中国人肠上皮细胞gamma射线损伤的差异表达基因

小肠是电离辐射的敏感组织．γ射线照射后小肠上皮干细胞的损伤，肠道组织形态和生理功能改变的文献报道较多［１］，但肠道辐射损伤分子机制的研究尚未见报道．我们采用ｍＲＮＡ差异展示技术，分离正常的和经６０Ｃｏγ射线照射的人肠上皮细胞株ＨＩＥＣ细胞的差异表达基因，拟从分子水平探讨电离辐射对人肠上皮细胞损伤的分子机制，为建立辐射损伤的分子诊断标准打下基础．１　材料和方法１．１　材料　①正常人肠上皮细胞株ＨＩＥＣ由Ｐａｔｅｒｓｏｎ肿瘤研究所Ｐｏｔｔｅｎ教授馈送．②ｍＲＮＡ提取试剂盒、ＰＣＲ产物纯化试剂盒皆购自…     

中国人肠上皮细胞γ射线损伤的差异表达基因

小、肠是电离辐射的敏感组织.γ射线照射后小肠上皮干细胞的损伤,肠道组织形态和生理功能改变的文献报道较多[1],但肠道辐射损伤分子机制的研究尚未见报道.我们采用mRNA差异展示技术,分离正常的和经60Coγ射线照射的人肠上皮细胞株HIEC细胞的差异表达基因,拟从分子水平探讨电离辐射对人肠上皮细胞损伤的分子机制,为建立辐射损伤的分子诊断标准打下基础.     

炎性细胞因子对人肾小管上皮细胞C3a受体表达的调控

目的:为更好了解肾脏组织中C3aR表达调控因素,我们研究了多种炎性细胞因子对人肾小管上皮细胞(HK2细胞)C3a受体(C3aR)表达的调控作用。方法:用多种炎性细胞因子[干扰素γ(IFN-γ)、C3a、白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)、转化生长因子β(TGF-β)]刺激HK2细胞,利用荧光定量PCR、免疫组化及免疫印迹的方法检测HK2细胞C3aR表达水平。结果:炎性细胞因子IFN-γ能够显著促进HK2细胞C3aR的表达上调[与正常对照组相比,C3aR mRNA表达水平的约增长5倍(P=0.001),蛋白水平表达上调大约1.6倍(P=0.046)],且HK2细胞C3aR mRNA水平表达上调与IFN-γ呈剂量及时间依赖关系;而其他的炎性细胞因子如TNF-α、TGF-β、IL-1β甚至生物活性多肽C3a均未观察到对HK2细胞C3aR表达造成影响。结论:肾小管上皮细胞表面可表达生物活性肽段C3aR,而且炎症细胞因子IFN-γ能够明显增强肾小管上皮细胞C3aR表达,预示C3a/C3aR轴可能参与了肾脏局部炎症反应的调节并且与肾脏疾病的发生、发展有一定的相关性。     

不同月龄大鼠大脑中低密度脂蛋白受体相关蛋白的表达

目的　探讨不同月龄大鼠大脑额叶、海马、纹状体低密度脂蛋白受体相关蛋白 (LRP 1)表达的变化。方法　雄性SD大鼠 15只 ,流式细胞技术检测并计算 1,3,10月龄大鼠大脑额叶、海马、纹状体匀浆单细胞悬液LRP 1表达的平均荧光指数 ;免疫组织化学技术测定大脑海马CA1区神经细胞、微血管LRP 1表达 ,并应用MIAS图像分析系统进行平均吸光度测定。结果　流式细胞研究显示 ,大鼠海马 1、3月龄LRP 1表达强度均显著高于 10月龄大鼠 ;而额叶及纹状体各月龄组间差异无显著性意义。 1、3月龄大鼠海马区LRP 1表达强度均显著高于额叶及纹状体。免疫组织化学结果显示 ,LRP 1在海马CA1区微血管、神经细胞均有表达。随着月龄的增加 ,LRP 1在微血管的表达显著下降 ,而在海马CA1区神经细胞的表达各月龄组差异无显著性意义。结论　随着月龄的增加 ,海马区LRP 1的表达有下降趋势 ,尤其是海马CA1区微血管LRP 1的表达显著下降。     

人肺上皮细胞C反应蛋白的表达

C反应蛋白(CRP)是一种急性时相蛋白,主要在肝脏由细胞炎症因子白细胞介素6(IL6)刺激肝细胞合成及分泌[1]。近来有文献报道,呼吸道本身可能存在有CRP的分泌现象[2,3]。我们的研究旨在探讨肺泡Ⅱ型细胞在脂多糖(LPS)诱导下是否有CRP的表达和分泌。材料与方法　人肺上皮细胞(A549)购于美国典型物保藏中心(ATCC)于含10%胎牛血清的DEME培养液中常规传代。LPS为美国Sigma公司产品。培养:肺泡上皮细胞株经解冻、复苏之后,用025%的胰蛋白酶消化分批传代至培养皿,调整细胞数目为每孔1×107个,给予不同的诱导浓度,在37℃、5%CO2条件下孵育…     

内毒素受体在肝星状细胞的表达及其作用

目的了解内毒素受体在肝星状细胞活化中的变化和作用。方法分离正常大鼠的肝星状细胞，以逆转录聚合酶链反应法检测其在体外培养过程中内毒素受体（CD14和TLR4）mRNA的表达变化。以细胞免疫染色法检测肝星状细胞内毒素受体CD14的表达。制作肝纤维化和肝硬化的大鼠模型，免疫组织化学法动态检测肝组织内CD14和α平滑肌肌动蛋白的表达变化和定位。结果初分离的肝星状细胞表达低水平的CD14 mRNA，不表达TLR4 mRNA，培养活化的肝星状细胞内毒素受体的表达增强，内毒素可上调这种表达。体外培养10d的肝星状细胞表达CD14蛋白，内毒素作用后CD14表达更明显。在肝纤维化的发展过程中，肝组织内CD14阳性细胞增多，阳性细胞多分布于肝窦周围，晚期CD14阳性细胞聚集在纤维隔内，与α平滑肌肌动蛋白阳性细胞的分布一致。结论肝星状细胞在体内外的活化过程中内毒素受体的表达增强，因此，内毒素受体可能参与肝星状细胞在肝脏炎症和纤维化中的作用。     

不同来源的CD34^＋细胞归巢相关分子的表达

目的 研究不同来源CD34^＋细胞归巢相关分子（HRM）的表达情况。方法采用免疫磁珠法（MACS）分选不同来源的CD34^＋细胞，免疫荧光标记流式细胞仪测定HRM的表达。结果骨髓（BM）、动员后的外周血（mPB）及脐血（UCB）来源的CD34^＋细胞均高表达细胞黏附分子CD49d、CD49e、CD54、CD11a、CD62L、CD44、CD31。UCB来源的CD34^＋细胞表面表达的细胞黏附分子中，CD49e表达显著低于BM和mPB来源的CD34^＋细胞（P〈0．05），CD54表达显著低于mPB来源者（P〈0．05），CXCR4表达显著低于BM来源者（P〈0．05）。结论UCB来源的造血干／祖细胞归巢能力低可能是UCB移植造血重建延迟的原因之一。     

白介素5受体α链在支气管上皮细胞的表达

目的白介素5(IL-5)作为参与嗜酸粒细胞(EOS)成熟、分化过程的最主要因子,其作用通过特异性受体白介素5受体α链(IL-5Rα)介导。本研究以支气管上皮细胞株为研究对象,发现支气管上皮细胞在炎症因子的刺激下可以表达IL-5Rα。方法通过促炎症因子肿瘤坏死因子α(TNF-α)和Th2型细胞因子IL-4,IL-5,IL-13以及白三烯成分之一的白三烯C4(LTC4)对细胞进行单独或协同刺激,以逆转录聚合酶链反应(RT-PCR)方法、流式细胞检测技术评价上皮细胞对IL-5Rα的基因和蛋白质的表达,同时评价在IL-5和TNF-α的协同刺激下IL-5Rα、细胞内黏附分子1(ICAM-1)和血管细胞黏附分子1(VCAM-1)的协同表达。结果正常支气管上皮细胞在Th2型细胞因子、LTC4和TNF-α的刺激下可以表达IL-5Rα基因,在TNF-α单独或与其他因子的协同刺激下表达显著增强,更可以表达IL-5Rα蛋白。流式细胞检测分析显示TNF-α单独或协同刺激后16hIL-5Rα蛋白表达最高,并随时间的延长表达下降。当TNF-α与IL-5协同刺激后上皮细胞可以同时表达VCAM-1和IL-5Rα,而ICAM-1呈持续表达。结论本研究通过对支气管上皮细胞的研究证实了支气管上皮细胞可以在炎症因子TNF-α和Th2型细胞因子的协同刺激下进一步表达IL-5特异性受体IL-5Rα,并在IL-5和TNF-α的刺激下进一步表达黏附分子VCAM-1,吸引EOS及其前体细胞浸润至气道参与炎症。说明支气管上皮细胞不仅在支气管哮喘炎症反应中是最主要的受累器官,也是导致炎症的参与者之一。     

Characterization of vasoactive intestinal peptide receptors in human colonic epithelial cells

Receptors, i.e. specific binding sites for vasoactive intestinal peptide (VIP), have been characterized in human colonic epithelial cells isolated by EDTA treatment using 125I-labeled porcine VIP. The binding was time and temperature dependent. Conditions of apparent equilibrium were obtained at 15 C after 45 min of incubation in the presence of 2.1-7.4 micrograms cell DNA-ml; these conditions minimized the degradation of the peptide and the binding sites. Native VIP competitively inhibited the binding of [125I]VIP in the range of 3 x 10(-11)-10(-7) M, and half-maximal inhibition was observed at 2 x 10(-9) M VIP. Scatchard analysis of these data was consistent with the existence of two classes of binding sites: 7.8 x 10(-9) high affinity sites/microgram DNA with a dissociation constant (Kd) of 1.4 x 10(-9) M, and 12.0 x 10(10) low affinity sites/microgram DNA with a Kd of 46 x 10(-9) M. Among the natural hormones structurally related to VIP, gastric inhibitory polypeptide (GIP) and glucagon had no effect on the binding of labeled porcine VIP. Porcine secretin inhibited [125I]VIP binding, but at doses 1000 times higher than those of porcine VIP. Studies of the coupling between the binding of VIP and the stimulation of cAMP formation indicated a nonlinear relationship between the two processes, with full activation of the cAMP-producing system with occupancy of only a limited number of the binding sites. The presence of binding sites with high affinity for VIP coupled with the cAMP production in human colonic epithelial cells support the concept that this peptide may contribute to the physiological regulation of the functions of the human colonic epithelium in normal and pathological conditions.     

Immunolocalization of a new intestinal antiproliferative factor in human intestinal epithelial cells

A new intestinal antiproliferative factor (IAF) with an approximate molecular weight of 120 kDa has been purified from the human small intestine. This factor blocks the progression of human colon adenocarcinoma cells HT-29 from the G₁ to the S phase. IAF, specific of the lower part of the digestive tract, was detected rather late in mouse embryonic development. For determination of the specific intestinal cell producing IAF, long-term differentiated mucus-secreting HT-29 Cl 16E and enterocytic HT-29 Cl 19A cell lines were used. IAF is synthesized exclusively in the intestinal goblet cells; it is processed in the RER and Golgi complex before being excreted in secretory vesicles independently of mucin secretion. IAF can be considered a growth inhibitor of intestinal proliferation for the same reason as TGF-. However, two features differentiate it from TGF-: (1) the intestinal cell type synthesizing it, and (2) the delay in its expression in embryonic development. Particular interest was paid to IAF expression in pathological conditions using human colon biopsies. IAF was consistently recovered in biopsies from patients with inflammatory bowel diseases and benign tumors, but it was never detected in malignant tumors. IAF could represent a marker of colon cancer owing to its absence from malignant tumors.     

Expression of nonclassical class I molecules by intestinal epithelial cells

It is well recognized that the nature of the immune response is different in the intestinal tract than in peripheral lymphoid organs. The immunologic tone of the gut-associated lymphoid tissue is one of suppression rather than active immunity, distinguishing pathogens from normal flora. Failure to control mucosal immune responses may lead to inflammatory diseases such as Crohn's disease (CD) and ulcerative colitis (UC) and celiac disease. It has been suggested that this normally immunosuppressed state may relate to unique antigen-presenting cells and unique T-cell populations. The intestinal epithelial cell (IEC) has been proposed to act as a nonprofessional antigen-presenting cell (APC). Previous studies have suggested that antigens presented by IECs result in the activation a CD8(+) regulatory T-cell subset in a nonclassical MHC I molecule restricted manner. We therefore analyzed the expression of nonclassical MHC I molecules by normal IECs and compared this to those expressed by inflammatory bowel disease (IBD) IECs. Normal surface IEC from the colon and, to a much lesser extent, the small bowel express nonclassical MHC I molecules on their surface. In contrast, mRNA is expressed in all intestinal epithelial cells. Surface IEC express CD1d, MICA/B, and HLA-E protein. In contrast, crypt IECs express less or no nonclassical MHC I molecules but do express mRNA for these molecules. Furthermore, the regulation of expression of distinct nonclassical class I molecules is different depending on the molecule analyzed. Interestingly, IECs derived from patients with UC fail to express any nonclassical MHC I molecules (protein and HLA-E mRNA). IECs from CD patients express HLA-E and MICA/B comparable to that seen in normal controls but fail to express CD1d. Thus, in UC there may be a failure to activate any nonclassical MHC I molecule restricted regulatory T cells that may result in unopposed active inflammatory responses. In CD only the CD1d-regulated T cells would be affected.     

Induction of apoptosis before shedding of human intestinal epithelial cells

OBJECTIVES: Human intestinal epithelial cells (IECs) derive from stem cells at the crypt base and migrate along the so-called crypt-villus axis toward the intestinal lumen. As they reach the luminal surface in the colon or the villus tip in the small intestine, IECs are shed and their life cycle is terminated. The role of apoptosis during IEC migration along the crypt-villus axis has been subject to studies with conflicting results. In this study we use a novel approach to identify the initiation of apoptosis within normal human IECs. METHODS: Normal mucosa from the large and small human intestine was analyzed employing a novel antibody directed against activated caspase-3--an early marker of apoptosis. RESULTS: IECs initiate the apoptotic cascade as they approach the area of shedding before displaying evident morphological features of apoptosis. IECs of the small bowel also show caspase-3 activation in the small intestinal crypt base, whereas IECs of the colononic crypt base rarely show evidence of ongoing apoptosis. CONCLUSIONS: These findings indicate that apoptosis is initiated in human IECs as they reach the luminal surface/villus tip and before shedding. Furthermore, they show that different sections of the intestinal tract vary significantly in the rate of IEC apoptosis, possibly reflecting their difference in susceptibility to epithelial cell neoplasia.     

Salmonella interactions with polarized human intestinal Caco-2 epithelial cells

Polarized monolayers of the human intestinal epithelial Caco-2 cell line were grown on permeable filters and infected apically with either Salmonella choleraesuis or Salmonella typhimurium. Both Salmonella species penetrated through the monolayer, requiring 2 h before appearing in the basolateral medium. Both species caused a loss in transepithelial resistance by 3-4 h, and the monolayer's integrity was completely disrupted by 6 h. Scanning and transmission electron microscopy revealed that the bacteria interacted with well-defined apical microvilli and caused disruptions in the brush border, including elongation and denuding of the microvilli. The cytoplasm was also disrupted locally, with blebs protruding from the apical surface. The bacteria entered (invaded) these cells and were enclosed in membrane-bound vacuoles within the cytoplasm. By 6 h there were many bacteria within most Caco-2 cells, and these organisms caused serious cytopathic consequences. These morphologic observations correlated well with animal infection models, indicating that this in vitro system will be useful to study pathogens that interact with human intestinal epithelia.