光合细菌2c菌株益生特性的实验研究

目的研究沼泽红假单胞菌2c菌株益生特性。方法利用耐酸、耐胆盐、体外黏附、平板抑菌试验及大鼠摄入2c菌株后在其体内的存活和持续时间实验研究其益生特性。结果pH2.5条件下处理30min及0.5%或0.9%牛胆盐处理对2c菌株活菌数无显著影响(P0.05);2c菌株体外对4株临床致病菌无抑制作用,体外黏附性能一般。2c菌株在大鼠体内存活和持续时间与摄入剂量有关,停止摄入3d后便不能富集出。结论2c菌株具有较好益生特性。     

胆盐水解酶产生菌的筛选及其益生潜力研究

目的从健康仔猪肠道及粪便中筛选具有胆盐水解酶活性的优良益生乳杆菌菌株,并对筛选菌株的体外、体内益生潜力进行初步探讨。方法用MRS培养基分别从仔猪新鲜粪便和小肠黏膜分离培养乳酸菌,用筛选鉴别培养基筛选阳性菌株,研究菌株的抗逆能力、黏附特性以及体内益生活性。结果从粪便和小肠黏膜共得到乳酸菌388株和97株,其中胆盐水解酶阳性菌株分别为291株(75%)和86株(89%)。选择5株胆盐水解酶活性最强、能水解游离胆酸的菌株,研究菌株的抗逆能力、黏附特性以及体内益生活性,并对最终选育得到的菌株进行生理生化及16S rRNA分子鉴定,发现菌株罗伊乳杆菌G22-2能耐受pH 3.0的酸度、1.0%的胆盐浓度,在小肠上皮细胞上的黏附效率超过15个以上;通过大鼠体内实验,筛选出的菌株G22-2对大鼠无毒无害,并能显著改善血脂水平。结论罗伊乳杆菌G22-2符合益生菌的要求,可以作为产胆盐水解酶的益生乳杆菌优良的候选菌株。     

几株饲用益生菌对体外模拟胃肠道环境的抗逆性评估

目的研究几株益生菌胃肠道环境下的抗逆能力。方法体外模拟正常猪的胃肠道环境,配制人工肠液和人工胃液,将实验室几株饲用益生菌在人工胃液和人工肠液中分别作用4、6 h,每2 h测一次活菌量。结果实验菌株对模拟胃液的耐受性都较强;除乳酸菌A外,其他菌株对模拟肠液的耐受性也较强。结论除乳酸菌A外的几株实验菌种作为饲用益生菌在抵御猪胃肠道的不良环境方面有很大优势。     

新型p1 基因型肺炎支原体分型方法的建立与应用

【目的】针对肺炎支原体新型p1基因型(V2c型)菌株检测工作的需要,建立相应PCR检测方法并进行评价。【方法】针对新型V2c型肺炎支原体菌株p1基因变异区域序列设计特异性扩增引物,建立对V2c型肺炎支原体菌株进行PCR检测的检测方法并用相关基因测序进行验证。使用所建立的巢式多重PCR对北京地区2008-2011年分离到的214株临床肺炎支原体进行分型分析。【结果】特异引物可有效检测出V2c菌株,在其它型别菌株均无阳性扩增。214株肺炎支原体临床分离株中1型菌株占90.2%(193/214),V2a型菌株占0.9%(2/214),V2c型菌株占8.9%(19/214);未检出2型菌株。【结论】针对V2c型肺炎支原体所建立的基于p1基因的PCR检测方法,能有效区分以往方法无法检测出的新型V2c型肺炎支原体菌株,对开展肺炎支原体流行病学调查和病原分析有重要意义。     

分子水平上益生菌研究进展

益生菌是指能在生物体内存活 ,对生物体的健康有益的一类微生物。目前已经对益生菌的作用机理进行了深入和广泛的研究 ,获得了许多使用益生菌的经验。近些年来 ,分子生物学特别是基因工程技术的发展将益生菌的理论和应用研究推向了一个新的高度。从益生菌菌株的鉴定、菌株的遗传学修饰、益生菌功能基因组学和安全性等几个方面 ,综述了近年来国际上在分子水平上研究益生菌的技术方法、获得的主要研究成果和面临的挑战 ,并提出了益生菌研究的发展方向。     

猪链球菌2型荚膜唾液酸对细菌毒力和宿主炎症反应的影响

【目的】阐明猪链球菌2型荚膜唾液酸是否影响细菌毒力以及宿主对其炎症反应应答,为研究猪链球菌2型的致病机制奠定基础。【方法】比较实验菌株对BLAB/c小鼠模型的致病性;通过涂板计数的方法检测实验菌株在小鼠体内的分布;观察小鼠脑组织病理改变,分析实验菌株感染小鼠后中枢神经系统的病变差异;从小鼠体外全血细胞水平,运用ELISA法检测实验菌株感染后细胞炎性因子的分泌水平。【结果】荚膜唾液酸合成基因neuB缺失突变株ΔneuB相比野生株05ZYH33株,对小鼠毒力显著降低,回复突变株cΔneuB毒力回复至野生株水平;野生株和突变株在血液及脑组织中分布具有显著差异,均可致BLAB/c小鼠脑组织不同程度的损伤;与野生株组相比较,细菌/细胞相互作用不同时间点后,突变株组体外刺激小鼠全血细胞分泌MCP-1、IL-6的水平显著提高;【结论】荚膜唾液酸影响细菌的毒力及宿主细胞对其的炎症反应应答,它是猪链球菌2型穿透血脑屏障导致脑膜炎的重要毒力因子。     

五种益生菌菌株对脐血单个核细胞免疫作用的实验研究

目的比较不同剂量的5种不同益生菌菌株的免疫调节作用,为选择适当的菌株进行治疗提供依据。方法取7例健康孕妇的脐血分离出的脐血单个核细胞(cord blood monocular cells,CBMC),分别与长双歧杆菌6-1株、婴儿双歧杆菌CGMCC313-1株、嗜酸乳杆菌YIT2004株、粪链球菌YIT0072株和酪酸梭状芽胞杆菌CGMCC313-2株,以菌和CBMC比例2∶1(低)、20∶1(中)和200∶1(高)共培养24~36 h,同时设阴性对照(PBS)和阳性对照(脂多糖,LPS)。然后采用流式细胞仪检测各组CBMC表面CD4、CD25分子表达情况,用ELISA方法检测培养上清中IL-10、IL-12、IL-4、TGF-β1和IFN-γ的水平。结果 (1)与阴性对照(PBS)组相比,除200∶1比例的酪酸梭状芽胞杆菌CGMCC313-2株能够显著提高CBMC表达CD4CD25(10.45±3.16 vs 5.84±2.32,P=0.009)以外,其余益生菌菌株对表达CD4CD25差异均无统计学意义(P0.05)。(2)长双歧杆菌6-1株在中高剂量下,能够刺激CBMC产生IL-10和IFN-γ,对产生IL-12无明显影响。(3)婴儿型双歧杆菌CGMCC313-1株在各个剂量下均能够刺激CBMC产生IL-10,对IL-12和IFN-γ产生无明显影响。(4)酪酸梭状芽胞杆菌CGMCC313-2株在中高剂量下,能够刺激CBMC产生IL-10,对IL-12和IFN-γ产生无明显影响。(5)粪链球菌YIT0072株在低中剂量下,能够刺激CBMC产生IL-10、IL-12和IFN-γ,而高剂量则无影响。(6)嗜酸乳杆菌YIT2004株在中高剂量下,能够刺激CBMC产生IL-10,在中剂量下,能够刺激CBMC产生IFN-γ,对IL-12无影响。(7)在本研究中均未能检测出IL-4和TGF-β1。结论在目前国内使用的益生菌菌株中,仅酪酸梭状芽胞杆菌CGMCC313-2株能够显著提高CBMC表达CD4CD25。5种菌株均能够刺激CBMC产生抗炎症因子IL-10;长双歧杆菌6-1株、粪链球菌YIT0072株和嗜酸乳杆菌YIT2004株能够刺激CBMC产生Th1型细胞因子INF-γ,仅粪链球菌YIT0072株能够刺激CBMC产生IL-12。各个菌株在不同的剂量下,具有不同作用。提示在应用益生菌治疗免疫等相关性疾病时,应该考虑不同菌株对免疫细胞的不同作用机制。     

8株光合细菌的鉴定及其系统进化关系分析

目的为8株光合细菌(photosynthetic bacteria,PSB)作为益生菌株提供系统资料。方法用常规方法对8株PSB菌株的形态、培养特性及生理生化特征进行鉴定,同时定性分析菌株产生的类胡萝卜素和CoQ,测定菌株16S DNA序列并分析其系统进化关系,在GenBank中获取了8个16S DNA序列号。结果菌株鉴定结果表明:菌株2C、2c和13ing为沼泽红假单胞菌,Ga、Il106、WS8N为类球红细菌,MT1131为荚膜红细菌,rub为深红红螺菌。基于菌株16S DNA序列的系统进化树显示,同一种菌并不总是聚为一簇,但相隔较近;种属完全不同的菌株,尽管序列相似性高达97%以上,在系统进化树上相隔较远。结论8株PSB菌株的鉴定和系统进化关系分析结果为后续研究提供了背景资料,同时菌株在GenBank中获得的16S DNA序列号为菌株作上了生物标记,也为菌株的产权保护提供了依据。     

弗氏志贺菌4c和2a型菌株的脉冲场凝胶电泳分型特征

目的：了解北京部分地区弗氏志贺菌4c型（F4c）和2a型（F2a）菌株的分子分型特征。方法：对2005年和2006年自北京部分地区腹泻监测分离的弗氏志贺菌菌株（4c型10株,2a型20株）进行生化鉴定和血清分型,用PCR检测侵袭性抗原基因ipaH,用改良Kirby-Bauer纸片法检测菌株的耐药谱,用脉冲场凝胶电泳（PFGE）对菌株进行分子分型。结果：10株血清型鉴定为F4c的菌株中,有7株间的PFGE图谱存在相当的差异,Dice系数为0.78～0.92,而F2a菌株与大部分F4c菌株间的距离较远（Dice系数小于0.8）;F4c和F2a菌株对14种抗生素的耐药谱略呈差异。结论：采用PFGE能够很好地辨别弗氏志贺菌4c型和2a型菌株;弗氏志贺菌4c型菌株具有易变性,可在流行过程中产生PFGE图谱的差异、血清亚型的转换、耐药谱的变化等。     

具有血管紧张素转化酶抑制活性益生菌筛选的研究

经体外血管紧张素转换酶（ACE）抑制活性实验,从保存的40株菌中筛选到7株具有降压活性的益生菌,其中菌株DM8319粗提样品经初步凝胶层析纯化后其IC50值从0．038mg／ml降到0．016mg／ml,达到了初步分离效果。后依据益生菌筛选标准,对上述菌株抗氧化性、耐酸、耐胆盐、抑菌和耐药性能进行考察。结果显示,菌株DM84013最适用于益生菌降压产品的研究,经初步鉴定该菌种为长双歧杆菌。     

Effects of Cytochrome c on Liver Functions of Old Rats

WE have shown^1,2 that cytochrome c, when orally administered, is absorbed intestinally. The effects of parenterally administered cytochrome c on the liver have been studied by Drabkin³ and Murano⁴. We have also studied the effects of orally administered cytochrome c on experimental liver injury⁵.     

Liposomes in leishmaniasis: Therapeutic effects of antimonial drugs, 8-aminoquinolines,and tetracycline

This study investigated the role of liposomes in changing the pharmacological efficacy of anti-leishmanial drugs in hamsters infected with visceral leishmaniasis. Enhanced anti-leishmanial activity could be accounted for only by liposome-encapsulated drugs. “Empty” liposomes (lacking anti-leishmanial drug) gave no therapeutic benefit by themselves, nor did they enhance the effectiveness of concurrently administered drugs. In the absence of additional drugs, empty liposomes actually resulted in a higher mortality due to endstage leishmaniasis. Mortality associated with chronic leishmaniasis, including that induced by empty liposomes, was reduced approximately 50% by orally administered unencapsulated tetracycline. Liposome-encapsulated tetracycline, given i.c., had no anti-leishmanial activity, thus indicating that tetracycline did not have inherent anti-leishmanial properties, and was beneficial because of its anti-bacterial effects. Liposomes containing an antimonial drug were effective when given i.c., i.p., or i.m., but not when given s.c. or p.o. Liposome-encapsulated antimonial drug had prophylactic activity and was effective when administered 8 days prior, but not 17 days prior, to infection. Unencapsulated antimonial drug had no prophylactic effect. In addition to antimonials, another class of compounds, 8-aminoquinolines, had marked anti-leishmanial activity in liposomes. One of these, WR 6026, was 700 to 1800 times more effective than an antimonial drug alone.     

Effect of a spirocyclic cyclodipeptide derivate of MIF on passive avoidance behavior and amnesia in rats

Cyclo (1-amino-1-cyclopentane-carbonyl-L-alanyl)-c(Acp-Ala), a derivative of MIF (prolyl-leucyl-glycinamide) affected passive avoidance behavior of rats when administered at different phases of the step-through type of experimental paradigm. c(Acp-Ala) given s.c. or orally in a 1 mg/kg dose increased avoidance latencies not only when administered before, or immediately after the shock trial but also when given before the pretraining trial, i.e. at the first exposure of animals to the experimental situation without shock treatment. The notion is discussed, that it is the influence of c(Acp-Ala) on processing of information received during the pretraining trial that manifests itself in the facilitation of avoidance response. The drug appears to have a long-term action since it was active when given 20 h before the pretraining trial or the shock trial or the test of retention. c(Acp-Ala) when administered immediately after the shock trial, attenuated amnesia in rats induced by electroconvulsive shock (ECS).     

Toxicity of Bacillus sphaericus crystal toxin to adult mosquitoes

Adult Culex quinquefasciatus mosquitoes were killed by alkaline-solubilized Bacillus sphaericus toxin when it was introduced by enema into the midgut of the insect but not when it was administered orally. Adult Aedes aegypti mosquitoes were not affected by the toxin.     

Toxicity of Bacillus sphaericus crystal toxin to adult mosquitoes.

Adult Culex quinquefasciatus mosquitoes were killed by alkaline-solubilized Bacillus sphaericus toxin when it was introduced by enema into the midgut of the insect but not when it was administered orally. Adult Aedes aegypti mosquitoes were not affected by the toxin.     

The use of ginger (Zingiber officinale Rosc.) as a potential anti-inflammatory and antithrombotic agent

The effect of an aqueous extract of ginger (Zingiber officinale) on serum cholesterol and triglyceride levels as well as platelet thromboxane-B(2) and prostaglandin-E(2) production was examined. A raw aqueous extract of ginger was administered daily for a period of 4 weeks, either orally or intraperitoneally (IP) to rats. Fasting blood serum was investigated for thromboxane-B(2), prostaglandin-E(2), cholesterol and triglycerides. A low dose of ginger (50 mg/kg) administered either orally or IP did not produce any significant reduction in the serum thromboxane-B(2) levels when compared to saline-treated animals. However, ginger administered orally caused significant changes in the serum PGE(2) at this dose. High doses of ginger (500 mg/kg) were significantly effective in lowering serum PGE(2) when given either orally or IP. However, TXB(2) levels were significantly lower in rats given 500 mg/kg ginger orally but not IP. A significant reduction in serum cholesterol was observed when a higher dose of ginger (500 mg/kg) was administered. At a low dose of ginger (50 mg/kg), a significant reduction in the serum cholesterol was observed only when ginger was administered IP. No significant changes in serum triglyceride levels were observed upon administration of either the low or high dose of ginger. These results suggest that ginger could be used as an cholesterol-lowering, antithrombotic and anti-inflammatory agent.     

Comparison of vitamin E derivatives alpha-TEA and VES in reduction of mouse mammary tumor burden and metastasis

A novel nonhydrolyzable ether derivative of RRR-alpha-tocopherol, RRR-alpha-tocopherol ether acetic acid analog [2,5,7,8-tetramethyl-2R-(4R,8R,12-trimethyltridecyl)chroman-6-yloxyacetic acid (alpha-TEA)], and a hydrolyzable ester derivative RRR-alpha-tocopheryl succinate (vitamin E succinate; VES) inhibited BALB/c mouse 66cl-4-GFP mammary tumor cell growth in vitro and in vivo. Treatment of 66cl-4-GFP cells in culture with alpha-TEA or VES induced dose-dependent DNA synthesis arrest and apoptosis and inhibited colony formation. Liposomal formulations of alpha-TEA delivered orally or by aerosol significantly reduced subcutaneous 66cl-4-GFP tumor burden and metastasis to lung and lymph nodes. Liposomal formulations of VES delivered by aerosol significantly reduced tumor burden and lung metastasis, but not lymph node metastasis. Unlike alpha-TEA, VES was ineffective in reducing tumor burden and metastasis to lungs and lymph nodes when administered orally. Analyses of tumor sections showed that alpha-TEA delivered by either method significantly reduced tumor cell proliferation as measured by Ki67, and increased apoptosis as measured by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL), whereas VES delivered by aerosol reduced tumor cell proliferation and increased apoptosis, but not significantly. In summary, the nonhydrolyzable ether vitamin E derivative alpha-TEA was effective in reducing tumor burden and metastasis when delivered either by aerosol or orally, whereas the hydrolyzable ester vitamin E derivative VES was effective only when delivered by aerosol.     

N-acetyl-cysteine: protective agent or promoter of gastric damage?

N-acetyl-cysteine (NAC), when given orally, has been shown to prevent gastric damage induced by ethanol, but when administered intraperitoneally, it appears to potentiate such damage. In an effort to resolve these seemingly discordant findings, fasted rats (six per group) received 1 ml of saline or 20% NAC orally or intraperitoneally (ip). Two hours or 15 min later, they received 1 ml of 100% ethanol orally. At sacrifice 5 min later, rats receiving oral pretreatment with 20% NAC at both 15 and 120 min prior to ethanol exposure demonstrated a significant reduction in the magnitude of gastric injury when compared with saline controls. In contrast, actual promotion of ethanol damage was noted when NAC was given intraperitoneally, but was more pronounced when NAC was administered 15 min prior to exposing the mucosa to 100% ethanol. In all animals receiving intraperitoneal NAC, large amounts of peritoneal fluid (4-6 ml/rat) were recovered at the time of sacrifice, most of which occurred within 15 min of NAC administration; these more pronounced peritoneal effects at 15 min after NAC correlated with the more severe injury from ethanol at this time period compared to 120 min after intraperitoneal NAC. Saline controls had no peritoneal fluid. Mucosal glutathione (GSH) levels generally paralleled these results in that a significant decrease in tissue GSH occurred at 15 min following intraperitoneal NAC when compared with controls; at 120 min after intraperitoneal NAC, GSH levels were similar to control values. Additional experiments demonstrated that within 15 min following NAC administration, systemic blood pressure dropped by approximately 20% and basically remained unchanged over the next 2 hr; intraperitoneal saline had no sustained adverse effects on blood pressure. It was concluded that the inability of NAC to prevent ethanol injury when given intraperitoneally in contrast to orally is related to the drop in blood pressure secondary to NAC's peritoneal irritant effects, which presumably altered gastric mucosal blood flow, thus obivating its ability to prevent ethanol damage under these conditions. Furthermore, the decreased levels in mucosal GSH following the hypotension induced by intraperitoneal NAC suggest that perturbations in GSH metabolism may also have contributed to the decreased resistance to ethanol injury.     

Intestine may be a major site of action for the apoA-I mimetic peptide 4F whether administered subcutaneously or orally

To determine if the dose of peptide administered or the plasma level was more important, doses of 0.15, 0.45, 4.5, or 45 mg/kg/day of the peptide D-4F were administered orally or subcutaneously (SQ) to apoliptotein (apo)E null mice. Plasma levels of peptide were ～1,000-fold higher when administered SQ compared with orally. Regardless of the route of administration, doses of 4.5 and 45 mg/kg significantly reduced plasma serum amyloid A (SAA) levels and the HDL inflammatory index (P < 0.0001); doses of 0.15 or 0.45 mg/kg did not. A dose of 45 mg/kg/day administered to apoE null mice on a Western diet reduced aortic atherosclerosis by ～50% (P < 0.0009) whether administered orally or SQ and also significantly reduced plasma levels of SAA (P < 0.002) and lysophosphatidic acid (P < 0.0009). Remarkably, for each dose administered, the concentration and amount of peptide in the feces was similar regardless of whether the peptide was administered orally or SQ. We conclude: i) the dose of 4F administered and not the plasma level achieved determines efficacy; ii) the intestine may be a major site of action for the peptide regardless of the route of administration.     

Chemotherapeutic Evaluation of Clotrimazole [Bay b 5097, 1 (o-Chloro-α-α-Diphenylbenzyl) Imidazole]

Clotrimazole has a broad spectrum of activity against yeast and filamentous fungi in vitro and also in vivo when given orally or parenterally to experimentally infected mice and when administered orally or topically to infected guinea pigs. In vitro a distinct inoculum effect has been observed with a number of strains of Candida and Torulopsis; minimal inhibitory concentrations have tended to increase with increased incubation time. With prolonged incubation times, resistance can be developed to clotrimazole in vitro, but this resistance is readily reversible upon passage in drug-free broth. The degree of in vivo activity of clotrimazole against Candida depends on the severity of infection used. Orally it appears to be more effective when administered by gavage than when given mixed in the diet. Pretreatment with the agent may decrease its efficacy because of drug inactivation. Against dermatophytes, clotrimazole has a degree of activity similar to griseofulvin when given orally, but it is less active than tolnaftate topically in cutaneous infection of Trichophyton mentagrophytes in guinea pigs. In vitro, but not in vivo, some gram-positive and gram-negative bacteria are inhibited by low concentrations of clotrimazole.