苹果轮纹病拮抗细菌T2、J4最优培养基的筛选

采用平板对峙培养法,通过T2、J4细菌发酵上清液对苹果轮纹病菌丝生长的抑制作用实验,筛选了T2、J4细菌培养的最优培养基。结果表明:4号培养基(黄豆粉30 g、玉米粉30 g、NaCl 0.6 g)是T2细菌的最优培养基;1号培养基(蛋白胨6g、酵母浸膏3g)和2号培养基(蛋白胨3g、牛肉浸膏1.8 g、葡萄糖1.5 g)是J4细菌的最优培养基。

苹果轮纹病拮抗细菌T2对病菌菌丝的抑制作用研究

采用对峙培养法和菌丝生长速率法,测定细菌发酵上清液对苹果轮纹病菌菌丝生长的抑制作用。结果表明:T2细菌发酵上清液对苹果轮纹病菌菌丝生长有较强的抑制作用,随着发酵上清液浓度的增大抑制作用越明显,浓度为1 L/mL的上清液对苹果轮纹病菌丝生长有促进作用。在浓度为100 L/mL的条件下抑制作用最强。浓度在5 L/mL、25 L/mL、50 L/mL的条件下,抑制作用不显著。细菌T2对苹果轮纹病菌菌丝生长有较强的抑制作用。

乌鲁木齐10号泉水体细菌群落对地震的响应

以无震时期细菌群落及水文化学元素信息为背景,阐明有感地震时的泉水细菌群落映震特征.2010年12月—2011年4月,乌鲁木齐周边先后发生了MS3.1和MS3.6两次有感地震.本文采用末端限制性片段长度多态性法(T-RFLP)分析了该时间段内乌鲁木齐10号泉中细菌群落结构、多样性、稳定性、优势类群及特殊类群变化特征.结果表明,水文地球化学指标对有感地震不敏感,泉水细菌群落结构稳定,地震对其优势类群及多样性影响均不显著,但无震时期部分类群(以82,170,176,462bp为代表)在前后两次地震中丰度变化趋势相似,即震前有小幅上升,震后1—12天内出现极大值,之后丰度逐渐恢复至较低水平,且表现出与震级、震中距具有一定相关性.这表明部分泉水细菌对有感地震映震灵敏.

T7 RNA聚合酶的克隆及其介导基因转录功能的鉴定

目的构建含T7RNA聚合酶(T7RNA Polymerase,T7RNP)的真核表达载体,同时构建含T7启动子、增强型绿色荧光蛋白(Enhanced Green Fluorescent Protein,EGFP)和T7RNP转录终止信号的载体,并通过观察EGFP的表达推测T7RNP表达和T7表达系统介导基因转录的功能。方法根据编码T7RNP的基因序列,应用PCR技术从含有T7RNP基因的E.coli BL21(DE3)基因组中扩增编码T7RNP的基因片段,克隆至载体pcDNA3·1(+)。同时从px8δt载体和pGEM-Teasy/EGFP上切下T7RNP识别的终止信号(terminator,TER)和编码EGFP的基因,克隆至载体pcD-NAII。将获得的两个重组质粒共转染BHK细胞。48h后,通过观察EGFP基因在真核细胞内的表达情况,推测T7RNP基因的表达及表达后识别T7启动子介导基因转录功能。结果成功构建了含T7RNP编码基因的真核表达载体pcDNA3·1(+)/T7RNP和原核表达载体pcDNAII/EG-FP/TER,共转染BHK细胞后,可观察到EGFP表达的绿色荧光。结论T7RNP能顺利在真核细胞内表达,并通过与T7启动子和TER的相互作用,成功实现了EGFP基因的转录和表达。

表皮生长因子受体为靶向重组T7噬菌体抗肿瘤疫苗的构建

目的:构建表皮生长因子受体(EGFR)为靶向的重组T7噬菌体抗肿瘤疫苗。方法:通过RT-PCR克隆EGFR基因膜外DNA片段,经测序鉴定正确,亚克隆3个不同的DNA片段插入T7噬菌体载体,构建重组T7噬菌体。用PCR、SDS-PAGE和Western印迹法鉴定构建的重组T7噬菌体。结果:目的DNA片段以融合蛋白形式表达,展示于噬菌体壳蛋白,Western印迹显示实验组重组T7噬菌体呈阳性信号。结论:构建的重组T7噬菌体融合表达了外源目的多肽,并保持了原有的功能活性。

细菌及生命起源的探索

生命的意义是什么?是科学家所说的生命只是最终死亡的开始,还是宗教教义所认为的死亡才是“永恒生命”的开始?或者生命是永无止境的生生死死地循环?

Granulocyte-macrophage colony-stimulating factor （GM-CSF） and T-cell responses： what we do and don＇t know

Granulocyte-macrophage colony-stimulating factor （GM-CSF） is an important hematopoietic growth factor and immune modulator. GM-CSF also has profound effects on the functional activities of various circulating leukocytes. It is produced by a variety of cell types including T cells, macrophages, endothelial cells and fibroblasts upon receiving immune stimuli. Although GM-CSF is produced locally, it can act in a paracrine fashion to recruit circulating neutrophils, monocytes and lymphocytes to enhance their functions in host defense. Recent intensive investigations are centered on the application of GM-CSF as an immune adjuvant for its ability to increase dendritic cell （DC） maturation and function as well as macrophage activity. It is used clinically to treat neutropenia in cancer patients undergoing chemotherapy, in AIDS patients during therapy, and in patients after bone marrow transplantation. Interestingly, the hematopoietic system of GM-CSF-deficient mice appears to be normal; the most significant changes are in some specific T cell responses. Although molecular cloning of GM-CSF was carried out using cDNA library oft cells and it is well known that the T cells produce GM-CSF after activation, there is a lack of systematic investigation of this cytokine in production by T cells and its effect on T cell function. In this article, we will focus mainly on the immunobiology of GM-CSF in T cells.

CagA+ H pylori infection is associated with polarization of T helper cell immune responses in gastric carcinogenesis

AIM:To characterize the immune responses including local and systemic immunity induced by infection with H pylori,especially with CagA+ H pylori strains and the underlying immunopathogenesis. METHODS:A total of 711 patients with different gastric lesions were recruited to determine the presence of H pylori infection and cytotoxin associated protein A (CagA),the presence of T helper (Th) cells and regulatory T (Treg) cells in peripheral blood mononuclear cells (PBMCs),expression of plasma cytokines,and RNA and protein expression of IFN-γ and IL-4 in gastric biopsies and PBMCs were determined by rapid urease test,urea 14C breath test,immunoblotting test,flow cytometry ,real time RT-PCR and immunohistochemistry. RESULTS:Of the patients,629 (88.47%) were infected with H pylori ; 506 (71.16%) with CagA+ and 123 (17.30%) with CagA- strains. Among patients infected with CagA+ H pylori strains,Th1-mediated cellular immunity was associated with earlier stages of gastric carcinogenesis,while Th2-mediated humoral immunity dominated the advanced stages and was negatively associated with an abundance of Treg cells. However,there was no such tendency in Th1/Th2 polarization in patients infected with CagA- H pylori strains and those without H pylori infection. CONCLUSION:Polarization of Th cell immune responses occurs in patients with CagA+ H pyloriinfection,which is associated with the stage and severity of gastric pathology during the progression of gastric carcinogenesis. This finding provides further evidence for a causal role of CagA+ H pylori infection in the immunopathogenesis of gastric cancer.

Bifidobacterium lactis attenuates onset of inflammation in a murine model of colitis

AIM: To assess the anti-inflammatory effect of the probiotic Bifidobacterium lactis (B. lactis) in an adoptive transfer model of colitis. METHODS: Donor and recipient mice received either B. lactis or bacterial culture medium as control (deMan Rogosa Sharpe) in drinking water for one week prior to transfer of a mix of naive and regulatory T cells until sacrifice. RESULTS: All recipient mice developed signs of colonic inflammation, but a significant reduction of weight loss was observed in B. lactis-fed recipient mice compared to control mice. Moreover, a trend toward a diminution of mucosal thickness and attenuated epithelial damage was revealed. Colonic expression of pro-inflammatory and T cell markers was significantly reduced in B. lactis-fed recipient mice compared to controls. Concomitantly, forkhead box protein 3, a marker of regulatory T cells, was significantly up-regulated by B. lactis. CONCLUSION: Daily oral administration of B. lactis was able to reduce inflammatory and T cells mediators and to promote regulatory T cells specific markers in a mouse model of colitis.

Increased presence of effector lymphocytes during Helicobacter hepaticus-induced colitis

AIM： To identify and characterize drosophila mothers against decapentaplegic （SMAD）3-dependent changes in immune cell populations following infection with He- Iicobacter hepaticus （H. hepaticus）. METHODS： SMAD3/ （n = L9） and colitis-resistant SMAD3＋/ （n = 24） mice （8-10 wk of age） were in- fected with/-/, hepaticus and changes in immune cell populations [T lymphocytes, natural killer （NK） cells, T regulatory cells] were measured in the spleen and mesenteric lymph nodes （MsLNs） at 0 d, 3 d, 7 d and 28 d post-infection using flow cytometry. Genotype-dependent changes in T lymphocytes and granzyme B＋ cells were also assessed after 28 d in proximal colon tissue using immunohistochemistry. RESULTS： As previously observed, SMAD3＋, but not SMAD3＋/- mice, developed colitis, peaking at 4 wk post-infection. No significant changes in T cell subsets were observed in the spleen or in the MsLNs between genotypes at any time point. However, CD4＋ and CD8＋/ CD62L＋＋ cells, an effector T lymphocyte population, as well as NK cells （NKp46/DX5＋） were significantly higher in the MsLNs of SMAD3/ mice at 7 d and 28 d post-in- fection. In the colon, a higher number of CD3＋ cells were present in SMAD3＋ compared to SMAD3＋/- mice at base- line, which did not significantly change during infection. However, the number of granzyme B＋ cells, a marker of cytolytic lymphoo/tes, significantly increased in SMAD3＋ mice 28 d post-infection compared to both SMAD3＋/- mice and to baseline values. This was consistent with more severe colitis development in these animals. CONCLUSION： Data suggest that defects in SMAD3 signaling increase susceptibility to H. hepaticus-induced colitis through aberrant activation and/or dysregulation of effector lymphoo/tes.

Detection of T lymphocyte subsets of children with Helicobacter pylori infection

AIM: To study the transformation of T lymphocyte subsets in children with Heliobacter pylori (H pylori) infection. METHODS: The H pylori infection status were determined by a combination of ELISA and Western blot (immunoblot) technique in 98 children and T lymphocyte subsets in peripheral blood were determined by flow cytometrical analysis.RESULTS: There were 75 children positive with H pylori infection and 23 negative in 98 children. Comparing the proportion of peripheral blood T lymphocytic subsets in children with H pylori infection and without H pylori infection, it was found that a higher proportion of CD4 T-cells in infected children (39.02±7.71 vs34.25±10.73,t = 2.246,P＜0.05) and higher value of CD4 to CD8 T-cells ratio (1.51±0.52 vs 1.25, t= 2.104,P＜0.05) were present, but there were not significant differences in CD3 T-cells and CD8 T-cells (73.11±10.02 vs69.49±17.08, 27.22±6.07vs 28.27±8.67, P＞0.05).CONCLUSION: Th1 cell-mediated immune responses may be induced by H pylori infection in children.

Fermentation Production of Ganoderma lucidum by Bacillus subtilis Ameliorated Ceftriaxone-induced Intestinal Dysbiosis and Improved Intestinal Mucosal Barrier Function in Mice

Objective This study aimed to develop a type of Ganoderma lucidum(G.lucidum)-probiotic fermentation broth that can effectively improve the intestinal mucosal barrier function of mice with ceftriaxone-induced intestinal dysbiosis.Methods By means of absorbance of optical density(OD)value and phenol-concentrated sulfuric acid measurement of polysaccharide content,the probiotic species can grow on the medium of G.lucidum were screened out,and the concentration of the medium of G.lucidum was determined,and the fermentation broth was prepared for subsequent experiments.Thirty-two SPF grade male BALB/c mice were randomly divided into four groups(eight mice in each group),namely control group(CON),intestinal mucosal barrier damage model group(CS),fermentation broth intervention group(FT)and G.lucidum medium intervention group(GL),respectively.The intestinal dysregulation model was induced by intra-gastric administration of 0.2 mL ceftriaxone sodium(twice a day for seven consecutive days).From day 8,the FT group and GL group were gavage with 0.2 mL fermentation broth and G.lucidum medium,respectively.On day 15,all mice were sacrificed.To draw the weight curve and measure the cecal index;pathological examination of colon tissues with HE staining;serum levels of LPS,IL-10,TNF and IL-6 were detected by ELISA.Flow cytometry was used to analyze the changes of CD3+T cells,CD4+T cells,CD8+T cells and macrophages in spleen.16S rRNA sequencing was performed to detect the intestinal microbiota structure of mice.Results Bacillus subtilis can decompose and utilize G.lucidum fruiting body medium,and the suitable concentration of G.lucidum fruiting body medium is 33.2 mg/mL.The effect of Bacillus subtilis-G.lucidum fermentation broth on the damage of intestinal mucosal barrier caused by ceftriaxone sodium was reduced,the body weight of mice recovered and colon swelling improved,colon histopathological injury was alleviated,inflammatory cell infiltration was alleviated,serum IL-10 increased significantly,LPS、TNF-αand IL-6 decreased significantly compared with model group,and the proportion of T cells and intestinal dysbiosis was improved.Conclusions The experimental results suggest that Bacillus subtilis-G.lucidum fermentation broth can effectively improve the intestinal barrier function damage,immune dysfunction and intestinal dysbiosis caused by antibiotic overdose,and has a certain regulatory effect on intestinal mucosal barrier function.

Lymphomatoidgastropathy mimicking extranodal NK/T cell lymphoma,nasal type:A case report

Extranodal natural killer(NK)/T-cell lymphoma,nasal type,exhibits aggressive tumor behavior and carries a poor prognosis.Recently,lymphomatoid gastropathy with NK/T cell infiltration into gastric mucosa has been recognized as a pseudo-malignant disease which regresses without treatment.Because the conventional immunohistochemical criteria of lymphomatoid gastropathy is similar to that of extranodal NK/T-cell lymphoma nasal type,it is difficult to distinguish between the two conditions by histopathological evaluation only.Here,we report a rare case of lymphomatoid gastropathy in a 57-year-old female.Gastroendoscopy on routine check-up revealed elevated reddish lesions < 1 cm in diameter in the gastric fornix and body.Although repeat endoscopies at 1 and 6 mo later revealed no gastric lesions at any locations without any treatments,at 12 mo later gastric lymphomatoid lesions recurred at gastric fornix and body.Histological examination of endoscopic biopsy specimens at 12 mo showed atypical NK cell infiltration with CD3+,CD4-,CD5-,CD7+,CD8-,CD20-,CD30-,CD56+,CD79a-and T-cell-restricted intracellular antigen-1+ into gastric mucosa.After treatment for Helicobacter pylori(H.pylori) eradication,the lesions disappeared in all locations of the gastric fornix and body over the subsequent 12 mo.Here,we report a case of H.pylori-positive lymphomatoid gastropathy with massive NK-cell proliferation,and also review the literature concerning newly identified lymphomatoid gastropathy based on comparison of extra nodal NK/T-cell lymphoma nasal type.In any case,these lesions are evaluated with biopsy specimens,the possibility of this benign entity should be considered,and excessive treatment should be carefully avoided.Close follow-up for this case of lymphomatoid gastropathy is necessary to exclude any underlying malignancy.

Stimulation of staphylococcal enterotoxin A combined with PML-RARα peptide on the specifical T-cells against NB4 cell line

Objective： To investigate the effects of staphyococcal enterotoxin A （SEA） on the cytotoxicity of T cells stimulated by PML-RARa peptide in vitro. Methods： Peripheral blood mononuclear cells （MNC） from healthy donor were obtained by density gradient centrifugation on Ficoll-Hypaque, MNC were cultured with PML-RARa peptide and SEA for 20 days. After induction, the cytotoxicity of T cells induced against NB4 and K562 cell lines were examined by Cell Counting Kit-8 （CCK-8）. The CD4 and CD8 surface markers on the harvested CD3^＋ T cells were detected by flow cytometry （FCM）. Results： The cytotoxicity of T cells induced by PML-RARa peptide with SEA was higher than that of T cells induced only by PML-RARa peptide against NB4 cells. The FCM assay showed that the ratio of CD4^＋/CD8^＋ T cells were gradually decreased in both groups of PML-RARα peptide whether with SEA or not at the intervals of day 5,10 and 20 day after induction, but the most significantly decreased by PML-RARe peptide with SEA. Conclusion： The specific cytotoxicity of T cells induced by PML-RARa peptide against NB4 cells could be enhanced with superantigen SEA.

噬菌体法检测宿主菌的侵染条件研究

目的初步建立一种确定寄生菌被完全侵染的方法。方法以细胞噬菌体T4（简称T4）侵染大肠杆菌为模型，通过3种方法测定不同滴度的T4在5min内侵染模拟样品中大肠杆菌的效果。结果在T4〉1．00×10^8PFU／mL的条件下侵染后，样液在培养中吸光度逐渐下降；在同样条件下侵染并清洗掉游离T4后，样液经平板培养无菌落产生，经液体培养不变混浊，并产生少量T4。结论采用1．0×（10^8-10^9）PFU／mL的T4能够在5min内有效而完全侵染模拟样品中大肠杆菌。

聚丙烯酰胺类油田添加剂生物毒性测试方法

为了有效解决明亮发光细菌T3价格暴涨且供货不稳定的问题,采用费氏弧菌为测试菌种,ZnSO4·7H2O为参比毒物,通过单因素实验探讨了不同时间内费氏弧菌的稳定性,考察了不同浓度的参比毒物对菌种发光强度和抑制率的影响,研究了费氏弧菌在油田添加剂生物毒性测试中的应用.结果表明:费式弧菌在测试过程中存活性高、稳定性好;参比毒物ZnSO4·7H2O浓度越大,发光强度越低,抑制率越高;费氏弧菌测得的降滤失剂的毒性大于包被剂,最佳测试时间为15~30 min.

噬菌体展示技术筛选乙型肝炎病毒X蛋白相互作用蛋白

目的利用T7噬菌体展示技术筛选与乙型肝炎病毒X蛋白相互作用的蛋白。方法构建X蛋白的原核表达载体,表达并纯化X蛋白,以X蛋白作为靶蛋白,应用T7噬菌体展示技术对人肝细胞cDNA文库进行筛选,对筛选到的阳性克隆进行DNA序列分析及同源性研究。结果表达并纯化了X蛋白,经过4轮筛选后,选择52个阳性克隆,采用T7特异性引物扩增插入片段,回收PCR产物进行测序。经过同源性分析,确定了7个与乙型肝炎X蛋白相互作用的蛋白。结论 T7噬菌体展示筛选是1种快速、简单的筛选蛋白质相互作用的工具,筛选的与X蛋白相互作用蛋白为进一步研究乙型肝炎病毒的致病机制奠定了基础。

噬菌体展示技术筛选人肠道病毒71型3A蛋白相互作用蛋白

目的利用T7噬菌体展示技术筛选与人肠道病毒71型3A蛋白相互作用的蛋白。方法构建3A蛋白的原核表达载体,表达并纯化3A蛋白,以3A蛋白为靶蛋白,应用T7噬菌体展示技术对人肝细胞cDNA文库进行筛选,对筛选到产物进行DNA序列分析及同源性研究。结果表达并纯化了3A蛋白,经过4轮筛选后,选择37个阳性克隆,采用T7特异性引物扩增插入片段,将PCR产物送公司进行测序。经过同源性分析,确定了2个与人肠道病毒3A蛋白相互作用蛋白。结论通过T7噬菌体展示技术可以得到与3A蛋白相互作用蛋白,通过研究此蛋白的功能就可以初步推测出3A蛋白可能的功能,为进一步研究人肠道病毒的致病机制鉴定了基础。

抗生素的药效学指标与给药方案优化

目的 :阐述依据抗生素的药效学指标制定给药方案在临床上可以获得最佳疗效。方法 :通过分析抗生素的药效学指标与临床疗效的相关性 ,阐明药效学指标在优化给药方案中起重要作用。结果 :应用药效学的特点可以把抗生素分为浓度依赖性和时间依赖性两类 ,不同抗生素具有不同的抗生素后效应 ,目前已有的药效学指标如AUC、AUIC、MIC、PAE可以客观地反映抗生素的疗效 ,为确定合理给药方案提供依据。结论 :临床根据各种抗生素的药效学指标制定给药方案 。

肾癌T7噬菌体展示肽库构建的实验研究

目的：构建肾细胞癌T7噬菌体展示肽库,为下一步筛选肾癌早期诊断分子标志群打下基础。方法：用传统Trizol方法分别抽取31例涵盖各种组织类型肾癌组织标本的总RNA,确定完整性后再根据OD值等量混合总RNA,用试剂盒完成mRNA的分离并电泳检测其质量,经反转录、末端平齐、片段长短筛选、加接头、双酶切、去除多余接头和〈300 bp的cDNA片段等步骤,再与T7Select10-3b载体连接、体外包装并扩增得到肾癌T7噬菌体展示cDNA文库。通过铺平板测定和PCR技术鉴定所建文库质量。结果：建成原始文库的库容量为5.0×10^7pfu/mL,扩增后文库滴度为3.5×10^12pfu/mL,重组率为95%,插入片段为300-2 000 bp。结论：成功用T7噬菌体构建了高质量的肾癌cD-NA文库,为筛选可用于临床早期诊断的肾癌特异标志奠定基础。