNA were significantly decreased in Tn 1.0 and Tn 2.0 groups (n P<0.01). Compared with Tn 1.0 group, the cell viability, apoptosis rate and expression of Cx43 and p-Cx43 were significantly decreased (n P0.05).n 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 activity of Cx43.