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目的:培育L型电压门控钙通道(LTCCs)α1D亚型的基因突变纯合子(α1D-/-)小鼠、杂合子(α1D+/-)小鼠及野生型(α1D+/+)小鼠模型,并利用这3种不同基因型小鼠研究α1D通道亚基在内耳听觉平衡功能及心脏起博传导中的作用。方法:利用同窝生不同基因型小鼠为实验对象,采用听觉脑干反应(ABR)和耳蜗内电位(EP)检测技术和心电图(ECG)检测技术,检测和观察各基因型小鼠听觉功能、EP及ECG的PP间期和PR间期。利用游泳实验和滚桶实验检测各个基因型小鼠的平衡功能。结果:α1D+/+小鼠的听力正常,ABR的短声(Click)阈值为(34.8±5.7)dBSPL;EP均值为(105.3±3.1)mV。α1D+/-小鼠听力低于同窝α1D+/+小鼠,ABR的短声阈值为(54.4±12.4)dBSPL,α1D+/-小鼠的EP为(75.8±9.9)mV,其平衡功能正常。α1D-/-小鼠呈现全聋,ABR在100dB无反应100dBSPL,其EP值为(48.6±19.3)mV;α1D-/-小鼠表现出听觉功能丧失但其平衡功能正常。心电图检测显示α1D+/+小鼠心律正常;α1D+/-小鼠和α1D-/-小鼠显示出心动过缓、RR间期延长;α1D-/-小鼠的心率最慢。α1D+/-小鼠出现窦性心动过缓(RR间期:146±1.4ms),与α1D+/+小鼠的相比较[(117±0.4)ms]其RR间期明显延长,差异有统计学意义(P<0.05)。α1D-/-小鼠和α1D+/-小鼠有窦性心动过缓和房室传导阻滞,RR间期和PR间期均延长。α1D-/-小鼠的PR间期延长[(53±0.5)ms],与α1D+/+小鼠的(PR间期:38±0.3ms)相比,差异有统计学意义(P<0.05);α1D-/-小鼠的RR间期[(244±2.9)ms]延长,与α1D+/-小鼠[(146±1.4)ms]相比,差异有统计学意义(P<0.05)。结论:LTCCsα1D亚型是维持内耳听觉生理的关键钙通道,α1D通道亚型的缺失或功能受限均可导致影响听觉生理功能。但α1D亚基的缺失不影响小鼠的平衡功能,表明α1D亚基在前庭系统中的功能作用有限。LTCCsα1D亚型在心脏起博传导系统中也具重要生理作用。
OBJECTIVE: To develop a genetically modified homozygous (α1D - / -), heterozygous (α1D +/-) and wild-type (α1D + / +) mouse model of α1D subtype of voltage-gated calcium channels (LTCCs) , And the role of the α1D channel subunit in the auditory homeostasis and cardiac pacing transduction was studied using these three different genotype mice. Methods: Using different genotypes of littermates as experimental subjects, auditory brainstem response (ABR) and intracochlear potential (EP) detection and electrocardiogram (ECG) detection techniques were used to detect and observe the auditory function , PP interval and PR interval of EP and ECG. The swimming test and drum experiment were used to test the balance function of each genotype mouse. Results: The hearing of α1D + / + mice was normal. The threshold of ABR was (34.8 ± 5.7) dBSPL. The mean of EP was (105.3 ± 3.1) mV. The audiometry of α1D +/- mice was lower than that of littermate α1D + / + mice, and the short-tone threshold of ABR was (54.4 ± 12.4) dBSPL. The α1D +/- mouse had an EP of (75.8 ± 9.9) mV and its balance was normal. The α1D - / - mice showed complete deafness. The ABR showed no response at 100dB and the EP value was (48.6 ± 19.3) mV. The α1D - / - mice showed loss of auditory function but their balance function was normal. The electrocardiogram showed normal heart rhythm in α1D + / + mice; α1D +/- mice and α1D - / - mice showed bradycardia with prolonged RR intervals; α1D - / - mice had the slowest heart rate. α1D +/- mice developed sinus bradycardia (RR interval: 146 ± 1.4 ms), with a significant prolongation of RR interval compared with α1D + / + mice (117 ± 0.4 ms) with statistically significant differences Significance (P <0.05). Both α1D - / - mice and α1D +/- mice had sinus bradycardia and atrioventricular block, with prolonged RR and PR intervals. The PR interval prolongation in [53 ± 0.5] ms α1D - / - mice was statistically significant (P <0.05) compared with that in α1D + / + mice (PR ± 38 ± 0.3 ms) ; RR interval of α1D - / - mice [(244 ± 2.9) ms] was longer than that of α1D +/- mice [(146 ± 1.4) ms], the difference was statistically significant (P <0.05). CONCLUSION: The LTCCs α1D subtype is a key calcium channel for the maintenance of auditory physiology in the inner ear. The absence or limited function of α1D channel subtypes may lead to the impact of auditory physiological function. However, the absence of the α1D subunit did not affect the balance function in mice, suggesting that the α1D subunit has a limited functional role in the vestibular system. LTCCs α1D subtypes also play important physiological roles in cardiac pacing transduction systems.