肥大细胞与肺成纤维细胞共育的形态学观察

用分离纯化的大鼠腹腔肥大细胞（ＰＭＣ）与同源胎鼠肺成纤维细胞（ＬＦｂ）共育，对ＬＦｂ进行形态学观察和形态计量分析。结果表明：与ＰＭＣ共育的ＬＦｂ呈复层生长，单位面积内细胞计数明显增加，粗面内质网（ＲＥＲ）增生，电镜见ＰＭＣ与ＬＦｂ紧密接触和部分ＰＭＣ有脱颗粒现象，提示ＰＭＣ可促进ＬＦｂ增值，并使其功能活跃。     

肥大细胞与成纤维细胞功能关系的光镜与电镜研究

通过对正常人体皮肤真皮与胶原纤维大量增生的疾患的真皮组织的光镜与电镜的比较观察,发现在病因不同,而都有胶原纤维大量增生的系统性硬皮病与皮肤纤维瘤的真皮组织中,肥大细胞都比正常时显著增多(P<0.01),而且肥大细胞常与成纤维细胞紧密相靠接,这里的肥大细胞脱颗粒现象也增多。从颗粒的超微结构特征所示,这些肥大细胞以未成熟的T型肥大细胞为主。以上提示:肥大细胞在调节成纤维细胞产生胶原纤维的功能上具有重要意义。     

干细胞因子反义寡核苷酸对小鼠成纤维细胞-肥大细胞相互作用的影响

目的探讨干细胞因子介导的成纤维细胞-肥大细胞相互作用在哮喘病理生理过程中的作用。方法以SCF反义寡核苷酸(SCF ASON)转染成纤维细胞NIH3T3,免疫组化和RT-PCR法检测SCF的表达;从小鼠骨髓中分离肥大细胞,建立与NIH3T3共育体系,加入10 mg/L SCF ASON,通过ELISA法和荧光测定法检测上清液中组织胺和Eotaxin的含量;绘制NIH3T3和肥大细胞生长曲线;丫啶橙染色、流式细胞仪检测肥大细胞的凋亡。结果SCFASON可显著抑制NIH3T3产生SCF;以SCF ASON干预共培养体系后,成纤维细胞生长受抑,肥大细胞出现凋亡(14.0%±0.81%,96 h);组胺[3.08±0.38]μg/L比较(3.83±0.4)μg/L,P<0.05]和Eotaxin的产生[(4.40±0.33)μg/L比较(5.79±0.40)μg/L,P<0.05]减少。结论SCF可能通过抑制肥大细胞凋亡,促进肥大细胞激活和成纤维细胞增殖,参与哮喘的发生、发展。     

虎杖苷对成纤维细胞生物学特性的影响

背景：虎杖多年来一直是烧伤、创伤创面愈合治疗方剂中的一味主药,因成分复杂,具体药理作用难以进一步研究,对于虎杖苷促愈合作用目前未见文献报道。 目的：分析不同浓度虎杖苷对成纤维细胞生物学特性的影响。 方法：取烧伤后行瘢痕切除植皮4例患者剩余小中厚皮片,原代培养人成纤维细胞。用含有10^-6,10^-5,10^-4,10^-3,10^-2 mol/L不同浓度虎杖苷培养液作用第2代人成纤维细胞,未加虎杖苷的培养液作为对照。MTT法检测细胞增殖情况;流式细胞仪检测细胞周期及凋亡;ELISA法检测上清中纤维结合蛋白、Ⅰ型胶原蛋白、Ⅲ型胶原蛋白的表达情况。 结果与结论：10^-5、10^-4 mol/L组促进成纤维细胞增殖最明显,10^-2 mol/L组吸光度值显著下降,细胞生长受抑制。10^-3 mol/L组G1期细胞大幅度下降,细胞有S期阻滞现象。10^-2 mol/L组有明确的促凋亡作用。10^-2 mol/L组上清中Ⅰ、Ⅲ型胶原蛋白较其他各组显著增高(P < 0.05);纤维结合蛋白较对照组显著增高(P < 0.05),较其他各组有所下降(P < 0.05)。说明低浓度虎杖苷有促进成纤维细胞增殖、保护细胞免于凋亡及促进纤维结合蛋白的表达与合成分泌的作用,促进成纤维细胞增殖的最适浓度应为10^-5~10^-4 mol/L。     

碱性成纤维细胞生长因子对瘢痕成纤维细胞生物学行为的影响

目的探讨碱性成纤维细胞生长因子（bFGF）对瘢痕成纤维细胞生物学特性的影响。方法采用细胞培养、MTT、ELISA法、氯胺T和RT—PCR法检测bFGF在不同作用剂量下对瘢痕来源的成纤维细胞生长增殖和细胞外基质（ECM）合成的影响。结果（1）MTT检测bFGF对瘢痕成纤维细胞有明确的促增殖作用，并具有剂量相关性；（2）氯胺T法显示实验组和对照组HPr含量无显著性差异，RT—PCR法显示各组Ⅰ、Ⅲ型胶原蛋白mRNA表达水平无明显变化；说明bFGF对瘢痕成纤维细胞胶原蛋白的合成无促进作用；（3）ELISA法表明随着bFGF作用浓度的升高，FN的表达表现为增高趋势，且以100ng／ml浓度下作用最显著。结论bFGF可以促进创面愈合，但不引起瘢痕的过度形成。     

骨髓间充质干细胞条件培养液对瘢痕成纤维细胞生物活性的影响

背景：间充质干细胞移植可通过多种调节机制促进皮肤创伤修复,抑制瘢痕形成,目前多数学者认为间充质干细胞分泌的生物活性因子起主要作用。 目的：观察骨髓间充质干细胞条件培养液对增生性瘢痕成纤维细胞增殖能力及胶原合成的影响。 方法：分离培养人骨髓间充质干细胞和增生性瘢痕成纤维细胞,制备骨髓间充质干细胞条件培养液。将12,24,48 h收集的条件培养液孵育体外培养的增生性瘢痕成纤维细胞24 h,并与空白对照组比较,采用CCK-8实验检测增生性瘢痕成纤维细胞的增殖活性,Real-time PCR检测增生性瘢痕成纤维细胞中Ⅰ型和Ⅲ型胶原的表达。 结果与结论：与空白对照组相比较,在24 h和48 h收集的骨髓间充质干细胞条件培养液对增生性瘢痕成纤维细胞的增殖具有明显的抑制作用(P < 0.01),对其胶原的合成也具有显著的抑制作用(P < 0. 01)。结果表明骨髓间充质干细胞条件培养液可通过分泌抗纤维化的生物活性因子抑制增生性瘢痕成纤维细胞的增殖和胶原产生,为细胞疗法策略减轻瘢痕提供了新的理论支持。 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;组织工程全文链接：     

成纤维细胞超微结构的研究

本文用实验组织学方法,系统观察了成纤维细胞6种基本细胞形态的超微结构。发现成胶原细胞有前、中、后期三个功能阶段,中期细胞又分为合成期和分泌期;破纤维细胞可区分出吞噬型和溶酶体型2种亚型;纤维细胞存在静止型和衰老型2种亚型。成胶原细胞和破纤维细胞分别是胶原合成和胶原降解的主要细胞。本文从超微结构方面,证实成纤维细胞来源于毛细血管旁未分化间充质细胞,少分化成纤维细胞是其他形态的成纤维细胞的来源。成纤维细胞各种成熟的细胞形态可以互相转化,在功能上互相协调。本文提出成纤维细胞系统的概念,认为该系统包括成纤维细胞的6种基本细胞形态,其主要机能是合成胶原蛋白和其他细胞间质,降解胶原,使结缔组织(包括瘢痕组织)收缩。     

维甲酸对成纤维细胞及脂肪源干细胞成骨诱导的影响

目的　探究维甲酸（RA）对乳鼠真皮成纤维细胞（DFB）及脂肪源干细胞（ADSCs）成骨诱导分化的影响。 方法　分别离体培养5~7 d龄乳鼠DFB及ADSCs。对DFB进行波形蛋白的免疫荧光鉴定,对ADSCs进行成骨、成脂肪诱导并做表面标记物鉴定。用RA及传统成骨诱导液分别对两类细胞进行成骨诱导2周,茜素红染色检测细胞内钙结节的形成,酶标仪检测细胞诱导后ALP 的OD值。 结果　两类细胞在常规成骨诱导及RA诱导成骨后,均有钙结节形成。传统成骨诱导与RA成骨诱导组与各自对照组相比,ALP活性均升高。在DFB组中,RA诱导的ALP活性比传统成骨诱导高,而在ADSCs组中,结果相反。 结论　RA可以促进DFB及ADSCs的成骨分化,并且因细胞种类不同而显著效果不同。     

碱性成纤维细胞生长因子对兔晶体上皮细胞的增殖作用

观察碱性纤维细胞生长因子（basic fibroblast growth factor,bFGF)对兔晶体上皮细胞（rabbit lens epithelial cells,RLECs)的促增殖作用,以及增殖细胞核抗原（proliferating cell nuclear antigen,PCNA)的表达,用第2－3代培养细胞,用MTT法测定细胞增殖,用免疫组织化学法观察PCNA的表达,流式细胞仪观察细胞周期的变化。结果显示bFGF可促进兔晶体上皮细胞的增殖,尤其是浓度为10μg/L作用最明显;正常细胞组PCNA表达呈阴性,添加bFGF组PCNA表达呈强阳性;并显示进入S期的细胞明显增加,提示bFGF是促进兔晶体上皮细胞增殖的重要因素。     

脐带血血浆对包皮成纤维细胞生长影响的实验研究

目的　探讨人脐带血血浆（HUP）能否维持人包皮成纤维细胞（HFF）的生长。 方法　将脐血浆经盐析、透析等处理后,按10%体积成分培养HFF,并与同样浓度的胎牛血清（FBS）进行比较,观察细胞的贴壁速度、生长曲线、细胞周期及增殖能力。 结果　 HFF在含10%HUP的培养液中能长期生长,其生长生长形态、细胞增殖能力及特征都与在含同体积FBS的培养液中无显著差异。 结论　人HUP能维持HFF的长期生长。     

青藤碱对RBL-2H3肥大细胞增殖凋亡以及活化脱颗粒的影响

目的 通过研究青藤碱对肥大细胞增殖凋亡以及活化脱颗粒的影响,深入探讨青藤碱的抗炎作用机理.方法 应用细胞培养,流式细胞术等方法,观察青藤碱等药物对体外培养的RBL-2H3肥大细胞增殖凋亡以及活化脱颗粒的影响.结果 青藤碱对肥大细胞增殖有明显的抑制作用,其IG50约为1 mmol/L,并能促进肥大细胞凋亡,且对RBL-2H3肥大细胞活化脱颗粒有较强的抑制作用.结论青藤碱可能部分通过下调肥大细胞功能发挥抗炎抗风湿作用.     

Airway smooth muscle proliferation and survival is not modulated by mast cells

Background Airway smooth muscle (ASM) hyperplasia and mast cell localization within the ASM bundle are important features of asthma. The cause of this increased ASM mass is uncertain and whether it is a consequence of ASM–mast cell interactions is unknown. Objective We sought to investigate ASM proliferation and survival in asthma and the effects of co‐culture with mast cells. Methods Primary ASM cultures were derived from 11 subjects with asthma and 12 non‐asthmatic controls. ASM cells were cultured for up to 10 days in the presence or absence of serum either alone or in co‐culture with the human mast cell line‐1, unstimulated human lung mast cells (HLMC) or IgE/anti‐IgE‐activated HLMC. Proliferation was assessed by cell counts, CFSE assay and thymidine incorporation. Apoptosis and necrosis were analysed by Annexin V/propidium iodide staining using flow cytometry and by assessment of nuclear morphology using immunofluorescence. Mast cell activation was confirmed by the measurement of histamine release. Results Using a number of techniques, we found that ASM proliferation and survival was not significantly different between cells derived from subjects with or without asthma. Co‐culture with mast cells did not affect the rate of proliferation or survival of ASM cells. Conclusion Our findings do not support a role for increased airway smooth proliferation and survival as the major mechanism driving ASM hyperplasia in asthma. Cite this as: D. Kaur, F. Hollins, R. Saunders, L. Woodman, A. Sutcliffe, G. Cruse, P. Bradding and C. Brightling, Clinical & Experimental Allergy, 2010 (40) 279– 288.     

Effects of mast cell-macrophage interactions on the production of collagenolytic enzymes by metastatic tumor cells and tumor-derived and stromal fibroblasts

Histological examination of the metastatic rat mammary adenocarcinoma line MTLn3 showed that macrophages and mast cells were frequently localized at the tumor periphery in the stromal tissues adjacent to the zones of tumor invasion. The interactions of these host cells with tumor cells and tumor-associated fibroblasts could be important in stimulating the production of extracellular matrix-degrading enzymes that facilitate tumor invasion and metastatic spread. Therefore, we examined the effects of isolated, activated macrophages and mast cells on the secretion of collagenolytic activities by normal fibroblasts, metastatic mammary adenocarcinoma cells and tumor-associated fibroblasts. Medium from activated macrophages or degranulated mast cells stimulated significant increases in production of collagenolytic activities by normal and tumor-associated fibroblasts and MTLn3 tumor cells. Medium from activated macrophages that had been pretreated with medium from degranulated mast cells, however, were less stimulatory to fibroblasts and tumor cell production of collagenolytic activities than medium from degranulated mast cells alone. We also examined the effects of two cytokines, interleukin-1 and tumor necrosis factor-a on activated macrophage- and degranulated mast cell-stimulation of fibroblast and tumor cell collagenolytic activities. The two cytokines alone or in combination stimulated increased production of collagenolytic activities by fibroblasts and tumor cells. Addition of the cytokines to degranulated mast cell products resulted in secretion of higher collagenolytic enzyme activities by normal fibroblasts (but not by tumor-derived fibroblasts or tumor cells) than with degranulated mast cell product-treatment of either target cell alone. Cytokines used in combination with macrophage-conditioned medium were less effective in stimulating fibroblast and tumor cell collagenase activities than cytokines alone. Thus normal infiltrating host cells such as macrophages and mast cells can have profound effects on the production of degradative enzymes by tumor cells and tumor-associated stromal fibroblasts.     

Effects of cholecystokinin-4 on secretory activity of rat mast cells

Intraperitoneal injection of cholecystokinin-4 in a dose of 100 mg/kg markedly increased secretory activity of mast cells in the mesentery and subcutaneous fat. These changes developed 5 min after treatment and progressed in time. Over the first 15 min we observed primarily merocrine secretion (granulolysis), while 60 min after cholecystokinin-4 administration apocrine secretion (degranulation) prevailed. In vitro cholecystokinin-4 had no effect on secretory activity of mast cells. Our findings suggest that stimulation of secretory activity of mast cells is determined by psychoemotional stress associated with activation of the sympathoadrenal and hypothalamic-pituitary-adrenal systems.     

碱性成纤维细胞生长因子和5-氮杂胞苷对大鼠骨髓间充质干细胞分化潜能和细胞增殖的影响

目的:探讨碱性成纤维细胞生长因子(bFGF)和5-氮杂胞苷(5-aza)对体外骨髓间充质干细胞(MSCs)细胞增殖与分化潜能的影响。方法:大鼠骨髓中获得MSCs,培养传代,在倒置相差显微镜和透射电镜下观察细胞形态变化;流式细胞仪检测细胞周期改变;MTT法检测bFGF和5-aga对MSCs生长的影响;免疫细胞化学方法检测actin、desmin、myoglobin、NSE、GFAP的表达。结果:有心肌样细胞和神经元样细胞的形态变化;MSCs经5-aza以及5-aza加bFGF联合诱导actin、desmin、myoglobin、NSE和GFAP的表达均较对照组显著增高;而经bFGF诱导后,前4种蛋白表达无明显变化,仅GFAP蛋白表达显著增高。结论:5-aza对MSCs细胞生长有抑制作用,而bFGF有明显促增殖作用。MSCs被5-aza诱导分化成心肌样细胞和神经元样细胞,bFGF可使MSCs分化为GFAP阳性的神经胶质样细胞。     

Gomisin N has anti-allergic effect and inhibits inflammatory cytokine expression in mouse bone marrow-derived mast cells

Gomisin N is a bioactive compound and a prominent anti-allergic agent found in the fruits of tree Schizandra chinensis. However, its effects on the bone marrow-derived mast cell (BMMC)-mediated allergy and inflammation mechanism remain unknown. In this study, the biological effects of gomisin were evaluated while focusing on its effects on the allergic mediator in PMA + A23187-stimulated BMMCs. The anti-allergic effect of gomisin has shown that inhibited PMA + A23187-induced interleukin-6 (IL-6) production. An investigation was also conducted to determine its effects on the production of several allergic mediators including prostaglandin D₂ (PGD₂), leukotriene C₄ (LTC₄), β-hexosaminidase (β-Hex), and cyclooxygenase-2 (COX-2) protein. The results revealed that gomisin inhibited the PMA + A23187-induced production of IL-6, PGD₂, LTC₄, β-Hex, and COX-2 protein. Taken together, these findings indicate that gomisin N has the potential for use in the treatment of allergy.     

Human mast cells stimulate fibroblast proliferation, collagen synthesis and lattice contraction: a direct role for mast cells in skin fibrosis

BACKGROUND: Mast cells, the key cells of immediate hypersensitivity type reactions, have also been postulated to have a central role in influencing tissue remodelling and fibrosis occurring in the skin. OBJECTIVE: Our aim was to investigate the direct role of human mast cells (HMC) in skin fibrotic processes, by assessing the effects of the addition of the human mast cell line HMC-1 to human skin fibroblasts, and to identify the responsible mediators. METHODS: HMC-1 sonicates were added to human skin fibroblasts and the following parameters were evaluated: proliferation ([3H]-thymidine), collagen synthesis ([3H] proline), activity of matrix metalloproteinases (MMPs) (zymography) and tissue inhibitors of metalloproteinases (TIMPs) (reverse zymography), and collagen gel contraction. RESULTS: HMC-1 sonicate increased significantly both proliferation and collagen production in the human skin fibroblasts and these properties were not affected by heating of the sonicate (56 degrees C, 30 min, or 100 degrees C, 3 min). Two main mast cell mediators, histamine and tryptase, were found to be responsible for the increase in fibroblast proliferation and collagen production. HMC-1 sonicate did not display any pre-formed gelatinase activity, and its addition to the fibroblasts did not change their pro-MMP-2 and MMP-2 activity. On the other hand, HMC-1 were found to possess TIMP-1 and TIMP-2. Addition of HMC-1 had no effect on fibroblasts TIMP-1 but induced a dose-dependent increase of TIMP-2 activity. In addition, HMC-1 sonicate seeded together with the fibroblasts in tri-dimensional collagen gel significantly enhanced their contraction. CONCLUSION: We have shown that human mast cells, by granule-stored and therefore quickly releasable mediators, increase human skin fibroblast proliferation, collagen synthesis, TIMP-2 and collagen gel contraction. Therefore, mast cells have a direct and potentiating role in skin remodelling and fibrosis.     

Differential staining of mast cells with toluidine blue

Abstract

Mast cells play a significant role in inflammatory diseases such as asthma, inflammatory bowel disease, and autoimmune diseases. Inhibition of c-kit receptor tyrosine kinase, a growth factor receptor, significantly reduces mast cell numbers. The purpose of this study was to determine the effect of Compound X (a c-kit inhibitor) on mast cell numbers in rats. Connective tissue mast cells (CTMCs) and mucosal mast cells (MMCs) have differing histochemical characteristics which presents a challenge when staining for quantification by semi-automated image analysis. CTMCs are present in tissues such as tongue and skin and will stain readily in tissues fixed routinely. In contrast, MMCs, such as those present in the intestinal mucosa, are sensitive to fixation. Brief fixation in Carnoy’s solution, although seldom used due to its composition (a mixture of ethanol, chloroform, and acetic acid), was employed to fix tissues for MMC staining, while tissues for CTMC demonstration were fixed in 10% neutral buffered formalin. An enzyme histochemistry method, napthol AS-D choloroacetate (specific esterase), was briefly considered for staining; however, granulocytes stained along with mast cells, requiring manual identification and exclusion, thereby rendering the method incompatible with automated means of quantification. Instead, staining was performed using two different toluidine blue methods which have proven conducive to semi-automated image analysis techniques. CTMCs were stained using Luna’s toluidine blue, while MMCs were stained with Matsson’s toluidine blue modification. In summary, the selected methods, based upon a conventional stain, were easy to do and successfully identified both populations of mast cells for quantification by image analysis.