高原鼢鼠和高原鼠兔心脏对低氧环境的适应

为了探讨高原鼢鼠和高原鼠兔心脏对低氧环境的适应机制,以Sprague-Dawley (SD)大鼠为对照,测量三者的心脏/体重比(HW/BW)、右心室/(左心室 室间隔)重量比[RV/(LV S)];应用免疫组织化学方法测定心肌微血管密度(microvessel density, MVD);通过显微体视学技术比较线粒体的面数密度(NA,单位面积中线粒体数目)、体密度(Vv,单位体积心肌纤维中线粒体的体积密度)、面密度(Sv,单位体积心肌纤维中线粒体外膜的面积密度)、比表面(δ,线粒体外膜面积与其自身体积的比);用分光光度法测定心肌中的肌红蛋白(myoglobin, Mb)含量、乳酸(lactic acid, LD)含量和乳酸脱氢酶(lactate dehydrogenase, LDH)活力;聚丙烯酰胺凝胶电泳观察LDH同工酶谱.结果显示:高原鼢鼠和高原鼠兔HB/WB显著大于SD大鼠(P<0.05), RV/(LV S)显著小于SD大鼠(P<0.05).高原鼢鼠、高原鼠兔和SD大鼠心肌MVD和线粒体NA依次递减(P<0.05);高原鼢鼠线粒体Vv显著低于高原鼠兔和SD大鼠(P<0.05),高原鼠兔与SD大鼠之间没有明显差异;高原鼢鼠线粒体Sv显著高于SD大鼠(P<0.05),与高原鼠兔相比无明显差异;高原鼠兔和SD大鼠的线粒体δ无显著差异,但均明显低于高原鼢鼠(P<0.05).高原鼢鼠和高原鼠兔心肌Mb含量显著高于SD大鼠(P<0.05);高原鼢鼠心肌LD含量显著高于高原鼠兔和SD大鼠(P<0.05);两种高原动物心肌LDH活力显著低于SD大鼠(P<0.05).同工酶谱显示,高原鼢鼠、高原鼠兔和SD大鼠的LDH中H亚基所占比例依次递减.以上结果提示,高原鼢鼠和高原鼠兔通过增加心肌线粒体Sv、MVD以及Mb含量提高其在低氧环境获取氧的能力;同时,由于生境和习性上的不同,两者线粒体指标又表现出差异性.     

高原鼢鼠和高原鼠兔骨骼肌糖酵解和肝脏乳酸代谢的差异(英文)北大核心CSCD

高原鼠兔(Ochotona curzoniac)和高原鼢鼠(Myospalax baileyi)是青藏高原地区特有的土著动物。本文旨在探讨高原鼠兔和高原鼢鼠骨骼肌糖酵解和肝脏乳酸代谢的不同生理机制。我们克隆出两种动物肝脏中的丙酮酸羧化酶(pytuvate carboxy-lase,PC)基因的部分序列;应用real-timePCR法测定两种动物肝脏和骨骼肌中PC、LDH-A和LDH-BmRNA的表达水平;使用苹果酸偶联法测定肝脏中PC酶活力,并测定两种动物骨骼肌和肝脏中乳酸含量、乳酸脱氢酶(lactate dehydrogenase,LDH)活力;用聚丙烯酰胺凝胶电泳观察肝脏和骨骼肌LDH同工酶谱。结果显示:(1)高原鼢鼠骨骼肌LDH-BmRNA的表达量极显著高于高原鼠兔(P<0.01),而LDH-AmRNA的表达量没有差异(P>0.05);(2)高原鼠兔肝脏中PC、LDH-A和LDH-BmRNA的表达量都极显著高于高原鼢鼠(P<0.01);(3)高原鼠兔肝脏和骨骼肌中LDH和乳酸含量以及肝脏中PC活力均极显著高于高原鼢鼠(P<0.01);(4)LDH同工酶谱显示,高原鼠兔骨骼肌以LDH-A4、LDH-A3B、LDH-A2B2为主,而高原鼢鼠骨骼肌以LDH-AB3、LDH-B4为主;在高原鼠兔肝脏中LDH以LDH-A3B,LDH-A2B2,LDH-AB3和LDH-B4为主,而高原鼢鼠肝脏只有LDH-A4。以上结果表明,高原鼠兔通过提高骨骼肌无氧糖酵解的水平为其快速奔跑提供能量,通过提高肝脏中糖异生水平快速将骨骼肌运动所产生的乳酸转化为葡萄糖和糖原,所以减少了在低氧环境中对氧的依赖,而高原鼢鼠尽管生活在低氧的地下洞道,它通过提高骨骼肌有氧糖酵解的水平,为其持续的挖掘活动提供能量。     

高原鼢鼠和高原鼠兔骨骼无机化学成分的研究Ⅰ.常量元素

本文对高原鼢鼠和高原鼠兔骨骼中的无机常量元素K、N a、Ca、M g、P 和A l 进行了比较分析。研究结果表明, 高原鼢鼠骨骼中Ca、P、A l 的含量极显著地高于高原鼠兔(P <0.01) ,K 含量高原鼢鼠极显著地低于高原鼠兔(P < 0.001) , N a 和M g 的含量两者间无显著差异(P > 0.05) ; 骨骼各部位元素总量的分布顺序为: 高原鼢鼠下肢骨> 头骨> 脊柱; 高原鼠兔头骨> 下肢骨> 脊柱。15个元素对中, 大部分元素之间线性相关非常显著, 其中高原鼢鼠10对呈显著的线性相关, 高原鼠兔13对呈显著相关。     

高原鼢鼠和高原鼠兔红细胞低氧适应特征

为探讨高原鼢鼠对低氧高二氧化碳洞道生境及高原鼠兔对高海拔低氧生境的适应机制,用Sysmex SF-3000血细胞分析仪及聚丙烯酰胺凝胶电泳对两种高原动物的血常规及血红蛋白类型进行分析,后者采用聚丙烯酰胺凝胶电泳法。结果表明,高原鼢鼠和高原鼠兔的红细胞数(RBC)、红细胞压积(HCT)及平均红细胞容积(MCV)组间无显著差异(P>0.05),但高原鼢鼠和高原鼠兔的红细胞数显著高于SD大鼠,红细胞压积及平均红细胞容积均显著低于SD大鼠(P<0.05);高原鼢鼠的血红蛋白浓度(HBC)与SD大鼠无显著差异(P>0.05),但显著高于高原鼠兔的HBC(P<0.05)。高原鼢鼠血红蛋白主要有2种类型,高原鼠兔血红蛋白主要有3种类型,而SD大鼠血红蛋白主要有5种类型。从血红蛋白电泳迁移来看,2种高原动物血红蛋白类型有明显的趋同特征并与SD大鼠具有明显的差异。上述结果提示,长期适应高海拔低氧环境的高原动物的红细胞和血红蛋白表现出趋同进化,同时因生境和习性的差异又表现出各自的特异性。     

不同季节高原鼢鼠乳酸脱氢酶活力和同工酶谱

目的:探讨高原鼢鼠对洞道低氧高二氧化碳环境的代谢适应机制。方法:用酶活力分析法,分析春季、夏季和秋季高原鼢鼠血清乳酸脱氢酶(LDH)活力、乳酸含量和组织LDH活力,用聚丙烯酰胺凝胶电泳法分析血清和组织LDH同工酶谱。结果:高原鼢鼠血清LDH活力在春夏秋三季具有明显的差异,春季高于夏季,夏季高于秋季,血清乳酸含量表现出同样的变化趋势;春季血清中五种同工酶条带都清晰可见,夏季血清中LDH5和LDH4清晰可见,秋季血清中只能看见LDH5带。骨骼肌、心肌和脑组织LDH活力较高,而且从春季到秋季显著降低;肝、肾和肺组织LDH活力较低,肝组织LDH活力春季显著高于夏季和秋季,夏秋两季之间没有明显差异;肾和肺组织LDH活力在春季与夏季之间没有明显差异,但秋季明显降低。心、肝、肺、肾、脑和肌肉组织LDH同工酶谱,在春夏秋三季都显示出五条带,并表现出明显的组织差异;各组织同工酶含量也有不同程度的季节差异。结论:高原鼢鼠体内糖酵解过程具有明显的季节性变化,从春季到秋季依次降低,这与它们的季节性活动特点和洞道中氧气和二氧化碳的季节性波动有关。     

高原鼢鼠、高原鼠兔和大鼠肺表面活性物质组成和含量的比较

本研究通过分析肺表面活性物质(pulmonary surfactant,PS)组成和含量探讨高原鼢鼠(Myospalax baileyi)和高原鼠兔(Ochotona curzoniae)的低氧适应机制。高原鼢鼠和高原鼠兔各36只,捕捉于海拔3600 m左右的青海省海南州贵德县拉脊山地区,36只Sprague-Dawley(SD)大鼠购自海拔1500 m左右的兰州大学实验动物中心。所有动物麻醉后通过肺循环进行肺灌洗,充分洗净肺组织中的血液,取出完整的肺组织灌洗得到肺泡灌洗液。应用高效液相色谱法分析三种动物PS中磷脂的组成和含量;应用G-250考马斯亮蓝法、聚丙烯酰胺凝胶电泳(PAGE)和质谱测序分析PS蛋白质的含量和种类;应用溶氧电极测定了PS溶液中的溶氧量。结果表明:高原鼢鼠、高原鼠兔和SD大鼠PS中,总磷脂含量依次显著增加(P<0.05),总蛋白含量依次显著减少(P<0.05);三种动物PS中均含有亚油酰棕榈酰胆碱(linoleic palmitoylphosphatidylcholine,LPPC)、二棕榈酰磷脂酰胆碱(dipalmitoylphosphatidylcholine,DPPC)、磷脂酰甘油(phosphatidylglycerol,PG)、磷脂酰肌醇(phosphatidylinositol,PI)和磷脂酰丝氨酸(phosphatidylserine,PSe)等5种磷脂成分,但其相对含量明显不同;在高原鼢鼠、高原鼠兔和SD大鼠中,LPPC所占比例依次显著增加(P<0.01);高原鼢鼠PS磷脂中,DPPC、PG和PI所占比例极显著高于高原鼠兔(P<0.01),高原鼠兔与SD大鼠之间没有显著差异(P>0.05);高原鼢鼠与高原鼠兔PS磷脂中,PSe所占比例没有显著差异(P>0.05),但均极显著高于SD大鼠(P<0.01)。三种动物PS中均含有血清白蛋白(serum albumin,SA),高原鼢鼠和高原鼠兔PS中含有血红蛋白β亚基构成的含血红素的同源四聚体蛋白,高原鼢鼠和SD大鼠PS中含有免疫球蛋白(immunoglobulin,Ig)重链残基,高原鼢鼠Ig重链残基含量极显著高于SD大鼠(P<0.01),含血红素蛋白含量显著高于高原鼠兔(P<0.05)。高原鼢鼠、高原鼠兔和SD大鼠PS溶液中溶氧量依次显著减小(P<0.01),但均显著高于生理盐水(P<0.01)。以上结果提示,高原鼢鼠和高原鼠兔PS中总蛋白含量显著增加,总磷脂含量显著减少,蛋白成分中含有高含量的含血红素同源四聚体蛋白,且高原鼢鼠主要磷脂成分DPPC的相对含量显著增加,这种PS成分和含量的变化增强了这两种高原动物对低氧环境的适应性。     

高原鼢鼠和高原鼠兔肺细叶的结构特征

高原鼢鼠和高原鼠兔是青藏高原土著动物,对低氧具有很好的适应性.为了探讨在低氧环境中两者肺细叶结构的适应特征,应用体视学方法测量了肺细叶相关指标.结果发现 :高原鼢鼠和高原鼠兔肺单位面积肺泡数显著高于SD大鼠,单个肺泡面积和弹性纤维/肺实质比显著低于SD大鼠;高原鼢鼠肺泡隔厚度最厚,高原鼠兔最薄,且三种动物具显著差异; 高原鼢鼠和高原鼠兔气-血屏障的算术平均厚度(Ta)和调和平均厚度(Th) 均显著低于SD 大鼠;在三个级别的微血管中,高原鼠兔中膜肌层厚度显著低于高原鼢鼠,两种高原动物均显著低于SD大鼠;高原鼢鼠和高原鼠兔的微血管密度(MVD)显著高于SD大鼠.以上结果表明,高原鼢鼠和高原鼠兔肺细叶结构特征表现出一定趋同,这些特征有利于在低氧条件下提高肺气体扩散容量;但是,肺泡隔厚度和微血管中膜肌层厚度/血管外径比又表现出明显的差异,可能是不同生境造成的[动物学报 54(3):531-539,2008].     

高原鼠兔乳酸脱氢酶同工酶对低氧环境的应答

用聚丙酰胺凝胶薄层电泳和紫外光谱法,研究与分析高原鼠兔(Ochotona curzoniae)在天然及模拟低氧条件下,心脏、肝脏、肾脏及骨骼肌4种组织的乳酸脱氢酶(LDH)同工酶的酶谱和其酶活力的变化。     

高原鼢鼠和高原鼠兔血液参数和血红蛋白亚型对不同海拔生境的响应

高原鼢鼠和高原鼠兔是青藏高原特有的物种,对高原低氧环境有很强的适应性。本研究测定了不同海拔的高原鼢鼠和高原鼠兔的红细胞数目、血红蛋白浓度、平均红细胞压积、平均红细胞体积等;通过质谱测序明确两种高原动物血红蛋白亚型;采用PAML4.8程序分析两种动物血红蛋白亚基中的正向选择位点;采用同源建模的方法分析正向选择位点对血红蛋白氧亲和力的影响,基于对两种高原动物血液特征的分析,探讨了它们适应低氧的策略。结果表明,随着海拔升高,高原鼢鼠通过增加红细胞数目、减小红细胞体积应答低氧,相反,高原鼠兔通过减少红细胞数目、增大红细胞体积应答低氧;高原鼠兔红细胞中具有α₂β₂成年型血红蛋白和α₂ε₂胎儿型血红蛋白,高原鼢鼠红细胞中只有α₂β₂成年型血红蛋白,但高原鼢鼠血红蛋白氧亲和力和别构效应显著高于高原鼠兔;高原鼢鼠与高原鼠兔的血红蛋白α和β亚基中,正向选择的氨基酸位点数目、位置以及它们的侧链基团极性和取向具有明显不同,这可能是造成两种动物血红蛋白氧亲和力不同的重要原因。总之...     

高原鼠兔和高原鼢鼠种群暴发区干扰斑块空间分布格局与演替规律

高原鼠兔(Ochotona curzoniae)和高原鼢鼠(Myospalax baileyi)是两种在高寒草甸广泛分布的小型哺乳动物,其暴发活动可能造成高寒草甸的严重退化。使用无人机分别拍摄典型的高原鼠兔和高原鼢鼠种群暴发区图像,解译高原鼠兔洞口、高原鼢鼠土丘和它们的干扰斑块,获得干扰斑块的周长和面积信息,计算干扰斑块形状指标,确定高原鼠兔与高原鼢鼠暴发活动对高寒草甸的影响。研究表明：(1)高原鼠兔与高原鼢鼠暴发活动形成的干扰斑块周长与面积比(PARA)、形状指数(SHAPE)和分形维数(FRAC)差异显著(P<0.05),其中高原鼢鼠斑块的FRAC是1.0124±0.0091,属于普通的规则几何圆形分布格局,高原鼠兔斑块的FRAC是1.1203±0.0546,属于分形几何不规则的分布格局。(2)高原鼠兔洞口数量与其斑块面积没有线性相关关系(P=0.9677),高原鼢鼠土丘数量与其斑块面积呈显著正相关(P<0.05)。(3)高原鼢鼠斑块面积统计学上呈长尾分布,不符合正态分布(P<0.01)。根据高原鼠兔斑块面积分布格局可将斑块演替分为三个阶段：土丘形成阶段、斑块连通...     

Muscle and Muscle Fiber Type Transformation In Clawed Crustaceans

SYNOPSIS. The first pair of thoracic limbs in many crustaceansis elaborated into claws in which the principal muscle is thecloser. Changes in the fiber composition of the closer muscleduring claw development, regeneration and reversal are reviewedhere and the hypothesis is advanced that such changes are nerve-dependent.In adult lobsters, Homarus amencanus, the paired claws and closermuscles are bilaterally asymmetric, consisting of a minor orcutter claw with predominantly fast fibers and a small ventralband of slow and a major or crusher claw with 100% slow fibers.Yet in the larval and early juvenile stages the paired clawsand closer muscles are symmetric consisting of a central bandof fast fibers sandwiched by slow. Differentiation into a cutteror crusher muscle during subsequent juvenile development isby appropriate fiber type transformation. Experimental manipulationof the claws or the environment in early juvenile stages whenthe claws are equipotent revealed that the determination ofclaw and closer muscle asymmetry is dependent on the convergenceof neural input from the paired claws: the point of convergencemost likely being the CNS. Bilaterally symmetrical input resultsin the development of paired cutter claws while bilaterallyasymmetric input gives rise to dimorphic, cutter and crusherclaws. In the northern crayfish, Orconectes rusticus, wherethe paired claws are bilaterally similar, the closer muscletransforms its central band of fast fibers to slow, both duringprimary development and regeneration. Whether these fiber typetransformations are nerve-dependent is unknown. In adult snappingshrimps, Alpheus sp., the paired claws and closer muscles areasymmetric: the minor or pincer claw has a central band of fastfibers flanked by slow while the major or snapper claw has 100%slow fibers. Claw reversal occurs with removal of the snapperresulting in the transformation of the existing pincer to asnapper and the regeneration of a new pincer at the old snappersite. Transformation of the closer muscle from pincer to snappertype is by degeneration of the fast fiber band and hypertrophyof the slow fibers. Claw transformation can be either preventedif the pincer nerve is sectioned at the time of snapper removalor promoted if the snapper nerve is sectioned: both resultsimplicating a neural basis for muscle transformation.     

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Body movements are mainly provided by mechanical function of skeletal muscle. Skeletal muscle is composed of numerous bundles of myofibers that are sheathed by intramuscular connective tissues. Each myofiber contains many myofibrils that run longitudinally along the length of the myofiber. Myofibrils are the contractile apparatus of muscle and they are composed of repeated contractile units known as sarcomeres. A sarcomere unit contains actin and myosin filaments that are spaced by the Z discs and titin protein. Mechanical function of skeletal muscle is defined by the contractile and passive properties of muscle. The contractile properties are used to characterize the amount of force generated during muscle contraction, time of force generation and time of muscle relaxation. Any factor that affects muscle contraction (such as interaction between actin and myosin filaments, homeostasis of calcium, ATP/ADP ratio, etc.) influences the contractile properties. The passive properties refer to the elastic and viscous properties (stiffness and viscosity) of the muscle in the absence of contraction. These properties are determined by the extracellular and the intracellular structural components (such as titin) and connective tissues (mainly collagen) ^1-2. The contractile and passive properties are two inseparable aspects of muscle function. For example, elbow flexion is accomplished by contraction of muscles in the anterior compartment of the upper arm and passive stretch of muscles in the posterior compartment of the upper arm. To truly understand muscle function, both contractile and passive properties should be studied.The contractile and/or passive mechanical properties of muscle are often compromised in muscle diseases. A good example is Duchenne muscular dystrophy (DMD), a severe muscle wasting disease caused by dystrophin deficiency ³. Dystrophin is a cytoskeletal protein that stabilizes the muscle cell membrane (sarcolemma) during muscle contraction ⁴. In the absence of dystrophin, the sarcolemma is damaged by the shearing force generated during force transmission. This membrane tearing initiates a chain reaction which leads to muscle cell death and loss of contractile machinery. As a consequence, muscle force is reduced and dead myofibers are replaced by fibrotic tissues ⁵. This later change increases muscle stiffness ⁶. Accurate measurement of these changes provides important guide to evaluate disease progression and to determine therapeutic efficacy of novel gene/cell/pharmacological interventions. Here, we present two methods to evaluate both contractile and passive mechanical properties of the extensor digitorum longus (EDL) muscle and the contractile properties of the tibialis anterior (TA) muscle.     

Lack of CFTR in Skeletal Muscle Predisposes to Muscle Wasting and Diaphragm Muscle Pump Failure in Cystic Fibrosis Mice

Cystic fibrosis (CF) patients often have reduced mass and strength of skeletal muscles, including the diaphragm, the primary muscle of respiration. Here we show that lack of the CF transmembrane conductance regulator (CFTR) plays an intrinsic role in skeletal muscle atrophy and dysfunction. In normal murine and human skeletal muscle, CFTR is expressed and co-localized with sarcoplasmic reticulum-associated proteins. CFTR–deficient myotubes exhibit augmented levels of intracellular calcium after KCl-induced depolarization, and exposure to an inflammatory milieu induces excessive NF-kB translocation and cytokine/chemokine gene upregulation. To determine the effects of an inflammatory environment in vivo, sustained pulmonary infection with Pseudomonas aeruginosa was produced, and under these conditions diaphragmatic force-generating capacity is selectively reduced in Cftr^−/− mice. This is associated with exaggerated pro-inflammatory cytokine expression as well as upregulation of the E3 ubiquitin ligases (MuRF1 and atrogin-1) involved in muscle atrophy. We conclude that an intrinsic alteration of function is linked to the absence of CFTR from skeletal muscle, leading to dysregulated calcium homeostasis, augmented inflammatory/atrophic gene expression signatures, and increased diaphragmatic weakness during pulmonary infection. These findings reveal a previously unrecognized role for CFTR in skeletal muscle function that may have major implications for the pathogenesis of cachexia and respiratory muscle pump failure in CF patients.     

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.Download video file.^{(90M, mov)}