北部湾海洋真菌Aspergillus fumigatus DL-p0m-g2的化学成分及药理活性研究

为获得具有生物活性的化合物,对一株分离于北部湾钉螺菌株Aspergillus fumigatus DL-p0m-g2的化学成分及药理活性进行研究。综合应用反相ODS(octadecylsilyl)柱色谱、半制备液相等多种色谱学方法进行分离纯化,根据化合物理化性质、质谱和核磁共振波谱学数据进行结构鉴定,并对分离得到的化合物进行细胞毒活性、抑菌活性和胆固醇转运蛋白NPC1L1(NPC1-like intracellular cholesterol transporter 1)蛋白结合等生物活性评价。实验共分离得到21个生物碱类化合物和1个甾体,分别鉴定为6-methoxyspirotryprostatin B(1)、spirotryprostatin A(2)、fumitremorgin C(3)、cyclotryprostatin A(4)、fumitremorgin B(5)、pseurotin A(6)、azaspirofuran A(7)、azaspirofuran B(8)、cephalimysin C(9)、cephalimysin B(10)、fumiquinazoline C(11)、fumiquinazoline B(12)、fumiquinazoline A(13)、fumiquinazoline D(14)、fumiquinazoline F(15)、tryprostatin B(16)、verruculogen(17)、chaetominine(18)、bisdethiobis(methylthio)glitoxin(19)、helvolic acid(20)、7-deacetylpyripyropene A(21)、terezine D(22)。其中化合物6对人肝癌细胞(Hep G2)、人肺癌细胞(A549)、人直肠癌细胞(HCT116)有一定的细胞毒活性;化合物1、3和20对金黄色葡萄球菌表现出抑菌活性;化合物14与NPC1L1蛋白具有较好的结合,显示其在降脂药物开发中的研究潜力。

CD3ε of a pan T cell marker involved in mouse Aspergillus fumigatus keratitis

AIM:To explore whether CD3ε is involved in the adaptive immunity of Aspergillus fumigatus(A.fumigatus)keratitis in mice and the role of innate and adaptive immunity in it.METHODS:Mice models of A.fumigatus keratitis were established by intra-stromal injection and corneal epithelial scratching.Subconjunctival injections of natamycin,wedelolactone,LOX-1 inhibitor(poly I)or Dectin-1 inhibitor(laminarin)were used to treat mice with A.fumigatus keratitis.Mice were pretreated by intraperitoneal injection of anti-mouse CD3ε.We observed the corneal infection of mice under the slit lamp microscope and made a clinical score.The protein expression of CD3ε and interleukin-10(IL-10)was determined by Western blotting.RESULTS:With the disease progresses,the degree of corneal opacity and edema augmented.In the intrastromal injection models,CD3εprotein expression began to increase significantly on the 2^(nd) day.However,in the scraping epithelial method models,CD3ε only began to increase on the 3^(rd) day.After natamycin treatment,the degree of corneal inflammation in mice was significantly attenuated on the 3^(rd) day.After wedelolactone treatment,the severity of keratitis worsened.And the amount of CD3ε protein was also reduced,compared with the control group.By inhibiting LOX-1 and Dectin-1,there was no significant difference in CD3ε production compared with the control group.After inhibiting CD3ε,corneal ulcer area and clinical score increased,and IL-10 expression was downregulated.CONCLUSION:As a pan T cell marker,CD3ε participate in the adaptive immunity of A.fumigatus keratitis in mice.In our mice models,the corneas will enter the adaptive immune stage faster.By regulating IL-10,CD3ε exerts antiinflammatory and repairs effects in the adaptive immune stage.

Identifying genetic susceptibility to Aspergillus fumigatus infection using collaborative cross mice and RNA-Seq approach

Background:Aspergillus fumigatus(Af)is one of the most ubiquitous fungi and its infection potency is suggested to be strongly controlled by the host genetic back-ground.The aim of this study was to search for candidate genes associated with host susceptibility to Aspergillus fumigatus(Af)using an RNAseq approach in CC lines and hepatic gene expression.Methods:We studied 31 male mice from 25 CC lines at 8 weeks old;the mice were infected with Af.Liver tissues were extracted from these mice 5 days post-infection,and next-generation RNA-sequencing(RNAseq)was performed.The GENE-E analysis platform was used to generate a clustered heat map matrix.Results:Significant variation in body weight changes between CC lines was ob-served.Hepatic gene expression revealed 12 top prioritized candidate genes differ-entially expressed in resistant versus susceptible mice based on body weight changes.Interestingly,three candidate genes are located within genomic intervals of the previ-ously mapped quantitative trait loci(QTL),including Gm16270 and Stox1 on chromo-some 10 and Gm11033 on chromosome 8.Conclusions:Our findings emphasize the CC mouse model's power in fine mapping the genetic components underlying susceptibility towards Af.As a next step,eQTL analysis will be performed for our RNA-Seq data.Suggested candidate genes from our study will be further assessed with a human cohort with aspergillosis.

海洋来源真菌烟曲霉(Aspergillus fumigatus)次级代谢产物研究(Ⅱ)

对一株海洋来源的微生物（Aspergillus fumigatus.）中的次级代谢产物进行了研究,采用硅胶柱色谱方法进行分离纯化,根据理化性质和光谱数据鉴定了化合物的结构,分别为环-（丙-缬）（1）,环-（色-脯）（2）,N-[2-（4-羟基苯基）乙基]乙酰胺（3）,对羟基苯乙酰胺（4）,4,8-二羟基-1-四氢萘醌（5）,3,4-二羟基苯甲酸甲酯（6）,麦角甾醇（7）和过氧化麦角甾醇（8）。化合物1~8均首次从该菌株中分离。

真菌Aspergillus fumigatus var .fumigatus的次生代谢产物(英文)

目的:研究西双版纳粗榧共生真菌Aspergillus fumigatusvar .fumigatus的次生代谢产物。方法:通过柱层析法分离发酵产物的化学成分,并利用波谱数据和文献数据对照鉴定化合物的结构。结果:分离到2个化合物,分别鉴定为trypacidin (1)及其衍生物1 ,2-seco-trypacidin (2)。结论:化合物2是新结构;并对文献中关于trypacidin的C-4和C-7a位碳谱数据的指定进行了更正。

海洋真菌Aspergillus fumigatus次生代谢产物研究

对来源于腔节藻属海藻Coelarthrumboergesenii（采自中国南海西沙群岛）的一株海洋内生真菌Aspergillusfumigatus的发酵液进行化学成分的分离及鉴定。采用硅胶柱色谱、凝胶柱色谱和高压液相色谱等分离手段，从乙酸乙酯萃取部分中分离得到16个次级代谢产物（1-16），经核磁共振、质谱等现代波谱技术鉴定了它们的结构，分别为gliotoxin （ 1 ）. bis （dethio） bis （methylsulfanyl） gliotoxin （ 2 ）, fumiquinazoline C （ 3 ）. （-） -chaetomium （ 4 ）.verruculogen（5） .fumitremorgin C（6） .brevianamide F（7） .fumigaclavine C（8） .decanedioic acid,2-methylene（9） .decanedioic acid, 2-methylene-, 1-methyl ester （ 10 ）. 2-undecenedioic acid （ 11 ）. 2-undecenedioic acid, 1-methyl ester （ 12 ）. helvolic acid （ 13 ）. di-2-ethyl hexyl phthalate （ 14 ）. physcion （ 15 ）. emodin （16）,其中化合物9、10和12为新天然产物。

冬虫夏草定殖菌Aspergillus fumigatus中一个新的聚酮类化合物

从冬虫夏草中分离得到一株定殖菌烟曲霉,对其发酵液的乙酸乙酯萃取部位进行化学成分研究,借助正反相硅胶柱色谱及HPLC等方法进行分离纯化,分离得到一个聚酮类新化合物1,利用多种波谱手段(HRESI-MS,1D NMR,2D NMR)确定其结构为为6-methoxy-3-methyl-1H-isochromen-8-ol。化合物1对H1299(人肺癌细胞)、H460(大细胞肺癌细胞)、HGC-27(人胃癌细胞)、A549(人非小细胞肺癌细胞)和mnk-43(人胃癌高分化细胞)五种细胞株均有弱的细胞毒活性。

水母共附生真菌Aspergillus fumigatus SCSIO41214中生物碱类化合物研究

从水母共附生真菌Aspergillus fumigatus SCSIO41214中分离得到8个已知生物碱类化合物。这些化合物主要采用包括正相硅胶、反相硅胶等常规柱层析技术,以及高效液相色谱进行分离纯化。首次通过核磁共振波谱等多种现代波谱技术确定了化合物1的平面结构,通过单晶衍射技术确定了其相对构型,化合物2-8是通过波谱分析并结合文献数据比对确定了化合物的结构。体外抗炎活性研究筛选发现,化合物4在10μmol·L^(-1)浓度下显示弱的抑制RAW264.7细胞中LPS引起的NO的释放。

越前水母共附生真菌Aspergillus fumigatus sp.次级代谢产物研究

采用硅胶柱层析、Sephadex LH-20凝胶柱层析、中压反相柱层析以及半制备高效液相色谱等方法对韩国南部海岸来源的越前水母共附生真菌A.fumigatus的大米发酵产物进行分离纯化,并利用核磁共振、质谱等波谱学方法结合其理化性质及文献数据鉴定了7个单体化合物的结构:sinulariapeptide C(1)、simplicilliumtide A(2)、simplicilliumtide B(3)、sinulariapeptide D(4)、4-羟基-17 R-甲基降甾醇(5)、3-羟基苯乙酸甲酯(6)、邻苯二甲酸二丁酯(7)。化合物1~7均为首次从越前水母来源的菌属中分离得到。

海洋真菌Aspergillus fumigatus YK-7次级代谢产物的研究

对一株海洋真菌Aspergillus fumigatus YK-7的次级代谢产物进行了分离纯化、结构鉴定和抗肿瘤活性测定。利用硅胶柱色谱、ODS柱色谱、凝胶柱色谱、高效液相色谱等方法从该菌株发酵液的乙酸乙酯萃取物和菌丝体的丙酮提取物中分离得到10个化合物。通过核磁与质谱数据分析结合理化常数鉴定化合物的结构,分别为:5α,8α-过氧-(22E,22R)-麦角甾-6,22-二烯-3β-醇(Ⅰ)、(22E,24R)-麦角甾-8,22-二烯-3β,5α,6β,7α-四醇(Ⅱ)、环(脯-色)二肽(Ⅲ)、环(苯丙-丝)二肽(Ⅳ)、4,8-二羟基-α-四氢萘酮(Ⅴ)、苔黑酚(Ⅵ)、苔黑酚羧酸(Ⅶ)、原儿茶酸(Ⅷ)、2-acetylamino-3,5,14-trihydroxyicos-6-en-4-olide(Ⅸ)、1-单亚油酸甘油酯(Ⅹ)。其中,化合物Ⅸ为新天然产物,化合物Ⅱ、Ⅶ、Ⅷ、Ⅹ从曲霉属真菌中首次分离得到,化合物Ⅳ、Ⅵ从该种真菌中首次分离得到。化合物Ⅳ、Ⅴ、Ⅸ对选定的人肿瘤细胞具有一定的体外抗增殖活性。

烟曲霉Aspergillus Fumigatus 3-甾酮-Δ~1-脱氢酶基因的克隆、异源表达和活性鉴定

微生物转化在甾体药物合成中起着不可替代的作用,其中C1,2位脱氢是最为重要的反应类型。以烟曲霉(A.fumigatus)为出发菌株,运用同源序列比对从烟曲霉酸基因簇中钓取出kstDF序列。该基因与玫瑰色热微菌(Ther-momicrobium roseum)DSM 5159的kstD一致性为51%。构建pET28a(+)-kstDF表达载体,转化大肠杆菌(Escherichiacoli)BL21(λDE3),获得高表达重组子菌株。经IPTG诱导、SDS-PAGE电泳分析,发现高温(37℃)有利于KstDF蛋白表达,低温(20℃)有利于酶活的保持。该重组菌显示出良好的甾体转化能力,在12 h内完全转化13.3 mg.L-1雄甾-4-烯-3,17-二酮,为构建高效转化3-酮基-4-烯类固醇的基因工程菌奠定了基础。

土壤真菌 Aspergillus fumigatus固体发酵产物的化学成分及抗氧化活性研究

为研究来源于哀牢山国家级自然保护区河谷的土壤真菌Aspergillus fumigatus固体发酵产物的化学成分及抗氧化活性研究,采用正相硅胶柱层析、反相硅胶柱层析和Sephadex LH-20分离纯化,借助核磁共振波谱等方法鉴定化合物结构,从中共得到13个化合物,分别鉴定为rubrofusarin B(1)、rubrofusarin A(2)、carbonarone A(3)、aspernigrin A(4)、flavasperone(5)、(22 E,24 R)-5α,8α-过氧麦角甾-6,22-二烯-3β-醇(6)、(22 E,24 R)-5α,8α-过氧麦角甾-6,9(11),22-三烯-3β-醇(7)、ourosperone A(8)、(22 E,24 R)-麦角甾-5,7,22-三烯-3β-醇(9)、stigmast-1,5-dien-3β-ol(10)、fonsecinones A(11)、asperpyrone C(12)、asperpyrones B(13)。其中,化合物1~5和7~13为从该菌种中首次分离。抗氧化活性结果显示,化合物8对DPPH(IC_(50)=3.453 mg/mL)、ABTS+(IC_(50)=0.155 mg/mL)、OH(IC_(50)=0.019 mg/mL)自由基都有一定的清除效果。

Inhibition of LOX-1 alleviates the proinflammatory effects of high-mobility group box 1 in Aspergillus fumigatus keratitis

AIM: To investigate the inflammatory amplification effect of high-mobility group box 1(HMGB1) in Aspergillus fumigatus(A. fumigatus) keratitis and the relationship between lectin-like oxidized low-density lipoprotein receptor 1(LOX-1) and HMGB1 in keratitis immune responses.METHODS: Phosphate buffer saline(PBS), and Boxb were injected into BALB/c mice subconjunctivally before the corneas were infected with A. fumigatus. RAW264.7 macrophages and neutrophils were pretreated with PBS and Boxb to determine the HMGB1 inflammatory amplification effects. Abdominal cavity extracted macrophages were pretreated with Boxb and Poly(I)(a LOX-1 inhibitor) before A. fumigatus hyphae stimulation to prove the the relationship between the two molecules. LOX-1, interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), macrophage inflammatory protein-2(MIP-2) and IL-10 were assessed by polymerase chain reaction and Western blot.RESULTS: Pretreatment with Boxb exacerbated corneal inflammation. In macrophages and neutrophils, A. fumigatus induced LOX-1, IL-1β, TNF-α and MIP-2 expression in Boxb group was higher than those in PBS group. Poly(I) treatments before infection alleviated the proinflammatory effects of Boxb in abdominal cavity extracted macrophages. Pretreatment with Boxb did not influence Dectin-1 mRNA levels in macrophages and neutrophils.CONCLUSION: In fungal keratitis, HMGB1 is a proinflammatory factor in the first line of immune response. HMGB1 mainly stimulates neutrophils and macrophages to produce inflammatory cytokines and chemokines during the immune response. LOX-1 participates in HMGB1 induced inflammatory exacerbation in A. fumigatus keratitis.

The roles of surfactant protein D during Aspergillus fumigatus infection in human corneal epithelial cells

AIM: To investigate roles of surfactant protein D (SP-D) and relative cytokines in human corneal epithelial (HCE) cells Exposed to aspergillus fumigatus (AF) antigens. METHODS: HCE cells cultured 47 in vitro with AF antigens and sampled at 0, 0.5, 1 hour, 2, 4, 6 and 8 hours. The Expression of SP-D mRNA was evaluated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR).The expression of SP-D protein was shown by ELISA and immunocytochemistry SP methods. The expression of NF-kappa B and relative downstream cytokines such as TNF-alpha, IL-1 beta, IL-8 and IL-10 in supernatant fluid were measured by ELISA. RESULTS: SP-D mRNA and protein were detected in untreated HCE cells. The expression of SP-D and the relative downstream cytokines rose after being stimulated with AF antigens. SP-D mRNA began to rise at 0.5 hour and the most significantly peak was in 2 hours. The protein of SP-D in supernatant fluid had the same trend with mRNA. Immunocytochemistry of SP-D showed positive expression and gradually increased to 6 hours, and then the expression began to decline. NF-kappa B was activated after treated by AF antigens and the changes had correlation with SP-D. TNF-alpha, IL-1 beta, IL-8 and IL-10 began to rise after given AF antigens 1 hour and were 1.82, 1.43, 1.12 and 1.28 times higher than the untreated HCE cells separately. The expression of TNF-alpha and IL-1 beta reached the peak at 2 hours, separately 2.80 and 2.86 times than the untreated. The expression of IL-8 and IL-10 gradually increased with a time-dependent manner. ' CONCLUSION: HCE cells exists SP-D and it may play a significant role in pathogenesis of keratomycosis. AF may induce human corneal epithelial cells to express inflammatory cytokines via SP-D and NF-kappa B pathway. SP-D possibly mediates the recognition to AF mycelium.

Comparison of four supports for adsorption of reactive dyes by immobilized Aspergillus fumigatus beads

Four materials, sodium carboxymethylcellulose （Na-CMC）, sodium alginate （SA）, polyvinyl alcohol （PVA）, and chitosan （CTS）, were prepared as supports for entrapping fungus Aspergillusfumigatus. The adsorption of synthetic dyes, Reactive Brilliant Blue KN- R, and Reactive Brilliant Red K-2BP, by these immobilized gel beads and plain gel beads was evaluated. The adsorption efficiencies of Reactive Brilliant Red K-2BP and Reactive Brilliant Blue KN-R by CTS immobilized beads were 89.1% and 93.5% in 12 h, respectively. The adsorption efficiency by Na-CMC immobilized beads was slightly lower than that of mycelial pellets. But the dye culture mediums were almost completely decolorized in 48 h using the above-mentioned two immobilized beads （exceeding 95%）. The adsorption efficiency by SA immobilized beads exceeded 92% in 48 h. PVA-SA immobilized beads showed the lowest adsorption efficiency, which was 79.8% for Reactive Brilliant Red K-2BP and 92.5% for Reactive Brilliant Blue KN-R in 48 h. Comparing the adsorption efficiency by plain gel beads, Na-CMC plain gel beads ranked next to CTS ones. SA and PVA-SA plain gel beads hardly had the ability of adsorbing dyes. Subsequently, the growth of mycelia in Na-CMC and SA immobilized beads were evaluated. The biomass increased continuously in 72 h. The adsorption capacity of Reactive Brilliant Red K-2BP and Reactive Brilliant Blue KN-R by Na-CMC immobilized beads was 78.0 and 86.7 mg/g, respectively. The SEM micrographs show that the surface structure of Na-CMC immobilized bead is loose and finely porous, which facilitates diffusion of the dyes.

Role of the IL-33/ST2/p38 signaling pathway in the immune response of corneal epithelial cells to Aspergillus fumigatus infection

AIM: To investigate the expression of interleukin(IL)-33 in the cornea and human corneal epithelial cells(HCECs) exposed to Aspergillus fumigatus(A. fumigatus), and to determine the function of IL-33/ST2/p38 signaling pathway in the immune response of corneal epithelial cells to A. fumigatus infection.METHODS: The mRNA and protein expression of IL-33 in HCECs and mice corneas were examined by quantitative real-time reverse transcription polymerase chain reaction(q RT-PCR) and Western blot analysis, respectively. IL-33 expression was also detected in cornea samples from healthy donors and patients with fungal keratitis with immunohistochemistry. The cultured HCECs were treated with inactive A. fumigatus hyphae at various concentrations with or without recombinant human IL-33 protein, soluble recombinant ST2 protein, specific ST2 neutralizing antibody, or the mitogen-activated protein kinase(MAPK) p38 inhibitor SB203580 for evaluation of the expression and activation of IL-33/ST2/p38 signaling in the regulation of proinflammatory cytokines. The production levels of IL-6 and IL-1β were determined by qR T-PCR and enzymelinked immunosorbent assay(ELISA). The proliferation of HCECs was determined by a Cell Counting Kit-8(CCK8) assay and cell count.RESULTS: IL-33 expression levels increased in the corneal tissues of patients with fungal keratitis and in mice corneas of experimental A. fumigatus infection,as well as in HCECs with infection of A. fumigatus. A. fumigatus strongly stimulated HCECs-generated proinflammatory cytokine(IL-6 and IL-1β) production at both the mRNA and protein levels. This production of proinflammatory mediators stimulated by A. fumigatus was further stimulated by IL-33 and was prevented by soluble ST2 protein or ST2 neutralizing antibody. Moreover, IL-33 naturally promoted the p38 phosphorylation induced by A. fumigatus, which was suppressed by soluble ST2 protein. The MAPK p38 inhibitor SB203580 also inhibited the A. fumigatus-induced proinflammatory cytokine production. IL-33 administration for 48 h and 72 h promoted the proliferation of HCECs, which was attenuated by treatment with soluble recombinant human ST2 protein.CONCLUSION: A. fumigatus elevates IL-33 expression in human and mice corneas and HCECs. Thus, IL-33/ST2/p38 signaling may play an important role in amplifying the immune response of corneal epithelial cells to A. fumigatus infection. Besides, IL-33 promotes the cell proliferation of HCECs via its receptor ST2. These findings suggest a novel autocrine mechanism of amplification of the fungalinduced inflammatory response in the corneal epithelium, highlighting a potential therapeutic target for fungal keratitis.

海南东寨港红树林内生真菌Aspergillus fumigatus SAS10吡喃酮类代谢产物

采用大米培养基,对无瓣海桑内生真菌Aspergillus fumigatus SAS10进行静置发酵,运用硅胶柱色谱、Sephadex LH-20凝胶柱色谱、以及半制备高效液相等分离手段对发酵产物进行分离纯化,获得7个化合物。经分析质谱、核磁等波谱数据,并通过与文献对比鉴定化合物为:aspergyllone(1),(R)-2-(羟基(苯基)甲基)-4H-吡喃-4-酮(2),carbonarone A(3),campyrone A(4),campyrone C(5),asperfurandione A(6)和dibutylterephthalate(7)。所得7个化合物均为首次报道为烟曲霉次级代谢产物。

Dectin-1 expression at early period of Aspergillus fumigatus infection in rat's corneal epithelium

AIM:To investigate the expression of dendritic cell-associated C-type lectin-1 (dectin-1) at the early period of Aspergillus fumigatus infection in rat’s corneal epithelium. ·METHODS:A total of 72 Wistar rats were randomly divided into three groups:A, B and C. The right eyes were chosen as experimental eyes. Group A was control group. Rats in group B were not inoculated with Aspergillus fumigatus. Group C was taken as Aspergillus fumigatus keratitis model. Rats in group B and C (six from each group) were executed randomly at 4, 8, 16 and 24 hours after experimental model being established to assess the expression of dectin-1 mRNA through real-time PCR. Another six rats in group B and C were executed randomly at 24 hours to assess the expression of dectin-1 protein through immunohistochemistry. ·RESULTS:The results of real-time PCR indicated that dectin-1 mRNA expression was low in corneal epithelium of normal rats’. There was no significantly difference of dectin-1 mRNA expression in group A and B (P 】0.05). The expression of Aspergillus fumigatus infected corneal epithelium increased gradually after 8 hours in group C. The synchronous expression of group A and C had significant difference (P 【0.01). Immunohistochemisty discovered that dectin-1 receptor existed in normal rat’s corneal epithelium . Dectin-1 protein increased after 24 hours in group C. There was a significant difference of synchronous expression in group B and C(P【0.01). · CONCLUSION:Dectin-1 exists in rat’s cornealepithelium and its expression significantly increases at the early period of Aspergillus fumigatus infection. Dectin-1 is a pattern recognition receptor that expresses in corneal epithelium and involves in immune response to Aspergillus fungal keratitis.

Expression and role of aryl hydrocarbon receptor in Aspergillus fumigatus keratitis

●AIM:To observe the expression and role of aryl hydrocarbon receptor(Ah R)in the immune response of mouse cornea infected with Aspergillus fumigatus(A.fumigatus).●METHODS:Murine models of A.fumigatus keratitis were established by scraping the central epithelium of mouse cornea,daubing A.fumigatus on the cornea and covering with a contact lens.The mice were randomly divided into the control group and the A.fumigatus-infected(A.F.)group for 1,3 and 5 d respectively,which corneas were daily monitored by a slit lamp microscope and the clinical scores were also recorded timely after infection.In this study,immunofluorescence staining was used to detect the expression and localization of Ah R in mouse corneas,and the m RNA and protein of Ah R were detected by reverse transcription-polymerase chain reaction(RT-PCR)and Western blot.In addition,mouse peritoneal macrophages were stimulated by A.fumigatus with or without the pretreatment of Ah R antagonist CH223191 and Ah R agonist FICZ,and the tumor necrosis factor alpha(TNF-α),inducible nitric oxide synthase(i NOS),interleukin-10(IL-10)and Arg-1 m RNA were detected by RT-PCR.●RESULTS:According to the results of the slit light photography,it was clearly indicated that the corneal inflammation were the most severe and the clinical score became the highest as well on the 3 rd day after the infection of A.fumigatus.Contrasted with the control group,the expression of Ah R in the corneal epithelial cells infected with A.fumigatus was significantly increased detected by immunofluorescence staining.Ah R mainly expressed in the nucleus and cytoplasm of corneal epithelial cells.Consistent with the transcriptional level of Ah R m RNA,the expression level of Ah R protein reached the peak on the 3 rd day after infection which was detected by Western blot.Furthermore,RT-PCR showed that CH223191 up-regulated the expression of TNF-αand i NOS and down-regulated the expression of IL-10 and Arg-1 in peritoneal macrophages;inversely,FICZ reduced the expression of TNF-αand i NOS while elevated the expression of IL-10 and Arg-1.●CONCLUSION:Ah R is involved in the pathogenesis of A.fumigatus keratitis and induced immune protection in anti-A.fumigatus immune response by inhibiting M1 and increasing M2 phenotype macrophage-related inflammatory factors.

烟曲霉菌(Aspergillus fumigatus)的耐热性研究

为明确烟曲霉菌引起烟草霉烂病的限制因子,采用菌株Aspergillus fumigatus Q-1为研究对象,于PCR仪上测试了其菌丝体、分生孢子在不同温度下的存活能力。结果表明,该菌株的菌丝体在PDA培养基及水中于95℃处理10 min仍能存活;分生孢子在无水时于90℃处理10 min能存活,但其在水相中处理5 min时,在68℃处理温度中,孢子存活率仅为6.25×10^(-6)%;在65℃处理温度中,孢子存活率为8.75×10^(-4)%;在60℃处理温度中,仍有2.69%孢子存活。在水相中处理10 min时,在65℃处理温度中,孢子存活率为1.25×10^(-4)%;在60℃处理温度中,有0.29%孢子存活;在50℃处理温度中,有63.67%孢子存活。在50~70℃温度处理时接种孢子液的烟草叶柄10 min后,移至37℃下培养的烟草叶柄仍能出现典型的霉烂病。