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目的克隆人C型凝集素受体dectin-2和dectin-3胞外区基因并进行原核表达及制备它们的单克隆抗体。方法利用PCR扩增编码人dectin-2和dectin-3胞外区的基因序列,分别克隆到原核表达载体pET28a(+)中,IPTG诱导基因的原核表达;纯化后免疫Balb/c小鼠,融合并筛选获得克隆化的杂交瘤细胞株,制备Dectin-2和Dectin-3的单克隆抗体;利用ELISA、FACS和Western印迹法检测抗体的效价和特异性;检测Dectin-2和Dectin-3的单克隆抗体是否能够特异性阻断相应受体的功能。结果成功构建原核表达质粒pET28a(+)-Dectin-2和pET28a(+)-Dectin-3,并在E.coli BL21(DE3)中诱导Dectin-2和Dectin-3胞外区的高效表达。纯化目的蛋白后免疫Balb/c小鼠,融合获得8株Dectin-2和12株Dectin-3的阳性杂交瘤细胞。进一步克隆化筛选获得7株Dectin-2和8株Dectin-3的单克隆细胞株,并且其分泌的抗体能够特异结合Dectin-2和Dectin-3胞外区的原核表达蛋白。利用单克隆细胞株制备小鼠腹水,纯化获得Dectin-2和Dectin-3的单克隆抗体。单克隆细胞株D2-7和D3-2产生的抗体能够特异结合RAW中表达的人Dectin-2和Dectin-3蛋白及小鼠骨髓来源巨噬细胞中Dectin-2和Dectin-3蛋白。抗体功能实验显示:单克隆细胞株D2-7和D2-10产生的抗体能够阻断Dectin-2受体介导白念珠菌菌丝态刺激引起的NF-κB激活,而单克隆细胞株D3-1和D3-2产生的抗体能够阻断Dectin-3受体介导白念珠菌菌丝态刺激引起的NF-κB激活,并且具有剂量依赖性及高度特异性。结论成功获得高纯度的Dectin-2和Dectin-3胞外区蛋白,并分别获得了特异、高效价的单克隆抗体。 相似文献
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固有免疫在隐球菌感染中发挥着至关重要的作用,宿主建立对隐球菌有效免疫应答的第一步是通过固有免疫应答细胞表达的模式识别受体(PRR)识别隐球菌病原体相关分子模式(PAMP),进一步触发涉及许多蛋白质(脾酪氨酸激酶和胱天蛋白酶募集域蛋白9等)的细胞内信号级联反应。本篇综述中笔者总结了固有免疫应答细胞模式识别受体Dectin-1与隐球菌病原体相关分子模式的相互识别机制,并讨论了Dectin-1信号通路在宿主抗隐球菌感染免疫中的作用及其在隐球菌病药物和疫苗佐剂研发中的潜在应用。 相似文献
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目的:建立免疫抑制小鼠侵袭性烟曲霉菌感染模型,为感染性疾病的研究和治疗提供科学依据。方法:给予昆明种健康小鼠腹腔内一次性注射环磷酰胺(CY)200mg/kg,观察其对小鼠一般状况和白细胞数的影响。4d后,经小鼠双侧鼻孔滴注烟曲霉菌孢子悬液30μl引起侵袭性肺烟曲霉菌病(IPA),并于不同时间点处死小鼠,进行组织培养及病理分析。结果:环磷酰胺注射后,小鼠白细胞数量明显降低,病理切片可见免疫抑制小鼠感染烟曲霉菌后肺组织大量烟曲霉菌聚集,组织坏死,形成肺脓肿。结论:成功建立了免疫抑制小鼠侵袭性肺烟曲霉菌病动物模型,为研究人类烟曲霉菌病的致病机制、诊断和防治奠定了实验基础。 相似文献
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目的:探讨烟曲霉菌感染大鼠角膜后Dectin-1和Toll样受体-4(TLR4)表达及炎症因子的分泌特点,阐明其在大鼠角膜炎发病中的作用。方法:40只SD大鼠随机分为对照组(n=10)和烟曲霉菌性角膜炎组(n=30),对照组不予任何干预,烟曲霉菌性角膜炎组给予双眼烟曲霉菌感染造模,分别于感染12 h(n=10,12h组)、24 h(n=10,24 h组)和48 h(n=10,48 h组)后取角膜组织,ELISA法检测IL-1、TNF-α、IL-6、IL-10和NF-κB水平,免疫组织化学法、RT-PCR法及Western blotting法测定角膜组织中Dectin-1和TLR4mRNA及蛋白表达。结果:大鼠造模均成功,眼角膜裂隙灯检测观察可见对照组眼睛清澈,感染烟曲霉菌后,12 h组眼睛表面有明显的一层薄膜,薄膜与健康的角膜边界清晰;24 h组可见眼部有一层白色的浸润灶形成,且表面粗糙;48 h组角膜白色浸润灶进一步加深,且范围明显扩大,基本覆盖了整个眼球。烟曲霉菌感染后,12 h组、24 h组和48 h组角膜组织中炎症因子IL-1、IL-6、IL-10、TNF-α和NF-κB水平明显高于空白对照组(P<0.05),其中24 h组和48 h组明显高于12 h组(P<0.05),48 h组明显高于24 h组(P<0.05);免疫组织化学检测,大鼠角膜组织中Dectin-1和TLR4表达水平随着感染时间的增加逐渐增加,RT-PCR法和Western blotting法检测,大鼠眼角膜组织烟曲霉菌性角膜炎组(12 h组、24 h组和48 h组)Dectin-1和TLR4蛋白及mRNA表达水平明显高于对照组(P<0.05),其中24 h组和48 h组明显高于12 h组(P<0.05),48 h组明显高于24 h组(P<0.05)。结论:Dectin-1和TLR4在受到烟曲霉菌感染后可过量分泌,进而造成大鼠炎性相关因子表达,最终促使角膜炎的发生。 相似文献
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Among the various drug induced dermatological entities toxic epidermalnecrolysis (TEN) and Stevens-Johnson syndrome (SJS) occupy a primary place in terms of mortality. Toxic epidermal necrolysis also known as Lyell’s syndrome was first described by Lyell in 1956. Drugs are by far the most common cause of toxic epidermal necrolysis, in which large sheets of skin are lost from the body surface making redundant the barrier function of the skin, with its resultant complications. Drug-induced toxic epidermal necrolysis are severe adverse cutaneous drug reactions to various precipitating agents that predominantly involve the skin and mucous membranes. Toxic epidermal necrolysis is rare but considered medical emergencies as they are potentially fatal. Drugs are the most common cause accounting for about 65%-80% of the cases. The most common offending agents are sulfonamides, NSAIDs, butazones and hydrantoins. An immune mechanism is implicated in the pathogenesis, but its nature is still unclear. There is a prodormal phase in which there is burning sensation all over the skin and conjunctivae, along with skin tenderness, fever, malaise and arthralgias. Early sites of cutaneous involvement are the presternal region of the trunk and the face, but also the palms and soles, rapidly spread to their maximum extent, the oral mucosa and conjunctiva being affected. Initial lesions are macular, followed by desquamateion, or may be from atypical targets with purpuriccenters that coalesce, from bullae, then slough. The earlier a causative agent is withdrawn the better is the prognosis. Several treatment modalities given in addition to supportive care are reported in the literature, such as systemicsteroids, high-dose intravenous immunoglobulins, ciclosporin, TNF antagonists. Recovery is slow over a period of 14-28 days and relapses are frequent. Mortality is 25%-50% and half the deaths occur due to secondary infection. Here we report a 50-year-old female of drug-induced toxic epidermal necrolysis. She was admitted to the dermatology ward with extensive peeling of skin over the trunk and limbs. She had taken alamotrigine for epilepsy. A week after taking the tablets, the patient developed a severe burning sensation all over the body and followed by a polymorphic erythematous dermatitis and widespread peeling of skin. We treated this patient with high dose corticosteroids, high-dose intravenous immunoglobulins and etanercept, but eventually she died of secondary aspergillus fumigatus infection. 相似文献
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核苷酸结合寡聚化结构域2在大鼠烟曲霉菌性角膜炎角膜局部的表达 总被引:1,自引:0,他引:1
目的 探讨大鼠角膜真菌感染后角膜局部核苷酸结合寡聚化结构域2(NOD2)的表达.方法 在大鼠左眼角膜上制作烟曲霉菌性角膜炎的模型(实验组),右眼作为对照组.在感染后第2天(早期)、第6天(中期)及第14天(晚期)取下角膜组织,采用逆转录聚合酶链反应(RT-PCR)检测NOD2在不同病程角膜组织中的表达.结果 在实验组角膜中,NOD2早期开始表达,中期达高峰,以后逐渐下降.实验组与对照组早、中期NOD2 mRNA表达的差异均有统计学意义(P值均<0.05),晚期的差异无统计学意义(P>0.05);且实验组早期与中期、中期与晚期表达的差异均有统计学意义(P值均<0.01).结论 NOD2参与角膜烟曲霉菌性感染后角膜局部的免疫反应. 相似文献
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目的:探讨大蒜素对烟曲霉菌感染的免疫抑制小鼠细胞免疫功能的影响。方法:将30只昆明种(KM)小鼠随机分为对照组、预防组、治疗组,每组10只。实验前均给予腹腔内注射环磷酰胺(CY)200 mg/kg,制备免疫功能抑制模型鼠。实验时,预防组灌服大蒜素溶液14 d,治疗组灌服大蒜素溶液7 d(第8~14天),对照组灌服等量生理盐水14 d,并于实验第8天时给各组小鼠麻醉后鼻腔滴入烟曲霉菌孢子悬液。采用MTT法检测各小鼠T细胞活性;采用ELISA法检测各小鼠脾细胞培养上清液中IL-12、IFN-γ的表达水平。结果:预防组T淋巴细胞增殖活性、IFN-γ、IL-12水平与对照组比较,差异有显著性(P<0.01);治疗组与对照组比较,两者间有显著性差异(P<0.01);预防组和治疗组比较,两组间也有差异(P<0.05)。结论:治疗组和预防组脾脏T淋巴细胞增殖活性、IL-12和IFN-γ的水平均有提高,提示大蒜素可提高小鼠细胞免疫功能,增强机体抵抗烟曲霉菌感染能力。 相似文献
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目的构建β2受体(β2R)下调哮喘动物模型,以研究β2R下调及其激素保护作用的机制。方法32只BALB/c小鼠随机分成对照组、哮喘组、β2R下调组和地塞米松组,每组8只。哮喘组、β2R下调组和地塞米松组分别于实验的第0、14和21d腹腔注射卵白蛋白(OVA)和氢氧化铝的混悬液200μL,第28d开始连续1周(30min/d)雾化吸入1%OVA(W/V)溶液;β2R下调组和地塞米松组在最后1周每天雾化吸入OVA之前腹腔注射60μg沙丁胺醇及雾化吸入0.01%沙丁胺醇30min;地塞米松组同时予以腹腔注射地塞米松5mg·kg-1.d-1,连续7d。对照组腹腔注射等量的磷酸盐缓冲液(PBS),以PBS雾化吸入。用体积描记箱测定各组小鼠气道阻力。测定BALF中白细胞总数和分类计数。ELISA法检测BALF中IL-4、IFN-γ以及血清总IgE浓度。提取小鼠肺组织总蛋白,免疫印迹实验测定β2R总量,放射配基结合实验测定β2膜受体数量。结果哮喘组、β2R下调组与对照组、地塞米松组比较,高剂量乙酰胆碱激发下的气道阻力明显增高(P0.01),BALF中嗜酸粒细胞、中性粒细胞和淋巴细胞明显增多(P0.01),BALF中IL-4和血清总IgE水平显著增高(P0.01),BALF中IFN-γ水平显著降低(P0.01)。β2R下调组肺组织β2R总量和β2膜受体数量均显著低于其余三组(P0.01),对照组、哮喘组、地塞米松组之间无显著差异。结论在传统的OVA致敏激发哮喘动物模型基础上,采用腹腔注射结合雾化吸入沙丁胺醇的方法成功构建了β2R下调的哮喘动物模型。地塞米松对β2R下调具有保护作用。 相似文献
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Cutaneous and eyes Aspergillus fumigatus infection 总被引:2,自引:0,他引:2
KANG Er-xun WU Jin-ying WANG Guo-ying WANG Fu-sheng GAO Dong XIA Xiu-juan YAO Xian-ping 《中华医学杂志(英文版)》2008,121(22):2366-2368
Aspergillus fumigatus infection is a clinically rare condition. Here we report a case of facial cutaneous Aspergillus fumigatus infection where the skin lesions gradually extended to the eyes forming corneal and conjunctival ulcers that developed to panophthalmia. Fungus cultures were repeatedly performed on specimen from the eyes and from skin lesions. Aspergillus fumigatus was the pathogen identified morphologically and molecular biologically. 相似文献
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巨噬细胞在烟曲霉感染中的作用 总被引:3,自引:3,他引:0
近年来,烟曲霉成为引起免疫力低下患者高病死率的一种条件性致病真菌,其已引起重视.巨噬细胞是机体固有免疫的主要成分,在抗烟曲霉感染中发挥着重要作用.文中就巨噬细胞在烟曲霉感染中的特点作一综述. 相似文献
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Background Aspergillus fumigatus (A. fumigatus) is a ubiquitous saprophytic fungus responsible for the majority of invasive mold infections in patients undergoing chemotherapy, organ transplantation or with persistent neutropenia. This study aimed to determine the role of E-cadherin for adhesion and endocytosis of A. fumigatus blastospores in the human epithelial cell line A549.
Methods A. fumigatus blastospores were incubated with the total protein of A549 to investigate the binding of E-cadherin and blastospores followed by an affinity purification procedure. After establishing the adhesion model, the adhesion and endocytosis of A. fumigatus blastospores by A549 cells were evaluated by down-regulating E-cadherin of A549 cells using blocking antibody or small interfering RNA (siRNA).
Results E-cadherin was adhered to the surface of A. fumigatus blastospore. Adhesion and endocytosis of the blastospores were reduced by blocking or down-regulating E-cadherin in A549 cells.
Conclusions E-cadherin is a receptor for adhesion and endocytosis of A. fumigatus blastospores in epithelial cells. This may open a new approach to treat this fungal infection.
相似文献
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M. Turner-Warwick 《Postgraduate medical journal》1979,55(647):642-644
The range of clinical presentations of lung diseases associated with Aspergillus spp. is great. The conditions are very frequently misdiagnosed but errors should be avoided if the possibility of a fungal cause is considered and simple immunological tests undertaken. Often no more than skin-prick tests and serum precipitins need to be done. In many cases, fungus is not isolated from the sputum and negative results do not exclude the possibility of A. fumigatus as the causal agent. Treatment often results in marked clinical improvement and there is evidence that suppression of recurrent episodes of bronchopulmonary aspergillosis prevents progressive lung damage. 相似文献
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烟曲霉是最常见的空气播散性真菌病原体,在免疫受损患者,烟曲霉常引起侵袭性曲霉病(invasive aspergillosis, IA).与大多数的原发性病原菌不同,烟曲霉没有在宿主相关组织生长的毒力特征,研究表明烟曲霉的毒力特性是在自然界中抵制不利因素而产生的.这些特性不仅增加了其在生态环境中的总体竞争力,而且促进了它在人类宿主中的存活和生长.尽管许多与这些毒力特性相关的基因不符合经典毒力因子的定义,但对IA的致病机制仍很重要,主要包括与耐热生长、细胞壁屏障、分泌毒性产物、对环境的感知及信号传导、获取营养等方面有关的因子. 相似文献
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目的:克隆烟曲霉角鲨烯环氧化酶基因并探讨其与特比萘芬抗烟曲霉活性的关系.方法:利用聚乙二醇-甘油法将0.5μg烟曲霉基因组DNA文库转化pyrG-烟曲霉AF293.1原生质体;在含有特比萘芬的最低培养基上筛选耐特比萘芬的pyrG 转化子,然后对介导特比萘芬耐药的烟曲霉基因进行序列分析并进行基因克隆;最后将克隆的基因重新转化到烟曲霉中以确认其与特比萘芬敏感性之间的关系.结果:从5×104个转化子中得到一个耐特比萘芬的pyrG 转化子,该转化子表现出特比萘芬特异性耐药性且与质粒高度相关;序列分析发现,是烟曲霉角鲨烯环氧化酶基因介导了特异性特比萘芬耐药;将该基因克隆后重新转化到烟曲霉中,结果敏感的烟曲霉再次获得特异性特比萘芬耐药性.结论:首次获得烟曲霉角鲨烯环氧化酶基因,并发现额外拷贝的该基因可以导致特异性特比萘芬耐药这一新型机制. 相似文献
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目的 揭示国内烟曲霉临床分离株对目前常用及新型抗真菌药物敏感性及不同地区烟曲霉对抗真菌药物的敏感性特点。方法 实验室保存的分离自2011—2015年中国不同地区的159株临床烟曲霉菌株为试验菌株。采用美国标准化实验室与研究所(CLSI)推荐的M38-A2微量液基稀释法为药敏方法,按规定的标准进行。结果 12种抗真菌药物伊曲康唑(ITC)、伏立康唑(VRC)、泊沙康唑(POS)、里氟康唑(RAV)、艾沙康唑(ISA)、氟康唑(FLC)、米卡芬净(MCF)、阿尼芬净(ANI)、卡泊芬净(CBF)、两性霉素B(AMB)、特比萘芬(TBF)和5-氟胞嘧啶(5-FC)对159株烟曲霉菌的MIC90分别为1、0.5、0.25、0.5、1、64、0.063、0.063、0.5、2、4、64mg/L。烟曲霉唑类耐药率分别为: ITC为4.4%,ISA和RAV均为3.77%,POS为3.14%,VRC为1.9%。除VRC、FLC和5-FC外,烟曲霉临床分离株对其他9种抗真菌药物的敏感性在不同地区间差异均有统计学意义(P<0.05)。结论 三唑类(除FLC外)和棘白菌素类体外抗烟曲霉菌活性较高,优于AMB和TBF;烟曲霉在体外对FLC、5-FC表现为高度耐药。目前我国烟曲霉临床株唑类耐药率相对较低。烟曲霉株体外抗真菌药物的敏感性存在地区差异。 相似文献
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Using rabbit polyclonal and mouse monoclonal antibodies against Aspergillus fumigatus, we analyzed their cross-reaction with Alternaria alteria and Penicillium sp. by enzyme immuno assay (EIA) and radioimmunoprecipitation (RIP). It was found that neither of these two categories of antibodies reacted with Alternaria alteria while both reacted with Penicillium sp.. When components from Penicillium sp. precipitated by rabbit antisera were analyzed on 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), a wide range of bands with molecular weights larger than 90 K and a single 70 K band were observed. No band was found after the rabbit antisera was adsorbed with Aspergillus fumigatus. Moreover, two monoclonal antibodies against Aspergillus fumigatus cross-reacted with high molecular weight (greater than 200K) components of Penicillium sp.. These results suggest that the antigen of Alternaria alteria is different from that of Aspergillus fumigatus, and that Penicillium sp. and Aspergillus fumigatus share some antigenic components. In another experiment, human allergic sera containing IgE antibodies to Aspergillus fumigatus were tested against Penicillium sp. by RIP. No cross-reaction was observed. This suggests that in spite of sharing common antigenic components between these two fungi, the allergenic components of Aspergillus fumigatus is different from those of Penicillium sp.. 相似文献