【作者】 牟娇；

【导师】 何作云；

【作者基本信息】 第三军医大学 ， 内科学， 2005， 博士

【摘要】 目的 过去,人们比较重视心肌细胞主动收缩力的产生和影响机制,而忽略了心肌细胞被动生物力学特性。然而,根据近年来大量的研究结果,我们提出在心肌细胞病理情况下,后者可能更为重要。本研究目的就在于观察肥大心肌细胞骨架、生物力学特性的变化规律,并进一步阐明细胞骨架改变对肥大心肌细胞生物力学特性的具体影响及其调节机制。 方法 利用分步消化—差速贴壁法培养新生Wistar大鼠心肌细胞。将肾上腺素加入心肌细胞建立肥大心肌细胞模型后,再分别加入细胞骨架干预剂秋水仙素(Col,细胞骨架微管解聚剂)、细胞松弛素D(Cyto-D,细胞骨架微丝解聚剂)和紫杉醇(Taxol,细胞骨架微管聚合剂)。同时,在培养的肥大心肌细胞中加入Genistein以抑制蛋白酪氨酸激酶(PTK)活性。光学显微镜下观察心肌细胞搏动频率;利用同步摄像技术和计算机图像分析系统测定心肌细胞收缩前后表面积,心肌细胞收缩幅度以心肌细胞收缩前后表面积的变化表示;通过微管吸吮法检测心肌细胞粘弹性。RT—PCR法检测心肌细胞骨架蛋白(包括微丝蛋白actin、α-actinin、微管蛋白α-tubulin以及结蛋白desmin)mRNA的表达。分别采用免疫细胞荧光化学与Western Blot检测细胞骨架蛋白的分布、密度及含量。同时,我们还通过Western Blot测定了细胞骨架蛋白磷酸化水平。此外,我们采用PTK活性检测试剂盒测定了细胞骨架相关蛋白激酶的活性。 结果 与正常心肌细胞比较,肾上腺素作用后12h心肌细胞的收缩频率增快,收缩幅度增强(P<0.05),其粘性系数显著增大(P<0.01),而弹性系数明显减小(P<0.01)。随着肾上腺素作用时间的延长(加入肾上腺素作用后12h、24h、48h及72h),心肌细胞的收缩频率及收缩幅度均不断增加,粘性系数持续增大,而弹性系数则不断减小;RT-PCR结果显示与正常心肌细胞比较,肥大心肌细胞内细胞骨架各成分mRNA表达显著增强(P<0.01)。免疫荧光化学及Western blot结果表明肥大心肌细胞骨架成分蛋白含量、密度与正常心肌细胞比较均明显增加(P<0.01),但细胞骨架的分布不均匀,排列紊乱而不规则;同时,肥大心肌细胞骨架各成分蛋白的磷酸化水平明显升高更多还原

【Abstract】 Objective In the past, investigators relatively attached importance to the produce of initiative contractibility of cardiomyocytes while neglected the passive biomechanic properties and the regulative mechanism. However, according to the much research of recent years, we supposed that the latter may be more important especially on the condition of cardiac pathologies. In the study, we investigated the change rule of the cytoskeleton and biomechanics of hypertrophic cardiomyocytes and furthermore illuminated the influence of the cytoskeletal change on biomechanics of cardiomyocyte and the regulative mechanism.Methods The neonatal rat cardiomyocytes were cultured by the method of digestion step by step and adherence with different speeds. After cultured with adrenalin, the neonatal rat cardiomyocytes were respectively incorporated with colchicine (Col, used to disrupt the microtubule cytoskeleton), cytochalasin D (Cyto-D, used to disrupt the actin cytoskeleton) and Taxol (used to stabilize the microtubules). On the other hand, the inhibitor of protein tyrosine kinases (PTKs), Genistein was added into the cardiomyocytes with adrenalin. The cell area before and after cell contraction was measured by the technique of photograph at equal pace and image analysis system of computer. The cardiomyocytes’ contractile amplitude was figured by the cell area changes before and after cell contraction. The viscoelastic biomechanic properties of the cardiomyocytes were measured by micropipette aspiration technique. RT-PCR analyzed the mRNA expression of the cytoskeletal protein (including actin, a -actinin, a -tubulin and desmin). The distribution, density and the content change of the myocardial cytoskeletal proteins were respectively observed by immunocytochemistry technique and Western blot. At the same time, we also detected the phosphorylation level of the myocardial cytoskeletal protein. Moreover, we measured the activity of the cytoskeletal relative protein kinase by the PTK measured kit.Results In comparison with with normal contrast, the contractibility and beating frequency of the hypertrophic cardiomyocytes increased slightly (P<0.05); the viscouscoefficient of the hypertrophic cardiomyocytes increased markedly (P<0.01)while the elastic coefficient decreased obviously (P<0.01).With the prolonging of adrenine function (12h, 24h, 48h, 72h after the adrenine being added), the contractibility and beating frequency of the hypertrophic cardiomyocytes increased continuously; the viscous coefficient of the hypertrophic cardiomyocytes also increased unceasingly while the elastic coefficient decreased uninterruptly. The result of RT-PCR showed that the mRNA expression of the hypertrophic cardial cytoskeletal protein increased obviously as compared with the normal contrast. The density and content of the hypertrophic myocardial cytoskeletal protein were much more than the normal ones(P<0.01). The distribution of the the hypertrophic myocardial cytoskeletal protein was irregular and the alignment was indiscriminate. In contrast with the unsettled hypertrophic cardiomyocytes, the content and density of microtubule protein a -tubulin of the hypertrophic cardiomyocytes incorporated with Col decreased obviously(P<0.01)and the alignment of microtubule was relatively well-arranged; meanwhile, the phosphorylation level of the myocardial cytoskeletal protein increased distinctly(P<0.01). In contrast with the unsettled hypertrophic cardiomyocytes, the content and density of microfilament protein actin and a -actinin of the hypertrophic cardiomyocytes incorporated with cytochalasin D decreased obviously(P<0.01)and the alignment of microfilament was relatively well-arranged. Compared with the unsettled hypertrophic cardiomyocytes, the content and density of microtubule protein a -tubulin of the hypertrophic cardiomyocytes incorporated with Taxol increased obviously (PO.Ol); the distribution of microtubule protein was more irregular while the alignment of microtubule was more indiscrimented . In contrast with the unsettled hypertrophic group, the viscous coefficient of hypertrophic cardiomyocytes incorporated with Col and Cyto-D decreased distinctly(P<0.01) while the elastic coefficient increased obviously(P<0.01). The contractibility also increased significantly as compared with the unsettled hypertrophic cardiomyocytes(P<0.01). With the prolonging of the drug intervention, the above-mentioned changes became more and more obvious. In contrast with the unsettled hypertrophic group, the viscous coefficient of hypertrophic cardiomyocytes incorporated with Taxol increased distinctly(P<0.01) while the elastic coefficient decreased obviously(P<0.01). The contractibility also decreased significantly compared with the unsettled hypertrophic cardiomyocytes(P<0.01). With the prolonging of the drugintervention, the above-mentioned changes became more and more obvious. In contrast with the hypertrophic group, the phosphorylation level of the myocardial cytoskeletal protein(P<0.01) and the activity of the cytoskeletal relative protein kinase in the hypertrophic cardiomyocytes incorporated with Genistein decreased markedly(P<0.01). The viscous coefficient of the cardiomyocytes incorporated with Genestein increased obviously (P<0.01) while the elastic coefficient reduced markedly(P<0.01)as compared with he unsettled hypertrophic cardiomyocytes. However, the contractibility of the cardiomyocytes decreased compared with the contrast group(P<0.01).Conclusion The result of the experiment showed that during the course of the cardiomyocytes’ switch from the normal to hypertrophic state, the passive biomechanics properties (viscoelasticity) of the cardiomyocytes changed obviously, that was, the stiffness of the cardiomyocytes augmented and the contractile resisting force increased. In this course, the contractile increasement may be a compensatory mechanism that the hypertrophic cardiomyocytes overcame the augmented contractile resistance. The result showed that the density and content of the hypertrophic myocardial cytoskeletal protein were much more than the normal ones. The distribution of the hypertrophic myocardial cytoskeletal protein was irregular and the alignment was indiscrimented. After the hypertrophic cardiomyocytes were intervened by Colchicine and cytochalasin D, the content and density of microfilament or microtubule protein of the cardiomyocytes decreased obviously and the alignment of microfilament or microtubule was relatively well-arranged. Meanwhile, the viscous coefficient of hypertrophic cardiomyocytes incorporated with Col and Cyto-D decreased distinctly while the elastic coefficient increased obviously. The contractibility also increased significantly compared by the hypertrophic contrast group. While the hypertrophic cardiomyocytes were intervened by Taxol, the content and density of microtubule protein a -tubulin increased obviously; the distribution of microtubule was more irregular and the alignment of microtubule was more confused. The viscous coefficient of hypertrophic cardiomyocytes incorporated with Taxol increased distinctly ; the elastic coefficient decreased obviously while the contractibility also decreased significantly. When the PTK inhibitor was added into the hypertrophic cardiomyocytes, viscous coefficient of the cardiomyocytes increased obviously while the elastic coefficient reduced markedly; however, the contractibility of the cardiomyocytesdecreased. The above-mentioned result strongly suggested that the change of passive biomechanic property (viscoelastic resistance) of cardiomyocytes should be more important than that of the contractibility on the condition of the cardiac hypertrophic pathology. Further more, the study also showed that the change of the cytoskeletal protein, in particular their chang of distribution, density and phosphorylation level may play an important role in the biomechanics regulation of the hypertrophic cardiomyocytes. Moreover, the result strongly suggested that Colchicine and cytochalasin D should improve the contractile disturbance of hypertrophic and failured myocardium such that it could apply a new train of thought to the prevention and cure of heart failure.更多还原