肌钙蛋白I3基因表达下调对大鼠胚胎心肌细胞H9C2生物学特性的影响  

Effect of silencing troponin I3 gene expression on biological property of rat embryonic H9C2 cardiomyocytes

作　　者：张璐璐[1] 郑春杨 刘红波 姜红堃[3] 邱广蓉[1] Zhang Lulu;Zheng Chunyang;Liu Hongbo;Jiang Hongkun;Qiu Guangrong(Department of Medical Genetics, School of Life Sciences, China Medical University, Shenyang 110122, China;Department of Medical Statistics, College of Public Health, China Medical University, Shenyang 110122, China;Department of Pediatrics, the First Affiliated Hospital, China Medical University, Shenyang 110001, China)

机构地区：[1]中国医科大学生命科学学院医学遗传学教研室,沈阳110122 [2]中国医科大学公共卫生学院卫生统计学教研室,沈阳110122 [3]中国医科大学附属第一医院儿科,沈阳110001

出　　处：《中华实用儿科临床杂志》2019年第9期698-702,共5页Chinese Journal of Applied Clinical Pediatrics

基　　金：国家自然科学基金(81070131);辽宁省高等学校优秀人才支持计划(LJQ2012069);辽宁省自然科学基金(201602852).

摘　　要：目的探讨肌钙蛋白I3(Tnni3)基因表达下调对大鼠胚胎心肌细胞H9C2生物学特性的影响。方法培养大鼠胚胎心肌细胞H9C2,分为2组:转染阴性对照小干扰RNA(NC-siRNA)组和转染Tnni3小干扰RNA(Tnni3-siRNA)组。分别于转染后48 h、72 h收集细胞,实时定量PCR检测Tnni3 mRNA和Caspase-3 mRNA表达,Western blot检测Tnni3蛋白、Cyclin A1蛋白和Cyclin B1蛋白表达,Annexin V-异硫氰酸荧光素(FITC)凋亡检测试剂盒检测细胞凋亡,细胞计数试剂盒(CCK-8)检测细胞增殖,流式细胞仪检测细胞周期变化。结果与转染NC-si-RNA组比较,转染Tnni3-siRNA 48 h,Tnni3基因mRNA(0.27±0.05比1.00±0.00)和蛋白(0.18±0.03比1.00±0.00)表达降低,差异均有统计学意义(t=25.26、47.40,均P<0.01)。转染NC-siRNA组和转染Tnni3-siRNA组H9C2细胞均出现细胞凋亡。与转染NC-siRNA组比较,转染Tnni3-siRNA 72 h,H9C2细胞凋亡率明显增高[(11.30±1.85)%比(0.33±0.15)%],Caspase-3基因mRNA表达升高(1.39±0.13比1.00±0.00),差异均有统计学意义(t=10.24、5.19,均P<0.01)。与转染NC-siRNA组比较,转染Tnni3-siRNA组表现为显著的时间依赖性细胞增殖能力降低(48 h:0.32±0.06比0.46±0.03;72 h:0.31±0.01比0.63±0.04;96 h:0.36±0.01比0.75±0.04),差异均有统计学意义(t=3.62、13.45、16.39,均P<0.05)。转染Tnni3-siRNA 72 h,G1期、S期和G2期细胞比例分别为(71.25±3.82)%、(18.28±2.78)%和(9.94±1.09)%。与转染NC-siRNA组比较,转染Tnni3-siRNA组G2期细胞比例明显增加[(9.94±1.09)%比(4.54±0.99)%],Cyclin A1蛋白表达升高(1.89±0.09比1.00±0.00),Cyclin B1蛋白表达降低(0.47±0.06比1.00±0.00),差异均有统计学意义(t=6.35、17.12、15.32,均P<0.01)。结论Tnni3基因表达下调能够诱导大鼠胚胎心肌细胞H9C2细胞凋亡,抑制细胞增殖,导致细胞周期阻滞于G2期。Objective To investigate the effect of silencing troponin I3 (Tnni3) gene expression on biological property of rat embryonic H9C2 cardiomyocytes. Methods The rat embryonic H9C2 cardiomyocytes were cultured and divided into 2 groups: control group transfected with negative control small interfering RNA (NC-siRNA group) and experimental group transfected with Tnni3 small interfering RNA (Tnni3-siRNA group). At 48 h, 72 h after transfection, the cells were collected, and real time quantitative polymerase chain reaction (qPCR)was used to detect the mRNA expressions of Tnni3 and Caspase-3, and Western blot was used to detect the protein expressions of Tnni3, Cyclin A1 and Cyclin B1.Annexin V-fluorescein isothiocyanate(FITC) apoptosis detection kit was used to analyze cell apoptosis.Cell proliferation was measured by Cell Counting Kit-8 (CCK-8) solution and cell cycle was detected by flow cytometry. Results At 48 h post-transfection with Tnni3-siRNA, H9C2 cells exhibited a significant decrease in Tnni3 mRNA (0.27±0.05 vs. 1.00±0.00) and protein (0.18±0.03 vs. 1.00±0.00) compared with those transfected with NC-siRNA, and the differences were statistically significant (t=25.26, 47.40, all P<0.01). Apoptotic cells were observed in the NC-siRNA group and the Tnni3-siRNA group.At 72 h post-transfection, the percentage of apoptotic cells significantly increased in H9C2 cells transfected with Tnni3-siRNA [(11.30±1.85)% vs.(0.33±0.15)%] compared with those transfected with NC-siRNA, an increased expression of Caspase-3 mRNA was also observed in Tnni3-siRNA-transfected H9C2 cells (1.39±0.13 vs. 1.00±0.00), and the differences were statistically significant (t=10.24, 5.19, all P<0.01). Compared with NC-siRNA-transfected H9C2 cells, a time-dependent reduction in cell proliferation was observed in Tnni3-siRNA-transfected H9C2 cells (48 h: 0.32±0.06 vs. 0.46±0.03;72 h: 0.31±0.01 vs. 0.63±0.04;96 h: 0.36±0.01 vs 0.75±0.04), and the differences were statistically significant (t=3.62, 13.45, 16.39, all P<0.01). At 72 h p

关 键 词：肌钙蛋白I3基因 细胞凋亡 细胞增殖 细胞周期 

分 类 号：R725.4[医药卫生—儿科]