摘要
目的探讨柯萨奇病毒B3(CoxsackieviruB3,CVB3)感染大鼠心室肌钙通道的基因表达特点及其功能改变。方法取1~3日龄新生SD大鼠15只行心室肌细胞原代培养,将培养48 h且已形成规律搏动的心室肌细胞随机分为病毒感染组(CVB3株感染培养,n=8)与正常对照组(n=7),比较两组大鼠的细胞形态、结构变化特点等;采用半定量逆转录聚合酶链反应(RT-PCR)检测两组大鼠L型钙通道亚单位mRNA的表达;应用全细胞膜片钳技术对CVB3感染前后心室肌细胞L型钙电流(ICa-L)变化进行测定,记录细胞膜电容(Cm)、电流密度、电压依赖性激活与失活等动力学参数。结果正常心室肌细胞主要表现为三角形、梭形、多边形等形态,搏动规律有力,搏动细胞百分比>95%;接种CVB3培养3 d后细胞搏动开始减慢且呈不规律搏动,细胞变圆、扁梭,折光性增强,最终停止搏动、脱落、死亡;CVB3感染3 d后,溶酶体增多,线粒体空泡变形,细胞核肿胀,核膜破裂。病毒感染组心室肌细胞L型钙通道α1和β亚单位mRNA表达水平分别为(4.02±0.07)、(2.08±0.06)均高于正常对照组(P<0.05);而两组α2/δ亚单位mRNA表达水平差异无统计学意义。在-20~+10 mV测试电压下,病毒感染组ICa-L密度为(-8.65±0.99),较正常对照组明显增加(P<0.05);但CVB3感染培养对大鼠心室肌细胞Cm、电压依赖性激活与失活特性无显著影响。病毒接种后第4天,病毒感染组CVB3特异蛋白基因片段RNA均为阳性,正常对照组CVB3特异蛋白基因片段RNA均为阴性,差异有统计学意义(P<0.05),病毒感染组病毒接种1~4 d CK-MB浓度均高于正常对照组(P<0.05)。结论 CVB3感染大鼠心室肌细胞L型钙通道α1和β亚单位mRNA表达上调,ICa-L密度增大,可能是CVB3感染对心室肌细胞产生损伤作用的病理机制。
OBJECTIVE To investigate the gene expression characteristics and functional changes of calcium channel in ventricular muscle of rat infected with coxsackievirus B3(CVB3). METHODS The ventricular myocytes of 15 newborn SD rats aged 1 to 3 days were taken for primary culture, and the ventricular myocytes that had been cultured for 48 hours and formed regular pulsation were randomly divided into virus infection group(CVB3 strain infection culture, n=8) and normal control group(n=7).The cell morphology and structural changes of the two groups of rats were compared;semi quantitative reverse transcription polymerase chain reaction(RT-PCR) was used to detect the expression of L-type calcium channel subunit mRNA in the two groups of rats;the whole cell patch clamp technique was used to detect the changes of L-type calcium current(ICa-L) in ventricular myocytes before and after CVB3 infection.The dynamic parameters of cell membrane capacitance(CM), current density, voltage dependent activation and inactivation were recorded. RESULTS The normal ventricular myocytes were mainly in the form of triangles, spindles and polygons, etc, with strong pulsating regularity, and the percentage of beating cells was more than 95% After 3 days of inoculation with CVB3, the pulsation of the cells began to slow down and showed irregular pulsation.The cells became round and flat, and the refractive index increased, and finally stopped pulsating, fell off and died.Three days after CVB3 infection, lysosomes increased, mitochondria vacuoles deformed, nucleus swelled and nuclear membrane ruptured.The mRNA expression levels of L-type calcium channel α 1 and β subunits of ventricular myocytes in the virus infection group were 4.02±0.07 and 2.08±0.06, respectively, higher than those in the normal control group(P< 0.05), while there was no significant difference in the mRNA expression of α 2/δ subunit between the two groups.Under the test voltage of-20~+10 mV, the density of ICa-L in the virus infected group was-8.65±0.99, significantly higher than that in normal control group(P<0.05), however, CVB3 infection had no significant effect on the Cm, voltage-dependent activation and inactivation characteristics of rat ventricular myocytes.On the 4 th day after virus inoculation, the RNA of CVB3 specific protein gene fragment in the viral infection group was all positive, and the CVB3 specific protein gene fragment RNA in the normal control group was negative(P<0.05), and the difference was statistically significant(P<0.05).The concentration of CK-MB in virus infected group was higher than that in normal control group for 1~4 days after virus inoculation(P< 0.05). CONCLUSION The mRNA expression of L-type calcium channel α 1 and β subunit was up-regulated, and the density of ICa-L was increased significantly in VB3 infected rat ventricular myocytes, which might be the pathological mechanism of CVB3 infection on ventricular myocytes.
作者
郭丽敏
张东华
史宁
王芳
赵微
GUO Li-min;ZHANG Dong-hua;SHI Ning;WANG Fang;ZHAO Wei(The Fourth Hospital of Hebei Medical University,Shijiazhuang,Hebei 050011,China)
出处
《中华医院感染学杂志》
CAS
CSCD
北大核心
2021年第2期161-165,共5页
Chinese Journal of Nosocomiology
基金
河北省卫生厅科研基金资助项目(20130549)。
作者简介
郭丽敏(1974-),女,本科,主管护师,研究方向:临床护理。
