Tyrosol和Farnesol对白念珠菌生物被膜形成作用初探

目的 研究密度感应分子(quorum sensing molecule)tyrosol(对羟基苯乙醇)和farnesol(法呢醇)对白念珠菌生物被膜形成的调控作用.方法 在tyrosol和farnesol干预下构建白念珠菌临床株和标准株生物被膜,在倒置显微镜下观察细胞形态,应用RT-PCR技术检测密度感应分子对白念珠菌HTA1和EFG1基因表达的调控作用,并采用MTT法观察密度感应分子对细胞活性的影响.结果 tyrosol对白念珠菌生物被膜的菌丝发生和细胞活性无明显促进作用,也无法中和farnesol对菌丝发生和细胞活性的抑制作用.tyrosol使白念珠菌生物被膜内细胞HTA1的表达增强,对EFG1的表达并无明显影响;tyrosol不能改变famesol对HTA1和EFG1表达的抑制作用.结论 tyrosol能在一定程度恢复口腔白念珠菌生物被膜内细胞的活跃状态,但当tyrosol与famesol同时存在时,tyrosol的作用被后者的抑制效应所掩盖,细胞对farnesol更敏感.     

在人扁桃体细胞中诱生γ干扰素

本文初步研究了用人扁桃体细胞诱生γ干扰素及其影响因素。试验结果显示:细胞浓度在5×10~7/ml时产生的γ干扰素效价最高;PHA、PWM、ConA等T细胞促分裂剂诱生γ干扰素的适宜浓度范围分别为50～400μg/ml,2.5～40μg/ml,5～80μg/ml;诱生时间均以72小时为宜;2-ME对人γ干扰素的产生没有影响。本文还将人扁桃体细胞和外周血白细胞对4种γ干扰素诱生利(PHA、PWM、ConA、SEA)和3种α干扰素诱生剂(NDV、SPA菌体、CP)的反应进行了比较。     

激活态雪旺细胞在胶元几丁糖膜上生长规律的实验研究

目的 探索成年SD大鼠激活态雪旺细胞(SC)在体外在胶元几丁糖膜上的生长规律。方法 成年SD大鼠的坐骨神经切断预变性7天,采用复合酶分步消化法,接种于胶元几丁糖膜上,通过相差显微镜和扫描电镜观察细胞生长情况,S-100染色鉴定细胞的纯化程度。结果 200μl浓度为2×10~4/ml的激活态的SCs接种于胶元几丁糖膜上和培养皿上,2周后细胞浓度分别达到30×10~4/ml和20×10~4/ml,相差显微镜和扫描电镜下观察显示SCs在胶元几丁糖膜上贴壁生长情况良好,体外倍增时间为4天。绘制其生长曲线。结论 胶元几丁糖膜和高度纯化的激活态雪旺细胞有良好的亲和性,以其为工艺材料制成的组织工程支架很有可能在促进周围神经再生中发挥优势作用。     

细胞密度对组织工程化三维支气管模型体外再造影响的研究

研究种子细胞接种密度对体外构建组织工程化三维支气管模型的影响,并筛选出适宜体外构建支气管模型的细胞密度.以人支气管上皮细胞系和人胎肺成纤维细胞系为种子细胞,胶原凝胶复合Matrigel为支架材料,将两种细胞按照6×104/ml、6×105/ml、6×106/ml和6×107/ml四个接种密度共培养,应用静态拉伸系统体外构建组织工程化三维支气管模型.通过宏观观察、相差显微镜观察和组织学检测的方法对模型中种子细胞的生长情况进行鉴定.结果表明:6×104/ml细胞密度未见到组织片层形成,其余三个细胞密度可以看到收缩良好的组织片层,并具有一定韧性;相差显微镜下观察6×105/ml和6×106/ml细胞密度的组织片层可以形成网状结构,6×107/ml细胞密度的组织片层结构疏松;HE染色证实6×105/ml和6×106/ml细胞密度的组织片层中有三维网状结构形成,免疫组织化学染色广谱CK和Vimentin均呈阳性,前者的细胞活性较后者更好,而6×107/ml细胞密度组织片层基本丧失细胞活性.体外构建组织工程化三维支气管模型其种子细胞密度在6×105/ml至6×106/ml之间较为适宜.     

小鼠骨髓间充质干细胞的分离与培养

探索在体外分离培养小鼠骨髓间充质干细胞 (Bone m arrow m esenchym al stem cells,BMSCs)的最适条件。以密度梯度离心技术及贴壁筛选法相结合 ,在体外分离 BAL B/C小鼠的 BMSCs,通过 MTT法测定了不同离心力、不同换液时间、不同血清浓度、不同种植密度等因素对 BMSCs分离和培养的影响。在 5 0 0 g× 30 min的离心力下分离细胞 ,加入 10 %的胎牛血清 ,原代按 (12～ 2 0 )× 10 5/m l的细胞密度接种 ,接种 2 4 h后换液 ,继代种植密度为 6 .4～ 2 5 .6× 10 4 /ml时最适宜细胞生长 ;在此条件下 ,细胞快速增殖 ,传代后 8h贴壁达 90 %以上 ,2 4 h便进入对数生长期 ,直至第 5 d,细胞约增殖 3倍。本研究建立了在体外分离培养骨髓间充质干细胞的细胞生物学方法和技术参数。     

国产和进口培养基发酵培养重组人SOD工程菌的比较研究

 目的 以国产培养基替代昂贵进口培养基发酵培养重组人超氧化物歧化酶（rhSOD）工程菌。 方法 利用国产 LB 培养基对自行构建的含重组质粒 pTK- rhSOD 的基因工程菌 E.coli DH5α 通过摇瓶进行发酵培养，以 SDS-PAGE 分析方法考察不同菌体接种量（1% ~ 5%）、菌体生长密度[波长 600 nm 处吸光度（A600）值]、诱导时间（1 ~ 8 h）、氨苄青霉素（Amp）浓度（终浓度150、200、250 μg/ml）及加入时机（分别于接种前和诱导前加入）、摇瓶装液量（10% ~ 40%）和诱导温度（42、43、44、45 ℃）对 rhSOD 表达的影响，优选最适培养条件，并与进口 LB 培养基进行比较。 结果 对于国产 LB 培养基，采用 3% ~ 5% 接种量，菌体密度生长至 A600 达 0.38 ~ 0.63，诱导前补加Amp由初始的 100 μg/ml 至 200 μg/ml，于 42 ~ 44 ℃ 诱导 3 ~ 5 h，rhSOD 表达量可达 30%；对于进口 LB 培养基，采用 2%接种量，菌体密度生长至 A600 达 0.55 ~ 0.93，诱导前补加Amp 由初始的 100 μg/ml 至 150 μg/ ml，于 42 ~ 43℃ 诱导 2 ~ 4 h，rhSOD 表达量可达 30%。两种培养基摇瓶装液量对 rhSOD 的表达均没有明显影响。 结论 国产 LB 培养基经条件优化后可获得与进口培养基相同的目的蛋白表达量，可以替代昂贵的进口产品，为 rhSOD 进一步规模化生产降低发酵成本提供了实验依据。     

克隆的人LAK细胞对骨髓粒单系造血祖细胞体外增殖的抑制作用

本文应用琼脂半固体培养法观察了克隆化的人LAK细胞对骨髓粒单系造血细胞(CFU-GM)增殖的影响。LAK细胞对CFU-GM集落形成有抑制作用:2×10~5/mlLAK细胞与1×10~5/ml骨髓单个核细胞BMMNC预孵育0.5小时可使集落数减少43.8%(P<0.01),LAK与BMMNC预孵育4小时对CFUGM的抑制更为明显,0.5×10~5/ml LAK细胞即可使集落数减少58.8%(P<0.01),随LAK浓度加大抑制效应增强;当LAK与BMMNC在双层培养体系中不接触时,高浓度(4×10~5/ml)亦可表现集落抑制活性。抑制率28.8%(P<0.05)。自体LAK细胞对CFU-GM的抑制效应与异体相比无显著差异(P>0.20)。作者认为LAK细胞对CFU-GM的抑制作用为细胞直接作用与分泌可溶性因子介导的双重效应。     

激活态雪旺细胞在胶元几丁糖膜上生长规律的实验研究

目的探索成年SD大鼠激活态雪旺细胞(SC)在体外在胶元几丁糖膜上的生长规律.方法成年SD大鼠的坐骨神经切断预变性7天,采用复合酶分步消化法,接种于胶元几丁糖膜上,通过相差显微镜和扫描电镜观察细胞生长情况,S-100染色鉴定细胞的纯化程度.结果200μl浓度为2×104/ml的激活态的SCs接种于胶元几丁糖膜上和培养皿上,2周后细胞浓度分别达到30×104/ml和20×104/ml,相差显微镜和扫描电镜下观察显示SCs在胶元几丁糖膜上贴壁生长情况良好,体外倍增时间为4天.绘制其生长曲线.结论胶元几丁糖膜和高度纯化的激活态雪旺细胞有良好的亲和性,以其为工艺材料制成的组织工程支架很有可能在促进周围神经再生中发挥优势作用.     

一体化灌注法体外构建活化人工骨的优化研究

目的探讨人胚成骨细胞在多孔β-磷酸三钙（β-TCP）支架内灌注性接种及培养的影响因素及其作用机制,对一体化灌注法体外构建活化人工骨进行优化研究。方法应用自行设计的灌注式生物反应器进行多孔β-TCP支架内人胚成骨细胞的一体化灌注性接种和培养,以静态接种为对照。通过细胞活力（Mrrr法）测定、活细胞接种率、组织形态学观察和计量学分析等分别检测细胞接种时间、接种密度、灌注速率等因素对支架内细胞黏附和生长的作用。结果细胞灌注接种效果优于静态接种;灌注接种-灌注培养法细胞生长及分布优于静态接种-灌注培养法。接种时间、接种密度、灌注速率等因素均显著影响细胞接种及培养效果。灌注法构建活化人工骨的最优条件：接种时间12h～24h,接种密度2×10^5／ml一5×10^5／ml,灌注接种速率1ml／min,灌注培养速率0．5ml／min～2ml／min（灌注初期24h）及2ml／min（灌注24h后）。结论一体化灌注法利于多孔β-TCP支架内人胚成骨细胞的黏附和生长。接种时间、接种密度、灌注速率等因素均影响活化人工骨体外构建效果,对其进行优化有助于人工骨移植物的临床转化和推广应用。     

外周血淋巴细胞LDL受体活性测定新法

混悬于RPMI-1640中的淋巴细胞分两组培养96小时,一组加入0.6μM mevinolin(实验组),另一组加入等量的二甲亚砜溶媒(对照组),两组均加入50μg/ml PHA及不同浓度的LDL-ch(0/10μg/ml)。细胞收获前6小时各孔加入0.5μCi ~3H-TdR,收获细胞于4.9型纤维滤膜上测cpm值。LDL受体活性以淋巴细胞分裂抑制率表示,后者=(1-cpm实验/cpm对照)×100%。结果,当LDL-ch浓度为5μg/ml时,正常人分裂抑制率小于20%,FH杂合子分裂抑制率为25%～55%,FH纯合子分裂抑制率大于60%。由此可将FH与正常人以及普通高脂血症病人区别开来。     

Comparison of Biofilms Formed by Candida albicans and Candida parapsilosis on Bioprosthetic Surfaces

Little is known about fungal biofilms, which may cause infection and antibiotic resistance. In this study, biofilm formation by different Candida species, particularly Candida albicans and C. parapsilosis, was evaluated by using a clinically relevant model of Candida biofilm on medical devices. Candida biofilms were allowed to form on silicone elastomer and were quantified by tetrazolium (XTT) and dry weight (DW) assays. Formed biofilm was visualized by using fluorescence microscopy and confocal scanning laser microscopy with Calcofluor White (Sigma Chemical Co., St. Louis, Mo.), concanavalin A-Alexafluor 488 (Molecular Probes, Eugene, Oreg.), and FUN-1 (Molecular Probes) dyes. Although minimal variations in biofilm production among invasive C. albicans isolates were seen, significant differences between invasive and noninvasive isolates (P < 0.001) were noted. C. albicans isolates produced more biofilm than C. parapsilosis, C. glabrata, and C. tropicalis isolates, as determined by DW assays (P was <0.001 for all comparisons) and microscopy. Interestingly, noninvasive isolates demonstrated a higher level of XTT activity than invasive isolates. On microscopy, C. albicans biofilms had a morphology different from that of other species, consisting of a basal blastospore layer with a dense overlying matrix composed of exopolysaccharides and hyphae. In contrast, C. parapsilosis biofilms had less volume than C. albicans biofilms and were comprised exclusively of clumped blastospores. Unlike planktonically grown cells, Candida biofilms rapidly (within 6 h) developed fluconazole resistance (MIC, >128 microg/ml). Importantly, XTT and FUN-1 activity showed biofilm cells to be metabolically active. In conclusion, our data show that C. albicans produces quantitatively larger and qualitatively more complex biofilms than other species, in particular, C. parapsilosis.     

The effect of oral bacteria on Candida albicans germ-tube formation

A total of eight bacterial isolates belonging to six species, and a select group of 12 oral Candida albicans isolates, were used to study the effect of bacteria on germ-tube formation. Briefly, each bacterial suspension (10(5-6) cells/ml) was mixed with a C. albicans suspension (10(7) cells/ml) and incubated at 37 degrees C for 90 min with bovine serum, and the percentage germ-tube-positive Candida cells was quantified using a haemocytometer, under light microscopy. In general, out of eight bacteria, Streptococcus sanguis SK21A, Streptococcus salivarius SK56, Escherichia coli ATCC 25922, and S. salivarius OBU3 suppressed germ-tube formation to varying degrees, with different C albicans isolates. Porphyromonas gingivalis Pg 50, Lactobacillus casei ATCC 7469 and Prevotella intermedia OBU4 elicited significant enhancement of germ-tube formation, whereas S. sanguis OBU 2 had no effect. E. coli ATCC 25922 was the only organism to show statistically significant suppression of germ-tube formation (p=0.0312). A significant increase in the germ tube production of C. albicans isolated from HIV-infected compared with HIV-free individuals was also noted. The current results tend to suggest that commensal and transient oral bacterial populations may selectively influence the differential expression of germ-tube-forming ability of C. albicans isolates.     

Effect of amphotericin B alone or in combination with rifampicin or clarithromycin against Candida species biofilms

Effectiveness of amphotericin B alone or in combination with rifampicin or clarithromycin on the killing of Candida species biofilms was investigated in vitro. Amphotericin B was assayed at 0.005 to 10 mg/ml. Rifampin and clarithromycin were assayed at 10 mg/ml. We studied 7 Candida albicans, 3 Candida parapsilosis, 3 Candida glabrata, 3 Candida krusei and 2 Candida tropicalis strains. Biofilms were developed in 96-well, flat-bottomed microtiter plates for 48 hours. A synergistic effect between amphotericin B and clarithromycin was demonstrated against 66.6% of C. parapsilosis, 66.6% of C. glabrata, and 42.8% of C. albicans biofilms. A synergistic effect between amphotericin B and rifampin was demonstrated against 66.6% of C. parapsilosis, 42.8% of C. albicans, and 33.3% of C. glabrata biofilms. No synergistic effect was observed against C. krusei or C. tropicalis biofilms with any of the combinations. Rifampin or clarithromycin alone did not exert any effect on Candida species biofilms. Rifampin or clarithromycin combinations with amphotericin B might be of interest in the treatment of Candida biofilm-related infections.     

Candida albicans triggers interleukin-8 secretion by oral epithelial cells

Oropharyngeal candidiasis is a frequent opportunistic infection associated with immunocompromised hosts. Candida albicans is the principal species responsible for this infection. Production of interleukin-8 (IL-8), by oral epithelial cells can be expected to play a major role in the recruitment and activation of professional phagocytes at the infected site. The purpose of this study was to determine whether C. albicans triggers secretion of IL-8 by oral epithelial cells in vitro and investigate mechanisms of host cell-fungal interactions that trigger such responses. Oral epithelial cell lines (SCC4, SCC15, and OKF6/TERT-2) as well as primary gingival epithelial cells were used. Epithelial cells were cocultured with C. albicans, strains SC5314, ATCC28366 or ATCC32077, for 24-48 hr, and supernatants were analyzed for IL-8 content by ELISA. A germination-deficient mutant (efg1/efg1 cph1/cph1), otherwise isogenic to strain SC5314, was used to assess the requirement for germination in triggering IL-8 responses. In order to ascertain whether direct contact of yeast with host cells is required to trigger cytokine production, epithelial cells were separated from yeast using cell culture inserts. To test whether IL-8 secretion is dependent on IL-1alpha activity, epithelial cells were challenged with viable C. albicans in the presence or absence of neutralizing anti-IL-1alpha antibody or IL-1ra, and IL-8 secretion was measured in the supernatants. All cell lines and primary cultures responded to C. albicans with an increase in IL-8 secretion. IL-8 responses were contact-dependent, strain-specific, required yeast viability and germination into hyphae, and were in part autoregulated by IL-1alpha.     

Detection of Als Proteins on the Cell Wall of Candida albicans in Murine Tissues

A murine model of disseminated candidiasis was utilized to determine whether Candida albicans Als proteins are produced in vivo. The kidneys, spleen, heart, liver, and lungs were collected from mice inoculated with one of three C. albicans strains (SC5314, B311, or WO-1). Immunohistochemical analysis of murine tissues by using a rabbit polyclonal anti-Als serum indicated that Als proteins were produced by each C. albicans cell in the tissues examined. Patterns of staining with the anti-Als serum were similar among the C. albicans strains tested. These data indicated that Als protein production was widespread in disseminated candidiasis and that, despite strain differences in ALS gene expression previously noted in vitro, Als protein production in vivo was similar among C. albicans strains. The extensive production of Als proteins in vivo and their presence on the C. albicans cell wall position these proteins well for a role in host-pathogen interaction.     

诱导干细胞分化的胰岛细胞形成胰岛样结构的实验研究

目的建立一种体外诱导干细胞分化的胰岛细胞形成胰岛样结构的技术方法。方法体外诱导人胚胎胰腺干细胞分化为胰岛细胞，将胰岛细胞制备成浓度分别为1×10^4／ml、3×10^4／ml、1×10^5／ml、3×10^5／ml（1×10^4／ml、3×10^4／ml、1×10^5／ml、3×10^5／ml组）单细胞悬液，在含有细胞外基质（ECM）的培养基中悬浮培养24h以诱导形成胰岛样结构，在荧光体视显微镜下观察胰岛样结构的形态和大小；采用免疫荧光染色方法检测胰岛样结构中ECM成分及其细胞组成与定位。将胰岛样结构分别在含5．6和30．0mmol／L葡萄糖的培养基中体外培养2h，采用放射免疫法测定上清中胰岛素水平。建立糖尿病大鼠模型，经门静脉将胰岛样结构移植至大鼠肝脏，移植后连续3d经鼠尾静脉检测血糖水平。结果90％人胚胎胰腺干细胞分化的胰岛细胞形成了胰岛样结构，其包膜、大小均与天然人胰岛组织结构相似；以形成直径150μm大小胰岛样结构的比例为标准，比较各组细胞浓度与胰岛样结构形成率的关系，结果显示分别与1×10^4／ml、3×10^4／ml、3×10^5／ml组（25．0％±2．5％、28．0％±3．0％、21．5％±2．5％）相比，1×10^5／ml组胰岛样结构形成率最佳（36．5％±4．0％，均P〈0．01）。胰岛样结构包膜上含有与天然人胰岛组织类似的ECM成分，且其中含有胰岛素、胰高血糖素和C-肽阳性细胞，其细胞比例与分布均与天然人胰岛组织类似。30．0mmol／L高糖浓度刺激后胰岛样结构胰岛素释放水平[（110±12）μIU／ml]较5．6mmol／L基础糖浓度[（59．5±8．0）pIU／ml]明显增加（P〈0，01），胰岛样结构移植后糖尿病大鼠血糖水平较移植前均明显降低（P〈0．01）。结论本研究建立的技术方法可以将干细胞分化的胰岛细胞在体外大量、快速、高效地诱导形成具有功能的胰岛样结构。     

Interaction of Candida albicans with adherent human peripheral blood mononuclear cells increases C. albicans biofilm formation and results in differential expression of pro- and anti-inflammatory cytokines

Monocytes and macrophages are the cell types most commonly associated with the innate immune response against Candida albicans infection. Interactions between the host immune system and Candida organisms have been investigated for planktonic Candida cells, but no studies have addressed these interactions in a biofilm environment. In this study, for the first time, we evaluated the ability of C. albicans to form biofilms in the presence or absence of adherent peripheral blood mononuclear cells (PBMCs; enriched for monocytes and macrophages by adherence). Our analyses using scanning electron and confocal scanning laser microscopy showed that the presence of PBMCs enhanced the ability of C. albicans to form biofilms and that the majority of PBMCs were localized to the basal and middle layers of the biofilm. In contrast to the interactions of PBMCs with planktonic C. albicans, where PBMCs phagocytose fungal cells, PBMCs did not appear to phagocytose fungal cells in biofilms. Furthermore, time-lapse laser microscopy revealed dynamic interactions between C. albicans and PBMCs in a biofilm. Additionally, we found that (i) only viable PBMCs influence Candida biofilm formation, (ii) cell surface components of PBMCs did not contribute to the enhancement of C. albicans biofilm, (iii) the biofilm-enhancing effect of PBMCs is mediated by a soluble factor released into the coculture medium of PBMCs with C. albicans, and (iv) supernatant collected from this coculture contained differential levels of pro- and anti-inflammatory cytokines. Our studies provide new insight into the interaction between Candida biofilm and host immune cells and demonstrate that immunocytes may influence the ability of C. albicans to form biofilms.     

Polymorphism of Candida albicans is a major factor in the interaction with human dendritic cells

Morphological plasticity of Candida albicans is a major virulence factor. Using pH-dependent dimorphism we show, that human dendritic cells (DC) recognize filamentous forms and blastoconidia of a virulent C. albicans isolate (strain SC5314). Heat inactivated and viable blastoconidia are rapidly phagocytosed by human DC. However, viable yeast cells start to filament inside the DC at later stages of infection, leading to penetration and loss of cellular integrity. The cytokine burst of human DC induced upon contact with Candida is dominated by the granulocyte-activating, chemotactic factor IL-8 and the proinflammatory mediator TNF-alpha. Blastoconidia induce markedly lower cytokine levels than filamentous forms. Whereas IL-8 secretion is mainly cell mass dependent, release of TNF-alpha, a major proinflammatory cytokine, is clearly dependent on the morphology of Candida.