大白鼠膈肌卫星细胞去神经后的变化与其它去神经效应的关系

电子显微镜观察表明正常大白鼠膈肌卫星细胞的平均长度在终板区明显地比非终板区大,而卫星细胞的数目无显著区别。去神经或秋水仙碱处理膈神经后3天,与肌纤维的乙酰胆碱敏感性增高的出现相伴随,肌纤维卫星细胞的平均长度比对照组增加,但卫星细胞的平均数目无明显变化;同时,卫星细胞长度增加的程度在非终板区都比终板区大。去神经同时给放线菌素 D3天后,在肌纤维卫星细胞长度变化受阻的同时乙酰胆碱敏感性增高的发展亦被抑制。     

睫状神经营养因子对体外培养骨骼肌细胞的伸增殖效应

目的：探讨睫状神经营养因子（ＣＮＴＦ）对骨骼肌细胞的直接营养作用,从而为神经肌肉系统损伤和进行性病变的治疗提供新的思路。结果：ＣＮＴＦ可以促进体外培养的Ｌ６－ＴＧ肌母细胞和新生ＳＤ大鼠原代骨骼肌细胞增殖。结论：ＣＮＴＦ对体外骨骼肌细胞具有营养作用。ＣＮＴＦ的神经和肌肉双重营养性能在神经肌肉损伤和退行性为的治疗上发挥重要作用。     

小鼠骨胳肌在切腱、协同肌切腱和肉毒杆菌毒素中毒后对辣根过氧化物酶(HRP)的胞纳作用

本文报道了用生物化学方法测定离体小鼠比目鱼肌对 HRP 的胞纳作用。结果表明,在切断神经或切腱后引起萎缩的肌肉侧或在协同肌切腱后引起代偿性肥大的肌肉侧,与它们各自对照的正常肌肉侧相比,都可发生对 HRP 胞纳的明显增加。而在肉毒杆菌毒素中毒后引起萎缩的肌肉侧,和它正常的对照肌肉侧相比,却意外地不发生这种胞纳摄取的明显增加。本文的实验结果进一步证实。肌肉的胞纳增加并不一定导致肌纤维的变性和萎缩(例如代偿性肥大的肌肉);而肌纤维的变性和萎缩亦不一定需要肌肉的胞纳增加为前提(例如肉毒杆菌毒素中毒的肌肉)。本工作结果还提示:肌肉的胞纳增加有其神经原性和肌原性因素。本文还对肌肉胞纳增加的可能机制进行了讨论。     

鸡前、后背阔肌去神经后对辣根过氧化物酶(HRP)的胞纳作用

本文报道了用辣根过氧化物酶(HRP)作为大分子标记物,以组织化学和生物化学方法观察了鸡前后背阔肌在去神经后的胞纳现象。结果表明,在去神经后发生肥大的前背阔肌和去神经后发生萎缩的后背阔肌同样都出现胞纳的明显增加。组织化学所观察的结果表明,浸泡在含 HRP 的任氏液中的有完整神经支配的前、后背阔肌只有极少数的肌纤维摄取 HRP,而在去神经的前、后背阔肌中则有不少的肌纤维内部出现 HRP 染色反应。这种反应在有的纤维表现为弥散性染色,有的表现为浓的 HRP 反应颗粒。生物化学的结果显示,去神经后的前、后背阔肌中 HRP 的相对含量分别比有神经支配的对照肌肉明显地增多54%和87%,我们在鸡前背阔肌用组织化学和生物化学所得的实验结果与 Thesleff 等人提出的关于肌肉萎缩机制的假设显然不符。本工作证实了肌肉的胞纳作用的增加并不一定最终导致肌纤维的变性和萎缩。     

白芷细胞外21kD钙调素结合蛋白的生理功能初探

利用Ｓｅｐｈａｄｅｘ Ｇ１００ 及ＣＭＳｅｐｈａｒｏｓｅ层析法,从白芷悬浮培养细胞的胞外盐提液中纯化了２１ ｋＤ 钙调素结合蛋白（２１ ｋＤＣａＭＢＰ） ,测其等电点的ｐＨ 为８ ．９ ,紫外薄层扫描显示其纯度达９４ ％ 以上。以此蛋白为抗原,用免疫小鼠腹水法制备了特异性抗体。由纯化的２１ ｋＤＣａＭＢＰ及其特异性抗体研究其对白芷悬浮培养细胞增殖的影响,结果表明：加入外源纯化的２１ ｋＤＣａＭＢＰ 抑制细胞增殖,半抑制浓度约８ μｇ／ｍｌ;而加入２１ ｋＤＣａＭＢＰ特异性抗体却促进细胞增殖效应。推测胞外内源存在的２１ ｋＤＣａＭＢＰ在生理条件下可能具有参与调节胞外活化ＣａＭ 信号分子的浓度,从而调节胞外ＣａＭ 的生物学功能。     

EGCG对LPS刺激人肺腺癌A549细胞凋亡及CUGBP1蛋白表达的影响

目的：研究表没食子儿茶素-3-没食子酸酯（epigallocatechin-3-gallate,EGCG）对炎性刺激的人肺腺癌A549细胞增殖和凋亡的影响及与CUGBP1表达的关系。方法：MTT法检测EGCG和LPS刺激A549细胞增殖活性的影响;流式细胞仪检测细胞凋亡;免疫细胞化学检测EGCG对LPS刺激人肺腺癌A549细胞内CUGBP1蛋白的表达。结果：与对照组相比,LPS体外显著促进A549细胞增殖,其胞核胞质内CUGBP1表达明显增强（P〈0.01）。加入EGCG可拮抗LPS促A549细胞增殖的作用,促进其凋亡,明显抑制LPS刺激的A549细胞内CUGBP1的表达（P〈0.01）。CUGBP1蛋白定量分析可知EGCG和LPS共同孵育A549细胞4h、24h时,细胞中的CUGBP1蛋白表达量较单纯LPS作用时降低。但EGCG和LPS共同孵育A549细胞24h,A549细胞中胞核CUGBP1蛋白表达量（1210.565±3.46）较4h时胞核CUGBP1蛋白表达量（67.344±3.68）高,差异有统计学意义（t=927.164,P〈0.001）。结论：EGCG可能通过干扰CUGBP1基因的表达抑制炎症刺激人肺腺癌细胞A549的增殖,促进其凋亡。     

白芷细胞外21kD钙调素结合蛋白的生理功能初探

利用Ｓｅｐｈａｄｅｘ Ｇ－１００及ＣＭ－Ｓｅｐｈａｒｏｓｅ层析法,从白芷悬浮培养细胞的胞外盐提液中纯化了２１ｋＤ钙调素结合蛋白（２１ｋＤ ＣａＭＢＰ）,测其等电点的ｐＨ为８．９,紫外薄层扫描显示其纯度达９４％以上,以此蛋白为抗原,用免疫小鼠得水法制备了特异性抗体。由纯化的２１ｋＤ ＣａＭＢＰ及其特异性抗体研究其对白芷悬浮培养细胞增殖的影响,结果表明：加入外源纯化的２１ｋＤ ＣａＭＢＰ抑制细胞增殖,     

RGDS肽对大鼠主动脉球囊内膜剥脱后血管壁增殖的影响

在大鼠主动脉球囊内膜剥脱术后血管壁细胞过度增殖模型上,用合成的血小板膜纤维蛋白原受体（ｇｌｙｃｏｐｒｏｔｅｉｎⅡｂ／Ⅲａｃｏｍｐｌｅｘ,ＧＰⅡｂ／Ⅲａ）拮抗剂ＲＧＤＳ（Ａｒｇ－Ｇｌｙ－Ａｓｐ－Ｓｅｒ,５０μｍｏｌ·ｋｇ－１·ｄ－１）治疗可有效地抑制损伤血管壁的细胞计数增加和内膜增厚以及血管平滑肌细胞增殖,显著降低其血管组织３Ｈ－ＴｄＲ和３Ｈ－Ｌｅｕ的参入增加程度。实验结果提示ＲＧＤＳ肽作为血管成型术的辅佐剂,对于防治血管再狭窄可能具有潜在的临床应用前景。     

血管平滑肌细胞增殖与Cdk抑制蛋白p27的表达

ｐ２７蛋白是细胞周期素依赖性激酶（Ｃｄｋ）抑制蛋白家族中的一种,主要对外部促进或抑制细胞增殖的信号起反应。本研究应用流式细胞仪（ＦＣＭ）双标记的方法观察血管紧张素Ⅱ（ＡｎｇⅡ）、血管加压素（ＡＶＰ）和血小板源生长因子（ＰＤＧＦ）对血管平滑肌细胞（ＶＳＭＣｓ）细胞周期百分比和ｐ２７蛋白表达量的影响。静止状态培养的ＶＳＭＣｓ加入ＡｎｇⅡ,ＡＶＰ,ＰＤＧＦＢＢ后,在不同时间收集细胞,用碘化丙啶（ＰＩ）标记细胞ＤＮＡ,以确定细胞所处的周期。用ｐ２７蛋白的单抗和标记了ＦＩＴＣ的二抗标记细胞,通过流式细胞仪测定被激发出的荧光量来确定细胞ｐ２７蛋白表达的相对量。结果显示,ＡｎｇⅡ刺激ＶＳＭＣｓ增生,其蛋白含量增加了４３６％（Ｐ＜００１）,但不抑制ｐ２７蛋白的表达;ＡＶＰ可轻度抑制ｐ２７的表达,有轻度促进ＶＳＭＣｓ增殖和增生的作用（Ｐ＜００５）;ＰＤＧＦ明显抑制ｐ２７的表达,引起细胞增殖。本研究结果提示,ｐ２７蛋白抑制ＶＳＭＣｓ通过Ｇ１期进入Ｓ期,是抑制ＶＳＭＣｓ增殖的重要调节因子。     

用荧光影像系统研究莲子叶细胞中核酸和蛋白质的含量

用酶解法解离受精后５ ｄ 和２０ ｄ 的红莲（ Ｎｅｌｕｍｂｏ ｓｐ．） 子叶细胞,应用冷却型科学级电荷偶联装置（ＣＣＤ） 摄像式显微荧光影像系统对单个５ ｄ 细胞和２０ ｄ 细胞的ＤＮＡ、ＲＮＡ和总蛋白相对含量进行了相关测量。实验结果表明：２０ ｄ 细胞ＤＮＡ成为多倍化,为４Ｃ～８Ｃ水平。视窗分析表明：同一４ＣＤＮＡ 视窗中,２０ ｄ 细胞比５ ｄ 细胞的ＲＮＡ和总蛋白含量增加１１ 倍以上。２０ ｄ 细胞的两个ＤＮＡ视窗（４Ｃ和８Ｃ） 中,随着ＤＮＡ倍性的增高,ＲＮＡ 和总蛋白含量也以大致上相应的倍数增高。这些数据表明：红莲子叶贮存蛋白的大量积累既与ＤＮＡ 倍性有关,又与发育过程中贮存蛋白亚基基因选择性的表达有关     

Skeletal muscle denervation increases satellite cell susceptibility to apoptosis

Peripheral motor nerve trauma severely compromises skeletal muscle contractile function. Satellite cells respond to denervation by dividing multiple times, ultimately fusing with other satellite cells or myocytes to form new muscle fibers. After chronic denervation, satellite cell numbers decline dramatically, impairing the ability to regenerate and repair myofibers. This satellite cell depletion may contribute to the mechanical deficit observed in denervated or reinnervated muscle. Apoptosis, an evolutionarily conserved form of cell suicide, is a potential mechanism for satellite cell depletion in denervated skeletal muscle. This work tested the hypothesis that skeletal muscle denervation increases satellite cell susceptibility to apoptotic cell death. Adult rats underwent sciatic nerve transection to denervate the distal hindlimb musculature; rats of similar age without the operation served as controls. Two, 6, 10, or 20 weeks after denervation (n = 6 each group), the gastrocnemius and soleus were excised, enzymatically digested, and plated for satellite cell culture. After reaching 95 percent confluence, satellite cells were treated for 24 hours with tumor necrosis factor-alpha (20 ng/ml) and actinomycin D (250 ng/ml), known pro-apoptotic agents. Immunostaining for activated caspases, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and hematoxylin and eosin staining were performed to identify apoptotic satellite cells. Percentages of apoptotic cells were quantified histomorphometrically. In addition, the presence or absence of bcl-2 and bax was determined by Western blot analysis of control, 6 weeks of denervation, and 10 weeks of denervation specimens. At 6 and 10 weeks after nerve transection, TUNEL and caspase activity were increased more than two-fold in satellite cells isolated from denervated muscle compared with those isolated from control muscle (p < 0.05). In all experimental groups, retention of adherence to the collagen-coated substrate was strongly associated with satellite cell survival. Western blot analysis revealed that adherent satellite cells from all groups expressed both bcl-2 and bax. These data support the authors' hypothesis that skeletal muscle denervation increases satellite cell susceptibility to apoptotic cell death. Apoptosis may play a causative role in the depletion of satellite cells in long-term denervated skeletal muscle.     

Sustained cell proliferation in denervated skeletal muscle of mice

Summary Cellular proliferation in skeletal muscle was measured throughout the first 4 weeks after denervation. Twenty four mice had one leg denervated, and 4 groups of 6 of these mice were injected with tritiated thymidine once daily for 7 days, either during the first, second, third or fourth week after denervation. Autoradiographic labelling of muscle and connective tissue nuclei in denervated muscles was compared with innervated muscles from the opposite innervated legs of the same mice. Labelling of connective tissue and muscle (myonuclear and satellite cell) nuclei was significantly higher in denervated muscles, compared with innervated muscles on the unoperated side. There were no significant differences among labelling of nuclei in muscles denervated for 1, 2, 3 or 4 weeks. However, connective tissue labelling after 1 week of denervation was significantly higher than at later times. This study shows that nuclei of muscle and connective tissue cells proliferate and turnover at high levels for at least one month after denervation.     

Lysosomes in skeletal muscle following denervation

Summary The in-vivo uptake of exogenously applied horseradish peroxidase and the activities of the lysosomal enzymes acid phosphatase and cathepsin D were studied histochemically and/or biochemically in innervated and 2–14 day-denervated tibialis anterior muscles of the mouse. The biochemically determined uptake of horseradish peroxidase showed a large increase already 4 days after denervation. The activities of the lysosomal enzymes increased in a more gradual fashion, and only cathepsin D showed an increase in activity when expressed as total activity per muscle. Histochemically horseradish peroxidase was found to be localized in muscle fibres in characteristic spindle-shaped segments after denervation. The main increase in the number of such segments per transverse section of the muscle occurred between 3 and 6 days after denervation. In serial sections these segments frequently showed positive staining also for acid phosphatase.It is concluded that exogenously applied horseradish peroxidase is taken up into the lysosomal system, which after denervation becomes organized into characteristic spindle-shaped segments in the muscle fibres. The endocytic activity of muscle fibres increases early after denervation. This is followed by a more gradual increase in activity of lysosomal enzymes and finally by an organization of the lysosomal system into characteristic spindle-shaped segments. The results are compatible with the working hypothesis that increased endocytosis may initiate lysosomal activation in denervated skeletal muscle.     

Increased endocytotic and lysosomal activities in denervated type I and type II muscle fibres.

Previous work has shown that increased endocytotic and lysosomal activities occur in the endplate region of denervated skeletal muscle fibres. This, however, does not engage all fibres of a muscle at a given time after denervation. The present study was carried out in order to determine if both type I (slow) and type II (fast) muscle fibres can react to denervation by increased endocytotic and lysosomal activities. Uptake of horseradish peroxidase as a marker for endocytosis was studied in conjunction with acid phosphatase staining for lysosomal activity in type I and type II fibres of the denervated mouse hemidiaphragm. Fibre typing was performed using a monoclonal antibody against fast skeletal myosin and by adenosine triphosphatase staining. The results show that increased endocytosis and lysosomal activation occur in both type I and type II fibres after denervation.     

Increased endocytotic and lysosomal activities in denervated type I and type II muscle fibres

Summary Previous work has shown that increased endocytotic and lysosomal activities occur in the endplate region of denervated skeletal muscle fibres. This, however, does not engage all fibres of a muscle at a given time after denervation. The present study was carried out in order to determine if both type I (slow) and type II (fast) muscle fibres can react to denervation by increased endocytotic and lysosomal activities. Uptake of horseradish peroxidase as a marker for endocytosis was studied in conjunction with acid phosphatase staining for lysosomal activity in type I and type II fibres of the denervated mouse hemidiaphragm. Fibre typing was performed using a monoclonal antibody against fast skeletal myosin and by adenosine triphosphatase staining. The results show that increased endocytosis and lysosomal activation occur in both type I and type II fibres after denervation.     

Nerve activity-independent regulation of skeletal muscle atrophy: role of MyoD and myogenin in satellite cells and myonuclei

Electrical activity is thought to be the primary neural stimulus regulating muscle mass, expression of myogenic regulatory factor genes, and cellular activity within skeletal muscle. However, the relative contribution of neural influences that are activity-dependent and -independent in modulating these characteristics is unclear. Comparisons of denervation (no neural influence) and spinal cord isolation (SI, neural influence with minimal activity) after 3, 14, and 28 days of treatment were used to demonstrate whether there are neural influences on muscle that are activity independent. Furthermore, the effects of these manipulations were compared for a fast ankle extensor (medial gastrocnemius) and a fast ankle flexor (tibialis anterior). The mass of both muscles plateaued at approximately 60% of control 2 wk after SI, whereas both muscles progressively atrophied to <25% of initial mass at this same time point after denervation. A rapid increase in myogenin and, to a lesser extent, MyoD mRNAs and proteins was observed in denervated and SI muscles: at the later time points, these myogenic regulatory factors remained elevated in denervated, but not in SI, muscles. This widespread neural activity-independent influence on MyoD and myogenin expression was observed in myonuclei and satellite cells and was not specific for fast or slow fiber phenotypes. Mitotic activity of satellite and connective tissue cells also was consistently lower in SI than in denervated muscles. These results demonstrate a neural effect independent of electrical activity that 1) helps preserve muscle mass, 2) regulates muscle-specific genes, and 3) potentially spares the satellite cell pool in inactive muscles.     

ON THE DIFFERENTIAL RESPONSE OF SARCOPLASM AND MYOPLASM TO DENERVATION IN FROG MUSCLE

Electron microscopic evidence is presented that the early response to denervation ("simple atrophy") of the semitendinosus m. of the frog is characterized by a greater prominence of the sarcoplasmic reticulum and by the presence, in the interfibrillar spaces, of mitochondria which are more numerous and smaller than in normal muscle. In contrast with the dynamic changes of the sarcoplasmic structural components, the myofibrils showed a progressive decrease in diameter after denervation and throughout the period studied. By carrying out tissue fractionation experiments, the yield of microsome-protein was found significantly greater in the denervated muscles, as compared with the contralateral controls, in this initial stage. Under the conditions attending the overdevelopment of the sarcoplasmic reticulum (SR), denervated semitendinosus m. incorporated valine-C¹⁴ into proteins more actively than the control pairs. The denervated muscles also showed an increase in the number of freely scattered and membrane-bound ribosomes and of polyribosomes, suggesting a more active synthesis of the SR membranes. Pronounced atrophy of the myofibrils, disorganization of the SR, and an increased number of ribonucleoprotein particles lying in the enlarged interfibrillar spaces were the main ultrastructural features of "degenerative atrophy" in frog muscle in the late periods after denervation. The probably adaptive character of the early changes occurring on denervation of frog muscle is discussed.     

Combined effects of tenotomy and denervation on the dynamic properties of soleus muscle of the rat

Isometric contraction time (CT), half relaxation time (1/2 RT), tetanus fusion frequency (TFF) and tetanus: twitch ratio (T : t ratio) were measured in the denervated (D) and tenotomized-denervated (TD) Soleus muscle of the rat. In D muscle there was an apparent speeding effect at the 2nd day after denervation, with a significant decrease of CT, which was followed by the usual slowing process of denervated muscle. In TD muscle, denervation was performed a week after tenotomy. Tenotomy "per se" was ineffective in modifying dynamic properties of muscle, but it accentuated the early shortening of CT caused by denervation, while reducing and delaying the subsequent slowing process. The results are discussed in the light of the hypothesis that muscle disuse has a speeding effect which counteracts the slowing effect of denervation, and/or that tenotomy modifies the effects of denervation by changing the pattern of fibrillation development.     

Fibroblasts that proliferate near denervated synaptic sites in skeletal muscle synthesize the adhesive molecules tenascin(J1), N-CAM, fibronectin, and a heparan sulfate proteoglycan

Four adhesive molecules, tenascin(J1), N-CAM, fibronectin, and a heparan sulfate proteoglycan, accumulate in interstitial spaces near synaptic sites after denervation of rat skeletal muscle (Sanes, J. R., M. Schachner, and J. Covault. 1986. J. Cell Biol. 102:420-431). We have now asked which cells synthesize these molecules, and how this synthesis is regulated. Electron microscopy revealed that mononucleated cells selectively accumulate in perisynaptic interstitial spaces beginning 2 d after denervation. These cells were identified as fibroblasts by ultrastructural and immunohistochemical criteria; [3H]thymidine autoradiography revealed that their accumulation results from local proliferation. Electron microscopic immunohistochemistry demonstrated that N-CAM is associated with the surface of the fibroblasts, while tenascin(J1) is associated with collagen fibers that abut fibroblasts. Using immunofluorescence and immunoprecipitation methods, we found that fibroblasts isolated from perisynaptic regions of denervated muscle synthesize N-CAM, tenascin(J1), fibronectin, and a heparan sulfate proteoglycan in vitro. Thus, fibroblasts that selectively proliferate in interstitial spaces near synaptic sites are likely to be the cellular source of the interstitial deposits of adhesive molecules in denervated muscle. To elucidate factors that might regulate the accumulation of these molecules in vivo, we analyzed the expression of tenascin(J1) and fibronectin by cultured fibroblasts. Fibroblasts from synapse-free regions of denervated muscle, as well as skin, lung, and 3T3 fibroblasts accumulate high levels of tenascin(J1) and fibronectin in culture, showing that perisynaptic fibroblasts are not unique in this regard. However, when they are first placed in culture, fibroblasts from denervated muscle bear more tenascin(J1) than fibroblasts from innervated muscle, indicating that expression of this molecule by fibroblasts is regulated by the muscle's state of innervation; this difference is no longer apparent after a few days in culture. In 3T3 cells, accumulation of tenascin(J1) is high in proliferating cultures, depressed in confluent cultures, and reactivated in cells stimulated to proliferate by replating at low density or by wounding a confluent monolayer. Thus, synthesis of tenascin(J1) is regulated in parallel with mitotic activity. In contrast, levels of fibronectin, which increase less dramatically after denervation in vivo, are similar in fibroblasts from innervated and denervated muscle and in proliferating and quiescent 3T3 cells.(ABSTRACT TRUNCATED AT 400 WORDS)