体外转染Cyclin A2基因对原代大鼠心肌细胞增殖的影响

目的探讨体外转染细胞周期素A2(Cyclin A2)基因对原代大鼠心肌细胞增殖的影响,为心脏再生提供细胞学依据。方法 SD乳鼠12只,分离、培养、鉴定原代乳鼠心肌细胞并分为3组,实验组:转染带有强化绿色荧光蛋白(GFP)和Cyclin A2基因的重组腺病毒(Ad-Cyclin A2-GFP);空病毒组:转染不含目的基因带有GFP的重组腺病毒(Ad-Null-GFP);阴性对照组:未做转染处理,仅加入等量的培养基。利用GFP示踪技术,评估原代心肌细胞转染效率;转染后的细胞继续体外培养3~5d,利用免疫荧光技术分别检测Cyclin A2、磷酸化组蛋白H3(H3P)、心肌肌钙蛋白-T(c Tn T)。结果 1.荧光GFP示踪表明原代心肌细胞的转染效率高达(97±0.74)%;2.免疫荧光标记显示,空病毒组和对照组结果相似;心肌细胞特异性标记蛋白c Tn T分布于细胞质,原代心肌细胞纯度高达(95±0.62)%;心肌细胞Cyclin A2主要在胞核内聚集,少数分布于细胞质。H3P为核蛋白在细胞核内分布。3.转染Cyclin A2后,实验组H3P阳性率明显高于空病毒组和对照组,差异具有统计学意义(P0.05);实验组可见大量多核细胞,以双核为主,伴少量3核细胞。结论腺病毒作为转染载体对原代心肌细胞有很好的侵染效率;Cyclin A2超表达促进原代心肌细胞形成双核。     

S波段高功率微波辐射对原代培养乳鼠心肌细胞的影响

目的研究S波段高功率微波（HPM）辐射后乳鼠心肌细胞的损伤效应及其机制。方法采用平均功率密度为5～30mW/cm^2的S波段HPM模拟源辐射原代培养的乳鼠心肌细胞，应用流式细胞术、原子力显微镜观察辐射后心肌细胞凋亡和坏死率、[Ca^2＋]。及细胞膜形态。结果10—30mW／cm^2的S波段HPM辐射后6h，心肌细胞凋亡和坏死率增加（P〈0．05或P〈0．01），且随辐射剂量增大，坏死细胞增多；30mW/cm^2 HPM辐射后即刻[Ca^2＋]i升高（P〈0．01），细胞膜发生穿孔。结论10—30mW/cm^2的S波段HPM辐射可造成心肌细胞凋亡和坏死，[Ca^2＋]；升高以及细胞膜穿孔，是S波段HPM辐射致心肌细胞损伤的重要机制。     

Growth promoting and metabolic activity of the human insulin analogue [Gly,Arg,Arg]insulin (HOE 901) in muscle cells

[Gly^A21,Arg^B31,Arg^B32]insulin (HOE 901) represents a biosynthetic human insulin analogue that, due to its isoelectric point, precipitates at neutral tissue pH leading to a retarded absorption rate and a corresponding longer duration of action. In the present investigation we have evaluated the growth promoting and metabolic activity of this analogue in muscle tissue using exponentially growing H9c2 cardiac myoblasts and adult rat ventricular cardiomyocytes. Equilibrium binding studies of ¹²⁵I-labelled IGF-I (insulin-like growth factor I) to differentiating myoblasts revealed the presence of 7×10³ IGF-I receptors per cell. In contrast, no specific binding of insulin could be detected. Competition binding experiments showed a slightly higher affinity of HOE 901 for the IGF-I receptor when compared to regular human insulin with IC₅₀ (half-inhibitory concentration) values of 70 and 101 nM, respectively. However, the supermitogenic insulin analogue [Asp^B10]insulin competed significantly more efficiently for IGF-I binding (IC₅₀: 44 nM). Maximum growth promoting activity of the peptides was then determined in serum-starved myoblasts by an incubation with the peptides (5×10⁻⁷ M) for 16 h in the presence of [³H]thymidine. [Asp^B10]Insulin produced a stimulation of DNA synthesis (about 3-fold) which was comparable to the effect of IGF-I and significantly (P<0.005) higher than the effect of HOE 901 with the latter being essentially equipotent to native insulin. Comparable results were obtained at lower concentrations of the peptides (10⁻⁹ to 10⁻⁸ M). Metabolic activity of HOE 901 was determined by measuring the dose-dependent stimulation of 3-O-methylglucose transport in adult cardiomyocytes. Maximum transport stimulation was identical for insulin and HOE 901 with EC₅₀ (half-effective concentration) values of 0.7×10⁻¹⁰ and 1.9×10⁻⁹ M, respectively. We concluded that the IGF-I receptor-mediated growth promoting activity of HOE 901 in muscle cells and the maximal metabolic activity of this analogue are not different from those of native human insulin. It is suggested that differential interaction with IGF-I receptors significantly contributes to the action profile of insulin analogues.     

核转录因子-κB在缺氧预处理抑制心肌细胞凋亡中的作用

目的 :研究缺氧预处理 (hypoxicpreconditioning ,HPC)对于心肌细胞蛋白激酶C(PKC)和核转录因子κB (NF κB)表达的影响 ,及其在缺氧复氧诱导心肌细胞凋亡中的作用。方法 :在培养的SD乳鼠心肌细胞制作缺氧 /复氧 (H/R)模型 ,以荧光素染料Hoechst3 3 2 5 8测定心肌细胞凋亡率 ;制备心肌细胞蛋白提取物 ,以新PKCε亚型 (nPKCε)特异性抗体测定nPKCε相对蛋白含量 ;以抗NF κB抗体检测NF κB的表达 ;并以PKC抑制剂H7与心肌细胞预孵育后 ,观察H7对于HPC诱导的PKC和NF κB表达上调以及心肌细胞保护作用的影响。结果 :缺氧复氧造成心肌细胞凋亡 ,HPC可以降低心肌细胞H/R后凋亡率 ,并诱导nPKCε和NF κB表达上调 ;PKC抑制剂H7可以消除HPC诱导的PKC、NF κB表达上调和心肌细胞保护作用。结论 :HPC可以提高乳鼠心肌细胞对于H/R的耐受性 ,其机制涉及PKC介导的NF κB表达上调。     

Spatiotemporal changes of coxsackievirus and adenovirus receptor in rat hearts during postnatal development and in cultured cardiomyocytes of neonatal rat

Coxsackievirus B is the most common cause of viral myocarditis and is particularly virulent in neonates and children. Adenovirus is also a leading cause of the disease. The determinant of tropism for both viruses is considered to be the expression of coxsackievirus and adenovirus receptor (CAR) in target organs. However, developmental change and physiological localization of CAR in the heart are unknown. We examined expression levels of CAR in rat hearts by quantitative real-time polymerase chain reaction and Western blot analysis and found that CAR decreased gradually during postnatal development, although CAR was detectable, even in adults. Immunohistochemistry revealed CAR on the whole surface of cardiomyocytes in immature rat hearts. In contrast, CAR was detected predominantly on intercalated disks in the adult heart and was accumulated especially at the contact point between the cultured cardiomyocytes, even though they were prepared from the neonatal rat heart. In conclusion, CAR was expressed abundantly on the whole surface of cardiomyocytes in immature rat hearts. Both the expression level and the localization of CAR are possible determinants of the susceptibility to viral myocarditis of neonates and children.     

胰岛素对新生大鼠缺氧/复氧心肌细胞凋亡的影响

目的:探讨胰岛素在新生大鼠缺氧/复氧心肌细胞损伤中对细胞凋亡的影响。方法:通过给原代培养乳鼠心室肌细胞行缺氧2 h/复氧4 h,建立缺氧/复氧(Anoxia/Reoxygenation)心肌细胞损伤模型。将培养心肌细胞随机分为:正常对照组(CON)、缺氧/复氧组(A/R)、缺氧/复氧+胰岛素组(I/R+INS)。于复氧4 h后,利用2,4二硝基苯肼显色法检测乳酸脱氢酶(LDH)活性,硫代巴比妥酸显色法检测丙二醛(MDA)含量,原位末端标记法(TINEL)和DNA梯带法(DNA Ladder)检测细胞凋亡,并比较各组间差异。结果:与A/R组相比,A/R+INS可明显降低MDA、LDH值(P<0.01),抑制心肌细胞凋亡(P<0.01)。结论:胰岛素具有减少新生大鼠缺氧/复氧心肌细胞损伤作用,其作用机制与抑制细胞凋亡作用有关。     

Quantification and preservation of lectin binding by isolated cardiomyocytes

During preparation of cells for experimentation a considerable amount of bound substance is lost. Our aim was to develop a protocol which retained lectin binding to an extent similar to living cells. This procedure would use fixation procedures suited for fluorescent lectin conjugates and gold-conjugates to be visualized by light- and electron microscopy, respectively. We tested glutaraldehyde and paraformaldehyde in different concentrations before and after lectin binding, different buffers and divalent cations, as additives, to determine the effects on preservation of lectin binding. Lectin binding was visualized and semiquantitatively evaluated by image analysis in the light microscope after silver enhancement of lectin-gold conjugates and by using tetramethyl rhodaminyl isothiocyanate (TRITC)-conjugated lectins. Preservation of lectin binding was best visualized with fluorescent lectin conjugates, whereas during silver enhancement procedures of gold-conjugated lectins, a considerable amount of bound lectins was lost. In general, lectin binding to living cells followed by fixation is superior to fixation before lectin binding. Unfavourable combinations of fixatives and buffers can cause a loss of more than 90% bound lectin. In our experiments with freshly isolated guinea pig cardiomyocytes, lectin binding was best when we used Na-cacodylate buffer with glutaraldehyde fixation (0.1%) after binding of lectins to the living cells.     

颅通定对烧伤早期大鼠心室肌细胞的保护作用

目的：探讨中药颅通定对烧伤早期心室肌细胞保护作用物机理。方法：应用Ｃａ＾２＋荧光控针及ＥＰＣ－９光电联合检测系统测定烧伤早期大鼠心室肌细胞胞浆Ｃａ＾２＋浓度变化及颅通定对其影响。结果：烧伤对照组心室肌细胞于伤后１小时胞浆Ｃａ＾２＋浓度明显升高,６小时达高峰,１２小时后渐恢复。正常对照组及颅通定治疗组（浓度：３００μｍｏｌ／Ｌ）心室肌细胞胞浆Ｃａ＾２＋浓度未见明显升高。结论：颅通定可显著抑制烧伤早期大鼠心室肌细胞内Ｃａ＾２＋超载。     

激光共聚焦显微镜比较观察甾体皂苷Trilliuo、Lc253、Lc239对培养心肌细胞内[Ca2+]i的作用

目的：薯蓣皂苷元的皂苷为许多抗心血管中药中的主要活性成分，具有重要研究价值。本研究以经人工合成获得的薯蓣皂苷元皂苷同系物甾体皂苷Trilliuo(署蓣皂苷元葡萄糖苷、Lc253[薯蓣皂苷鼠李糖(1→2)葡萄糖苷]、Lc239(薯蓣皂苷元[鼠李糖(1→2)，葡萄糖(1→3)]葡萄糖苷)为研究对象，通过考察它们对培养心肌细胞钙离子释放的影响，分析其构效关系。方法：利用激光共聚焦显微镜测定加入fluo3—AM培养的心肌细胞中的钙离子变化情况。结果：Lc253能明显增强心肌细胞节律性和呈浓度依赖性增加心肌细胞静息钙离子水平，加入CaCl2 10mmol/L or KCl 60 mmol/L后钙离子荧光峰强度仍维持在较高水平；而Trilliuo and Lc239在10^—5mmol/L浓度时，对心肌细胞静息钙离子水平作用不明显，在高浓度胞外Ca^2 或K^ 情况下，抑制心肌细胞内钙离子升高；其作用特点与verapamil相似，具有钙离子通道阻断作用。结论：研究结果显示三种甾体皂苷对细胞内钙离子浓度有所差异，结构中连接糖的数目与调孔钙内流的活性有重要的相关性。这些皂苷类化合物对钙离子通道的活性有潜在的开发价值。     

二硫化碳对小鼠心肌细胞肌浆网Ca2+-ATP酶基因表达的影响

目的探讨二硫化碳(CS2)对小鼠心肌细胞肌浆网Ca2 -ATP酶(SERCA2 a)表达的影响机制。方法将48只昆明小鼠分为4组给予不同剂量CS2(0~800 mg/m3)吸入染毒5周后,提取心肌的总RNA,运用逆转录聚合酶链反应技术来检测染毒小鼠心肌细SERCA2 a表达水平的变化。结果染毒前后体重差异无显著性(P>0.05),随着染毒剂量的增大,小鼠心肌细胞SERCA2 a的表达逐渐降低,与对照组比较差异有显著性(P<0.05)。结论CS2能降低小鼠心肌SERCA2 a的mRNA表达水平。