西南三省一市解剖学会第七届学术年会议论文摘要

SD大鼠骨骼肌卫星细胞的成脂、成骨、成心肌样细胞的诱导 王凡，石钊（四川大学基础医学与法医学院解剖学教研室，四川成都610041）目的体外分离并成功诱导SD大鼠骨骼肌卫星细胞（SCs）分化为脂肪细胞，成骨细胞，心肌样细胞，证实了骨骼肌卫星细胞的多向分化潜能，为临床组织工程学提供可靠的种子细胞。     

聚合物表面生物修饰对转化人胚肌腱细胞粘附力学特性的影响

目的探讨增强转化人胚肌腺细胞与聚合物材料粘附力学特性的措施。方法采用生物可降解聚合物一乳酸与羟基乙酸共聚物85／15（PLGA85／15）,制成造光的薄膜,表面被衬细胞gbe质（l型胶原蛋白、纤维粘连蛋白,以及相应的抗体）和生长因子（类胰岛素生长因子1）,接种转化人胚肌健细胞后,利用微吸管实验技术测定转化人胚肌腱细胞与聚合物薄膜的粘附力。结果（1）随着I型胶原蛋白裤衬浓度从0μpg／ml增加到10μg／ml,转化人胚肌位细胞与聚合物薄膜的粘附力从1．57×10-8N增至4.17×10-8N,增幅达170％;若在I型胶原蛋白祛衬基础上,复合核…     

细胞外基质对气道平滑肌细胞增殖活性的促进作用及其机制

目的 观察纤维粘连蛋白（FN），层粘连蛋白（LN），及胶原蛋白I（ColI)等主要细胞外基质成分，对气道平滑肌细胞（ASMCs)增殖活性的影响，并探讨其作用机制。方法 将原代培养的大鼠ASMCs，接种于不同细胞外基质蛋白包被的培养板，观察各时间点细胞的贴壁数量。在培养的ASMCs中分别加入两种浓度（20和60mg/L)的不同细胞外基质蛋白溶液，即FN组，LN组和Col I组，采用^3H-TdR掺入法，检测作用8，12和24h后各组ASMCs增殖活性的变化。间接免疫荧光染色法检测RAS蛋白在各组ASMCs中的表达。结果 与无包被组相比较，FN，ColI包被组及LN包被组ASMCs的贴壁数量明显高（P＜0．01或P＜0．05）。与正常对照组相比较，FN和Col I各浓度组ASMCs的增殖活性增高显著（P＜0．01），且与浓度和作用时间成正比；而LN组ASMCs的活性无明显差异。同时，FN和Col I组ASMCs的RAS染色呈强阳性；对照组及LN组ASMCs的染色则为弱阳性。结论 ASMCs对FN，LN和Col I均有良好的粘附性，但只有FN的ColI 能明显提高ASMCs的增殖活性，其作用是通过RAS信号转导途径而实现的。表明细胞外基质是哮喘气道重塑的重要刺激因子之一。     

雌激素对骨骼肌缺血再灌注损伤的保护作用

目的观察17β-雌二醇对大鼠骨骼肌缺血再灌注损伤的作用。方法制作右后肢缺血再灌注模型，30只大鼠随机分为三祖，A组：假手术组；B组：骨骼肌缺血再灌注（I／R）＋生理盐水组；C组；I／R＋雌激素干预组。测定血浆肌酸磷酸激酶（CPK）、乳酸脱氢酶（LDH）、丙二醛（MDA）和小腿三头肌组织中髓过氧化酶（MPO）活性及组织结构变化。结果B组、C组与A组比较，血浆CPK、LDH、MDA及MPO含量明显升高，B组、C组均可见骨骼肌损伤，B组明显重于C组。结论雌激素对肢体骨骼肌缺血再灌注损伤具有保护作用。     

排斥性导向分子a抑制剂6FNⅢ对大鼠大脑皮质神经元缺血再灌注后自噬的作用

目的 探讨排斥性导向分子a(RGMa)特异性抑制剂6FNⅢ对大鼠大脑皮质神经元缺血再灌注引发的自噬及对神经功能恢复的影响。 方法 雄性成年SD大鼠立体定位侧脑室注射不同浓度6FNⅢ后建立大脑中动脉闭塞（MCAO）/再灌注模型,Western blotting检测RGMa下游分子脑衰反应调节蛋白-2（CRMP-2）蛋白表达水平,确定最佳6FNⅢ干预浓度。将72只雄性成年SD大鼠随机分成4组：假手术组（sham）、脑缺血再灌注组（I/R）、生理盐水干预组（I/R+NS）、6FNⅢ干预组（I/R+6FNⅢ）,每组18只。缺血2h再灌注后24h,采用神经功能缺损评分评估各组大鼠神经功能恢复情况;Western blotting检测自噬相关蛋白LC3Ⅱ/Ⅰ水平;免疫荧光双标检测LC3在神经元内的表达情况;透射电子显微镜观察自噬体的形成情况。 结果 根据各浓度6FNⅢ干预组CRMP-2表达,确定中浓度（0.5 g/L）为本实验的最适浓度。缺血再灌注后24h,I/R组较sham组神经功能评分降低(P＜0.05)、自噬相关蛋白LC3Ⅱ/Ⅰ水平升高(P＜0.05)、自噬体形成增多;I/R+6FNⅢ组较I/R组神经功能评分增高(P＜0.05)、自噬相关蛋白LC3Ⅱ/Ⅰ水平降低(P＜0.05)、自噬体形成减少;而I/R+NS组与I/R组以上各项均无统计学意义(P＞0.05)。 结论 RGMa特异性抑制剂6FNⅡ可在缺血再灌注急性期抑制自噬发生,促进神经功能恢复。     

左旋卡尼汀对缺氧/复氧诱导的子鼠心肌细胞凋亡的影响

目的：探讨左旋卡尼汀（L-carnitine）对缺氧/复氧（I/R）诱导的乳鼠心肌细胞凋亡的影响及其可能的作用机制。方法： 采用原代培养大鼠子鼠心肌细胞作为模拟缺血再灌注损伤实验模型，实验分3组：①正常对照组（control）；②I/R组:缺氧120 min,复氧240 min;③L-carnitine组：I/R前2 h加用L-carnitine设4个浓度梯度。观察各组细胞经I/R损伤后，细胞内超氧化物歧化酶（SOD）活性、丙二醛（MDA）含量的变化情况，并用流式细胞仪（FCM）检测细胞凋亡率。结果： I/R组SOD活性低于对照组，MDA含量、细胞凋亡率均高于对照组，与control组相比均有显著差异(P＜0.05)；在L-carnitine用药组，SOD活性高于I/R组，MDA含量、细胞凋亡率均显著高于I/R组，P＜0.05。结论： 左旋卡尼汀对I/R损伤诱导的子鼠心肌细胞凋亡有抑制作用，其作用在一定范围内呈剂量依赖关系。     

缺血再灌注诱导大鼠骨骼肌组织蛋白质组变化的初步研究

目的：研究缺血再灌注(ischemia/reperfusion, I/R)对大鼠骨骼肌组织蛋白质表达谱的影响。方法：健康雄性Wistar大鼠12只随机分为2组(n=6)：假手术组和I/R组，无创动脉夹夹闭右侧股动脉4 h，松夹再灌注24 h建立I/R模型；实验结束时提取骨骼肌组织蛋白质，双向电泳技术分离骨骼肌组织蛋白质，分析差异显示的蛋白质并选取7个差异显著的蛋白点进行质谱分析。结果：双向电泳可分离(354±13)个蛋白质，点匹配率为(78.7±1.4)%，I/R诱导骨骼肌组织10种蛋白质出现明显差异表达，其中6种表达上调，3种表达下调，1种在I/R组为2个点。质谱鉴定出5个蛋白质为：线粒体醛脱氢酶(mitochondrial aldehyde dehydrogenase，ALDH)前体、热休克蛋白27(heat shock 27 kD protein，HSP27) 和一未命名蛋白（I/R组表达升高）、α-肌动蛋白(α- actin，I/R组表达下降）、核转移因子2 (nuclear transport factor 2，NTF - 2) 在I/R组发生突变。结论：I/R损伤引起大鼠骨骼肌蛋白质表达发生改变，其中α-肌动蛋白、ALDH和HSP27表达及NTF-2突变可能与I/R损伤有关。     

烧伤大鼠粒细胞与内皮细胞粘附力学特性的研究

本文以大鼠作为烧伤的实验动物模型，以微管吸吮技术研究了烧伤情况下粒细胸（ＰＭＮ）与肺毛细血管内皮细胞（ＰＣＥＣ）的粘附力学特性．作者首先测定了ＰＭＮ与ＰＣＥＣ间的粘附力Ｆａ，进而在实验条件下定义了相对粘附应力Ｓ１．实验结果表明：在烧伤后２４小时内，粒细胞与肺毛细血管内皮细胞间的粘附力及相对粘附应力均显著升高，且在烧伤后１小时达到最大值。结合烧伤早期的病理变化，作者对实验结果进行了一定的分析和讨论．     

钙调神经磷酸酶的抑制参与大鼠心脏缺血后处理的保护作用

目的：研究缺血后处理（I-postC）对缺血/再灌注(I/R)大鼠心肌钙网蛋白（CRT）及其下游钙调神经磷酸酶（CaN）信号转导途径的影响，探讨I-postC保护I/R心脏的机制。方法：采用Wistar大鼠在体心脏I/R模型，检测血流动力学及血浆乳酸脱氢酶（LDH）和肌酸激酶（CK-MB）含量，以TTC法和TUNEL法分别检测心肌梗死面积和细胞凋亡，发色底物法测定心肌CaN活性，免疫印迹法检测心肌组织CaN和CRT蛋白表达。结果：CaN抑制剂环孢霉素A显著缩小I/R所致的心肌梗死面积(P<0.05)，抑制细胞凋亡(P<0.01)，但对心功能无明显改善(P>0.05)；与I/R组比较，I-postC组心功能改善（P<0.01），心肌梗死范围缩小（P<0.01），LDH和CK-MB漏出减少（P<0.01），细胞凋亡率降低（P<0.01），并显著抑制I/R诱导的心肌组织CaN活性升高（P<0.05）及CaN和CRT表达上调（P<0.05），与缺血预处理组比较差异无显著，但对I/R心肌的保护作用强于单纯环孢霉素A组。结论： I-postC至少部分通过抑制CRT-CaN信号途径，减轻大鼠心肌I/R损伤。     

IGFBP-rP1对肝星状细胞活化及核因子-κB活性的影响

目的：明确胰岛素样生长因子结合蛋白相关蛋白1（IGFBP-rP1）是否具有活化肝星状细胞（HSC）、增加细胞外基质（ECM）合成的作用；并探讨可能的信号转导途径。方法：大鼠肝星状细胞株（HSC-T6）体外培养，分别设立空白对照组（加入等量PBS）和不同浓度的 IGFBP-rP1 处理组，干预因素处理24 h后收集细胞爬片或制备单细胞悬液，采用免疫细胞化学染色观察HSC-T6中α-平滑肌肌动蛋白（α-SMΑ）、I型胶原（collagen I）、纤维连接蛋白（FN）的表达变化；流式细胞仪检测HSC-T6中核因子-κB p65（NF-κB p65）的DNΑ结合活性。结果：免疫细胞化学染色结果发现，IGFBP-rP1各处理组α-SMΑ、collagen I、FN的表达均较空白对照组显著增强，且在一定范围内呈剂量依赖关系；流式细胞仪检测结果显示，IGFBP-rP1各处理组NF-κB p65阳性细胞百分数均较空白对照组明显增高。结论：IGFBP-rP1可以激活HSC，且在一定剂量范围内随着IGFBP-rP1剂量的增加HSC活化的程度逐渐增强；IGFBP-rP1可使ECM的重要组成成分collagen I和FN的合成增加；IGFBP-rP1可增强HSC中NF-κB p65的DNΑ结合活性。     

The effect of self-designed bifunctional RGD-containing fusion protein on the behavior of human keratinocytes and dermal fibroblasts

In this study, self-designed bifunctional RGD-containing fusion protein (BFP) was grafted on the petri dish to evaluate its cytotoxicity and attachment efficiency on primary cultured keratinocytes and dermal fibroblasts. Two lengths of the GRGDS sequences were separately fused to the N-terminus and C-terminus of the Trichoderma koningii cellobiohydrolase I gene cellulose-binding domain, to serve as linking molecule between the cell and the substrate. The grafting procedure was no more labor-intensive and could be done just in aqueous condition itself. The epidermal keratinocytes and dermal fibroblasts, harvested and separated from human foreskin, were cultured in serum-free keratinocyte culture medium and DMEM, respectively. The BFP was dissolved in double-deionized water, and was prepared at different concentrations. The BFP solution was subsequently added into the petri dish for grafting. MTT assay, total DNA measurement, and lactate dehydrogenase analysis were used to evaluate the cell viability, cell proliferation, and cytotoxicity. The immunochemical stain and SEM examination were chosen to make sure that the cultured cells still kept in phenotype. The results showed that the self-designed BFP was successfully coated on the petri dish to improve the cells' adhesion. The whole coating procedure was just done in aqueous solution without any organic solvent being involved. This method was much simpler than the traditional one, and there was no possibility to damage the immobilized biomolecules. From the results of the study, BFP could enhance attachment of keratinocytes and dermal fibroblasts without losing normal cell morphology and keep keratinocytes on the desired differentiation pathway. We believe that coating BFP on petri dish not only enhanced the keratinocyte attachment but also promoted keratinocytes proliferation. We suggest that the self-designed BFP has a great potential to apply on surface modification for the tissue-engineering scaffolds in the future.     

Immunocytochemistry of M-cadherin in mature and regenerating rat muscle

Background: Cadherins are transmembrane proteins mediating calcium-dependent cell–cell adhesion in a cell type-specific manner by means of homophilic binding. M(muscle)-cadherin is a recently detected member of the cadherin family. Methods: We have investigated the localization of M-cadherin innormal and aneurally regenerating skeletal muscle of rat by means of pre-embedding immunocytochemistry. The antibody was directed against the extra-cellular domain of M-cadherin. Results: Myoblasts and myotubes in regenerating muscles tended to be arranged in clusters enclosed by a common basal lamina. Satellite cells of mature muscle fibers were attached to the underlying fiber without separating basal lamina. Reactivity for M-cadherin was restricted to the plasma membranes of myoblasts and satellite cells, and was most intense at the membrane areas facing adjacent myotubes or myofibers. Myoblasts interposed between two myotubes were stained on the entire surface. The adjacent plasma membrane of the otherwise negative myotube or muscle fiber, was stained as well, and extracellular reaction product was in the gap between the cells. Conclusion: The cellular localization of M-cadherin may indicate that M-cadherin is involved in calcium-dependent adhesion between satellite cells and muscle fibers or, by means of interposed myoblasts, between the clustered myotubes in a regenerating muscle. © 1994 Wiley-Liss, Inc.     

Selective promotion of neuronal adhesion by target tissue-derived materials

Chick sympathetic nerves densely innervate expansor secundariorum muscle, but not skeletal muscle. By contrast, parasympathetic ciliary nerves innervate striated muscle, but no parasympathetic innervation occurs in expansor secundariorum muscle. The present study revealed accordingly that sympathetic ganglionic neurons from chick embryos adhered firmly to a dish precoated with expansor secundariorum muscle-conditioned medium (ECM), but not to one precoated with skeletal muscle-conditioned medium (SCM), while parasympathetic ciliary ganglionic neurons adhered to a dish precoated with SCM, but not to one precoated with ECM. These results indicate that there are certain materials which mediate specific adhesion between neurons and their target cells.     

krox-20/egr-2 is up-regulated following non-specific and homophilic adhesion in rat macrophages

Macrophages are known to adhere to a plastic dish via beta2 integrin (CR3) and scavenger receptors. Although their functions such as phagocytosis, endocytosis, and nitric oxide production have been investigated on adherent macrophages in vitro, very little is known about intracellular signals triggered by adhesion to a plastic dish. Recently we reported that the mRNA level of krox-20/egr-2 was significantly increased in rat alveolar macrophages following exposure to fibrous titanium dioxide particles. In the present study we report that up-regulation of krox-20/egr-2 gene expression following adhesion to a plastic dish and homophilic adhesion in rat alveolar macrophages and rat macrophage cell line, NR8383. The mRNA level of krox-20/egr-2 increased with a peak 1 hr after adhesion to a plastic dish in both cell types. Piceatannol inhibited tyrosine-phosphorylation of Syk and decreased both adhesion and krox-20/egr-2 mRNA level. In contrast staurosporine, a serine/threonine kinase inhibitor, increased adherence of macrophages and yet prohibited the adhesion-dependent increase in krox-20/egr-2 gene expression. When NR8383 cells are cultured in suspension, the cells aggregated naturally and produced cell clumps. The mRNA level of krox-20/egr-2 also increased in response to the homophilic intercellular adhesion. The increased mRNA level of krox-20/egr-2 was not caused by inflammatory stimuli, because lipopolysaccharide did not affect the aggregation-dependent up-regulation of krox-20/egr-2 gene. The up-regulation of krox-20/egr-2 gene due to the homophilic cell aggregation was also inhibited either by piceatannol or staurosporine. Those results suggest that krox-20/egr-2 gene expression is triggered by sensing non-specific and homophilic cellular adhesion and the following phosphorylation of signal transducing proteins including Syk and staurosporine-inhibitable kinases.     

Nanopillar sheets as a new type of cell culture dish: detailed study of HeLa cells cultured on nanopillar sheets

Nanopillar sheets were fabricated with high-aspect ratio structures with a diameter of 160–1000 nm and a height of 1 μm by nanoimprinting. The suitability of nanopillar sheets as a new type of cell culture dish was examined by studying the behavior of HeLa cells cultured on the sheets using light microscopy, scanning electron microscopy, and fluorescence microscopy observing actin and vinculin molecules. The nanopillar structure enabled easy subculture of the cells from the sheets without conventional trypsinization. Moreover, the HeLa cells divided and proliferated on the sheets in a different way to that found on petri dish because of the manner in which the cells adhered to the materials.     

羊水细胞原位培养与产前诊断镶嵌体分析

目的了解载片培养瓶对羊水细胞原位培养的效果,并探讨产前诊断羊水染色体中镶嵌体不同分级水平与临床意义。方法使用载片培养瓶（研究组）与载片培养皿（对照组）对1274例有产前诊断指征的孕妇进行羊水细胞原位培养并G显带分析。结果研究组与对照组培养时间平均为6.7和8.9d,细胞克隆数平均为11.5和6.0个,两组培养天数和细胞克隆数比较差异有统计学意义（χ2=1814.4,P〈0.01;χ2=2049.04,P〈0.01）。羊水镶嵌体检出34例,Ⅰ级水平25例,Ⅱ级水平7例,Ⅲ级水平2例,其中真性镶嵌体3例。结论载片培养瓶对羊水细胞原位培养优于载片培养皿,羊水原位培养镶嵌体分级水平的研究对产前诊断有非常重要的临床意义,Ⅱ级水平不能认为全是假性镶嵌体。     

Quantitative study of the effects of denervation and castration on the levator ani muscle of the rat.

The levator ani muscle (LA) of the rat is highly androgen-sensitive and, like all skeletal muscles, deteriorates structurally and functionally when denervated. In order to elucidate the interplay of neural and endocrine influences, the separate and combined effects of denervation and castration on myofiber cross-sectional area and nuclear populations were quantitatively studied. In one group of 4-month-old male rats (A), the LA was denervated. Another group (B) was surgically castrated and a third group (C) was both denervated and castrated. The control rats (D) remained both gonad- and nerve-intact. After two months, the LA was obtained for myofiber and nuclear enumeration, cross-sectional area and satellite cell frequency determination. In the denervated muscle of gonad-intact rats (Group A), myofiber cross-sectional area was markedly diminished (265.84+/-11.38 microm2; compared with controls [Group D]: 1519.98+/-79.41 microm2; P < 0.05). Satellite cell nuclei, as a percentage of total sublaminar nuclei (i.e., satellite cell ratio), increased significantly (4.26%, from a control value of 1.91%). Castration alone (Group B) resulted in pronounced myofiber atrophy (mean cross-sectional area: 754.03+/-89.63 microm2) but had no significant effect on satellite cell ratio (2.36%). The combination of castration and denervation (Group C) elicited the same degree of myofiber atrophy as denervation alone (Group A) but had no significant impact on satellite cell ratio. Instead, the nuclear count per myofiber declined to about a third of the control level (300.5+/-38.49 compared with 861.7+/-24.8; P < 0.05). The results indicate that the atrophic effects of denervation and castration on the LA are non-synergistic and mechanistically similar. They also show that the inability of satellite cells to respond mitotically to the withdrawal of neural input under disandrogenized conditions is a factor in the myonuclear depletion of the denervated muscle of castrated rats.     

A modified method to culture human osteoblasts from bone tissue specimens using fibrin glue

INTRODUCTION: To establish primary osteoblast cultures is a challenge. The methods for isolation mostly comprise digestion with extracellular matrix degrading enzymes after mincing the bone samples. These methods are labour intensive and lead to an inefficient recovery of cells. Therefore, the aim of this study was to develop a more reliable method for culturing human osteoblasts. MATERIALS AND METHODS: Bone tissue specimens were obtained from 20 patients undergoing reconstructive operations. Bone specimens were dissected and put into petri dishes with the bottom covered with fibrin glue. To identify the nature of the outgrowing cells, cytological staining was performed, i.e. Von Kossa, Azan, Dahl's, alkaline phosphatase, and collagen type I. RESULTS: Mean time interval of cellular outgrowth was 12 days after preparing the bone tissue specimens. Confluence of the cell cultures was reached after four to five weeks on average. All cells were positively stained using Von Kossa, alkaline Phosphatase and collagen type I. The matrix consisted of lime, calcium and collagens. CONCLUSION: A simplified method to culture osteoblasts from all kinds of bone tissue specimens is presented. The fibrin glue allows firm adhesion of the specimens to the petri dish. This allows the cells to grow out without disturbance. Normally, due to movements during medium exchange the adhesive bonds are disrupted. The fibrin glue retains the adhesive bonds. This method allows studying human osteoblasts in different clinical settings.     

Bio-Gide胶原膜对兔骨髓间充质干细胞增殖和成骨分化的影响

背景：相关实验表明Bio-Gide胶原膜与细胞有良好的生物相容性,但有关与其复合培养干细胞成骨分化能力的报道少见。 目的：观察Bio-Gide胶原膜对骨髓间充质干细胞增殖及成骨分化的影响。 方法：全骨髓贴壁法体外分离培养兔骨髓间充质干细胞,将第3代兔骨髓间充质干细胞分别接种于覆盖Bio-Gide胶原膜的培养板(实验组)与单纯培养板(对照组)培养。于培养1,4,7,14 d利用CCK-8试剂盒检测细胞增殖;成骨分化诱导培养1,4,7,14 d收集细胞培养液上清,检测细胞碱性磷酸酶活性。 结果与结论：两组细胞数量均随着培养时间的增加而不断增加,对照组培养7 d细胞数量明显多于实验组(P < 0.05),其他时间点组间比较差异无显著性意义。两组细胞碱性磷酸酶活性均随着培养时间的增加而不断增加,实验组成骨诱导14 d细胞碱性磷酸酶活性高于对照组(P < 0.05),其他时间点组间比较差异无显著性意义。表明Bio-Gide胶原膜可促进兔骨髓间充质干细胞的增殖及成骨分化。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

Comparative activity of deet and AI3-37220 repellents against the ticks Ixodes scapularis and Amblyomma americanum (Acari: Ixodidae) in laboratory bioassays

The repellents N,N-diethyl-3-methylbenzamide (deet) and racemic 2-methylpiperidinyl-3-cyclohexene-1-carboxamide (AI3-37220) were evaluated using two different laboratory bioassays to determine their relative effectiveness against host-seeking nymphs of the blacklegged tick, Ixodes scapularis Say, and the lone star tick, Amblyomma americanum (L.). In a petri dish bioassay, ticks were released within a ring of repellent on a horizontal filter paper disk. In the second bioassay, ticks were allowed to climb a vertical strip of filter paper whose central portion was treated with a repellent. Deet and AI3-37220 were more effective against I. scapularis than A. americanum nymphs. In the petri dish bioassay, none of the concentrations of deet or AI3-37220 tested confined A. americanum within the treated ring. However, in the vertical bioassay, both species exhibited avoidance of the repellents, and I. scapularis was repelled by much lower concentrations than A. americanum. I. scapularis were repelled by lower concentrations in the vertical bioassay than in the petri dish bioassay. Deet was slightly more effective against I. scapularis than AI3-37220 in both bioassays, but AI3-37220 was significantly more effective than deet against A. americanum in the vertical bioassay.