大鼠小梁细胞的体外培养及弹性模量的测量

目的小梁网是由小梁薄片和其上的小梁细胞构成的网状结构,它对眼压和房水流出具有重要的调节作用,同时小梁细胞的力学特性和生物学特性与房水流出阻力密切相关。因此本研究主要探讨体外培养的大鼠小梁细胞的生物学特性并应用原子力显微镜测量其弹性模量,为今后建立高眼压动物模型并探究原发性开角型青光眼的发病机制提供理论依据。方法取3只SD大鼠双眼小梁网组织,应用消化法对小梁细胞进行体外混合培养。倒置相差显微镜和免疫组化SABC染色的方法确定小梁细胞并观察其生物学特性。应用原子力显微镜压痕方法测量细胞的弹性模量。结果大鼠小梁细胞10 d左右达到融合,细胞形态多样,免疫组化检测结果显示层粘连蛋白、纤维连接蛋白和神经元特异性烯醇化酶染色阳性。小梁细胞弹性模量为1. 02 kPa±0. 66 k Pa。结论消化法成功培养出大鼠小梁细胞,并利用原子力显微镜测得小梁细胞的弹性模量,为之后研究青光眼小梁细胞的特性奠定基础。     

基于双光子共聚焦成像的大鼠小梁网三维重建及有限元分析

青光眼是一组威胁视神经视觉功能、与眼压升高密切相关的眼病。目前认为临床上大部分眼压的升高与房水流出阻力增加有关,特别是小梁网流出阻力的增加。因此,研究高眼压下影响房水外流阻力的重要区域小梁网的形态学信息尤为重要。通过前房灌注的方法制造大鼠急性高眼压动物模型,将6只SD大鼠分成两组(A组和B组),B组大鼠处死后于左眼球加压60 mmHg处理,其余眼球均为未加压对照组,利用双光子共聚焦成像系统采集正常眼压和高眼压下小梁网组织的断层序列图,通过图像处理方法,定量分析眼压对小梁网组织孔隙率变化的影响。通过三维重建获取正常眼压下的小梁网结构模型,并利用有限元方法,探讨眼压对于小梁网组织形态结构的影响。结合实验数据与模拟计算的结果,综合分析眼压的变化对于小梁网外流阻力的影响。高眼压组数据显示,部分小梁与周围组织融合,胶原纤维出现塌缩,越是靠近前房的小梁组织损伤越严重。有限元分析结果表明,孔隙越多的区域变形越大,而且压力越大,小梁变形程度越明显。眼内压升高可能会引起房水外流通道结构发生异常,主要表现为小梁网胶原纤维发生塌缩。高眼压与正常眼压情况相比,小梁网区域外流阻力增大的可能性较大。     

肾上腺素抑制培养牛眼小梁细胞AQP1表达

目的测定肾上腺素对体外培养牛眼小梁细胞水通道蛋白-1(aquaporin1,AQP1)表达的影响,探讨肾上腺素降低眼压的机制。方法将传三代的牛眼小梁细胞随机分为对照和肾上腺素处理组。将不同浓度的肾上腺素(10-4、10-5和10-6mol/L)分别加入培养液持续培养14天后,免疫细胞化学方法检测牛眼小梁细胞AQP1表达水平,计算机图像分析软件测量细胞表面积。结果发现经不同浓度肾上腺素处理后的小梁细胞AQP1表达量吸光度值A和细胞表面积显著低于对照组(P<0.05)。结论肾上腺素对牛眼小梁细胞AQP1表达的抑制可能参与了肾上腺素降眼压作用。     

改良组织块培养法体外培养人角膜上皮干细胞

文题释义：角膜上皮干细胞：属于单能干细胞,具有细胞周期长、低分化状态、增殖潜力大、不对称分裂等特点,定位于角膜缘基底细胞层,又称之为角膜缘干细胞,对角膜上皮细胞更新及维持角膜透明起着重要作用。角膜缘干细胞的体外培养方法：主要包括酶消化培养法和组织块培养法。酶消化培养法是利用DispaseⅡ酶破坏角膜缘上皮细胞与基底膜之间的半桥粒连接,然后剥取角膜缘上皮层,再使用胰酶将其消化为单个细胞进行培养。组织块培养法没有经过酶的双重消化,将剖取的角膜缘组织块进行贴壁,细胞游离出组织块进行贴壁生长,需要一个漫长的过程。  摘要背景：角膜上皮干细胞定位于角膜缘,又称之为角膜缘干细胞,临床上由于眼表严重热烧伤、化学性烧伤、慢性炎症等原因引起的角膜缘干细胞缺乏或功能障碍治疗较为棘手。目前利用组织工程技术体外培养角膜上皮干细胞并进行临床移植成为新型有效的治疗方向。目的：探讨在无血清培养条件下采用改良组织块培养法培养人角膜上皮干细胞的可行性。方法：人角膜缘组织来自河南省眼库,植片直径小于8 mm角膜移植术后的供者剩余眼球材料,手术显微镜下剖取角膜缘上皮层外2/3区域,采用2种方法培养人角膜上皮干细胞,常规组织块培养组是将组织块上皮面向上贴壁,加入K-SFM培养液后置于 37 ℃、体积分数为5%CO₂细胞培养箱中培养;改良组织块培养组是先将组织块浸泡于K-SFM培养液中,置于细胞培养箱中孵育12 h,然后组织块上皮面向下贴壁培养。组织块周边有细胞游离出贴壁生长记作“培养第1天”,每日相差显微镜下观察细胞生长变化。利用免疫荧光染色技术检测改良组织块培养第5,10,14天时原代细胞中p63及K3的表达。结果与结论：①改良组织块培养组出膜时间明显短于常规组织块培养组(P < 0.05),出膜率明显高于常规组织块培养组(P < 0.05);②改良组织块培养组细胞生长状态良好,培养第10天可见小体积细胞较多,聚集成灶状分布;培养第14天可见细胞克隆灶,克隆灶内细胞体积较小,形态均一;③培养第5天,K3表达量较多,p63表达量较少;培养第10天,K3和p63表达量均增多;培养第14天,K3表达量未见明显增多,p63表达量明显增多;④在无血清培养条件下,改良组织块培养法能显著促进角膜上皮干细胞的游离,提高体外培养细胞数量,为人角膜缘上皮组织片的构建提供种子细胞。ORCID: 0000-0001-8370-174X(许中中) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

悬浮组织块法分离培养兔成纤维样滑膜细胞

目的:建立兔成纤维样滑膜细胞悬浮组织块分离培养法。方法:取正常兔膝关节滑膜组织,用悬浮组织块法分离培养兔成纤维样滑膜细胞,观察细胞生长状况及形态特征,取第3 代对数期细胞,CCK鄄8 法绘制其生长曲线,免疫荧光法检测波形蛋白的表达情况,并与组织块贴壁法进行比较。结果:两种方法体外培养得到的兔成纤维样滑膜细胞形态为长梭形纤维样,细胞生长力较旺盛,生长曲线为典型的“S冶形,免疫荧光检测显示第3 代细胞强表达波形蛋白。结论:悬浮组织块法可以分离培养出兔成纤样维滑膜细胞,方法简便,效率高,为体外分离培养兔成纤维样滑膜细胞提供了一种新的方法。     

Myocilin蛋白在小梁网组织中的作用

Myocilin基因是与原发性开角型青光眼成因有关的基因.其蛋白产物myocilin蛋白是一种分泌型糖蛋白,具有特征性区域:N端亮氨酸拉链区,中央链接区,C端类嗅质蛋白(嗅素)区.眼组织中,小梁网myocilin蛋白表达水平最高且在细胞内外均可检测到.细胞内myocilin蛋白由小梁网细胞以外泌体样囊泡形式释放至胞外,...     

改良Alamar Blue法检测结直肠微小组织块的活力***☆

背景：Alamar Blue法是测定组织细胞活性的方法之一,目前多数应用在细胞系活力测定方面,尚未见应用于微小组织块活力的评价。 目的：采用改良Alamar Blue法评价结直肠正常黏膜组织块和癌组织块的活力。 方法：以细胞活力测定的Alamar Blue法为基础,应用单位质量吸光度来评价结直肠正常黏膜组织块和癌组织块的活力。根据检测试剂盒说明书和预实验结果,实验选择0,6,12,18,24,30,36 h作为检测时间点,每一例标本分为7组,设空白对照孔。 结果与结论：单位质量吸光度能够随培养时间的推移,很好地反映组织块增殖活力的变化趋势,结直肠正常黏膜组织块和癌组织块培养12~24 h达到良好的增殖状态,证实改良Alamar Blue法是评价结直肠微小组织块灵敏有效的新型检测方法之一。     

低强度超声对激素诱导的兔高眼压的作用

目的探讨低强度超声对兔激素性高眼压的影响及其机制。方法采用超声直接接触法对兔激素性高眼压连续治疗5d并监测眼压。观察眼压、小梁组织的HE染色,透射电镜、免疫组化法及图像分析观察小梁外基质层黏连蛋白、纤维连接蛋白和胶原Ⅰ的变化。结果低强度超声处理后眼压显著下降,小梁网间隙增大,胶原I、层黏连蛋白及纤维连接蛋白的表达低于对照组。结论低强度超声所致眼压降低与小梁组织超微结构改变及超声机械震荡等使异常增多的小梁外基质减少有关。     

小梁细胞转移生长因子β_1 mRNA的表达和分泌

作者取猪眼球,在显微镜下分离虹膜、睫状体、小梁细胞,部分小梁组织培养。各组织块通过匀浆、离心得到mRNA。培养的小梁组织用磷酸缓冲液冲洗。细胞在avzian myelblastosis病毒逆转录酶作用下合成cDAN,PCR扩增,30bp的寡核苷酸探针行Southein blot。收集小梁细胞培养液,无血清培养8h,3次;再培养24h,3次。离心后用~(125)I-TGFβ_1测定。 结果发现,猪的小梁细胞含有TGF_1β_1 mRNA,能分泌和产生TGFβ_1。在磷酸缓冲液冲洗,无血清反复培养后培养液中测得的TGFβ_1不是血清携带的,而是小梁细胞自身分泌所致。     

体外培养条件下兔脊柱运动节段髓核组织的变化

背景：椎间盘器官体外培养模型作为体内试验和细胞培养的联系,能够在原生基质中维持细胞活性,从而保留了重要的细胞与细胞及细胞与基质的相互作用。 目的：观察体外培养兔脊柱运动节段模型髓核组织的变化。 方法：将13只新西兰白兔处死后在无菌条件下取出脊柱运动节段50个,在高渗培养基中进行整体培养  (410 mOsm/kg),于培养前及培养后第3,7,14,21天,各取10个椎间盘分别进行苏木精-伊红染色、Ⅱ型胶原免疫组织化学、蛋白多糖含量和髓核细胞活力测定。 结果与结论：培养14 d苏木精-伊红显示椎间盘组织结构基本保持完整,21 d椎间盘组织形态学破坏;Ⅱ型胶原免疫组织化学染色强度14 d无显著差异(P > 0.05),21 d染色变浅,与之前各时间点相比有显著差异(P <   0.05);蛋白多糖PAS/AB染色7 d内强度无降低,14 d强度有所减弱,21 d染色强度进一步减弱;髓核细胞荧光检测21 d髓核细胞荧光强度减弱,细胞活性降低,与之前各时间点比较差异明显(P < 0.05)。结果表明,培养14 d兔脊柱运动节段椎间盘髓核组织退变不显著,培养21 d模型髓核退变明显,脊柱运动节段目前在14 d内可作为研究生物力学对椎间盘影响的体外实验模型。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程      

Evaluation of the MB/BACT automated mycobacteria culture system versus culture on Lowenstein medium

Objective: To evaluate the growth time and recovery rate of mycobacteria, and the percentage of contamination in the MB/BACT system versus traditional culture, on Lowenstein medium.
Methods: One thousand one hundred and fifty-nine samples for mycobacterial analysis were cultured in Lowenstein medium and the MB/BACT system: 0.5-mL aliquots of the sample were inoculated into tubes containing either medium and incubated for 49 days. The mycobacterial isolates were identified by means of the Accu-Probe (Gen-Probe) or a system based on chromogenesis, time and temperature of growth and principal biochemical differentiation analyses. Fischer's test was performed.
Results: Ninety-three mycobacterial strains were isolated: 80 Mycobacterium tuberculosis , seven strains of the M. avlum-intracellulare complex, four M. gordonae and two M. smegmatis. For M. tuberculosis , 76 of 80 isolates grew in MB/BACT, while 60 of 80 grew on Lowenstein medium. A total of 88 of the mycobacterial strains grew in the MB/BACT system, while 66 grew on the Lowenstein solid medium. Growth in the MB/BACT averaged 16.51 days, as opposed to 22.71 days on Lowenstein medium.
Conclusions: The MB/BACT system is a suitable complement to culturing on Lowenstein medium, while the workload does not significantly increase in comparison with culturing on solid media only and still allows major recovery of mycobacteria with a significant time-saving.     

不同培养基对人脐静脉内皮细胞体外培养效果的影响

目的研究不同培养基对人脐静脉内皮细胞体外培养生长的影响,探讨内皮细胞的最佳体外扩增培养条件。方法取健康产妇分娩后脐带,用胶原酶Ⅰ消化后得脐静脉内皮细胞,进行原代培养,并用免疫荧光染色的方法对细胞进行鉴定,传代培养时则分别采用RPMI-1640培养基和EGM-2培养基,倒置显微镜观察两种培养条件下细胞的生长状态,同时利用流式细胞仪检测其生长周期,对比培养效果。结果 EGM-2组内皮细胞生长良好,2d后贴壁生长的细胞可达90%。EGM-2组S期细胞比例为（29.07±1.48）%,RPMI-1640培养基组S期细胞为（17.58±3.49）%,二者比较差异有统计学意义（P〈0.01）。结论 EGM-2培养基更适合人脐静脉内皮细胞的体外传代扩增培养。     

A simple and convenient culture chamber conversion for use with standard disposable culture dishes

Summary A tissue culture chamber is described for the observation of living cells utilizing inverted phase-contrast microscopy. This chamber is easily assembled from component parts already available in most tissue culture facilities. Rapid assembly and low unit cost are the principle advantages of this system.     

Laboratory preparation of plasticware to support cell culture

Summary Many plastics, including polystyrene and poly(ethylene terephthalate), are unsuitable for cell culture applications as formed because they do not support cell growth. Although cells may attach to these materials, the attached cells typically round up and detach or die after a short time. However, plastics can be made to support normal cell attachment and growth through surface modification by glow discharge processes that produce ionized gas species which react with the surface of the plastic. This article describes radiofrequency glow discharge (RFGD) modification of plastics in the presence of organic vapors such as acetone, methanol and ethylene oxide. These treatments render laboratory plastics amenable to in vitro cell culture. Successful modification is a function of RFGD reaction parameters (position within the reactor, discharge power, system pressure, flow rate, and reaction time), and can be confirmed by electron spectroscopy for chemical analysis (ESCA). Identification by high resolution ESCA of functional groups introduced onto the surface by the RFGD process can be used to correlate cell growth with surface chemistry. A brief discussion of other processes thought to be used for preparation of commercial tissue culture ware is also provided.     

Cell culture propagation of rotaviruses

Summary Rotaviruses are ubiquitous intestinal pathogens that infect the young of many species. Their adaptation to cell culture has met with variable success depending on the strain and serogroup of virus. Successful cultivation of rotaviruses has been achieved through sequential use of animal passage and kidney cell cultures. Roller tubes were beneficial for adaption of several strains as opposed to use of stationary cell cultures. Activation of rotaviruses with proteolytic enzymes has increased their adaptability to culture and is standard practice with many strains. Alternative methods for cultivation of rotavirus, particularly human strains, have involved use of reassortants between animal and human rotaviruses. Continuous cell lines for rotavirus passage include rhesus monkey kidney (MA-104 & LLC-MK₂), African green monkey kidney (BSC-1 & CV-1), and Madin-Darby bovine kidney. Fluorescent foci, cytopathic effects, and plaque assays are used to titrate infectious virus. Enzyme-linked immunosorbent assay, hemagglutination, dsRNA electropherotyping, and electron microscopy are used to detect rotavirus growth. Optimal conditions for the growth of two group A rotaviruses (OSU & NCDV) in MA-104 cells are described in this report.     

一种组合式组织工程动态培养仪

随着组织工程技术应用的多样化,为不同的目标组织提供不同的培养条件,改进工程组织的化学和生物力学性能,或通过不同的培养条件诱导干细胞向不同方向分化,这些得到了学者们越来越多的关注。结合中国现有的实验室条件,中国医学科学院,北京协和医学院整形外科医院自主研发了一种组合式组织工程动态培养仪,以为不同的目标工程组织提供不同的动态培养环境。     

Whole embryo culture and teratogenesis

Whole embryo culture (WEC) is under evaluation in numerous laboratories to determine the role it should play in teratogen testing. There is general agreement that its role is to complement the Segment II Teratology studies required by regulatory authorities. Currently it is used as a 'screen' in order to minimize the number of compounds used in animal tests and also to determine the mechanism(s) of teratogenesis. This brief review focuses on the study of retinoid teratogenicity in WEC in order to illustrate these points.     

Isolated osteoclasts in primary culture: first observations on structure and survival in culture media

Summary Osteoclasts were isolated mechanically from the medullary bone of laying hens kept 7 days on a low calcium diet. Osteoclast enrichment was achieved with 3–4 sedimentations of the cell suspension in test-tubes prepared by layering on the bottom with BSA 10% in MEM-HEPES or PBS, above which the cells were suspended in MEM-HEPES or PBS. The final suspension of osteoclasts was cultivated in MEM with 10% FCS for 3 weeks. The cultures were observed by phase-contrast and scanning electron microscopy (SEM). By the third day, the osteoclasts were completely spread onto the plastic dishes and a variety of morphologies and of intercellular contacts was established. Osteoclasts in culture do not lose their morphology; they survive for long periods and can be used in many experimental systems.     

Primary culture of mammalian nephron epithelia

Nephron segments were dissected from fetal or from early postnatal rabbit kidneys (n=86) and explanted individually into primary tissue culture. Outgrowth of epithelioid cells and proliferation in monolayers from the collecting tubule and the thick ascending limb of Henle's loop occurred regularly if a special substratum and matrix were used. Media were either supplemented with non-proteins (defined media) or with embryo extract and fetal sera (undefined media). Outgrowth and monolayer spreading were recorded by Differential Interference Contrast Microscopy. Monolayer cells resemble those seen with the same recording technique during tubule in vitro of convoluted tubule segments from embryonic Nephron Anlagen and outgrowth of cells from proximal straight tubules, in contrast to the distal nephron segments. The specific functional and morphological properties of cells derived from primary culture of nephron segments are under study.