物理性刺激对骨膜软骨生成的影响

目的探讨物理性刺激对骨膜软骨生成方面的影响,以期培养出一种与正常关节软骨更相似的软骨组织。方法从新西兰大白兔胫骨近端内侧取下骨膜,将骨膜固定在支架上,然后将细胞支架悬吊在旋转瓶内,用水流产生的剪切应力去刺激骨膜。通过宏观观察、体积大小测量、组织切片染色与细胞外基质(ECM)成分的比较及生物力学测试分析软骨体外生长的最佳环境。结果宏观观察发现软骨生长的方向与水流的方向相同。组织切片染色可见有两层不同形态的软骨细胞和不同密度的ECM,免疫组织化学染色见在剪切应力的刺激下,软骨表面可分泌浅层蛋白质及润滑剂,且在不同大小的剪切应力刺激时,软骨表面还会产生不同厚度的表层。结论剪切应力刺激能使骨膜上的干细胞分化形成软骨,同时证明力学环境不仅影响细胞的分化与生长,而且影响细胞的形态与ECM的分泌。     

Cytoskeletal and muscle-like elements in cochlear hair cells

Summary Monospecific antibodies to actin and to tubulin were used as immunofluorescent probes to evaluate the distribution of microtubules and actin filaments in the organ of Corti in mouse and guinea pig. The results indicate that in cochlear receptor cells actin and actin filaments as well as tubulin and microtubules are integral cytoskeletal elements. The presence of actin suggests a possible contractile mechanism within the sensory cilia whereas tubulin is thought to play an important role in the stability of sensory cells. Both proteins are discussed to form structural elements required for the mechano-chemical coupling in hearing.

---

Abbreviations ATP adenosin-tri-phosphate - SDS sodium-dodecyl-sulphate - PBS phosphate-buffered saline Dedicated to Professor Dr. A. Herrmann on the occasion of his 80th birthday     

Calmodulin stimulates the degradation of brain spectrin by calpain

Brain spectrin has been shown to be a preferential substrate of calcium-dependent proteases (Baudry, Bundman, Smith, and Lynch: Science 212:937-938, 1981) and a major calmodulin-binding protein (Kakiuchi, Sobue, and Fujita: FEBS Lett. 132:144-148, 1981). Since calmodulin, spectrin, and a proteolytically derived spectrin fragment are all components of isolated postsynaptic density preparations (Grab, Berzins, Cohen, and Siekevitz: J. Biol. Chem. 254:8690-8696, 1979; Carlin, Bartelt, and Siekevitz: J. Cell Biol. 96:443-448, 1983), we investigated the functional role of calmodulin binding to brain spectrin with respect to its susceptibility to digestion by proteases. We report that calmodulin's interaction with brain spectrin results in a marked acceleration of the rate of spectrin degradation by calcium-dependent proteases (calpains I and II), but not by chymotrypsin. The cleavage of erythrocyte spectrin (which lacks a high-affinity calmodulin binding site) by calpain I is unaffected by the presence of calmodulin. The stimulatory effect of calmodulin is blocked by trifluoperazine, a calmodulin antagonist, which by itself does not modify brain spectrin proteolysis by calcium-dependent proteases. These results suggest a novel role for calmodulin in neuronal function--namely, a synergistic interaction with calcium-dependent proteases in the regulation of cytoskeletal integrity.     

烧伤血清刺激对大鼠肠上皮细胞结构和粘弹性的影响

目的　动态观察烧伤血清对体外肠上皮细胞 (IEC)骨架和细胞生物力学 (粘弹性 )的影响。　方法　培养大鼠肠上皮细胞株IEC 6 ,用烧伤血清刺激后 ,通过细胞骨架免疫组化、细胞ELISA法定量分析以及粘弹性测定技术 ,动态观察IEC致伤前后的变化。　结果　IEC在烧伤血清作用早期 ,骨架蛋白的表达即明显降低 ,微丝、微管蛋白阳性信号减弱 ,细胞粘弹性下降。　结论　细胞骨架的损伤可引起细胞脆性增加、粘弹性下降 ,导致细胞生物力学特性的改变。这种变化 ,可能直接参与烧伤后肠上皮细胞损伤的发生。     

Gene structure and genetic localization of the PCLO gene encoding the presynaptic active zone protein Piccolo

Piccolo belongs to a family of presynaptic cytoskeletal proteins likely to be involved in the assembly and function of presynaptic active zones as sites of neurotransmitter release. Given that abnormalities in the formation of synaptic junctions are thought to contribute to cognitive dysfunction during brain development, we have analyzed and compared the gene structure of the Piccolo gene, PCLO, from humans and mice and determined their chromosomal localization. A comparison of the deduced amino acid sequence of cDNA clones encoding Piccolo from human, mouse, rat and chicken reveals the presence of distinct homology domains. Only subsets of these are also present in the structurally related active zone protein Bassoon indicating that Piccolo and Bassoon perform related but distinct functions at active zones. Characterization of the PCLO gene reveals the presence of 25 coding exons spread over 380kb of genomic DNA. The human PCLO gene maps to 7q11.23-q21.3, a region of chromosome 7 implicated as a linkage site for autism and Williams Syndrome suggesting that alterations in the expression of Piccolo or the PCLO gene could contribute to developmental disabilities and mental retardation.     

骨组织工程研究进展

组织工程学是应用生命科学和工程学的原理及技术，构建、培育活组织，研制生物替代物，以修复或重建组织器官的结构，维持或改善功能的一门新兴学科。目前组织工程学研究的主要问题是：①种子细胞的体内、体外培养；②支架材料的研究与开发；③种子细胞与基质材料相互作用的调控等。骨创伤、肿瘤和炎症等导致的骨缺损是目前骨科临床的常见病和难治病，惟一的方法是通过骨移植进行修复。利用骨组织工程培养的人工骨不仅可修复大面积的骨缺损，而且可按需塑形及大量制备，是一种理想的创伤修复及功能重建的材料。     

Microcystin-LR affects cytoskeleton and morphology of rabbit primary whole embryo cultured cells in vitro

Microcystin-LR is the most frequently studied cyclic heptapeptide produced by different genera of cyanobacteria and is hepatotoxic to livestock and human populations. The adverse effects of microcystin-LR on morphology and cytoskeletal elements in different stages of early embryonal development have been studied in vitro. Embryos and whole embryo cultures have been exposed to microcystin-LR (10–100 μM). Actin filaments were visualized by fluorescence staining and the microtubular network labelled by immunostaining. Growth, development and cytoskeleton organization of the embryos embedded in zona pellucida are not affected by microcystin-LR in concentrations up to 100 μM, while whole embryo cell cultures are affected by the presence of microcystin-LR in the culture medium. High microcystin-LR concentrations (100 μM) cause cells to be detached and destroyed, while lower concentrations (10–20 μM) profoundly affect actin and microtubule organization. These effects are confirmed also by the presence of transformed microcystin-LR in all the media at the lowest concentrations. It seems that the changes to the cells are far more serious than that expressed in cell morphology. From our experiments we conclude that the presence of zona pellucida is an effective way of embryo protection against xenobiotics like microcystin-LR.     

Functional linkage of the cardiac ATP-sensitive K+ channel to the actin cytoskeleton

The role of the cytoskeleton in the rundown and reactivation of adenosine triphosphate (ATP) sensitive K⁺ channels (KATP channels) was examined by perturbing selectively the intracellular surface of inside-out membrane patches excised from guinea-pig ventricular myocytes. Actin filament-depolymerizing agents (cytochalasins and desoxyribonuclease I) accelerated channel rundown, while actin filament stabilizer (phalloidin) or phosphatidylinositol biphosphate (PIP₂; inhibitor of F-actin-severing proteins) inhibited spontaneous and/or Ca²⁺-induced rundown. When rundown was induced by cytochalasin D or by long exposure to high Ca²⁺, channel activity could not be restored by exposure to MgATP, but application of F-actin with MgATP could reinstitute channel activity. The processes of rundown and reactivation of cardiac K_ATP channels may thus be influenced by the assembly and disassembly of the actin cytoskeletal network, which provides a novel regulatory mechanism of this channel.     

细胞松弛素B对内皮细胞损伤修复的影响

目的观察细胞松弛素B对内皮细胞损伤修复过程中微丝骨架系统的形态结构变化,研究阻断微丝功能对内皮细胞损伤修复的影响。方法以培养单层内皮细胞损伤模型,采用免疫荧光染色和3H-TdR掺入法,研究微丝功能对创面愈合及细胞增殖的影响。结果内皮细胞在损伤修复过程中伴随微丝特殊而有序的变化。用细胞松弛素B破坏微丝,可不同程度抑制创面的愈合及细胞增殖,并呈一定的时间-剂量依赖关系。结论微丝功能在促进内皮细胞修复过程中起重要作用,可通过直接或间接效应影响DNA合成,从而影响修复过程。