Silica, hyaluronate, and alveolar macrophage functional differentiation.

BACKGROUND: Silicosis is mediated by macrophages, their soluble mediators, and extracellular matrix molecules. In this study, we investigated the effects of silica and/or hyaluronate (HA) on several alveolar macrophage responses. METHODS: We evaluated glycosaminoglycan (GAG) production by radiolabeled precursors, nitric oxide (NO) release by its oxidation product, phagocytic activity by Candida albicans internalization, and the secretion of two fibrogenic cytokines, tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta, by specific assays. RESULTS: Silica significantly reduced GAG secretion, particularly HA secretion. Alone, it decreased Candida uptake; associated with HA, it enhanced the reduction. Silica and Candida reduced NO release, which was not significantly affected when silica- or Candida-exposed cells were also treated with HA. TNF-alpha and TGF-beta activities were stimulated by silica but reduced by HA. CONCLUSIONS: The results suggest that silica and HA modify alveolar macrophage functional differentiation. Silica- and HA-induced modifications of the microenvironment could determine whether the response proceeds toward healing and repair or toward lung chronic pathology.     

Comparison of live human neutrophil and alveolar macrophage elastolytic activity in vitro. Relative resistance of macrophage elastolytic activity to serum and alveolar proteinase inhibitors.

Elastin is an extracellular matrix protein critical to the normal structure and function of human lung. Recently reported data indicate that live human alveolar macrophages can degrade purified elastin in vitro. In this study, we directly compared the elastolytic activity of alveolar macrophages with that of human neutrophils. In the absence of proteinase inhibitors, human neutrophils degrade much more elastin than do human alveolar macrophages. However, macrophages cultured in 10% human serum and in contact with purified 3H-elastin degraded 4.7 micrograms elastin/10(6) cells per 24 h, as compared to less than 1 microgram/10(6) cells/24 h for neutrophils. We observed a similar pattern when the two cells were cultured in human alveolar fluid. We determined that the relative resistance of macrophage elastolytic activity to serum or alveolar proteinase inhibitors was not simply due to phagocytosis of substrate by the larger macrophages. Live macrophages as well as neutrophils degrade 125I-elastin coupled to noningestible sepharose beads. Again in serum-free media, neutrophils degraded eight-fold more elastin than macrophages but only macrophages degraded sepharose-coupled elastin in the presence of 10% serum. Because of these findings, we compared the enzymatic mechanisms of elastin breakdown by macrophages with that of neutrophils. Macrophage elastolytic activity is largely (65-80%) due to a cysteine proteinase(s), at least part of which is Cathepsin B. Approximately half of the cysteine proteinase activity appeared to be expressed at or near the cell surface. These experiments defined two enzymatically distinct pathways of elastin breakdown by human inflammatory cells: the classic, neutrophil derived soluble elastase(s) that is sensitive to serum and alveolar proteinase inhibitors, and a macrophage-mediated pathway that is largely cell associated and relatively resistant to inhibitors. The function of the two pathways depends on the relative excess or deficiency of soluble inhibitors. At inflammatory sites rich in proteinase inhibitors, tissue macrophages may degrade more extracellular matrix elastin than neutrophils. In smokers without antiproteinase deficiency, pulmonary macrophages, which are known to be increased in number, may be the more important cause of elastin breakdown and emphysema.     

外胚间充质干细胞的体外分离培养与鉴定

背景：外胚间充质干细胞不仅可以作为组织工程研究的一种新种子细胞,而且可以为临床神经系统肿瘤提供一种新的治疗策略。目的：体外分离外胚间充质干细胞,对其进行磁珠筛选、形态及超微结构观察、神经干细胞表型鉴定及生长增殖能力检测。方法：从SD胎鼠的上下颌突分离出外胚间充质干细胞进行原代培养,培养液中加入磁珠包被的低亲和力神经生长因子受体抗体,进行磁珠筛选纯化。结果与结论：经磁珠筛选后的外胚间充质干细胞呈“成纤维细胞样”细胞,为长梭形,漩涡状生长：透射电子显微镜显示主要为间充质样细胞,形状小规则,细胞增殖活性高,处于未分化状念;免疫组织化学染色结果显示人自然杀伤细胞1、波形蛋白、S-100在细胞浆中呈阳性表达,而细胞角蛋白则为阴性;生长曲线显示呈“S”形,第3天进入对数生长期,第6天进入平台期,其倍增时问TD=40．28h。提示体外分离经磁珠筛选纯化扩增的细胞证实为外胚间充质干细胞,其具有干细胞特征。     

人脐静脉血管内皮细胞分离与原代培养体会

目的:探讨分离人脐静脉血管内皮细胞(HUVEC)和原代培养的可靠方法与经验.方法:利用0.1%胶原酶消化、分离、体外原代培养脐静脉血管内皮细胞,0.125%胰酶-0.02%EDTA溶液消化传代,根据细胞特有的形态学特征和Ⅷ因子、CD31细胞免疫组化及对乙酰化LDL高摄取试验进行内皮细胞鉴定.结果:脐静脉血管内皮细胞的体外原代培养、传代生长均良好,呈典型的铺路石样形态学表现,Ⅷ因子、CD31细胞免疫组化显示阳性反应,及Dil-Ac-LDL摄取荧光检测提示其具有强摄取能力,从而鉴定为血管内皮细胞.结论:本方法获得的HUVEC量多,方法简单、可靠,为血管内皮细胞的研究提供实验模型.     

Mitogenic effects of Brazilian arthropod venom on isolated islet beta cells: in vitro morphologic ultrastructural and functional studies.

BACKGROUND: One of the major pitfalls associated with use of isolated adult islets of Langerhans' cells is their minimal mitotic capacity. Consequently, maintenance of a steady viable islet cell mass is very difficult. To explore how to enhance beta-cell mitogenesis, we have examined the effects of venom fractions extracted from a Brazilian scorpion on morphologic and functional beta-cell patterns. The venom was previously known to induce nesidioblastosis-like effects with chronic hypoglycemia and pancreatitis in animal models. METHODS: Venom fractions purified from Tityus bahiensis were incubated with batches of isolated rat islets, while a morphologic examination, glucose-stimulated insulin release, insulin content, and insulin messenger ribonucleic acid (mRNA) were carried out early during incubation. On fixation and double fluorescence immunolabeling (rhodamine for anti-insulin monoclonal antibodies; fluorescein for anti-5-bromodeoxyuridine), the preparations were imaged by confocal laser microscopy (CLM) for morphometric quantification of the mitoses. Insulin recovery and mRNA were also assessed at 21 days of culture. RESULTS: Under CLM examination, the beta-cell mitotic rate significantly rose from 1 to 12.8% for the venom-exposed islets. At day 7, insulin release and content were significantly lower for the venom-exposed than the control islets. However, at day 21 of culture, insulin release in response to static incubation with glucose and insulin mRNA from the venom-exposed islets was higher than controls (p < .05). CONCLUSIONS: Incubation with the scorpion venom induced a rapid and significant increase in the beta-cell proliferation not associated with a short-term increase in insulin secretion. The latter fully resumed and overcame controls later in culture, possibly after completion of the beta-cell expansion process.     

体外分离与培养组织工程皮肤附属物:发育标记物检测及调控和增殖

背景:现有各类组织工程皮肤面临着共同的难题,突出表现在没有完整的皮肤功能,尤其是缺乏皮肤附属物如毛囊、汗腺、皮脂腺等.如何培养具有皮肤附属物的"功能性组织工程皮肤"即完整的皮肤器官,成为皮肤组织工程迫在眉睫的研究方向.目的:文章通过综述皮肤附属物相关干细胞的分离与培养以及向皮肤附属物诱导分化的研究进展,为解决当前人工复合皮肤中难以形成皮肤附属物的难题提供帮助.方法:应用计算机检索Pubmed数据库(1999-01/2009-12),以"skin tissue engineering,skin appendages"为检索词;应用计算机检索中国期刊全文数据库(2004-01/2009-12),以"皮肤组织工程、皮肤附属物、毛囊、汗腺、皮脂腺"为检索词.纳入标准:文章所述内容应与利用组织工程技术使干细胞分化、增殖形成毛囊、汗腺、皮脂腺等皮肤附属物研究相关.排除标准:重复研究或荟萃分析类研究.结果与结论:计算机初检得到97篇文献,排除因研究目的与此文无关的35篇,内容重复性的研究26篇,共保留36篇文献进行综述.人体多种细胞都有向皮肤附属物分化增殖的能力,进一步阐明胚胎发育的机制,深入研究干细胞定向分化的调控机制,在皮肤修复的同时能够诱导毛囊、皮脂腺和汗腺的发生,无疑是构建理想组织工程皮肤的重要途径.将生长因子、干细胞生物技术与生物材料工程学相结合,才可能研制成功真正意义上的皮肤永久替代物,但体外研制成功的皮肤永久替代物应用于人体的安全性及效果还有待进一步分析.     

Subcellular localization of angiotensin-converting enzyme in the human alveolar macrophage

The present investigation was performed in order to elucidate the subcellular localization of angiotensin-converting enzyme (ACE) in human alveolar macrophages. A pure population of alveolar macrophages was obtained by centrifugal elutriation of bronchoalveolar lavage (BAL) fluid from seven sarcoid patients. The cells were homogenized by sonication and the postnuclear supernatant was fractionated on a discontinuous sucrose gradient. Fractions of particulate material were collected and characterized by marker enzymes. The distribution pattern of ACE closely resembled that of NADPH-cytochrome-c-reductase and sialyltransferase, markers of the endoplasmic reticulum and the Golgi complex, respectively, indicating a common localization. This localization is compatible with synthesis taking place in the alveolar macrophage.     

The etiologic agents of varicella and herpes zoster; isolation,propagation, and cultural characteristics in vitro

Fourteen strains of virus derived from the cutaneous lesions of cases of varicella and eight from patients with herpes zoster were propagated serially in primary explant cultures of human preputial or embryonic skin-muscle tissue. Infectious material could not be demonstrated in the fluid phase of infected cultures and inocula for passage therefore consisted of suspensions of infected tissue. Such tissue suspensions when stored in the frozen state did not regularly retain infectivity. The cytopathic process was focal and appeared to develop as the result of transfer of infectious material from cell to contiguous cell. Optimum development of the focal lesions in vitro related directly to conditions favoring optimum tissue growth and was further influenced by the spatial relationship of the tissue outgrowth. A variety of types of cells of human origin and several of monkey origin were susceptible to infection and responded with the formation of intranuclear inclusion bodies. The cellular response otherwise was variable, ranging from simple rounding with little change in size to the formation of large multinucleated cytoplasmic syncytia. Strains of virus recovered from patients with varicella and from patients with herpes zoster could not be distinguished on the basis of their cultural attributes.     

Immunoglobulin G-induced single ionic channels in human alveolar macrophage membranes.

While it is well known that the engagement of IgG Fc receptors on the macrophage surface triggers a number of cellular responses, including particle ingestion, secretion, and respiratory burst activity, the mechanism of signal transmission following ligand binding remains poorly understood. To acquire more data in this area, we studied the electrical properties of the macrophage membrane and its response to oligomeric immunoglobulin G (IgG) using the patch-clamp technique on human alveolar macrophages that were obtained by bronchoalveolar lavage and maintained in short-term tissue culture. The results showed that cell resting potentials, as determined from whole-cell tight seal recordings, increased from -15 mV on the day of plating to -56 mV after the first day in culture and remained stable at this hyperpolarized level. Macrophages revealed an input resistance of 3.3 G omega, independent of age in culture. Extracellular application of heat-aggregated human IgG to cells voltage-clamped at -70 mV resulted in peak inward currents of approximately 470 pA. We identified an IgG-dependent, nonselective channel in both cell-attached and isolated membrane patches, with a unitary conductance of approximately 350 pS and a predominant subconductance level of 235 pS in symmetrical NaCl solutions. Single channel open times were observed to be in the range of seconds and, in addition, were dependent upon membrane voltage. Channel opening involved transitions between a number of kinetic states and subconductance levels. Channel events recorded in cell-attached patches showed characteristic exponential relaxations, which implied a variation in membrane potential as a result of a single ion channel opening. These data suggest that the IgG-dependent nonselective cation channel that we have characterized may provide the link between Fc receptor engagement and subsequent cellular activation.     

人脑神经干细胞对不同储存方式及分离培养和诱导分化条件的要求

背景近年来发现神经干细胞可于体外增殖并分化为神经细胞,是用来修复替代损伤神经组织的较为理想的来源,但是神经干细胞的培养中如何提高它的增殖能力,诱导分化成需要的表现型,尚处于研究探讨阶段.目的探讨细胞冻存和非冻存方法分离培养人脑神经干细胞及诱导其向成熟神经细胞分化的培养条件.设计以细胞为观察对象,单一样本研究.单位江西医学院泌尿外科研究所,江西医学院第二附属医院神经外科.材料实验于2003-12/2004-06在江西医学院泌尿外科研究所完成.取16周龄新鲜人胚脑组织.方法用胰蛋白酶消化法从人胚脑中分离单个细胞,细胞冻存1个月后复苏或/和新鲜细胞用无血清培养基进行体外培养,碱性成纤维细胞生长因子和表皮生长因子刺激细胞生长,用血清诱导其分化;采用免疫荧光细胞化学染色方法检测培养的细胞神经巢蛋白抗原和分化后成熟神经细胞抗原神经元特异性烯醇化酶的表达.观察碱性成纤维细胞生长因子和表皮生长因子及血清对分离培养的人脑神经干细胞增殖与分化的影响.主要观察指标①冻存与新鲜细胞后续培养的生长状况.②细胞形态变化.③神经干细胞标志物神经巢蛋白及成熟神经细胞标志神经元特异性烯醇化酶的鉴定.结果①从胚龄16周的新鲜人胚脑中成功分离出神经干细胞.人胚脑原代细胞为圆形亮球状,培养3d后可见细胞开始聚集成神经球,部分呈悬浮生长,2周后神经球增大,部分细胞增大数倍,具有极强折光性,可见分裂增殖.经传代后细胞仍保持原有特征,细胞形态不变.克隆细胞在有血清培养基中培养,24 h后可见大部分细胞贴壁生长,细胞从神经球游出分散,形态不规则;48 h后多数细胞分化为大量形态不一的、分散成片的多突起星状细胞和神经元样细胞,突起相互交织成网.②分离出的神经干细胞在体外培养条件下传代培养,并表达神经于细胞标志巢蛋白;含血清培养基培养48h分化后的细胞主要表达成熟神经细胞标志神经元特异性烯醇化酶.③冻存复苏的细胞95%以上为活细胞,与新鲜细胞比较,细胞生长状况无明显差别.结论用无血清培养条件下结合碱性成纤维细胞生长因子和表皮生长因子的作用,使干细胞体外得到了分离培养、增殖和纯化,证实其方法可行,同时冻存和新鲜细胞的比较,说明可用冻存方法保存人胚脑细胞,复苏后使用.     

人脐静脉内皮细胞体外培养、鉴定与形态学特征观察

目的:建立人血管内皮细胞的培养模型,为体外研究动脉硬化发病机制提供重要的实验手段。方法:采用1.25g/L胰蛋白酶(2.5g/L胰蛋白酶:0.2g/LEDTA=1∶1V/V)消化、分离和传代人脐静脉内皮细胞,并用光镜、电镜和免疫组化等方法进行内皮细胞的形态观察和鉴定。结果:原代培养的内皮细胞在接种后约2h开始贴壁生长,光镜下内皮细胞呈铺路石状镶嵌排列;免疫组化可见内皮细胞胞浆中人第VIII因子相关抗原呈阳性反应;电镜下可见胞浆中有W-P小体,证实培养的细胞为内皮细胞。结论:用胰酶灌注消化脐静脉可获得高纯度的内皮细胞,细胞存活率高。本方法为血管内皮细胞的研究提供了实验模型。     

人颌骨骨膜成骨细胞的体外分离与培养

目的探讨人下颌骨骨膜分离培养成骨细胞的可行性。方法切取人健康的下颌骨骨膜,用胰酶、胶原酶分步消化法分离出成骨细胞,利用差异贴附原理对分离出的细胞进行纯化,对成骨细胞进行体外培养与扩增,倒置显微镜下观察细胞的生长、增殖与黏附情况,利用碱性磷酸酶染色和免疫组织化学方法检测人成骨细胞Ⅰ型胶原表达来对成骨细胞进行鉴定。结果人下颌骨骨膜成功分离出成骨细胞并进行体外培养与扩增,免疫组化学方法检测出Ⅰ型胶原表达,碱性磷酸酶染色阳性反应率达95％以上。结论自人下颌骨骨膜分离培养成骨细胞的方法切实可行。     

人脑神经干细胞的分离培养及鉴定的研究

目的探讨分离培养人脑神经干细胞的方法及条件.方法用胰蛋白酶消化法从人胚脑中分离单个细胞,用无血清培养基进行体外培养,bFGF和EGF刺激细胞生长,用血清诱导其分化;采用免疫荧光细胞化学染色方法检测培养的细胞神经巢蛋白(Nestin)抗原和分化后成熟神经细胞抗原神经元特异性烯醇化酶(NSE)的表达.结果从胚龄16周的新鲜人胚脑中成功分离出神经干细胞.该细胞可在体外培养条件下传代培养,并表达神经干细胞标志Nestin;分化后的细胞主要表达成熟神经细胞标志NSE.结论在体外培养条件下可从人胚脑中分离培养出神经干细胞.     

人脑神经干细胞的分离培养及鉴定的研究

目的探讨分离培养人脑神经干细胞的方法及条件。方法用胰蛋白酶消化法从人胚脑中分离单个细胞,用无血清培养基进行体外培养,bFGF和EGF刺激细胞生长,用血清诱导其分化;采用免疫荧光细胞化学染色方法检测培养的细胞神经巢蛋白(Nestin)抗原和分化后成熟神经细胞抗原神经元特异性烯醇化酶(NSE)的表达。结果从胚龄16周的新鲜人胚脑中成功分离出神经干细胞。该细胞可在体外培养条件下传代培养,并表达神经干细胞标志Nestin;分化后的细胞主要表达成熟神经细胞标志NSE。结论在体外培养条件下可从人胚脑中分离培养出神经干细胞。     

Platelet storage at 22°C; metabolic, morphologic, and functional studies

Platelets stored at 22 degrees C for transfusion purpose have been examined with metabolic, morphologic, and functional studies. Evaluations were made of platelet-rich plasma (PRP) stored for 3-4 days and platelet concentrates (PC) stored for 24 hr. During these periods, lactate accumulated continuously without significant change in platelet count, pH, or plasma glucose. Platelet glycogen fell dramatically both chemically and by electron microscopy, but adenosine triphosphate (ATP), adenosine diphosphate (ADP), and intracellular potassium did not change. After storage, the cell's capacity for glucose utilization through glycolysis, the hexose monophosphate shunt, and the tricarboxylic acid cycle appeared to be intact. Although platelet volume during storage did not change, disc to sphere transformation was observed by phase microscopy. Platelet aggregration with ADP was reduced even after 1 day of storage. After transfusion of stored platelets to thrombocytopenic recipients, recovery of platelet glycogen and capacity for aggregation occurred within 24 hr. In summary, the platelet remains surprisingly intact during the intervals studied; those defects which do develop are reversible in the circulation of a thrombocytopenic recipient if viability has been maintained. A "storage lesion" responsible for loss of viability has not been defined.     

Immunophenotypical, morphologic, and functional characterization of maturation-associated plasmacytoid dendritic cell subsets in normal adult human bone marrow

BACKGROUND: Information about maturation of plasmacytoid dendritic cell precursors (pre-pDCs) in normal bone marrow (BM) remains limited.
STUDY DESIGN AND METHODS: Immunophenotypical, morphologic, and functional changes associated with maturation of pre-pDCs were analyzed in adult normal human BM (n = 45).
RESULTS: Three pre-pDC maturation stages, with an increasingly higher degree of maturity, were systematically identified: CD34++/HLA-DR++/+++/CD123++/CD45+/++ (Stage I), CD34+/HLA-DR+/++/CD123++/+++/CD45+/++ (Stage II), and CD34−/HLA-DR++/CD123++/+++/CD45++ (Stage III) cells. Lymphoid- and early myeloid-associated molecules, as well as CD86, were coexpressed in Stage I pre-pDCs, being down regulated afterward. CD304 and CD85j appeared in Stage I, progressively increasing their levels thereafter. Interestingly, cutaneous lymphocyte-associated antigen was heterogeneously expressed throughout the maturation, particularly in Stage I pre-pDCs. The morphologic appearance of Stage I pre-pDCs was consistent with their undifferentiated stage, while Stage II/III cells showed morphologic features of more differentiated cells. From the functional point of view, only Stage II and Stage III pre-pDCs were capable of inducing allogeneic T-cell proliferation, the later subset also showing interferon-γ secretion; in contrast, Stage I pre-pDCs showed the highest endocytic ability.
CONCLUSION: In summary, three maturation stages of pre-pDCs can be identified in adult normal BM, which show unique phenotypical, morphologic, and functional characteristics; these results provide a frame of reference for a better understanding of pDC malignancies.     

Young's syndrome: morphologic, functional and genetic aspects of the mucociliary system

The article presents analysis of functional-morphological and genetic aspects and condition of reproductive system in Young's syndrome. On the basis of the study of the mucociliary system a working conception is proposed of Young's syndrome pathogenesis in terms of mucociliary systems of the airways.