冻融联合灌注法优化大鼠肾脏脱细胞支架的制备

本研究旨在探索优化肾脏脱细胞支架的制备方法,为肾脏组织工程及肾脏体外病理、毒理研究提供实验基础。取大鼠肾脏灌注PBS作为对照组 (Control组),在不同流速下分别以十二烷基磺酸钠 (Sodium dodecyl sulfate,SDS) 灌注 (S组),Triton X-100联合SDS灌注 (TS组),反复冻融后Triton X-100联合SDS灌注(FTS组),制备肾脏脱细胞支架,并测定其流体分布及脉管阻力。HE染色、DAPI染色、DNA定量检测脱细胞支架脱细胞程度,Masson染色、PAS染色、免疫组织化学染色检测脱细胞支架主要成分的保留和结构的完整,扫描电镜检测支架的超微结构,MTT法检测支架的细胞毒性,ELISA检测支架中生长因子的含量。结果显示,FTS组脱细胞用时较S组、TS组少,10 mL/min组支架脉管阻力较低,S组、TS组、FTS组流体分布与Control组存在差异。HE染色和DAPI染色显示各组支架未见细胞成分残留,DNA含量＜50 ng/mg。Masson染色和PAS染色可见细胞外网状胶原及多糖,免疫组织化学染色见Ⅰ型胶原 (CollagenⅠ)、Ⅳ型胶原蛋白 (Collagen Ⅳ)、纤维连接蛋白 (Fibronectin)、层粘连蛋白 (Laminin) 表达。扫描电镜见支架呈蜂窝状结构。MTT法检测支架细胞毒性分级在0–1级之间。ELISA检测提示FTS组VEGF、EGF、IGF-1、PDGF含量明显高于S组和TS组。综上,联合冻融和灌注法能够制备更为理想且有效的大鼠肾脏整器官脱细胞支架,为肾脏组织工程及肾脏体外病理、毒理学研究奠定基础。     

组织器官脱细胞支架的制备及研究进展

脱细胞基质(decellularized extracellular matrix, dECM)旨在去除引起免疫排斥的细胞,保留原组织结构和成分。由于其具有与原组织器官相似的结构和成分,在组织工程和生物医学的应用上受到广泛关注,已成为一种很有前景的生物医学材料。通过适当的脱细胞方法,dECM很容易能够从组织器官中获得。文中总结了脱细胞的方法及最新研究进展,同时对脱细胞后支架灭菌、交联和保存的方式进行综述,概括了不同组织器官获得的脱细胞支架的最新应用及进展。最后对脱细胞支架目前面临的问题和挑战进行分析,并展望了未来的发展趋势。     

软骨基质来源定向结构支架复合软骨细胞体外构建组织工程软骨的研究

目的：探讨采用软骨细胞外基质材料制备的定向结构软骨支架复合软骨细胞,在体外静态培养条件下生成组织工程软骨的可能性。方法：制备牛关节软骨细胞外基质材料,利用温度梯度热诱导相分离技术构建具备垂直定向孔道结构的软骨支架,同时采用传统冷冻干燥方法制备非定向支架,检测两组支架的力学性能;提取兔关节软骨细胞,分别接种两组支架,体外静态培养2周及4周后取材,对构建的组织工程软骨进行组织切片染色、生物化学分析及生物力学检测。结果：定向软骨支架的压缩弹性模量数值明显高于非定向软骨支架,体外培养时定向支架上种子细胞在3-9d内增殖高于非定向支架,差异有统计学意义（P〈0．05）;体外静态培养4周后形成的两组新生组织工程软骨进行软骨特异性染色均呈阳性,在定向组新生软骨切片中在垂直方向上可见大量呈规则平行排列的粗大胶原纤维,两组新生软骨的生物化学检测包括总DNA、总GAG及总胶原含量差异无统计学意义（P〉0．05）。定向组织工程软骨压缩弹性模量在2周及4周时均高于非定向组织工程软骨,差异有统计学意义（P〈0．05）。但两组组织工程软骨上述指标均显著低于正常关节软骨（P〈0．05）。结论：软骨细胞外基质材料制备的定向结构软骨支架复合软骨细胞,在体外静态培养条件下能够成功生成具有定向纤维结构的组织工程软骨,并可以有效促进新生软骨组织力学性能的提升,在软骨组织工程中具有良好的应用前景。     

细胞支架研究进展

目前细胞培养通常采用二维平面培养技术,但由于在培养板和培养瓶二维细胞培养并不能完全模拟体内细胞的三维生长环境,因此所得的试验数据与在体情况有偏差。然而细胞支架材料却能为细胞提供一个良好的三维生长环境,更利于细胞粘附、生长和增殖。目前可用于细胞支架材料的来源有天然和人工两大类,现将细胞支架研究进展综述如下。     

细胞支架研究进展

目前细胞培养通常采用二维平面培养技术,但由于在培养板和培养瓶二维细胞培养并不能完全模拟体内细胞的三维生长环境,因此所得的试验数据与在体情况有偏差。然而细胞支架材料却能为细胞提供一个良好的三维生长环境,更利于细胞粘附、生长和增殖。目前可用于细胞支架材料的来源有天然和人工两大类,现将细胞支架研究进展综述如下。     

兔组织工程同种异体气管支架三种制备方法的比较

目的:探讨深低温冷冻-酶洗法制备的气管支架在去除抗原性、维持生物力学及保护细胞外基质方面的效果。方法:健康新西兰兔24只随机分为气管未处理作为对照A组,深低温冷冻法处理B组,玻璃化法处理C组,深低温冷冻-酶洗法D组,各组样本数均为6。处理后将各组标本行HE染色后光镜观察,戊二醛固定后电镜扫描,并测量气管最大拉伸力、破裂力和变异率等生物力学性能。结果:组织学观察显示对照A组有大量完整的粘膜上皮细胞;B组和C组可见部分气管粘膜上皮细胞;D组标本未见气管粘膜上皮细胞,且细胞核碎裂。电镜显示A、B、C、D组气管支架可见丰富的细胞基质,未暴露胶原纤维。组间两两比较,气管支架的最大拉伸力、最大破裂力和变异率均无统计学差异。结论:综合组织学、扫面电镜和生物力学分析,应用深低温冷冻-酶洗法制备气管支架D组可以有效地去除抗原,维持生物力学性能,并具有较完整的细胞外基质。     

生物支架材料——组织工程连载之二

具有三维结构的支架材料是组织工程的核心内容之一。现有组织工程支架可分为天然生物材料、合成有机材料和无机材料三类。支架材料近年来研究十分活跃,不仅在组织工程的最早产品人工皮肤领域进行了更为完善的研究和开发,同时在诸如人工骨、软骨、神经、血管、皮肤、肝、脾、肾、膀胱等方面进行了大量研究和探索。与普通组织工程支架需要预先制备并在体外成型不同,近年来在骨和软骨组织工程实践中兴起的可注射支架具有许多优势,是未来组织工程支架发展的重要方向之一。     

骨组织工程脱细胞骨基质的研究进展及存在问题

随着骨科学的发展,骨组织缺损治疗这一难题尤显突出,急需一种更为有效的疗法.骨组织工程是采用组织工程学的原理与方法,研制具有修复骨缺损能力的骨替代物的一门科学.经过20余年的发展,骨组织工程最终确立了将骨再生相关分子、成骨活性细胞与支架材料三者复合来构建组织工程骨的基本模式.支架材料是骨组织工程的核心,而作为支架材料之一的脱细胞骨基质(Acellular bone extracellular matrix,ABECM),近年来发展迅猛,展示出强大的生命力及临床应用前景.并且ABECM已有应用于临床试验的报道.本文将就此做一综述.     

内部结构可控的大体积三维细胞支架制备研究

目的：研究一种可以控制三维细胞支架内部孔隙结构的实验技术,用于制备孔隙结构可控的三维细胞支架,以满足组织工程对支架孔隙结构的要求。方法：均匀混合粘结剂与致孔剂,在离心力作用下去除混合物中多余的粘结剂,应用溶剂浇注/颗粒沥析方法制备三维细胞支架。结果：致孔剂粘结块的结构非常均匀,粘结程度可以通过实验条件控制。例如,直径为100~220μm的致孔剂,在离心力为161g,粘结剂浓度分别为20％和40％时,颗粒间粘结程度分别为33.78±556 (134)μm和42.89±5.87 (132) μm。并且,利用该技术制备的三维多孔支架,其内部孔隙大小取决于致孔剂颗粒大小,孔隙间的通道直径取决于致孔剂的粘结程度,即离心粘结与溶剂浇注/颗粒沥析技术相结合,能够方便地控制三维支架的孔隙结构。例如,当粘结程度为33.78±556 (134) μm时,支架的通道直径为33.34±5.21(12)μm,两者之间无显著差异。 结论：利用离心粘结与溶剂浇注/颗粒沥析技术结合,获得了孔隙呈球形、孔隙间完全连通的、结构均匀的大体积三维细胞支架,并且支架的孔隙以及孔隙间通道大小均可以实现人为控制。     

利用脱细胞血管基质体外构建小口径组织工程血管

目的探讨利用犬的间充质干细胞诱导分化种子细胞,以异种脱细胞血管基质为基础体外构建小口径血管移植物。方法采用密度梯度离心和贴壁培养的方法从犬骨髓中分离出间充质干细胞并体外培养,诱导分化成内皮样细胞和平滑肌样细胞;采用非离子型去垢剂和胰蛋白酶去除猪颈动脉血管壁结构细胞,对脱细胞基质进行组织学、力学检测及孔隙率评估。在生物反应器内采用旋转种植的方法将犬骨髓间充质干细胞诱导的内皮样细胞种植到脱细胞基质上,体外构建小口径组织工程血管。结果犬的骨髓间充质干细胞体外能够定向诱导分化为平滑肌样细胞和内皮样细胞,可以作为血管组织工程的种子细胞。经过脱细胞处理后,光镜和电镜观察证实血管壁的细胞成分完全去除。具有良好的孔径和孔隙率。支架在生物力学、孔隙率等方面符合构建组织工程血管支架的要求。在生物反应器内剪切力条件下可以初步构建出组织工程血管。结论小口径血管移植物可以将间充质干细胞诱导种子细胞,以异种脱细胞血管支架作为基质,在搏动性生物反应器内培养的方法进行构建。     

Serial Expression Analysis of Liver Regeneration-Related Genes in Rat Regenerating Liver

We investigated the gene expression changes in rat hepatic restoration with Rat Genome 230 2.0 chip containing 11,789 known genes and 13,231 unknown genes (taking up 90 percent of rat whole genome) following a 2/3 hepatectomy. The expression profiles and roles of these genes in rat liver regeneration (LR) were assayed using bioinformatics and systems biology method. Among the above genes, 1,004 known genes and 857 unknown genes were found to be associated with rat LR. The numbers of the known genes up-regulated, down-regulated, and up/down-regulated were 622, 443, and 15, respectively; that of the unknown genes were 367, 400, and 14, respectively. Out of the above two groups of genes, the ones up- and down-regulated 20 times or more were 62 and 38, 8, and 14, respectively. Notably, The highest expression level of dehydrogenase/reductase member 7 (DHRS7) was more than 968-fold compared to control, and alpha-1-B glycoprotein (A1BG), the product of gene with the lowest expression abundance, was 58 times lower than control. During rat liver regeneration, 467 up–regulated, 282 down–regulated, 10 up/down-regulated genes, and 1,031 undetected genes in our study interacted with each other and formed a network with a total of 4,014 connectivities. Among them, the genes for the regulation, synthesis, transport, signal transduction, protein modification, and physiological response formed 630, 290, 691, 373, 2010, and 20 connectivities, respectively; and the genes jun, fos, myc, ptgs2, ccna2, ccl2 had relatively higher degree of connectivity. The results indicated that cell apoptosis and inflammatory response were enhanced in the initial phase and the early part of progressive phase in LR.     

大鼠肝线粒体中的脂肪酸分析

用双 2 乙基己基酚酞酸酯 (DEHP)诱导大鼠肝过氧化物酶体增殖 ,然后用蔗糖密度梯度离心法分离大鼠肝线粒体 ,用毛细管气相色谱法测定肝线粒体中的脂肪酸含量。测定结果 :所测 1 4种脂肪酸的总量 ,青年正常组大于青年诱导组 (P <0 .0 1 ) ,青年正常组大于老年正常组 (P <0 .0 5 )。不饱和脂肪酸与脂肪酸总量的比例 ,老年诱导组大于老年正常组 (P <0 .0 5 ) ,青年正常组大于老年正常组 (P <0 .0 5 )。长链脂肪酸与脂肪酸总量的比例 ,老年正常组小于老年诱导组 (P <0 .0 5 )。结果表明 ,用DEHP诱导大鼠肝过氧化物酶体增值 ,影响肝线粒体脂肪酸正常代谢 ,使线粒体膜结构发生变化 ,这种变化 ,青年鼠与老年鼠不同     

Elevation of Substance P-like Immunoreactivity in Rat Central Nervous System by Protease Inhibitors

Abstract: Several substance P-rich areas in rat CNS had increased levels of substance P-like immunoreactivity following the intraventricular injection of the protease inhibitors SQ 20881, SQ 14225, and leupeptin. There were significant differences in response patterns from region to region, possibly on account of an interaction of anatomical, biochemical, or physiological variables. Although the compound SQ 14225 appeared to be the most potent of the inhibitors examined, it had no apparent effect on CNS substance P-like immunoreactivity when administered peripherally.     

Proteomic Analysis of Membrane-Associated Proteins from Rat Liver Autophagosomes

Proteins associated with membranes from purified rat liver autophagosomes were separated by two-dimensional (2D) gel electrophoresis (zoom gels, pI 4-7 and 6-9), silver-stained and identified by MALDI-TOF mass spectrometry. Among >1,500 detectable protein spots, 58 (derived from 39 different known proteins) were at least twofold (and significantly) enriched in autophagosomal membranes relative to cytoplasmic membranes. All of these membrane-associated proteins were also present in the cytosol, many of them being truncated enzyme variants that would be expected to serve a binding rather than an enzymatic function.     

The Regional Distribution and Chromatographic Characterisation of Neurotensin-Like Immunoreactivity in the Rat Central Nervous System

Abstract: Carboxy- and amino-terminal specific neurotensin antisera have been characterized and used to determine the nature of neurotensin-like immunoreactivity in the rat central nervous system. Using these antisera, together with the separation of neurotensin-like immunoreactivity on reversephase HPLC columns, it is clear that the majority of rat central nervous system neurotensin-like immunoreactivity is indistinguishable from the synthetic tridecapeptide. However, smaller amounts of carboxy- and amino-terminal neurotensin-like immunoreactivity were detected, which may correspond to carboxy- and amino-terminal fragments of neurotensin. In addition, using the amino-terminal directed neurotensin antiserum, a detailed distribution of neurotensin-like immunoreactivity in the rat central nervous system is described. Highest amounts were found in the hypothalamus, central amygdaloid nucleus, bed nucleus of the stria terminalis and the substantia gelatinosa of the spinal cord and of the trigeminal region.     

Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at submicrometer resolution. One of the first such instruments producing whole-brain-scale data is the Knife-Edge Scanning Microscope (KESM)^{7, 5, 9}, developed and hosted in the authors'' lab. KESM has been used to section and image whole mouse brains at submicrometer resolution, revealing the intricate details of the neuronal networks (Golgi)^{1, 4, 8}, vascular networks (India ink)^{1, 4}, and cell body distribution (Nissl)³. The use of KESM is not restricted to the mouse nor the brain. We have successfully imaged the octopus brain⁶, mouse lung, and rat brain. We are currently working on whole zebra fish embryos. Data like these can greatly contribute to connectomics research¹⁰; to microcirculation and hemodynamic research; and to stereology research by providing an exact ground-truth. In this article, we will describe the pipeline, including specimen preparation (fixing, staining, and embedding), KESM configuration and setup, sectioning and imaging with the KESM, image processing, data preparation, and data visualization and analysis. The emphasis will be on specimen preparation and visualization/analysis of obtained KESM data. We expect the detailed protocol presented in this article to help broaden the access to KESM and increase its utilization.     

Characterization of FMRF Amide-Like Immunoreactivity in Rat Spinal Cord by Region-Specific Antibodies in Radioimmunoassay and HPLC

Material in rat spinal cord extracts that reacts with antibodies to the molluscan tetrapeptide FMRF amide (Phe-Met-Arg-Phe-NH2) has been characterized by HPLC and radioimmunoassay using region specific antibodies. An antibody to the N-terminally extended analogue, Tyr-Gly-Gly-Phe-Met-Arg-Phe-NH2 (YGGFMRF amide), did not react with the rat material. Two antibodies to FMRF amide were characterized that differed markedly in their affinities for analogues with substitutions in the second and third positions from the C-terminus; both required the C-terminal amide, and neither showed appreciable sensitivity to substitutions in the fourth position from the C-terminus. With both antibodies the relative potency of the avian brain peptide, LPLRF amide, was about 0.1. Both antibodies revealed similar concentrations of immunoreactive material in rat spinal cord extracts. On reversed-phase HPLC using Techsil C18 and Spherisorb-phenyl columns, two peaks were separated that could be distinguished in retention times from FMRF amide, Leu-Pro-Leu-Arg-Phe-NH2 (LPLRF amide), and YGGFMRF amide. The results suggest that the rat spinal cord peptides are structurally related to the C-terminal tripeptide of FMRF amide and are probably extended at the N-terminus by sequences immunochemically distinct from other known peptides.     

Quantitative Autoradiography of Tyrosine Hydroxylase Immunoreactivity in the Rat Brain

Levels of tyrosine hydroxylase (TH) were quantified in discrete areas of unfixed rat brain tissue sections using a rapid and sensitive radioimmunohistochemical method. The immunological reaction with the TH monoclonal antibody was revealed by a 35S-labelled secondary antibody and thus permitted autoradiographic detection of the enzyme. Autoradiograms were generated by apposition of tissue sections to high-sensitivity films or by dipping into autoradiographic emulsion. A detailed analysis of antibody concentration, incubation time, tissue section thickness, and exposure time of the film was undertaken to determine optimal conditions to produce a linear radiolabelling intensity with respect to the amount of antigen. Quantification of the antigen at regional levels was assessed by computer-assisted image analysis. Autoradiographic optical density of radiolabelling in brain areas was converted to enzyme concentrations by interpolation with a constructed TH calibration curve processed in parallel with tissue sections. The specificity of the labelling and the validity and reproducibility of the quantification were investigated. The distribution of TH radiolabelling was comparable to that described using immunofluorescence histochemistry or measuring TH enzymatic activity on homogenates. Using a 35S-labelled antibody, the detection of TH could be performed at the cellular level.     

The Reaction Mechanism of Rat Liver Glyoxalase I and Its Inhibition by MS-3

Glyoxalase I from rat liver was purified about 25-fold by acetone fractionation and ion-exchange chromatography on CM-Sephadex and DEAE-cellulose columns. The kinetic study of the enzymatic reaction supported the one-substrate mechanism : the hemimercaptal adduct produced nonenzymatically from methylglyoxal and glutathione is the substrate. The Km value determined was 0.1 mm and similar to that of porcine erythrocytes enzyme but differed significantly from that of yeast enzyme. It was inhibited by free glutathione competitively (Ki 1.2 mm). Kinetic studies on inhibition of glyoxalase I by MS–3 which was obtained from a cultured mushroom, Stereum hirsutum, indicated the inhibition type was competitive with the hemimercaptal adduct (Ki 4.6 × 10^?6 m). By the graphical study of the multiple inhibition kinetics free glutathione and MS–3 were shown to bind at the same sites of the enzyme.