摘要
目的探讨pH值降低对大鼠心肌细胞活力的影响并分析其可能机制。方法取新生SD大鼠5只，分离心脏培养原代心肌细胞，进行以下实验。（1）取原代心肌细胞，按照随机数字表法分为pH值7.4＋6 h组、pH值7.0＋6 h组、pH值6.5＋6 h组、pH值6.0＋6 h组、pH值6.5＋1 h组和pH值6.5＋3 h组，每组4孔。pH值7.4＋6 h组、pH值7.0＋6 h组、pH值6.5＋6 h组、pH值6.0＋6 h组细胞常规培养48 h后（下同）分别更换pH值为7.4、7.0、6.5、6.0的DMEM-F12（下同）培养基，培养6 h；pH值6.5＋1 h组、pH值6.5＋3 h组细胞更换pH值为6.5的培养基，分别培养1、3 h。噻唑蓝法检测细胞活力。（2）取原代心肌细胞，按照随机数字表法分为pH值7.4组、pH值6.5组和pH值6.5＋紫杉醇组，每组2孔。pH值7.4组细胞更换pH值为7.4培养基，pH值6.5组和pH值6.5＋紫杉醇组细胞更换pH值为6.5培养基，pH值6.5＋紫杉醇组细胞另加入终物质的量浓度为0.2 μmol/L紫杉醇，培养6 h。免疫荧光法检测细胞微管形态及密度。（3）取原代心肌细胞，同实验（2）分组处理，每组2孔，蛋白质印迹法检测细胞聚合态和游离态微管蛋白表达。（4）取原代心肌细胞，同实验（2）分组处理，每组4孔，噻唑蓝法检测紫杉醇处理后细胞活力。对数据行单因素方差分析、LSD-t检验。结果（1）pH值7.4＋6 h组、pH值7.0＋6 h组、pH值6.5＋6 h组、pH值6.0＋6 h组、pH值6.5＋1 h组、pH值6.5＋3 h组细胞活力分别为1.00±0.08、0.90±0.08、0.85±0.06、0.83±0.04、0.91±0.10、0.89±0.10。与pH值7.4＋6 h组比较，pH值7.0＋6 h组、pH值6.5＋6 h组、pH值6.0＋6 h组、pH值6.5＋1 h组、pH值6.5＋3 h组细胞活力均有不同程度下降（t＝2.476、4.002、4.996、2.168、2.400，P〈0.05）。（2）pH值7.4组细胞微管围绕细胞核呈放射状分布，管状结构清晰；与pH值7.4组比较，pH值6.5组细胞微管骨架破坏明显，表现为管状结构�
ObjectiveTo explore the effects of decline of pH value on cardiomyocyte viability of rats, and to analyze the possible mechanism.MethodsHearts of five newborn Sprague-Dawley rats were isolated, and then primary cardiomyocytes were cultured and used in the following experiments. （1） The primary cardiomyocytes were divided into pH 7.4＋ 6 h, pH 7.0＋ 6 h, pH 6.5＋ 6 h, pH 6.0＋ 6 h, pH 6.5＋ 1 h, and pH 6.5＋ 3 h groups according to the random number table, with 4 wells in each group. After being routinely cultured for 48 h （similarly hereinafter）, cells in pH 7.4＋ 6 h, pH 7.0＋ 6 h, pH 6.5＋ 6 h, and pH 6.0＋ 6 h groups were cultured with pH 7.4, pH 7.0, pH 6.5, and pH 6.0 DMEM-F12 medium （similarly hereinafter）, respectively, and then they were cultured for 6 h. Cells in pH 6.5＋ 1 h and pH 6.5＋ 3 h groups were cultured with pH 6.5 medium, and then they were cultured for 1 h and 3 h, respectively. Viability of cells was detected by methyl-thiazolyl-tetrazolium （MTT） method. （2） The primary cardiomyocytes were divided into pH 7.4, pH 6.5, and pH 6.5＋ taxol groups according to the random number table, with 2 wells in each group. Cells in pH 7.4 group were cultured with pH 7.4 medium, while cells in pH 6.5 and pH 6.5＋ taxol groups were cultured with pH 6.5 medium. Cells in pH 6.5＋ taxol group were added with taxol of a final molarity of 0.2 μmol/L in addition, and then they were cultured for 6 h. Morphology and density of microtubule of cells was detected by immunofluorescence assay. （3） The primary cardiomyocytes were grouped and treated as in experiment （2）, with 2 wells in each group. The expressions of polymerized microtubulin and free microtubulin were determined with Western blotting. （4） The primary cardiomyocytes were grouped and treated as in experiment （2）, with 4 wells in each group. Viability of cells after treated with taxol was detected by MTT method. Data were processed with one-way analysis of variance and LSD-t test.Results（1） The viabilit
作者
杨雷
赵利平
崔琳
黄耀
叶晶莹
张琼
张东霞
黄跃生
Yang Lei;Zhao Liping;Cui Lin;Huang Yao;Ye Jingying;Zhang Qiong;Zhang Dongxia;Huang Yuesheng(State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Burn Research, the First Affiliated Hospital of Army Medical University （the Third Military Medical University） , Chongqing 400038, Chin)
出处
《中华烧伤杂志》
CAS
CSCD
北大核心
2018年第5期303-308,共6页
Chinese Journal of Burns
基金
国家自然科学基金重点项目（81430042）
国家自然科学基金面上项目（81571903）
关键词
酸中毒
肌细胞
心脏
微管
细胞活力
Acidosis
Myocytes, cardiac
Microtubules
Cell viability
