大鼠肺泡巨噬细胞吞噬煤尘颗粒的实验研究

本实验用相差显微电影、溶酶体荧光定位和透射电镜等方法,研究了大鼠离体肺泡巨噬细胞吞噬煤尘的作用,并用二氧化矽和酵母菌作实验对照。结果表明,煤尘颗粒同二氧化矽类似,经过附着,迅速以吞噬体形式被摄入胞浆,而酵母菌呈典型的伪足包围和缓幔吞噬过程。实验证明,用吖啶橙作活细胞溶酶体定位是有效的。被标记的溶酶体发出强橙色荧光。当煤粒吞噬体进入胞浆后,常被多个溶酶体接触包围,形成次级溶酶体。电镜观察提示,煤尘可引起次级溶酶体膜的损害。     

硒肽Se-ZnCu-65P增强小鼠腹腔巨噬细胞吞噬功能并抑制一氧化氮和过氧化氢的分泌

目的研究硒肽Se-Zn Cu-65P对小鼠腹腔巨噬细胞吞噬能力与NO和H2O2分泌水平的影响。方法以不同剂量的具有超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GPx)双抗氧化酶活性的硒肽Se-Zn Cu-65P作用于脂多糖(LPS)诱导的小鼠腹腔巨噬细胞,培养24 h,采用MTT法检测巨噬细胞的相对活力,中性红摄入法检测巨噬细胞的吞噬能力,硝酸还原酶法测定NO分泌水平,钼酸铵比色法测定H2O2分泌水平。结果 Se-Zn Cu-65P单独作用于巨噬细胞时,可增强细胞的相对活力,却不影响细胞的吞噬能力;能降低巨噬细胞分泌H2O2的水平,而不能抑制NO的分泌。而LPS诱导的巨噬细胞吞噬能力增强,同时NO和H2O2的水平也显著上升。当Se-Zn Cu-65P作用于LPS诱导的巨噬细胞时,细胞的吞噬能力进一步增强,细胞NO和H2O2的水平却显著降低。结论 Se-Zn Cu-65P能有效提高LPS诱导的小鼠腹腔巨噬细胞的相对活力,增强细胞的吞噬能力,降低NO和H2O2的分泌水平。     

小鼠小肠派伊尔结巨噬细胞吸附乳杆菌的研究

研究小鼠小肠派伊尔结巨噬细胞吸附乳杆菌及其与乳杆菌免疫调节作用之间的关系,可以为免疫调节性乳杆菌或活菌疫苗载体的筛选提供理论依据。提取了小鼠小肠派伊尔结(PP)巨噬细胞,研究其吸附9株乳杆菌的性能。结果发现,PP巨噬细胞吸附不同乳杆菌的能力差别很大,其中PP巨噬细胞对植物乳杆菌Lp6(L.plantarumLp6)有最强的吸附性能。用中性红吞噬法测定了不同乳杆菌对PP巨噬细胞吞噬活性的调节作用,结果发现乳杆菌对巨噬细胞吞噬活性的增强作用与PP巨噬细胞吸附它们的能力有正相关关系。9株乳杆菌的细胞表面物理化学性质和其巨噬细胞吸附性之间没有直接关系,植物乳杆菌Lp6细胞表面的甘露糖特异性凝集素参与了PP巨噬细胞吸附该细菌的过程。     

用血红蛋白酶释放法检测巨噬细胞的吞噬能力

以鸡红细胞作为靶细胞,小鼠腹腔巨噬细胞为效应细胞研究了巨噬细胞的吞噬能力。经低渗处理后,血红蛋白酶释放的多少以它氧化邻苯二胺所产生的颜色反应表示吞噬百分比。对红细胞数与OD值关系、不同粘附时间和不同吞噬时间与吞噬能力的关系进行了研究。方法灵敏、可靠、客观,有推广应用的价值。     

活细胞标记和流式细胞术应用于体外检测巨噬细胞吞噬活性的方法建立

目的:通过活细胞标记和流式细胞术的联合应用,建立一种方法研究体外巨噬细胞的吞噬活性。方法:研究采用活细胞示踪染料CFSE标记对照组巨噬细胞,对未标记CFSE的细胞使用免疫刺激剂LPS处理,两组细胞混匀后与预先超声处理过的Alexa594标记的大肠杆菌孵育,在一个体系中完成吞噬过程,流式细胞术检测双荧光信号来评价对照组细胞和LPS处理后细胞的吞噬活性。结果:对照组和LPS处理组巨噬细胞的吞噬率分别为48.6%和62.3%,LPS处理后巨噬细胞的吞噬活性明显升高,与文献报道一致。实验表明,通过活细胞标记区分,在同一体系中完成LPS处理和未处理细胞的吞噬活性检测是非常可行的。也正因为待比较吞噬活性的两组细胞处在同一个吞噬环境中,吞噬率反映的吞噬活性更为准确。结论:本研究应用活细胞标记和流式细胞术,成功建立了一种实现体外处理的巨噬细胞吞噬活性的检测方法,简便、准确、可信度高。     

邻苯二甲酸二丁酯对巨噬细胞吞噬能力的影响研究

目的:近期台湾地区食品及饮料中添加邻苯二甲酸酯类(塑化剂)的安全问题受到广泛关注,已有研究证实邻苯二甲酸酯类物质能影响人类生殖系统发育,但其对人体免疫系统影响尚不清楚。本文研究邻苯二甲酸二丁酯对巨噬细胞吞噬能力的影响,以期揭示其对人类健康的危害。方法:使用不同浓度邻苯二甲酸二丁酯体外处理小鼠腹腔巨噬细胞24小时,然后进行巨噬细胞对细菌E.coli以及诱导凋亡的小鼠胸腺细胞的吞噬实验,并检测其吞噬率和吞噬指数,从而评价其对巨噬细胞吞噬能力的影响。用流式细胞仪检测邻苯二甲酸二丁酯对巨噬细胞表面吞噬相关分子CD11b、CD54、CD36、TLR4和CD64的影响;利用siRNA技术对吞噬相关受体CD36进行表达干扰,验证其在邻苯二甲酸二丁酯抑制巨噬细胞吞噬能力中的作用。结果:经过24小时体外处理,不同浓度邻苯二甲酸二丁酯对巨噬细胞吞噬能力均有抑制作用,并且具有剂量依赖性。此外,邻苯二甲酸二丁酯能显著降低巨噬细胞清道夫受体CD36的表达。结论:研究结果证实邻苯二甲酸二丁酯通过降低细胞膜表面CD36表达从而抑制巨噬细胞吞噬能力。     

BLP耐受通过上调MARCO增强巨噬细胞的吞噬能力

目的:探讨胶原样结构巨噬细胞受体(MARCO)在细菌脂蛋白(BLP)耐受巨噬细胞吞噬能力增强过程中的作用。方法:分离、分化和培养骨髓来源的巨噬细胞(BMDMs),并制备BLP耐受细胞;利用流式细胞术和荧光显微镜检测并比较BLP耐受与非耐受细胞吞噬细菌的能力,同时用流式细胞术和qPCR检测BLP耐受对MARCO蛋白及mRNA表达的影响;进一步利用小干扰RNA下调MARCO表达,通过流式细胞术及荧光技术探讨MARCO对BLP耐受巨噬细胞吞噬能力的影响。结果:BLP耐受巨噬细胞吞噬细菌的量较非耐受巨噬细胞明显增加(P0.05);同时BLP耐受巨噬细胞膜表面MARCO蛋白表达水平较非耐受细胞明显升高,并且在细菌刺激后进一步增加,MARCO的mRNA水平变化与蛋白水平变化一致;下调MARCO表达后,BLP耐受巨噬细胞对细菌的吞噬能力明显下降,提示BLP耐受巨噬细胞吞噬能力增强与巨噬细胞膜表面MARCO蛋白的表达上调有关。结论:BLP耐受通过上调巨噬细胞膜表面MARCO蛋白的表达,增强其对细菌的吞噬能力。     

调衡方多糖增强体外培养巨噬细胞的吞噬能力

目的研究调衡方多糖对巨噬细胞吞噬功能的影响,探讨其多糖对巨噬细胞吞噬作用的免疫调节机制。方法依照中药多糖的制备方法从调衡方中提取粗多糖;从小鼠腹腔分离提取并培养巨噬细胞;(500、200、10)μg/m L调衡方多糖处理巨噬细胞48 h,光学显微镜下观察活细胞的形态,墨汁吞噬实验以及金黄色葡萄球菌吞噬实验观察巨噬细胞的吞噬能力,酶细胞化学染色观察巨噬细胞胞内酸性磷酸酶的变化。结果与对照组比较,(500、200、10)μg/m L调衡方多糖处理巨噬细胞后,巨噬细胞体积显著增大,对墨汁和金黄色葡萄球菌的吞噬能力均显著提高,酸性磷酸酶的活性也明显增强,并与多糖浓度呈正相关。结论调衡方多糖能增强巨噬细胞的吞噬功能,并提高细胞内酸性磷酸酶的活性。     

B淋巴细胞吞噬功能研究进展

以往的观点认为只有巨噬细胞、单核细胞、粒细胞和树突状细胞是专职的吞噬细胞,而B细胞作为免疫系统的抗体产生细胞,虽然能够产生特异的免疫球蛋白与抗原结合,却不具备吞噬能力。但最近的研究指出硬骨鱼类（彩虹鳟鱼）和两栖类（爪蟾）的B细胞可以吞噬颗粒抗原,首次揭示了进化上较为原始的B细胞同样具有吞噬能力。众多研究提示哺乳动物B-1 B细胞很可能具有吞噬能力。     

生长抑素和血管活性肠肽对腹腔巨噬细胞吞噬能力的调节和细胞内钙离子浓度的调节

生长抑素(SS)和血管活性肠肽(VIP)是两种具有多种生物活性的神经肽,在胃肠道内含量丰富,近年来研究发现,在淋巴细胞和巨噬细胞的表面有SS和VIP的特异性受体,它们对免疫的调节作用日益受到重视。本实验的目的是观察SS和VIP对体外培养的腹腔巨噬细胞吞噬能力的影响和对细胞内钙离子浓度的调节作用。方法:1巨噬细胞吞噬中性红后,溶解细胞,用分光光度计测定光密度,反映巨噬细胞的吞噬能力;2巨噬细胞与45Ca共同培养后,用液体闪烁计数测定其放射性,来反映巨噬细胞内Ca2+的浓度;3Fluo-3/AM作为荧光探针,应用激光共聚焦扫描显微镜观察巨噬细…     

活卡介苗对家兔肺泡巨噬细胞溶酶体杀伤和消化酵母菌的实验研究

本实验用吖啶橙标记溶酶体,用荧光显微镜观察肺泡巨噬细胞吞噬活酵母菌的细胞内杀伤和消化过程。根据细胞的形态学改变,将此过程分为融合前期、融合与杀伤期和消化期。结果表明,活卡介苗活化家兔离体肺泡巨噬细胞的吞噬率、吞噬指数、融合率和融合指数,均较对照组提高(P<0.01),本研究从形态学角度证实,活化细胞溶酶体的细胞内杀伤和消化作用有提高。     

Impaired chemotactic responses of bronchoalveolar leukocytes in experimental pneumoconiosis

Rats were exposed to clouds of the following pneumoconiotic dusts: quartz, coal-mine dust, and chrysotile asbestos at 10 or 50 mg/m3 for 8, 32, and 75 days; for comparison, rats were also exposed to the non-pathogenic dust titanium dioxide (TiO2). The bronchoalveolar leukocytes (macrophages and neutrophils) from dust-exposed and control rats were obtained by lavage and tested for their ability to migrate toward zymosan-activated serum. Varying amounts of neutrophils were present depending on the ability of the dust to cause inflammation and the length of exposure. There was a marked loss of chemotactic ability in leukocytes from rats inhaling the pneumoconiotic dusts compared with controls; TiO2-exposed leukocytes had some impairment of chemotaxis, but this was substantially less than that found with the pneumoconiotic dusts. The loss of chemotactic activity did not correlate with the percentage of neutrophils in the lavage cells except when there were very high levels of neutrophils, and there was substantial impairment of chemotaxis with negligible numbers of neutrophils, showing that macrophage chemotaxis was impaired. A phagocytic burden within the leucocytes was not sufficient alone to inhibit chemotaxis, nor was the loss of chemotactic activity due to occupied receptors, since incubation failed to restore chemotaxis. Loss to chemotactic activity by leukocytes from pneumoconiotic dust-exposed lung could be an important factor in the development of pneumoconiosis.     

The chemiluminescent response of human phagocytic cells to mineral dusts

Luminol-dependent chemiluminescence (CL) was used to assess the in vitro production of reactive oxygen species by human neutrophils and monocytes on exposure to six standard respirable mineral dusts. Every dust caused CL production in both phagocytic cell types, although, for each dust, the two cells showed a different pattern of response. Light output was markedly affected by the presence of serum in the system. While the results illustrated the complexity of the interaction between mineral dusts and monocytes and neutrophils, they did not support the hypothesis that pathogenic dusts would induce the production of more reactive oxygen species than non-pathogenic dusts.     

The chemiluminescent response of human phagocytic cells to mineral dusts.

Luminol-dependent chemiluminescence (CL) was used to assess the in vitro production of reactive oxygen species by human neutrophils and monocytes on exposure to six standard respirable mineral dusts. Every dust caused CL production in both phagocytic cell types, although, for each dust, the two cells showed a different pattern of response. Light output was markedly affected by the presence of serum in the system. While the results illustrated the complexity of the interaction between mineral dusts and monocytes and neutrophils, they did not support the hypothesis that pathogenic dusts would induce the production of more reactive oxygen species than non-pathogenic dusts.     

异形矿物粉尘巨噬细胞毒性研究

为研究粉尘样品对兔肺泡巨噬细胞（AM）产生的不同毒性。探讨巨噬细胞受损的机制，采用体外细胞培养技术，观测兔肺泡巨噬细胞死亡率，丙二醛（MDA）、乳酸脱氢酶（LDH）及超氧化物歧化酶（SOD）的活性及变化。结果表明．沸石、硅灰石无细胞毒性，而其它的纤维状及颗粒状矿物粉尘则表现出不同程度的细胞毒性。纤维状矿物尘对AM毒性大于颗粒状矿物，毒性程度与粉尘中的活性OH^-含量正相关，但并不一定与SiO2含量相关。粉尘所形成的高pH值不利于细胞的生存，低生物持久性的粉尘对人体是安全的。粉尘中变价元素的含量可能影响其毒性。表面电位是粉尘毒性的非稳定因素。矿物尘的微形态是影响其毒性的因素之一．而矿物尘的毒性主要依赖于其特性。     

An investigation into the cytotoxicity of respirable dusts from British collieries.

A series of respirable dusts from British collieries was collected and analysed for mineral content and physical characteristics. Where possible 2 samples of dust were collected from the same site at 8-month intervals. All dusts were tested for their cytotoxic potential using a permanent line of macrophage-like cells (P388D1). In addition, for some dusts, a haemolytic technique was used. With both techniques a positive overall correlation was found between cytotoxicity and the total ash content of the dusts. When the results from collieries producing high- and low-rank coals were considered separately, however, it was found that the ash content of high-rank dusts (r=0.75) showed a much closer correlation with cytotoxicity than low-rank dusts (r=0.40). With the cell test system the ash components, kaolin and mica (r=0.58) and to a lesser extent quartz (r=0.48) showed significant positive correlations with cytotoxicity for high-rank coal dusts but not for low. Using the haemolytic system, however, only the quartz content of the high-rank dusts showed a significant relationship (r=0.69) to levels of haemoglobin release. Both the results of mineralogical analysis of dust samples and cytotoxicity tests showed that the mineral content and cytotoxic potential of dusts collected from the same colliery, and even from the same underground site, at different times, varied considerably. A poor correlation was found between cytotoxicity and various measurements of pneumoconiosis risk but this may well be partly due to this great variation of dust composition with time. In general, the overall results of this study were in good agreement with those of previous work on coal dust toxicity in that both the rank and composition of colliery dusts were found to be of importance, whereas the role of quartz remained enigmatic.     

An investigation into the cytotoxicity of respirable dusts from British collieries

A series of respirable dusts from British collieries was collected and analysed for mineral content and physical characteristics. Where possible 2 samples of dust were collected from the same site at 8-month intervals. All dusts were tested for their cytotoxic potential using a permanent line of macrophage-like cells (P388D1). In addition, for some dusts, a haemolytic technique was used. With both techniques a positive overall correlation was found between cytotoxicity and the total ash content of the dusts. When the results from collieries producing high- and low-rank coals were considered separately, however, it was found that the ash content of high-rank dusts (r=0.75) showed a much closer correlation with cytotoxicity than low-rank dusts (r=0.40). With the cell test system the ash components, kaolin and mica (r=0.58) and to a lesser extent quartz (r=0.48) showed significant positive correlations with cytotoxicity for high-rank coal dusts but not for low. Using the haemolytic system, however, only the quartz content of the high-rank dusts showed a significant relationship (r=0.69) to levels of haemoglobin release. Both the results of mineralogical analysis of dust samples and cytotoxicity tests showed that the mineral content and cytotoxic potential of dusts collected from the same colliery, and even from the same underground site, at different times, varied considerably. A poor correlation was found between cytotoxicity and various measurements of pneumoconiosis risk but this may well be partly due to this great variation of dust composition with time. In general, the overall results of this study were in good agreement with those of previous work on coal dust toxicity in that both the rank and composition of colliery dusts were found to be of importance, whereas the role of quartz remained enigmatic.     

Glucose Stimulates Phagocytosis of Unopsonized Pseudomonas aeruginosa by Cultivated Human Alveolar Macrophages

Glucose has previously been shown to increase the in vitro phagocytosis of unopsonized Pseudomonas aeruginosa by freshly explanted murine peritoneal macrophages (PM) and cultivated alveolar macrophages (AM). This study examined the effect of glucose on the same phagocytosis process in human AM in order to determine whether this phenomenon is conserved among species. Freshly explanted human AM phagocytosed unopsonized P. aeruginosa at a low level (2 bacteria/macrophage/30 min), whereas mouse AM ingested a negligible number of P. aeruginosa (0.01 bacterium/macrophage/30 min). Glucose had no effect on this or other phagocytic processes in freshly explanted mouse or human AM. However, following in vitro cultivation for 72 h, human AM phagocytosed three to four times more unopsonized P. aeruginosa than did freshly explanted cells, but only in the presence of glucose. This glucose-inducible phagocytic response had also been observed in cultivated murine AM. Although similar increases were also detected for the phagocytosis of latex particles and complement-coated sheep erythrocytes by cultivated human AM, these processes were not glucose dependent. The lack of response to glucose in freshly explanted mouse AM was attributed to insufficient glucose transport; however, freshly explanted human AM exhibited significant facilitative glucose transport activity that was inhibitable by cytochalasin B and phloretin. Taken together, these results suggest that the process of glucose-inducible phagocytosis of unopsonized P. aeruginosa is conserved among macrophages from different species, including humans, and that AM, but not PM, required cultivation for this glucose effect to occur. Glucose transport by AM appears to be necessary but not sufficient for phagocytosis of unopsonized P. aeruginosa.