pH值对应用mATP酶法进行梭外肌纤维分型的影响

目的 探讨酸性和碱性预孵育处理，对应用mATP酶法进行梭外肌纤维分型的影响。方法 mATP酶法。结果 ①用pH4．3的预孵育液孵育后，梭外肌纤维可分为Ⅰ，ⅡA，ⅡAB及ⅡB型；pH4．6预孵育后，梭外肌纤维可分为Ⅰ，ⅡA和ⅡB型；pH9．4和pH10．4预孵育液条件下，梭外肌纤维均可分为Ⅰ型和Ⅱ型肌纤维，且Ⅰ型肌纤维的比例略低于酸法。②预孵育液和作用液的pH值随时间的延长均有下降的趋势。结论 用mATP酶法研究梭外肌纤维的分型时应当说明并严格把握预孵育液的pH值。     

纤维粘连蛋白在人滋养上皮的不同定位与功能

从正常人不同发育时期,不明原因流产,增殖型和侵蚀型葡萄胎滋养细胞角度,用免疫组织化学方法观察纤维粘连蛋白（FN）的显微定位,比较研究其不同定位与滋养上皮增殖,生长，分化,凋亡，迁移和浸润的关系。结果显示 正常人不同发育时期,FN在早孕合体滋养细胞基底膜和绒毛外滋养细胞膜呈阳性着色,在中期非合体结处的滋养细胞质呈免疫反应阳性,在足月滋养细胞呈阴性着色; 不明原因流产,FN在合体滋养细胞核内呈阳性着色; FN在增殖型葡萄胎滋养细胞膜和绒毛外滋养细胞质呈强阳性着色;FN在侵蚀型葡萄胎滋养细胞质呈阳性着色。提示FN胞质定位与滋养上皮迁移和侵蚀密切相关,FN基底膜定位与滋养细胞分化密切相关,FN胞膜定位与滋养细胞增殖相关,FN的阴性着色与滋养细胞衰老,FN胞核转位与滋养细胞凋亡可能相关。     

蒙古羊脂肪来源间充质干细胞的分离培养及体外分化诱导

建立蒙古羊脂肪间充质干细胞（Adipose-derived mesenchymal stem ceils,ADSCs）体外分离培养方法,并对其生物学特性和多向分化潜能进行鉴定。利用I型胶原酶将蒙古羊脂肪组织消化后,离心得到单核细胞,并进行传代培养,测定其倍增时间。采用甲苯胺蓝染色和PAS染色法以及RT-PCR法,分别从组织学水平和基因水平对第3代蒙古羊ADSCs向成神经和成心肌的诱导分化情况进行鉴定。结果显示,分离得到的脂肪间充质干细胞大小较为均匀,呈梭形或星形的成纤维细胞样;传代接种后第4天细胞进入指数生长期,第8天进入平台期,前10代ADSCs的倍增时间平均为34．1h;经成神经诱导后,细胞呈胶质细胞状,RT-PCR检测EN02和GFAP基因表达呈阳性;心肌诱导后,细胞体积增大,多呈长梭形,平行排列,诱导15d后部分细胞可见类肌管样结构,PAS染色可见明显的糖原沉积,RT-PCR检测NKX2．5和GATA-4基因表达呈阳性。表明获得的蒙古羊ADSCs具有多向分化潜能。     

H+对非洲爪蟾卵母细胞ATP-激活电流的调制作用

研究H^＋对非洲爪蟾卵母细胞内源性ATP受体的调制作用。采用双极电压钳技术记录卵母细胞膜ATP受体介导的电流。实验检测的细胞全部对ATP敏感，应用pH为7．4的ATP（10^-3mol／L）后引起一特征性电流，呈浓度依赖性。改用pH为6．5，6．0，5．5的ATP（10^-3mol／L）后，电流幅值明显减弱。结果表明，pH值降低，可抑制ATP诱导的卵母细胞膜电流。     

利用生物酶法制备土豆高纯膳食纤维粉

利用生物蛋白酶和脂肪酶对生产土豆淀粉过程中产生的土豆漆在近中性条例上进行水解，制备了食用性高纯土豆纤维粉。该实验结果表明，对蛋白酶的适宜水解温度应为60℃，最佳PH值为7．1，时间为10－12h；对脂肪酶的适宜水解温度应为45℃，最佳PH为7．5，时间为8－10h，通过酶法处理后得到的纤维素具有纯度高，口感好等特点，同时，也为制备其他纤维性食品提供了一种参考途径。     

人颞肌亚部化及其肌内神经分布研究

目的:查明人颞肌肌内神经、肌梭的配布特征以及相互关系,为临床成功进行肌移植提供解剖学依据.方法:20侧成人尸体颢肌标本,每具尸体选1侧,共10侧采用改良Sihler氏神经染色观察颞肌肌内神经分布.另10侧标本,依据颞肌肌纤维排列方向分前、中、后3部份,分别制作组织块,HE染色后应用图像分析仪对颞肌肌梭的形态结构、空间构象以及分布密度进行体视学分析.结果:改良Sihler氏神经染色显示颞肌有3条独立的神经分支呈放射状由颢肌下缘向肌远端分布.颞肌肌梭分布呈不均质性.在颞肌前部近喙突处神经分支密集区,肌梭分布密度高,空间构象多样;而在颞肌后部以及颞肌的远端缺乏肌梭的分布.结论:颞肌可划分为3个功能完整和独立的肌亚部.     

3个年龄组的腓肠肌纤维型分布

实验采用肌球蛋白ATP酶染色法检测了 8例男性 (3～ 16岁 )、8例男性 (18～ 45岁 )、5例男性 (5 0～ 6 5岁 )腓肠肌各亚部肌纤维型的构成和分布 ,结果表明 ,不同年龄组Ⅰ型纤维比例不同 ,分别为 (5 2 .5± 9) %、(5 6 .5±6 ) %、(5 8.4± 4.8) %。其中 3～ 16岁年龄组的Ⅰ型纤维比例显著高于其他两年龄组 (P <0 .0 5 ) ;而后两年龄组的Ⅰ型纤维比例在统计学上无显著差异 ,但随着年龄的增长 ,Ⅰ型纤维比例增加。从外侧头和内侧头亚部分析 ,只有内侧头深部 (3～ 16岁 )的Ⅰ型纤维比例 (5 2 .9± 8) %显著高于其它两年龄组 (P <0 .0 5 ) ;外侧头深部 (3～ 16岁 )的Ⅰ型纤维比例 (5 2 .3± 7.2 ) %显著高于其它两年龄组 (P <0 .0 5 )。而其它内、外侧头亚部 3年龄组在统计学上无显著差异。说明 3～ 16岁年龄组和后两年龄组肌纤维型比例有较大差异 ;18～ 45岁、5 0～ 6 5岁年龄组之间Ⅰ型纤维比例和内、外侧头各亚部之间Ⅰ型肌纤维比例差异不显著。本文还对不同年龄组肌纤维构成与肌功能适应的问题作了讨论。     

家兔在发育过程中外侧腓肠肌内MyHCs变化与诱发电位的关系

 为研究在生后不同年龄组家兔外侧腓肠肌各亚体肌纤维MyHCs变化与诱发电位的关系,探讨生后发育期间肌纤维MyHCs组成和作用差异与表型之间的相关联系,采用电生理记录仪结合十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS PAGE）检测外侧腓肠肌各亚体。结果表明中间亚体在刺激时各年龄组的峰值电压基本相同,而外侧亚体和内侧亚体分别有些变化。同时发现成年家兔的持续时间都较幼年的长,这可能与各亚体的肌纤维型构成比例有很大关系。外侧腓肠肌各亚体的肌球蛋白重链异构体（MyHCs）电泳条带分别显示MyHCsⅡa、Ⅱd（或Ⅱx）、Ⅱb、Ⅰ共4种,对应于骨骼肌纤维表型ⅡA型（FOG型）、ⅡX（FO型）、ⅡB型（FG型）及Ⅰ型（SO型）4类。     

小鼠胚胎干细胞建系新方法的初步研究

通过对小鼠胚胎着床初期子宫组织块的培养及内细胞团的分离，探讨利用该方法获得胚胎干细胞的可能性，我们用这种方法已建立了一株小鼠胚胎干细胞系，传至第10代，AKP染色细胞呈强阳性，RT—PCR证实Qct-4表达强阳性。结果表明，利用这种方法获得胚胎干细胞是可行的，与传统方法相比，这种新方法极大简化了实验步骤，降低了实验技术难度。     

体外诱导人骨髓间充质干细胞向胰岛β样细胞分化的研究

目的：探讨体外诱导人骨髓间充质干细胞（ＭＳＣs）向胰岛β样细胞分化。方法：在无菌条件下从正常成人骨髓中分离间充质干细胞,体外培养传3代后用表皮生长因子（epidermal growth factor,EGF）、β-巯基乙醇和高糖培养基诱导MSCs向胰岛β样细胞分化。观察MSCs在诱导前后的形态变化;用胰岛素免疫细胞化学染色检测胰岛素的表达;用双硫腙染色鉴定胰岛β样细胞。结果：未经诱导的MSCs在培养体系中呈贴壁生长,长梭形,经诱导分化后,细胞逐渐变圆,并聚集成团;胰岛素免疫细胞化学表明细胞团内的细胞呈胰岛素染色强阳性反应：双硫腙染色阳性。结论：人骨髓间充质干细胞可在体外被定向诱导分化为胰岛β样细胞。     

Synthesis of fast myosin induced by fast ectopic innervation of rat soleus muscle is restricted to the ectopic endplate region

Skeletal muscle fibres, long multinucleated cells, arise by fusion of mononucleated myoblasts to form a myotube that matures into the adult fibre. The two major types of mature fibre, fast and slow fibres, differ physiologically in their rate of isotonic shortening. At the molecular level these type-specific physiological properties are ascribed to different isoforms of myosin, a major protein involved in shortening. Differentiation of fast and slow fibres seems to be under the control of motoneurones, and mature fibres are innervated by only one motoneurone. When rat soleus muscle (SOL, a slow muscle) is dually innervated with a fast nerve, it acquires some properties of a fast muscle, that is, low sensitivity to caffeine and high glycogen content. We report here that in dually innervated soleus muscle the foreign fast nerve induces synthesis of fast isoforms of myosin, but only in the segment of the muscle fibre that is close to the foreign endplate. The localized influence of the nerve endplates suggest that factors controlling the phenotypic expression of the muscle fibre have a short range of activity.     

Fibre type composition of single motor units during synapse elimination in neonatal rat soleus muscle

Skeletal motor neurones innervate the specialized 'types' of fibres comprising most mammalian muscles in a characteristic fashion: each motor neurone forms a 'motor unit' by innervating a set of fibres all of the same type. Because the type expression of adult muscle fibres is plastic and apparently controlled by their innervation, each motor neurone is thought to impose a common type differentiation on all the fibres in its motor unit. However, the situation in developing muscles cannot be this simple. Muscle fibres in neonates receive synaptic input from several motor neurones and achieve the adult, single innervation only after a period of 'synapse elimination. Despite this polyneuronal innervation, differentiated fibre types are present in neonatal muscles. This means either that the motor neurones polyneuronally innervate fibres in a random fashion and type expression is not determined by innervation or that the polyneuronal innervation is ordered in such a way that each fibre could receive unambiguous instructions for type differentiation. We have investigated these possibilities here by determining the fibre type composition of motor units in neonatal rat soleus muscle. We find that even during the time of polyneuronal innervation each motor neurone confines its innervation to largely one of two fibre types present in the muscle. Therefore, some mechanism during early development segregates the synapses of two groups of soleus motor neurones onto two separate populations of soleus muscle fibres.     

Migration of myoblasts across basal lamina during skeletal muscle development

Basal lamina is a sheet of extracellular matrix that separates cells into topologically distinct groups during morphogenesis and is thought to form a barrier to cell migration. We have examined whether, during normal muscle development, myoblasts--mononucleate muscle precursor cells--can cross the basal lamina that surrounds each multinucleate muscle fibre. We marked myoblasts in vivo by injecting replication-defective retroviral vectors encoding LacZ into muscle tissue and analysed the fate of their progeny by the expression of beta-galactosidase. A dual labelling method with broad application to retroviral lineage-marking studies was developed to ensure that most clusters of labelled cells were clones derived from a single precursor cell. Most of the myoblasts that were infected at a late stage of rat hindlimb development, when each fibre with its satellite myoblasts is individually encased in a basal lamina sheath, gave rise to clones that contributed to several labelled fibres. Our results show that myoblasts from healthy fibres migrate across basal lamina during normal development and could contribute to the repair of fibres damaged by injury or disease.     

Embryonic acetylcholine receptors guarantee spontaneous contractions in rat developing muscle

Many proteins are expressed in distinct embryonic and adult forms. However, in most cases we do not know why the embryonic form of proteins is required. This question can be readily addressed for the acetylcholine receptor (AChR) because developmentally specified modifications of this ligand-gated ion channel can be directly related to changes in membrane currents. In developing rat soleus muscle, spontaneous transmitter release causes miniature end-plate currents (m.e.p.cs) to flow into the muscle cell. We show here that these m.e.p.cs in neonatal soleus trigger spontaneous contractions. By injecting m.e.p.cs into young fibres, we showed that only embryonic m.e.p.cs can trigger such contractions; adult m.e.p.cs do not last long enough. Developing muscle fibres must be active for synapse and muscle differentiation. Our experiments indicate that the embryonic form of the AChR is essential for spontaneous contractile activity and may therefore be required for normal neuromuscular development.     

Imprinting of acetylcholine receptor messenger RNA accumulation in mammalian neuromuscular synapses

IN mammalian muscle, the subunit composition of the nicotinic acetylcholine receptor (AChR) and the distribution of AChRs along the fibre are developmentally regulated. In fetal muscle, AChRs are distributed over the entire fibre length whereas in adult fibres they are concentrated at the end-plate. We have used in situ hybridization techniques to measure the development of the synaptic localization of the messenger RNAs (mRNAs) encoding the alpha-subunit and the epsilon-subunit of the rat muscle AChR. The alpha-subunit is present in both fetal and adult muscle, whereas the epsilon-subunit appears postnatally and specifies the mature AChR subtype. The synaptic localization of alpha-subunit mRNA in adult fibres may arise from the selective down-regulation of constitutively expressed mRNA from extrasynaptic fibre segments. In contrast, epsilon-subunit mRNA appears locally at the site of neuromuscular contact and its accumulation at the end-plate is not dependent on the continued presence of the nerve terminal very early during synapse formation. This suggests that epsilon-subunit mRNA expression is induced locally via a signal which is restricted to the end-plate region and is dependent on the presence of the nerve only during a short period of early neuromuscular contact. Evidently, several mechanisms operate to confine AChR mRNAs to the adult end-plate region, and the levels of alpha-subunit and epsilon-subunit mRNAs depend on these mechanisms to differing degrees.     

Why animals have different muscle fibre types

Animals have different muscle fibre types: slow fibres with a low maximum velocity of shortening (Vmax) and fast fibres with a high Vmax. An advantage conferred by the use of different fibre types during locomotion has been proposed solely on the basis of their in vitro properties. Isolated muscle experiments show that force generation, mechanical power production and efficiency are all functions of V/Vmax, where V is the velocity of muscle shortening. But it is not known whether animals actually use the different fibres at shortening velocities that are optimal for mechanical power production and efficiency. Here we compare the V of muscle fibres during locomotion with their Vmax. This comparison shows that during slow locomotion, the slow fibres shorten at a velocity that gives peak mechanical power and efficiency and the fast fibres shorten at their optimal velocity when powering maximal movements. Our results also show that maximal movements are impossible without fast fibres because the slow ones cannot shorten rapidly enough.     

An agrin minigene rescues dystrophic symptoms in a mouse model for congenital muscular dystrophy

Congenital muscular dystrophy is a heterogeneous and severe, progressive muscle-wasting disease that frequently leads to death in early childhood. Most cases of congenital muscular dystrophy are caused by mutations in LAMA2, the gene encoding the alpha2 chain of the main laminin isoforms expressed by muscle fibres. Muscle fibre deterioration in this disease is thought to be caused by the failure to form the primary laminin scaffold, which is necessary for basement membrane structure, and the missing interaction between muscle basement membrane and the dystrophin-glycoprotein complex (DGC) or the integrins. With the aim to restore muscle function in a mouse model for this disease, we have designed a minigene of agrin, a protein known for its role in the formation of the neuromuscular junction. Here we show that this mini-agrin-which binds to basement membrane and to alpha-dystroglycan, a member of the DGC-amends muscle pathology by a mechanism that includes agrin-mediated stabilization of alpha-dystroglycan and the laminin alpha5 chain. Our data provides in vivo evidence that a non-homologous protein in combination with rational protein design can be used to devise therapeutic tools that may restore muscle function in human muscular dystrophies.     

Is resistance of a muscle to fatigue controlled by its motoneurones?

The original experiment of Buller et al. and the many subsequent confirmatory reports clearly show that the time-to-peak tension and many other speed-related parameters of slow and fast muscle fibres are dictated by the motoneurone. It has been concluded that the motoneurone exerts this control of the physiological and associated biochemical properties by the frequency at which it excites the muscle fibre. However, no studies have been reported on the fatigue properties and the associated biochemical characteristics after cross-reinnervation. Based on the 'size principle' of motoneurones, it would be reasonable to assume that a muscle fibre reinnervated by a small motoneurone would be active often and that this would be manifested biochemically as an elevated oxidative capacity. Also, it has been shown repeatedly that the mitochondrial content of a muscle fibre can be modified by daily endurance type exercise. Thus, it would seem that the motoneurone at least indirectly also controls the mitochondrial content of a muscle fibre by controlling the degree of activity. We have now tested this hypothesis using self- and cross-reinnervated muscles in cats. We found that fast- and slow-twitch muscles retained their characteristic fatigue resistance properties regardless of whether the nerve to which they had become connected had originally innervated a fatigue-resistant or relatively fatiguable muscle.     

Contractile force measured in unskinned isolated adult rat heart fibres

A number of investigators have succeeded in preparing isolated cardiac cells by enzymatic digestion which tolerate external [Ca2+] in the millimolar range. However, a persistent problem with these preparations is that, unlike in situ adult ventricular fibres, the isolated fibres usually beat spontaneously. This spontaneity suggests persistent ionic leakage not present in situ. A preferable preparation for mechanical and electrical studies would be one which is quiescent but excitable in response to electrical stimulation and which does not undergo contracture with repeated stimulation. We report here a modified method of cardiac fibre isolation and perfusion which leaves the fibre membrane electrically excitable and moderately resistant to mechanical stress so that the attachment of suction micropipettes to the fibre is possible for force measurement and length control. Force generation in single isolated adult rat heart fibres is consistent with in situ contractile force. The negative staircase effect (treppe) characteristic of adult not heart tissue is present with increased frequency of stimulation. Isometric developed tension increases with fibre length as in in situ ventricular tissue.