MSCs定向诱导移植修复关节软骨的实验研究

目的 用组织工程方法修复软骨损伤.方法 兔骨髓间充质干细胞体外扩增诱导分化后,移植于兔关节损伤区,术后进行大体及x线观察.结果 实验组移植8周后,软骨损伤区由透明软骨样组织填充,软骨修复良好.结论 自体间充质干细胞移植可修复关节软骨损伤.     

脊髓损伤患者骨髓间质干细胞形态学观察

目的骨髓间充质干细胞具有自我复制能力,可以经过不可逆的终末分化过程产生子代细胞,其潜在的多向分化潜力以及可作为细胞治疗这一特点,极有可能成为组织工程较为理想的种子细胞.本实验是对脊髓损伤患者骨髓间充质干细胞形态学观察.方法抽取脊髓损伤患者的骨髓,经密度梯度离心分离、纯化和培养,观察原代和传代细胞的细胞形态.结果原代培养的BM-MSCs最佳的贴壁时间为3 d,生长性状不一,呈散在圆形细胞群、克隆圆形细胞群、散在梭形细胞群、花带状细胞群、旋涡状细胞群,而传代培养的细胞,增殖速度较快,性状一致,排列规则,呈饱满的梭行.结论体外非诱导培养的BM-MSCs方法可能为临床治疗脊髓损伤患者提供种子细胞.     

小型猪骨髓间充质干细胞经心导管冠状动脉移植及其迁移和分化的研究

目的建立小型猪心导管介入冠状动脉治疗模型，为骨髓间充质于细胞（MSCs）移植治疗心血管疾病提供新的方法，观察骨髓间充质干细胞经心导管介入冠状动脉移植后在心肌内的迁移及分化。方法选用冠状动脉解剖生理特点与人类相似的小型猪，应用心导管介入技术将体外培养扩增的小型猪自体MSCs移植进入左冠状动脉前降支，用免疫荧光检测即刻移植和移植后6W移植细胞的肌钙蛋白T（cTnT）、缝隙连接蛋白43（Cx43）、anti－Ⅷfactor的表达。结果成功的完成了小型猪心导管介入冠状动脉进行自体骨髓间充质干细胞移植。移植细胞存活，向心肌组织迁移，并向心肌细胞和毛细血管方向分化。结论该方法稳定，技术先进。可进行准确的定位和动态观测，为临床应用提供了一个可靠的技术平台。MSCs移植后在心肌内发生迁移及分化。     

间充质干细胞治疗缺血性脑血管病的研究现状

缺血性脑血管病是一种缺血性疾病,正逐渐成为全世界发病和致死的主要原因之一。在动物模型和临床试验中,间充质干细胞(MSCs)已被用于缺血性脑血管病。大量研究表明,MSCs的治疗是安全的,能改善缺血性疾病患者的症状,但其疗效与多种因素有关,其中包括细胞移植途径、移植时间窗和剂量及MSCs的来源等。本文综述了这些影响因素及采用MSCs治疗缺血性脑血管病的作用机制和临床试验的安全性,为进一步的研究和临床治疗提供帮助。     

试验证明干细胞心脏修复疗法可能有利有弊

韩国科学家公布的一项最新研究表明,患者自身的干细胞能够修复心脏病发作造成的心肌损伤,但同时也可能带来无法预料的副作用。该结果再一次引起了人们对干细胞心脏修复疗法利与弊的关注。韩国汉城国立大学的研究人员在近日出版的最新一期《柳叶刀》杂志上报告说,他们向2 0名曾经出现过心肌梗塞的患者体内注射了一种名为粒系集落刺激因子(G -CSF)的化合物,它能够刺激患者骨髓产生血液干细胞。试验中,研究人员将其中10名患者血液中的干细胞分离出来,并将其直接导入患者心脏。6周后,导入干细胞的心脏病患者心肌机能有所增强,能够在跑步机上运…     

组织工程骨膜修复兔大段骨缺损的可行性实验研究

目的探索组织工程骨膜体内成骨修复兔大段骨缺损的可行性。方法培养新西兰大白兔骨髓间充质干细胞(BMSCs),以成骨诱导剂诱导成骨分化后,与猪小肠粘膜下层(SIS)复合构建组织工程骨膜。扫描电镜(SEM)观察细胞与材料复合情况。选4月龄新西兰大白兔24只,制备单侧桡骨干4 cm缺损模型。随机选12只植入组织工程骨膜,作为实验组;另12只骨缺损旷置,作为对照组。术后6周后摄x线片观察,切取整段桡骨作为标本行HE及Masson染色观察。结果BMscs诱导14 d后可成骨分化。SEM显示构建的组织工程骨膜上黏附大量种子细胞。x线片观察:实验组骨缺损处有长柱状新生骨形成,并与截骨端骨性融合,密度与正常骨相近;对照组骨缺损处无成骨征象,密度同周围软组织影。组织学观察:实验组骨缺损处有新骨形成,新生骨组织中可见丰富的血管腔及不规则髓腔样结构;对照组骨缺损处仅为纤维结缔组织,无骨组织形成。结论以SIS和BMSCs构建的组织工程骨膜有修复兔大段骨缺损的可行性。组织工程骨膜有进一步深入研究、开发的价值和前景。     

组织工程骨膜的构建及重组人骨形态发生蛋白-2对其体外成骨能力的影响

目的探讨重组人骨形态发生蛋白-2对组织工程骨膜体外成骨能力的影响,以期构建性能更加优良的产品用于临床。方法兔骨髓间充质干细胞经成骨诱导培养,接种于猪小肠黏膜下层构建组织工程骨膜,后分别对其使用含有100 ng/ml、50 ng/ml、10 ng/ml及不含有重组人骨形态发生蛋白-2的成骨诱导培养基进行培养,7天后行电镜扫描,并对各组在多个时间点采用MTT法绘制生长曲线、测定碱性磷酸酶和骨钙素含量,分析重组人骨形态发生蛋白-2对组织工程骨膜成骨能力的影响。结果加入重组人骨形态发生蛋白-2培养的组织工程骨膜在细胞增殖速度、碱性磷酸酶和骨钙素素表达量均高于未加入重组人骨形态发生蛋白-2组,且与浓度成正相关。结论重组人骨形态发生蛋白-2可提高组织工程骨膜体外成骨能力。     

骨髓间充质干细胞的培养及其体外标记跟踪的实验研究

目的研究大鼠骨髓间充质干细胞(BMSCs)的体外分离培养;观察Brdu、DAPI以及Hoechst33342体外染色的合适时间和最佳剂量,比较三种方法的优缺点。方法 BMSCs取自SD雄性大鼠的股骨和胫骨,然后采用贴壁分离培养法对BMSCs进行分离培养,当BMSCs培养至第三代时,采用流式细胞仪对其表面抗原CD34、CD44、CD45进行测定;同时分别用Brdu、DAPI以及Hoechst33342对BMSCs进行染色标记,Brdu标记效果用免疫细胞化学方法检测,DAPI和Hoechst33342的标记率用荧光显微镜观测,最后用MTT检测BMSCs的细胞增值率,观察三种不同方法体外染色的合适时间和最佳剂量,同时比较其优缺点。结果流式细胞仪检测发现BMSCs表达CD44阳性,CD34和CD45阴性;BMSCs染色前后其表面标志表达无明显统计学差异(P0.05);Brdu染色标记BMSCs的最佳浓度和时间是10μmol/L、48小时;DAPI染色标记BMSCs的最佳浓度和时间是20μg/ml、30分钟;Hoechst33342染色标记BMSCs的最佳浓度和时间是5μg/ml、1小时。结论采用粘附贴壁分离培养法能够有效获取高纯度的BMSCs;运用Brdu、DAPI以及Hoechst33342三种方法标记BMSCs效果良好,方法可靠、合理,为研究BMSCs体内追踪打下了基础。     

大鼠骨髓内皮祖细胞分离与体外培养条件研究

目的 探讨大鼠骨髓单个核细胞的最佳分离方法,观察不同培养条件对内皮祖细胞的生长情况、抗原表达的影响.方法 健康SD大鼠水合氯醛麻醉后断尾放血,从其骨髓中分离单个核细胞,重悬于添加不同条件培养基中,分别在预涂或未涂FN的培养瓶中进行培养,记录细胞贴壁情况、集落生成时间和数量及细胞特异性抗原的表达.结果VEGF诱导是内皮祖细胞生长分化的必需条件,在2～10 ng/mL之间,浓度越高,其对骨髓单个核细胞诱导分化的能力越强;bFGF能够协同VEGF诱导分化的作用;纤连蛋白能够明显促进骨髓单个核细胞贴壁.贴壁细胞均表迭内皮祖细胞和内皮细胞的特异性标志,各组之间没有明显差异.结论 大鼠骨髓中单个核细胞可以诱导分化为内皮祖细胞,VEGF(10 ng/mL)、bFGF(4 ng/mL)和纤连蛋白组合更有利于内皮祖细胞的增殖分化.     

人类红细胞胞内束缚水的EPC与DSC方法比较研究及其应用

使用新颖的差示扫描量热仪方法测定了人类红细胞从0℃冷冻至–40℃这一过程终结时的体积值, 为等渗体积的53%. 使用一种新型的颗粒粒度及计数分析仪 (Multisizer^TM 3, Beckman Coulter Inc, USA)测定了人类红细胞在各种非等渗溶液中的平衡体积, 溶液渗透压增加至3186 mOsm (mol·kg_水^–1）, 红细胞的最终体积值为等渗体积的57%. 两种实验方法均表明大约34%~40%的红细胞胞内水被束缚在细胞内, 不能参与渗透性迁移. 实验结果和文献结论“至少20%~32%的等渗胞内水被保留在红细胞内”相符. 基于这一实验结果, 改进了传统的细胞冷冻模型, 并使用改进后的模型预测冷冻过程中红细胞在不同降温速率下的体积变化过程. 随后对这一结果在细胞低温冷冻保存和冻干保存中的应用进行了探讨.     

Alteration of capsular type of encapsulated strains of Staphylococcus aureus during freeze-drying and storage

Remarkable alteration was shown in capsular type antigen production in encapsulated strains of Staphylococcus aureus stored by lyophilization for 10 years. This alteration was further elucidated by antibody production in rabbits immunized with the altered strain and by absorbing the antibodies with representative capsular type strains.     

Auxin dependence and auxin oxidase of cultured sycamore cells

Summary Quantitative and qualitative differences in auxin-oxidases extracted from auxin-dependent (S) or auxin-independent (MB) sycamore cells were analyzed. MB auxin-oxidases have a higher activity, but the molecular weight of this enzymatic complex is lowered by freeze-drying, without loss of the activity. Correlations with auxin-independence are discussed in this context.     

Alteration of capsular type of encapsulated strains ofStaphylococcus aureus during freeze-drying and storage

Summary Remarkable alteration was shown in capsular type antigen production in encapsulated strains ofStaphylococcus aureus stored by lyophilization for 10 years. This alteration was further elucidated by antibody production in rabbits immunized with the altered strain and by absorbing the antibodies with representative capsular type strains.     

Un développement nouveau de la lyophilisation: La cryodessiccation des systèmes non aqueux

Summary A new development is made to enlarge the field of lyophilization using non-aqueous media. Unstable compounds are preserved by freezing and drying their solutions in organic solvents. The same technique applied to chemicals dissolved in liquid ammonia enables the preparation of stable concentrated free radicals, due to the very low temperature of sublimation of frozen NH₃. Finally, experiments are made demonstrating the possibilities of high velocity, low temperature sublimation of solutions in solid CO₂ at normal atmospheric pressure.      

Preservation ofBordetella pertussis

Summary Bordetella pertussis has been shown to remain viable for a period of 13 years on freeze-drying. The revived cultures had unaltered antigenic composition and biological activity.Thanks are due to Dr M. Balasubrahmanyan for his guidance and Dr M.S. Zakharova, Gamaleya Institute of Microbiology and Epidemiology, Moscow, USSR for supplyingB. pertussis factor sera. We thank Mr Ram Jas for his technical assistance.     

Ultrastructure of 30–40 million year old leaflets from Dominican amber (Hymenaea protera,Fabaceae: Angiospermae)

Hymenaea protera leaflet fossils entombed in amber, dated at 30 to 40 million years (mine strata and exomethylene dating) were observed by both light and transmission electron microscopy. Ultrastructure preservation in these leaflets shows the presence of chloroplasts with thylakoid membranes, cell walls, mitochondria with associated endoplasmic reticulum, nuclei, and xylem tissue. Tissues show varying degrees of degradation; however, natural resin, which has perfused the cells, seems to maintain the structural integrity of the membranes and walls. We conclude that preservation of amber entombed organisms results from dehydration and slow fixative properties leaving the ultrastructure in excellent condition. These findings parallel reports on the exceptional preservation of amino acids and of DNA in amber-entombed organisms.     

Scientific heritage: Reflections on its nature and new approaches to preservation,study and access

Scientific heritage can be found in every teaching and research institution, large or small, from universities to museums, from hospitals to secondary schools, from scientific societies to research laboratories. It is generally dispersed and vulnerable. Typically, these institutions lack the awareness, internal procedures, policies, or qualified staff to provide for its selection, preservation, and accessibility. Moreover, legislation that protects cultural heritage does not generally apply to the heritage of science. In this paper we analyse the main problems that make scientific heritage preservation so difficult to address. We discuss the concept and present existing preservation tools, including recent surveys, legislation, policies, and innovative institutional approaches. We briefly analyse two recent initiatives for the preservation of scientific heritage, at the Universities of Lisbon and Cambridge.     

Histidine-induced injury to cultured liver cells,effects of histidine derivatives and of iron chelators

The amino acid histidine is an excellent buffer and is therefore included in several organ preservation solutions used in transplantation medicine. However, when used at concentrations as in these solutions, histidine has a marked injurious potential. Therefore, we here assessed the mechanism of histidine-induced cell injury and searched for ways to use the buffering power of histidine but avoid histidine toxicity. When cultured hepatocytes were incubated in HTK solution or in modified Krebs-Henseleit buffer containing 198 mM L-histidine at 37°C, most cells lost viability within 3 h (LDH release 86 ± 7% and 89 ± 5%, respectively). This injury was accompanied by marked lipid peroxidation, and was strongly inhibited by hypoxia, by the antioxidants trolox, butylated hydroxytoluene and N-acetylcysteine and by the membrane-permeable iron chelators 2,2′-dipyridyl, 1,10-phenanthroline, LK 614, LK 616 and deferoxamine. Thus, histidine-induced cell injury appears to be mediated by an iron-dependent formation of reactive oxygen species. D-Histidine, imidazol and L-histidine methyl ester also elicited marked injury, while the N-substituted derivatives Nα-acetyl-L-histidine and tert-butyl-oxycarbonylhistidine and histidine-containing dipeptides showed almost no toxicity. Histidine toxicity, its iron dependence and the superiority of Nα-acetyl-L-histidine were also evident during/after cold (4°C) incubations. Therefore, we suggest the addition of iron chelators to histidine-containing solutions, and/or replacing histidine with Nα-acetyl-L-histidine in organ preservation solutions. Received 23 October 2006; accepted 21 November 2006     

Eine Methode zur Trockenfixation grossflächiger Kryostatschnitte

Summary By exposure to formaldehyde vapor after freeze-drying large cryostat sections are brought into a stable form. These sections can be used for documentation and may be compared easily with autoradiograms made of the same slices. Furthermore, a method is shown of evaluating these sections with a thin-layer scanner and a 2-flow counter, or by measuring quantitatively the radioactivity of single, excised organs or parts of tissues after automatic combustion by liquid scintillation counting.     

A small chimerically bifunctional monomeric protein: <Emphasis Type="Italic">Tapes japonica</Emphasis> lysozyme

The lysozyme of the marine bilave Tapes japonica (13.8 kDa) is a novel protein. The protein has 46% homology with the destabilase from medicinal leech that has isopeptidase activity. Based on these data, we confirmed hydrolysis activity of T. japonica lysozyme against three substrates: L--Glu-pNA, D--Glu-pNA, and -(-Glu)-L-Lys. The optimal pH of chitinase and isopeptidase activity was 5.0 and 7.0, respectively. The isopeptidase activity was inhibited with serine protease inhibitor, but the lytic and chitinase activities were not. Moreover, only isopeptidase activity is decreased by lyophilization, but lytic and chitinase activities were not. We conclude that T. japonica lysozyme expresses isopeptidase and chitinase activity at different active sites.Received 25 February 2003; received after revision 29 May 2003; accepted 12 June 2003