辛伐他汀对平滑肌祖细胞分化、增殖、迁移及黏附能力的影响

目的：观察辛伐他汀对骨髓源性平滑肌祖细胞（SPC ）分化及增殖的影响,筛选新一代包被洗脱支架药物。方法：采用密度梯度离心法从大鼠骨髓获取单个核细胞,接种于纤维连接素包被的培养板,培养8d后,以平滑肌肌动蛋白（α-SM A ）免疫荧光染色鉴定骨髓源性SPC ,并在倒置荧光显微镜下计数。收集培养8d单个核细胞、贴壁细胞,分别加入不同浓度辛伐他汀（0.01-10μmol/L ）培养8d、24h。分别采用氚标记胸苷（3 H-TdR）摄入法、改良的Boyden小室和黏附能力测定实验观察SPC的分化、增殖、迁移和黏附能力。结果：与对照组（无辛伐他汀干预）相比,0.01μmol/L辛伐他汀显著抑制骨髓单个核细胞向SPC分化[（85±4）个比（79±5）个]、增殖[（4070±184）个比（3833±126）个]、迁移[（44±3）个比（39±3）个]和黏附[（59±5）个比（52±4）个], P均＜0.05,且随辛伐他汀浓度增加S PC数量显著减少（ P＜0.01）。结论：辛伐他汀可抑制骨髓源性平滑肌祖细胞分化、增殖、迁移及黏附能力。     

兔左室楔形心肌组织块对药物致心律失常作用评估的价值

目的探讨冠状动脉灌流的兔左室楔形心肌组织块模型对药物致心律失常作用评价的价值。方法对6种临床上致心律失常发生率已知的药物,应用冠状动脉灌流的兔左室楔形心肌块对其致心律失常危险度进行评价。采用浮置玻璃微电极法同步记录心内膜、心外膜心肌细胞跨膜动作电位及跨室壁心电图。观察记录QT间期、复极离散度和心律失常来量化每种药物的相对危险度。结果临床上无致心律失常作用的药物其危险度评分小于等于0.1,临床上有致心律失常作用的药物其危险度评分介于1.2至9.8之间。结论应用冠状动脉灌流的兔左室楔形心肌组织块模型评价药物致心律失常作用具有较高的价值。     

Protection of reoxygenated cardiomyocytes against sarcolemmal fragility: the role of glutathione

This study addressed the question of whether the sarcolemmal fragility of cardiomyocytes after anoxia and subsequent reoxygenation can be altered by modulation of the cellular glutathione state. Isolated ventricular cardiomyocytes (from adult rats) were exposed to 120 min anoxia and subsequently to 30 min reoxygenation. Osmotic stress was generated by reduction of medium osmolarity from 270 to 80 mosmol/l and sarcolemmal fragility assessed by the leakage of lactate dehydrogenase (LDH). Under normoxic conditions 6.7±1.0 % of total LDH activity was found extracellularly. Hyposmolar reoxygenation, but not hypoosmolar anoxia, increased LDH release (17.9±2.7% of total, P<0.05). Increasing cellular glutathione content by pretreatment with N-acetylcysteine (1 mM) reduced LDH release following hyposmolar reoxygenation (12.3±1.9% vs. 18.2±2.9% of LDH in medium, P<0.05). Depletion of glutathione content by pretreatment with buthionine sulphoximine (BSO, 200 μM), increased LDH release following osmotic stress already in normoxia (10.5±1.8% of LDH in medium; P<0.05 vs. no BSO), and even further after reoxygenation (21.8±3.2%, P<0.05 vs. normoxia). We conclude that the increased sarcolemmal fragility in reoxygenated cardiomyocytes is due to reoxygenation in the presence of reduced antioxidant defence. Received: 7 January 1999 / Received after revision: 23 March 1999 / Accepted: 31 March 1999     

心肌干细胞移植可短期内改善心肌梗死心电生理稳定性和提高室颤阈值

背景：课题组前期研究已证实心肌干细胞移植中期(6周)能有效改善心肌梗死大鼠的心电生理稳定性和室颤阈值。目的：比较心肌干细胞和骨髓间充质干细胞移植对心肌梗死大鼠心电生理学稳定性和室颤阈值的短期疗效差异。方法：取30只健康雄性SD大鼠,开胸结扎大鼠的左前降支冠状动脉建立心肌梗死动物实验模型,随机分成3组,分别为心肌干细胞移植组,骨髓间充质干细胞移植组,PBS对照组,每组10只。分别于建模成功2周后在梗死区心肌内局部注射0.1 mL PBS悬浮的5×10⁶ PKH26标记的心肌干细胞、0.1 mL PBS悬浮的5×10⁶ PKH26标记的骨髓间充质干细胞及0.1 mL PBS。植入2周后,再次开胸检测大鼠心脏梗死区、梗死边缘区、非梗死区的心电生理特性和室颤阈值。实验结束后,分离心脏梗死边缘区进行病理切片及荧光显微镜检查PHK26标记的心肌干细胞和骨髓间充质干细胞缝隙连接蛋白43表达情况。结果与结论：心肌干细胞移植组单极电图纠正的激动恢复时间离散度、电刺激所激发的恶性心律失常及梗死区、梗死边缘区、非梗死区的室颤阈值与PBS组、骨髓间充质干细胞移植组相比差异有显著性意义(P < 0.05);骨髓间充质干细胞移植组单极电图纠正的激动恢复时间离散度及非梗死区的室颤阈值与PBS组相比差异有显著性意义。病理结果提示在心肌干细胞组、骨髓间充质干细胞组梗死边缘区发现有心肌干细胞、骨髓间充质干细胞的存在。通过组织免疫荧光检测,PKH26标记的心肌干细胞表达较多的缝隙连接蛋白43,而PKH26标记的骨髓间充质干细胞很少表达缝隙连接蛋白43,PBS组不表达缝隙连接蛋白43。上述结果提示心肌干细胞移植短期内心电生理学特性改善的效应较骨髓间充质干细胞优越,且改善效应与缝隙连接蛋白43相关。 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

利拉鲁肽对缺氧/复氧诱导的乳鼠心肌细胞损伤的影响

[目的]观察新型降糖药物利拉鲁肽对缺氧/复氧(H/R)诱导的乳鼠心肌细胞损伤的影响.[方法]将体外培养Wistar大鼠乳鼠心肌细胞进行分组:单纯H/R组(A组)、利拉鲁肽组+H/R组(B组)、正常对照组(C组),将A,B两组细胞同时进行H/R损伤,复氧后分别检测3组的乳酸脱氢酶(LDH)、丙二醛含量(MDA)、超氧化酶歧化酶(SOD)活性及各组心肌细胞凋亡率.[结果]A组与C组比较,其细胞培养液中LDH、MDA含量、细胞凋亡率均增加,SOD活性降低,且差异有显著性(P＜0.01).与A组相比,B组LDH、MDA含量、细胞凋亡率则明显降低(P＜0.01),但仍高于C组,且差异有显著性(P＜0.01);SOD活性较A组增高,差异亦有显著性(P＜0.01).[结论]H/R可以造成心肌细胞的损伤,增加细胞的凋亡;利拉鲁肽作为胰高血糖素样肽-1类似物,其直接作用于心肌细胞,可减轻H/R造成的心肌细胞损伤,抑制心肌细胞的凋亡,具有潜在的心脏保护作用.     

硝苯地平对自发性高血压大鼠左心室肥厚心肌细胞蛋白偶联受体激酶的表达及亚细胞分布的影响

目的观察硝苯地平对自发性高血压大鼠(SHR)左心室肥厚心肌细胞GRK2的表达及亚细胞分布的影响,探讨硝苯地平逆转心肌肥大的可能机制。方法以SHR为研究对象,分为对照组(SHRC)、低剂量硝苯地平干预组(SHRL:10mg·kg-1.d-1)及高剂量硝苯地平干预组(SHRH:30mg·kg-1.d-1)(n=6),由6月龄喂养至8月龄,处死后分离心脏,通过免疫荧光标记及Western Blot等方法,观察左心室心肌细胞GRK2的表达及其亚细胞分布的变化。结果 GRK2于左心室心肌组织总蛋白、浆蛋白中表达水平SHRL、SHRH较SHRC组显著减少,而SHRL与SHRH组中GRK2表达水平差异无统计学意义,于膜蛋白表达水平按对照组、低剂量组、高剂量组的顺序呈递减的趋势,即SHRC>SHRL>SHRH(P<0.05);激光共聚焦显微镜观察显示:与SHRC组比较,SHRL组及SHRH组GRK2在心肌细胞膜上分布减少,SHRH组较SHRL组又进一步减少。结论通过硝苯地平的干预,SHR左心室心肌细胞GRK2的表达减少且出现亚细胞分布的变化,提示硝苯地平对GRK2的影响可能是逆转心肌肥大及心室重塑的机制之一。     

参麦注射液保护氧化损伤心肌细胞线粒体的机制研究

目的: 探究参麦注射液对氧化损伤心肌细胞的保护作用及其对线粒体能量代谢过程的影响。方法: 采用叔丁基过氧化氢（t-BHP）诱导构建H9c2心肌细胞氧化损伤模型。分别采用MTT法和化学发光法检测参麦注射液对细胞存活率和ATP水平的影响;采用Clark氧电极检测参麦注射液对细胞有氧呼吸速率的调节作用;采用ELISA法检测丙酮酸及丙酮酸脱氢酶（PDH）的含量及活性;采用蛋白质印迹法检测丙酮酸脱氢酶亚基（PDHA1）含量;采用实时定量RT-PCR检测丙酮酸脱氢酶激酶1（PDK1）mRNA的表达。结果: 与氧化损伤模型比较,参麦注射液可有效提高心肌细胞存活率和ATP水平（均P < 0.01）,提高细胞氧呼吸速率,减少丙酮酸含量,并提高PDH活性（P < 0.05或P < 0.01）。在分子水平上,参麦注射液可提高PDHA1蛋白表达（P < 0.05）,并具有降低PDK1 mRNA表达的趋势（P>0.05）。结论: 参麦注射液可有效保护氧化损伤心肌细胞,其机制可能与提高PDH活性、减少丙酮酸堆积,从而维持线粒体功能稳定和细胞能量代谢稳定有关。     

Isoflurane induces expression of vascular endothelial growth factor through activating protein kinase C in myocardial cells

Objective： Vascular endothelial growth factor （VEGF） plays important roles in establishing collateral circulation of ischemic myocardium. This study aimed to investigate the effect of isoflurane on VEGF expression and the potential intracellular signal transduction pathway in cultured rat myocardial cells in order to further reveal the molecular mechanism of myocardial preservation of isoflurane. Methods： Primary myocardial cells of Sprague-Dawley rats were isolated and cultured. They were divided randomly into control group, isoflurane group, protein kinase C （PKC） inhibitor group and PKC inhibitor＋isoflurane group where cells were respectively incubated without any treatment, treated by 0.5, 1.0 and 1.5 minimum alveolar concentration （MAC） of isoflurane for 6 hours, by PKC inhibitor calphostin C at a final concentration of 50 nmol/L and by 50 nmol/L calphosfin C＋ 1.0 MAC isoflurane for 6 hours. VEGF expression was detected by enzyme-linked immunosorbent assay （ELISA） and the expression levels of PKC isoforms were determined by Western immunoblotting method. Results： Isoflurane increased the VEGF expression in myocardial cells in a dose-dependent way. VEGF levels were significantly higher in 1.0 and 1.5 MAC isoflurane groups than in the control group （both P〈0.01）. The effect of isoflurane on upregulating VEGF expression was blocked by PKC inhibitor calphostin C （P〈0.01）, but calphostin C did not alter VEGF expression （P〉0.05）. Isoflurane induced the activation and translocation of PKC Immunoblotting analysis revealed that the immunoreactivity of PKC ε increased significantly in the membrane fractions and deceased significantly in the kytoplasm fractions for cells treated with 1.0 MAC isoflurane as compared with the untreated cells, but not of PKC a, PKCα and PKCζ （P〈0.01）. Conclusion： Isoflurane induces myocardial cells to release VEGF through activating PKCε from the endochylema to the cytomembrane, suggesting a possible novel mechanism of isoflurane protecting myocardial cells.     

锌指蛋白A20抑制ox-LDL诱导的平滑肌细胞迁移的实验研究

目的 探讨锌指蛋白A20对ox-LDL诱导的血管平滑肌细胞作用的可能机制.方法 用锌指蛋白A20基因转染大鼠主动脉血管平滑肌细胞后提取核蛋白用western blot的方法检测NF-κB核移位的量;进行Boyden小室迁移实验,观察平滑肌细胞的迁移能力变化.结果 ox-LDL刺激大鼠主动脉血管平滑肌24 h后NF-κB的核移位明显增加,迁移能力也显著增强;而转染含A20基因质粒的平滑肌细胞NF-κB的核移位及迁移能力达到正常水平.结论 ox-LDL能明显激活平滑肌细胞NF-κB信号系统,并增强平滑肌细胞的迁移能力,可能由此激活动脉壁的炎症反应,诱导平滑肌迁移至内膜,触发动脉粥样硬化发生;A20明显抑制了ox-LDL诱导下的NF-κB活化水平,同时抑制了的平滑肌细胞迁移能力的增强,A20可能是通过抑制NF-κB信号而起作用,并可能由此阻断了动脉粥样硬化的发生发展.     

A laser diffraction system with improved sensitivity for long-time measurements of sarcomere dynamics in isolated cardiac myocytes

To measure the mechanical activity of enzymatically isolated mammalian myocytes the principle of laser light diffraction was used. Since the viability of isolated cardiac myocytes showed a marked dependence on the laser power used, an opto-electronic system with improved light sensitivity and low susceptibility to optical noise was developed. The high sensitivity was achieved by a novel approach in the detection of diffraction patterns, that provides a significant reduction of the amount of laser power required. This improvement rendered possible the application of laser diffraction during extended experiments including pharmacological interventions. The static performance of the system, as assessed by means of calibration gratings, showed a resolution in the order of 5 nm for small changes in sarcomere length in the range from 1.2 m to 2.0 m. Examples of measurements on resting and contracting cells are presented, and the limitations of the application of the system to biological specimens are discussed.