香烟对小鼠C2C12成肌细胞分化的影响

目的:探讨香烟烟雾浓缩物(CSC)对小鼠骨骼肌C2C12成肌细胞分化的影响。方法:小鼠骨骼肌成肌细胞C2C12传代后分为对照组、0.3%CSC孵育组、3%CSC孵育组,细胞在含有马血清的DMEM培养基第6-7d可分化成熟的肌细胞。细胞分别在第1d、第3d在相应组中加入CSC,并在第6d进行各项检测:通过光学显微镜来观察细胞形态的变化,通过酶联免疫检测仪检测细胞内谷胱甘肽(GSH)和肌酸磷酸激酶(CK)的浓度,应用Western印迹检测肌球蛋白重链(MYHC)的蛋白表达,通过凝胶迁移率阻滞实验来检测转录因子核因子-κB(NF-κB)的活性。结果:在细胞形态上0.3%和3%的CSC能够抑制骨骼肌肌管的形成;0.3%和3%CSC处理组细胞中GSH和CK的含量较对照组明显减少,在0.3%和3%的CSC孵育组中MYHC蛋白表达明显减少,0.3%和3%的CSC孵育组中NF-κB的活性较对照组明显增强。结论:CSC一方面加重细胞氧化应激,使细胞的GSH减少,同时导致细胞的炎症反应增加,导致NF-κB的活性增加。二者相互作用引起骨骼肌细胞的分化明显减弱。     

Smooth muscle and epithelial cells express specific binding sites for the C1q component of complement.

In injury and inflammation, interactions of complement C1q with C1q receptors may provide attachment sites for cell localization and tissue regeneration. Cultured smooth muscle cells (58%), epithelial cells (26%), and endothelial cells (25%) attach to C1q-coated surfaces, while only 6% of cultured B cells (Raji) attach. Endothelial and Raji cells express C1q receptors, but C1q receptors (C1qR) on smooth muscle cells and epithelial cells have not previously been demonstrated. Evidence is provided that smooth muscle cells express an average of 1.5 x 10(6) C1qR/cell (K alpha = 10(8) M-1) and that epithelial cells express an average of 0.7 x 10(6) C1qR/cell (K alpha = 1.4 x 10(8) M-1). Binding properties of C1qR, and immunoreactivity to anti-C1qR antibodies, are characterized. The antibodies specifically recognize a 67-kDa component of smooth muscle cell lysates and inhibit cell attachment to C1q substrates. We conclude that distribution of C1qR may be ubiquitous; binding properties, size, and antigenicity of various C1qR may be related, but adhesive function may be tissue specific.     

Glutathione depletion impairs myogenic differentiation of murine skeletal muscle C2C12 cells through sustained NF-kappaB activation

Skeletal muscle differentation is a complex process regulated at multiple levels. This study addressed the effect of glutathione (GSH) depletion on the transition of murine skeletal muscle C2C12 myoblasts into myocytes induced by growth factor inactivation. Cellular GSH levels increased within 24 hours on myogenic stimulation of myoblasts due to enhanced GSH synthetic rate accounted for by stimulated glutamate-L-cysteine ligase (also known as gamma-glutamylcysteine synthetase) activity. In contrast, the synthesis rate of GSH using gamma-glutamylcysteine and glutamate as precursors, which reflects the activity of the GSH synthetase, did not change during differentiation. The stimulation of GSH stores preceded the myogenic differentiation of C2C12 myoblasts monitored by expression of muscle-specific genes, creatine kinase (CK), myosin heavy chain (MyHC), and MyoD. The pattern of DNA binding activity of NF-kappaB and AP-1 in differentiating cells was similar both displaying an activation peak at 24 hours after myogenic stimulation. Depletion of cellular GSH levels 24 hours after stimulation of differentiation abrogated myogenesis as reflected by lower CK activity, MyHC levels, MyoD expression, and myotubes formation, effects that were reversible on GSH replenishment by GSH ethyl ester (GHSEE). Moreover, GSH depletion led to sustained activation of NF-kappaB, while GSHEE prevented it. Furthermore, inhibition of NF-kappaB activation restored myogenesis despite GSH depletion. Thus, GSH contributes to the formation of myotubes from satellite myoblasts by ensuring inactivation of NF-kappaB, and hence maintaining optimal GSH levels may be beneficial in restoring muscle mass in chronic inflammatory disorders.     

The distribution of α-bungarotoxin binding sites on mammalian skeletal muscle developing in vivo

1. The distribution of α-bungarotoxin binding sites on embryonic and neonatal rat skeletal muscle fibres was determined by autoradiography. Most of the bungarotoxin binding could be inhibited by curare. This observation, together with the spatial distribution of toxin-binding sites, indicates that the distribution of bound toxin reflects that of acetylcholine (ACh) receptors on these developing muscle cells.

2. At 15 days of embryogenesis, muscle fibres showed an essentially uniform distribution of receptors. By 16 days, many fibres showed an accumulation of receptors in their mid-region. This accumulation was at the same location as histochemically demonstrated cholinesterase activity.

3. At 16 days ACh receptors were distributed over the entire length of the fibres, with a gradient of increasing density as the accumulation was appoached. The density of toxin binding sites in the accumulation was greater than the general level on 15 day cells, suggesting that the high junctional density does not develop solely by the loss of extrajunctional receptors.

4. The accumulations of ACh receptors became more pronounced and circumscribed with embryonic development, and after birth the extent of the localizations appeared to follow the size of the neuromuscular junction. The extrajunctional receptor density decreased with development, and by 1 week after birth was undetectable by the methods used.

5. The results suggest that the high junctional receptor density found on adult, innervated skeletal muscle fibres develops after the formation of the neuromuscular junction.

     

TAZ激活剂衍生物对C2C12细胞肌分化的作用

目的目的应用C2C12肌肉分化模型,探讨9种TAZ激活剂IBS008738衍生物对肌分化的作用。方法设计合成IBS008738衍生物,C2C12细胞按分化培养液中相应终浓度处理细胞,荧光显微镜下观察分化后肌管并行免疫染色,用Image J软件测量并计算融合指数。结果设计合成的9种衍生物经1H NMR确认结构及纯度,作用于分化C2C12细胞3d后,IBS008738衍生物中KI-1表现出了强于IBS008738的促肌分化的作用,显示肌管粗,数量增多,融合指数增高,且呈现剂量-效应关系。结论衍生物KI-1与IBS008738共同结构可能是IBS008738的促进肌肉分化的功能成分,对其强化可开发出更具效能的抗肌萎缩药物。     

The age-dependent changes in the number of3H-ouabain binding sites in mammalian skeletal muscle

The influence of age on the binding of³H-ouabain in skeletal muscle has been characterized in rats, mice and guinea pigs. Measurements performed using biopsies and intact fibers obtained from different types of rat muscles showed that from birth to the 4th week of life, the number of³H-ouabain binding sites per unit weight increases up to 5-fold, followed by almost the same relative decrease to a plateau around 250 pmol/g wet wt at an age of 22 weeks. These changes were not associated with any major alterations in apparentK _D (1.7–3.1×10^–7M) dissociation rate or heterogeneity in binding characteristics. Measurements of 3-O-methylfluorescein phosphatase activity, an enzyme activity which is closely correlated to the Na–K-ATPase activity, confirmed the³H-ouabain binding data.In mice, the number of³H-ouabain binding sites showed similar, albeit less pronounced changes with age, a maximum being reached at the 4th week of life. In guinea pigs, the number of³H-ouabain binding sites per unit weight decreased by 60% from birth to maturity.The results indicate that the early development and differentiation of individual skeletal muscles is associated with a marked increase in the number of Na–K-pumps (when expressed as pmol/muscle), until at maturity a plateau is reached. However, when expressed as pmol/g wet wt the increase is followed by a decrease to a plateau. This may in part account for the relatively low digitalis sensitivity seen in infants as compared to newborn and mature individuals.     

Localization of voltage-sensitive sodium channels on the extrasynaptic membrane surface of mouse skeletal muscle by autoradiography of scorpion toxin binding sites

Summary Voltage-dependent sodium channels (Na⁺ channels) were localized by autoradiography on mouse skeletal muscle using both light and electron microscopy.¹²⁵I-scorpion toxins (ScTx) of both the a and type were used as probes. The specificity of labelling was verified by competitive inhibition with unlabelled toxin and by inhibition of ScTx labelling in depolarizing conditions. Under light microscopy, the labelling of the myocyte surface appeared randomly distributed with both the a and toxins. No difference in the labelling density obtained with ScTx was observed between a 2 mm central segment of the fibre containing the endplate and an adjacent segment not containing the endplate. At the endplate, however, the ScTx binding site density was about seven fold higher at the edge of the synaptic primary clefts. This density decreased with distance from the synaptic cleft reaching the extrasynaptic value at 30–40 m. An analysis of myocyte labelling using electron microscopy provided evidence for a specific, but very low labelling of the myocyte interior which can be attributed to the T-tubules. These results confirm a relatively high density of Na⁺ channels in a perijunctional zone about 50 m in width, which could ensure the initial spread of the surface depolarization with a high safety factor, and a homogeneous distribution over the remaining surface with a low density evaluated at 5–10 per m². However, the very low labelling of T-tubules could be attributed mainly to a low density of tubular Na⁺ channels.     

C2C12细胞诱导构建三维骨骼肌组织

目的 利用修饰并铸型后的Sylgard 184凹槽与C2C12细胞复合培养、诱导分化,获取三维极性骨骼肌组织. 方法 Sylgard 184双组分以10∶1的比例均匀混合并倒板,室温下静置固化并对其表面压槽铸型,Hank液冲洗凹槽,Matrigel和胶原的混合液均匀铺被凹槽底部,置生物安全柜待细胞基质自然干燥、紫外线照射消毒1h以上时接种C2C12细胞悬液,细胞增殖约80%汇合时改用分化培养基进行分化诱导,倒置显微镜下观察肌管的分化状态, RT-PCR方法检测肌管内myogenin和desmin基因mRNAs的表达,免疫荧光检测生肌转录因子myogenin和desmin蛋白的表达,扫描电镜观察肌管形态和肌管间的连接. 结果 C2C12细胞在Sylgard 184弹性体铸型压槽中培养分化7d后,倒置显微镜下可见肌管呈极性分化,且肌管之间融合紧密;21d后,扫描电镜检测可见肌管之间排列紧密且相互重叠,形成膜样结构,厚度可达0.15mm,具有三维性;RT-PCR、免疫荧光检测证实极性分化肌管内具有myogenin和desmin的阳性表达. 结论 修饰并铸型的Sylgard 184凹槽具有一定的方向引导效应,能促进C2C12细胞分化形成多核肌管,且肌管呈极性重叠排列,形成三维极性骨骼肌组织结构.     

Quantitative changes in ouabain binding after denervation and during reinnervation of mouse skeletal muscle

Among the earliest changes following denervation of mammalian skeletal muscle fibres is a reduction of the resting membrane potential. This depolarization has been attributed to (a) a loss of the electrogenic effect of the active Na-K-transport across the plasma membrane (Locke & Solomon 1967); (b) a decrease in the potassium permeability of the resting plasma membrane (Klaus, Lüllmann & Muscholl 1969); (c) an increase in its sodium permeability (Robbins 1977). The purpose of the present study was to examine whether denervation affected the number of active Na-K-transport sites by determining the specific oubain binding to innervated, denervated and reinnervating muscles in vitro     

D600 binding sites on voltage-sensors for excitationcontraction coupling in frog skeletal muscle are intracellular

Summary Charge movements were measured in frog cut twitch fibres mounted in a double Vaseline gap chamber at 14° C with 30M D600 in the external solution. TEST-minus-CONTROL current traces appear normal with a hump current component (I) embedded in the decay phase of the early current component (I) in the ON-segment and an exponentially decaying current transient in the OFF-segment. When a conditioning depolarization to 0 mV is applied at around 6° C, charge movement is greatly reduced but not completely suppressed and no hump component can be visualized in the ON-segment. In addition, an extra capacitive component is generated having a time course slower than, and a polarity opposite to, that of the usual charge movement. This extra component makes the transients in both the ON- and OFF-segments appear bisphasic. When temperature is restored to 14° C, the bisphasic nature is greatly enhanced. After the application of a conditioning hyperpplarization, the shape of the TEST-minus-CONTROL current trace is converted back to that before paralysis, but the total amount of charge reprimed is less than 100% of control. In general, more Q is reprimed than Q, and the amount of Q reprimed varies over a wider range from fibre to fibre than that of Q. Extracellularly applied D890 cannot reproduce the blocking effect of D600 whereas intracellularly applied D890 can. As D890 is permanently charged and cannot permeate through the plasma membranes, it can be concluded that the binding sites for D600/D890 on the charge movement macromolecules must be on the myoplasmic side. This adds another parallelism between the charge movement entities and L-type calcium channels. However, the specific prerequisites for the blockage of the former not shared by the latter differentiates the two physiological units.     

香烟烟雾对C2C12小鼠成肌细胞HDAC2及炎症介质的影响

目的:探讨香烟烟雾提取物(CSE)对C2C12小鼠成肌细胞组蛋白去乙酰化酶2(histone deacetylase 2,HDAC2)和炎症介质的影响。方法:CSE刺激C2C12细胞,用质粒脂质体法将构建好的HDAC2 siRNA转染细胞,实时荧光定量PCR和Western blotting法检测HDAC2的表达情况,ELISA检测细胞培养上清白细胞介素-8(IL-8)和肿瘤坏死因子-α(TNF-α)的含量。结果:CSE组细胞HDAC2的mRNA及蛋白表达明显低于对照组(P0.05),细胞上清IL-8和TNF-α的水平明显高于对照组(P0.05);应用HDAC2 siRNA转染细胞后再用CSE刺激细胞,细胞HDAC2的mRNA及蛋白表达均明显低于CSE组及对照组(P0.05),细胞释放IL-8和TNF-α的量均明显高于CSE组及对照组(P0.05)。结论:氧化应激条件下C2C12小鼠成肌细胞通过下调HDAC2表达使IL-8和TNF-α表达增多。     

The use of CalciumOrange-5N as a specific marker of mitochondrial Ca2+ in mouse skeletal muscle fibers

We report the use of the fluorescent dye CalciumOrange-5N (CaOr-5N) as a specific mitochondria Ca²⁺ marker in enzymatically dissociated mouse FBD muscle fibers. Using laser scanning confocal microscopy and the dyes Mitotracker Green (MTG), di-8-ANEPPS and endoplasmic reticulum tracker green (ERTG), we determined the relative position of mitochondria, transverse tubules and sarcoplasmic reticulum in the sarcomere. Comparison with electron micrographies showed that mitochondria are mostly present at both sides of Z lines and near the triads located at the A-I band border. CaOr-5N fluorescence was mainly distributed in mitochondria, highly co-localised with MTG and basically excluded from the A band space. ERTG localised mostly between the two t-tubules present in each sarcomere. We studied the effect of the protonophore FCCP using CaOr-5N to measure mitochondrial Ca²⁺ and JC-1 dye to measure mitochondria inner membrane potential (ΔΨ _m). After FCCP treatment, the CaOr-5N fluorescence diminished by about 33% in 80 s, while JC-1 fluorescence diminished by 36% in 200 s. Our results show the loss of Ca²⁺ from mitochondria when ΔΨ_m is depolarised and demonstrate the usefulness of CaOr-5N to mark mitochondrial [Ca²⁺]_m. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Human myometrial cells in culture express specific binding sites for urinary trypsin inhibitor

Urinary trypsin inhibitor (UTI), which is present in amniotic fluid, prevents uterine contractility during pregnancy possibly via specific binding protein mechanisms. To test for the presence of UTI binding sites on the cell surface, we prepared cultured myometrial cells obtained at biopsy from 12 pregnant women and performed binding, competition, and cross-linking experiments using a specific radiolabelled UTI as a ligand. We report for the first time two classes of binding sites of differing affinities. Scatchard analysis at 4 degrees C, using radioiodinated UTI, revealed that UTI binds to 35 000 high affinity binding sites/cell (K(d) = 9.1x10(-9) mol/l) and 450 000 lower affinity binding sites/cell (K(d) = 3.5x10(-7) mol/l) in cultured myometrial cells. It appears to be the low affinity site that is internalized, and this has been identified as a protein of approximately 45 kDa by cross-linking and immunoaffinity labelling studies. Monoclonal antibodies against the NH(2)-terminal fragment of UTI abrogated specific binding of this protein to the cells. Treatment of the cells with hyaluronidase resulted in >80% inhibition of the [(125)I]-labelled UTI binding to the cells. These data show that the UTI binding site, which is hyaluronidase sensitive, is expressed on the surface of human uterine myometrial cells to accumulate the UTI molecule during pregnancy.     

3H-Ouabain binding sites in porcine skeletal muscle as influenced by environmental temperature and energy intake

The influence of environmental temperature and energy intake on³H-ouabain binding sites in skeletal muscle has been investigated in young growing pigs at 8 weeks of age. Animals lived for several weeks at 35 or 10°C on a high (H) or low (L) level of energy intake. The four treatment groups were thus: 35H, 35L, 10H and 10L. The total number of³H-ouabain binding sites (B _max) in longissimus dorsi muscle (mean values ± SEM) were 221±66, 214±61, 350±76 and 486±114 pmol/g wet weight for the 35H, 35L, 10H and 10L groups respectively.B _max was significantly greater in those living in the cold than the warm (P<0.001). Moreover, at 10°C energy intake had a significant effect, withB _max being greater in the 10L than the 10H group (P<0.005). Level of energy intake had no influence onB _max at 35°C. The apparent dissociation constant was not affected by either temperature or intake. The elevatedB _max and hence the increase in number of Na⁺, K⁺-pumping sites in the cold is probably related to increased muscular activity associated with shivering. However, thyroid status also influences the number of Na⁺, K⁺-pumping sites and this may have been a contributory factor in the present study. In addition, the elevatedB _max suggests a greater potential for non-shivering thermogenesis associated with increased Na⁺, K⁺-ATPase concentration in the cold. Differences in relative stage of development between the four groups may help to explain the results forB _max in relation to level of energy intake.     

Soluble factors from neuronal cultures induce a specific proliferation and resistance to apoptosis of cognate mouse skeletal muscle precursor cells

The mechanisms or the physiological events, which control the regeneration of skeletal muscle through muscle precursor cell multiplication and differentiation, are still largely unknown. To address the question of the involvement of neurons in this process, skeletal muscle progenitors were grown in the presence of conditioned media obtained from 3-day-old cultures of embryonic neurons (derived from either the dorsal or the ventral region of 11-day-old mouse embryos) or media conditioned with satellite cells. Strikingly, only satellite cells cultured in medium conditioned from ventral embryonic neurons exhibited increased proliferation, as well as resistance to staurosporine (STS)-induced apoptosis. Our results suggest the existence of specific anti-apoptogenic neural soluble signals, which could be involved in skeletal muscle regeneration pathways.     

High level stable expression of human interleukin-2 receptors in mouse cells generates only low affinity interleukin-2 binding sites

A bovine papilloma virus-derived vector was used to direct the high level expression in mouse C127 cells of three different cDNAs encoding the human interleukin-2 receptor. These were: the previously described cDNA clone isolated from the T-cell lymphoma, HUT-102; a cDNA clone isolated from mitogen-activated, normal peripheral blood T cells; and an altered version of the HUT-102 receptor in which Ser247, believed to be the site of protein kinase C-mediated phosphorylation, has been changed to an Ala residue. Fluorescence-activated cell-sorting using a monoclonal antibody directed against the human IL-2 receptor was used to derive stable lines of C127 cells expressing from 2-6 X 10(6) IL-2 binding sites per cell. However, all of these receptors bound IL-2 with low affinity.     

Characterization of mouse interleukin-12 p40 homodimer binding to the interleukin-12 receptor subunits.

Interleukin-12 (IL-12) is a heterodimeric cytokine composed of two disulfide-bonded subunits, p35 and p40, which has important regulatory effects on T cells and natural killer (NK) cells. In contrast to heterodimeric IL-12, a homodimer of the p40 subunit, designated (p40)2, has been shown to be a potent IL-12 antagonist. To study the interaction between (p40)2 and the known IL-12 receptor (IL-12R) subunits, IL-12Rbeta1 and IL-12Rbeta2, we directly measured the binding activity of mouse (p40)2 to ConA-activated lymphoblasts and purified B cells from splenocytes of C57BL/6J mice. These results demonstrated the presence of both high (Kd about 5 pM) and low affinity (Kd about 15 nM) binding sites for mouse 125I-labeled (p40)2. To elucidate which of the IL-12R subunits binds mouse (p40)2, binding studies of mouse 125I-labeled (p40)2 to Ba/F3 cells expressing recombinant mouse IL-12Rbeta1 and/or mouse IL-12Rbeta2 were carried out. Mouse IL-12Rbeta1 bound mouse 125I-labeled (p40)2 with high and low affinities, comparable to that observed on Con A blasts and B cells. In contrast, mouse IL-12Rbeta2 bound mouse 125I-labeled (p40)2 very poorly. These data demonstrate that similar to IL-12, mouse (p40)2 binds with both high and low affinity to Con A blasts and B cells, and that IL-12Rbeta1 is responsible for mediating the specific binding of mouse (p40)2.     

Myogenic progenitor cells express filamin C in developing and regenerating skeletal muscle

The regenerative capacity of skeletal muscle is due to the myogenic progenitor cell population that is resident in adult skeletal muscle. To enhance our understanding of this cell population, we examined the temporal-spatial expression pattern for filamin C during murine embryogenesis, adult muscle regeneration and in selected myopathic models of human disease. Using in situ hybridization, we observed filamin C to be restricted to mesodermal lineages including the developing heart and skeletal muscle during embryogenesis. Following cardiotoxin-induced muscle injury of adult skeletal muscle, filamin C expression was dynamically regulated in activated myogenic progenitor cells and newly regenerated myotubes. This expression pattern was further supported using RT-PCR analysis of filamin C expression in differentiating C2C12 myotubes. These results support the paradigm that the regulatory mechanisms of muscle regeneration largely recapitulate the fundamental events observed during muscle development and that filamin C may function in signal transduction or cellular migration of the myogenic progenitor cell population.     

Characterization of lactogen binding sites in choroid plexus

Prolactin binding sites have been demonstrated previously in rat choroid plexus using in vivo radioautography (Walsh et al. 1978). In the present study we have employed this procedure to characterize further the binding specificity of these sites. Following the injection of 125I-hGH or 125I-oPRL an intense radioautographic reaction was observed over the choroid plexus. The reaction was significantly reduced by coinjecting excess unlabeled hGH or oPRL but not bGH. The specific binding of 125I-oPRL to choroid plexus from rat, rabbit, sheep and pig was demonstrated by in vitro assays. Subsequently a survey of 125I-oPRL specific binding in a number of regions of pig brain indicated that the highest binding was in choroid plexus. A detailed study of the characteristics of 125I-oPRL binding to pig choroid plexus was undertaken. Specific binding increased with choroid plexus homogenate protein to a maximum of 30% (3.0 mg protein/tube). Binding was maximum at 4 degrees C by 30-40 h of incubation. During the incubation the integrity of 125I-oPRL in the incubation medium declined steadily to 50% after 20 h and 35-40% after 48 h. Radioactivity eluted from binding sites was fully intact as judged by rebinding to lactogen receptor-enriched membranes. Binding showed a broad pH optimum of 5.5-7.5. On cell fractionation of choroid plexus binding sites were enriched in microsomes. The binding of 125I-oPRL and 125I-hGH was inhibited in a dose-dependent manner by unlabeled lactogens and was of high affinity. hGH and oPRL were equipotent inhibitors of the binding of both radioligands whereas bGH and a variety of structurally unrelated peptides were non-inhibitory.