AIM:To investigate the effects of autoantibodies against β_1-adrenoceptor in hepatitis virus myocarditis on action potential and L-type Ca^(2+) currents. METHODS:Fifteen samples of autoantibodies against β_1- adreno...AIM:To investigate the effects of autoantibodies against β_1-adrenoceptor in hepatitis virus myocarditis on action potential and L-type Ca^(2+) currents. METHODS:Fifteen samples of autoantibodies against β_1- adrenoceptor positive sera of patients with hepatitis virus myocarditis were obtained and IgGs were purified by octanoic acid extraction.Binding of autoantibodies against β_1- adrenoceptor to guinea pig cardiac myocytes was examined by immunofluorescence.Using the patch clamp technique, the effects on the action potential and I_(ca-L) of guinea pig cardiac myocytes caused by autoantibodies against β_1-adrenoceptor in the absence and presence of metoprolol were investigated. Cell toxicity was examined by observing cell morphology and permeability of cardiac myocytes to trypan blue. RESULTS:The specific binding of autoantibodies against β_1-adrenoceptor to guinea pig cardiomyocytes was observed. Autoantibodies against β_1-adrenoceptor diluted at 1:80 prolonged APO_(20),APD_(50)and APD_(90) by 39.2%,29.1% and 15.2% respectively,and only by 7.2%,5.3% and 4.1% correspondingly in the presence of 1 μmol/L metoprolol. Autoantibodies against β_1-adrenoceptor diluted at 1:80, 1:100 and 1:120 significantly increased the I_(ca-l) peak current amplitude at 0 mV by 55.87±4.39%,46.33±5.01% and 29.29±4.97% in a concentration-dependent manner.In contrast,after blocking of β_1-adrenoceptors (1 μmol/L metoprolol),autoantibodies against β_1-adrenoceptor diluted at 1:80 induced a slight increase of I_(ca-L) peak amplitude only by 6.81±1.61%.A large number of cardiac myocytes exposed to high concentrations of autoantibodies against β_1- adrenoceptor (1:80 and 1:100) were turned into rounded cells highly permeable to trypan blue. CONCLUSION:Autoantibodies against β_1-adrenoceptor may result in arrhythmias and/or impairment of myocardiums in HVM,which would be mediated by the enhancement of I_(ca-L.展开更多
目的探讨miR-155-5p对骨关节炎软骨细胞增殖和凋亡的影响及分子机制.方法分离培养软骨细胞,设置对照(NC)组、白细胞介素-1β(IL-1β)组、1、2、3、4、5组.实时荧光定量PCR(RT-qP-CR)检测miR-155-5p和Ⅲ型纤维连接蛋白结构域蛋白3B(FNDC3...目的探讨miR-155-5p对骨关节炎软骨细胞增殖和凋亡的影响及分子机制.方法分离培养软骨细胞,设置对照(NC)组、白细胞介素-1β(IL-1β)组、1、2、3、4、5组.实时荧光定量PCR(RT-qP-CR)检测miR-155-5p和Ⅲ型纤维连接蛋白结构域蛋白3B(FNDC3B)mRNA表达水平;蛋白质印迹(West-ern blot)法检测FNDC3B、细胞周期蛋白D1(Cyclin D1)、裂解的半胱氨酸天冬氨酸蛋白酶-3(Cleaved-cas-pase-3)、磷酸化p65(p-p65)、磷酸化核因子kappaB抑制蛋白α(p-IκBα)蛋白表达;细胞计数试剂盒8(CCK-8)检测细胞存活率;流式细胞术检测细胞凋亡;双荧光素酶报告实验验证miR-155-5p和FNDC3B的靶向关系.结果IL-1β诱导的软骨细胞中miR-155-5p高表达(3.12±0.31 vs 1.00±0.10),FNDC3B mRNA(0.36±0.03 vs 1.03±0.11)和蛋白(0.40±0.04 vs 0.88±0.08)低表达(P<0.05),且IL-1β可诱导软骨细胞凋亡(20.46±2.05 vs 6.33±0.60).抑制miR-155-5p表达,IL-1β诱导的软骨细胞中细胞存活率升高(83.12±8.30 vs 49.05±4.91),细胞凋亡率降低(9.86±0.98 vs 18.26±1.80)(P<0.05);过表达FNDC3B,细胞存活率升高(89.05±8.90 vs 50.77±5.10),细胞凋亡率降低(10.66±1.08 vs 19.89±1.95)(P<0.05).miR-155-5p靶向调控FNDC3B;抑制miR-155-5p表达可抑制p-p65、p-IκBα表达;低表达FNDC3B可以部分逆转miR-155-5p低表达对IL-1β诱导的软骨细胞增殖、凋亡及NF-κB信号通路的影响.结论抑制miR-155-5p表达可能通过上调FNDC3B促进IL-1β诱导的软骨细胞增殖,抑制细胞凋亡,且其可能与NF-κB信号通路有关.展开更多
基金Supported by the National Natural Science Foundation of China,NO.39970306
文摘AIM:To investigate the effects of autoantibodies against β_1-adrenoceptor in hepatitis virus myocarditis on action potential and L-type Ca^(2+) currents. METHODS:Fifteen samples of autoantibodies against β_1- adrenoceptor positive sera of patients with hepatitis virus myocarditis were obtained and IgGs were purified by octanoic acid extraction.Binding of autoantibodies against β_1- adrenoceptor to guinea pig cardiac myocytes was examined by immunofluorescence.Using the patch clamp technique, the effects on the action potential and I_(ca-L) of guinea pig cardiac myocytes caused by autoantibodies against β_1-adrenoceptor in the absence and presence of metoprolol were investigated. Cell toxicity was examined by observing cell morphology and permeability of cardiac myocytes to trypan blue. RESULTS:The specific binding of autoantibodies against β_1-adrenoceptor to guinea pig cardiomyocytes was observed. Autoantibodies against β_1-adrenoceptor diluted at 1:80 prolonged APO_(20),APD_(50)and APD_(90) by 39.2%,29.1% and 15.2% respectively,and only by 7.2%,5.3% and 4.1% correspondingly in the presence of 1 μmol/L metoprolol. Autoantibodies against β_1-adrenoceptor diluted at 1:80, 1:100 and 1:120 significantly increased the I_(ca-l) peak current amplitude at 0 mV by 55.87±4.39%,46.33±5.01% and 29.29±4.97% in a concentration-dependent manner.In contrast,after blocking of β_1-adrenoceptors (1 μmol/L metoprolol),autoantibodies against β_1-adrenoceptor diluted at 1:80 induced a slight increase of I_(ca-L) peak amplitude only by 6.81±1.61%.A large number of cardiac myocytes exposed to high concentrations of autoantibodies against β_1- adrenoceptor (1:80 and 1:100) were turned into rounded cells highly permeable to trypan blue. CONCLUSION:Autoantibodies against β_1-adrenoceptor may result in arrhythmias and/or impairment of myocardiums in HVM,which would be mediated by the enhancement of I_(ca-L.
文摘目的探讨miR-155-5p对骨关节炎软骨细胞增殖和凋亡的影响及分子机制.方法分离培养软骨细胞,设置对照(NC)组、白细胞介素-1β(IL-1β)组、1、2、3、4、5组.实时荧光定量PCR(RT-qP-CR)检测miR-155-5p和Ⅲ型纤维连接蛋白结构域蛋白3B(FNDC3B)mRNA表达水平;蛋白质印迹(West-ern blot)法检测FNDC3B、细胞周期蛋白D1(Cyclin D1)、裂解的半胱氨酸天冬氨酸蛋白酶-3(Cleaved-cas-pase-3)、磷酸化p65(p-p65)、磷酸化核因子kappaB抑制蛋白α(p-IκBα)蛋白表达;细胞计数试剂盒8(CCK-8)检测细胞存活率;流式细胞术检测细胞凋亡;双荧光素酶报告实验验证miR-155-5p和FNDC3B的靶向关系.结果IL-1β诱导的软骨细胞中miR-155-5p高表达(3.12±0.31 vs 1.00±0.10),FNDC3B mRNA(0.36±0.03 vs 1.03±0.11)和蛋白(0.40±0.04 vs 0.88±0.08)低表达(P<0.05),且IL-1β可诱导软骨细胞凋亡(20.46±2.05 vs 6.33±0.60).抑制miR-155-5p表达,IL-1β诱导的软骨细胞中细胞存活率升高(83.12±8.30 vs 49.05±4.91),细胞凋亡率降低(9.86±0.98 vs 18.26±1.80)(P<0.05);过表达FNDC3B,细胞存活率升高(89.05±8.90 vs 50.77±5.10),细胞凋亡率降低(10.66±1.08 vs 19.89±1.95)(P<0.05).miR-155-5p靶向调控FNDC3B;抑制miR-155-5p表达可抑制p-p65、p-IκBα表达;低表达FNDC3B可以部分逆转miR-155-5p低表达对IL-1β诱导的软骨细胞增殖、凋亡及NF-κB信号通路的影响.结论抑制miR-155-5p表达可能通过上调FNDC3B促进IL-1β诱导的软骨细胞增殖,抑制细胞凋亡,且其可能与NF-κB信号通路有关.
