大肠杆菌-长双歧杆菌穿梭载体的构建及PTEN在长双歧杆菌中的表达

长双歧杆菌可特异地定植于实体瘤低氧区,可用做肿瘤靶向性基因治疗的载体,而构建大肠杆菌-长双歧杆菌穿梭质粒则被证明是外源基因在长双歧杆菌中稳定表达的有效途径。为了构建能在长双歧杆菌中稳定表达外源基因的穿梭质粒并检测携带抑癌基因的工程菌对小鼠实体瘤的抑制效果,利用软件设计并合成了48条部分序列相互重叠的引物,通过PCR合成了长双歧杆菌质粒pMB1序列及长双歧杆菌HU启动子区序列,插入克隆载体pMD18-T,构建穿梭载体pMB-HU,该载体可在大肠杆菌DH5α及长双歧杆菌L17中稳定复制。PTEN基因编码具有蛋白质和酯类双重特异性磷酸酶活性的抑癌因子。将PTEN基因cDNA序列插入载体pMB-HU中HU启动子下游,构建重组质粒pMB-HU-PTEN,电击转化长双歧杆菌后,Western blot检测表明,表达产物中存在55kDa的PTEN蛋白特异条带。抑癌试验表明:与对照组相比,携带PTEN基因的长双歧杆菌可显著抑制小鼠实体瘤的生长。上述结果为以长双歧杆菌为载体的实体瘤靶向性基因治疗研究奠定了基础。     

培菲康双歧三联活菌制剂中三株菌对抗菌药物敏感性检测

目的：检测培菲康活菌制剂中双歧杆菌,嗜酸乳杆菌与粪链球菌三株菌对30种抗菌药物的敏感性。方法：用纸片琼脂扩散法（disc agar diffusion,即Kirby-Bauer法）和微量稀释法（microdilution bro9th method)进行检测。结果：这三株菌对大多数抗菌药物敏感,对少数抗菌药物耐受。与文献报导相似,这三株菌与相应的国际参考菌株及临床菌株比较,没有发现明显耐药变异。结论：为避免抗菌药物对双歧三联活菌制剂功效的影响。每天两药宜分开时间服用。服用双歧三联活菌不会引起耐药性的扩散。     

Consecutive oral administration of Bifidobacterium longum MM‐2 improves the defense system against influenza virus infection by enhancing natural killer cell activity in a murine model

Bifidobacterium, one of the major components of intestinal microflora, shows anti‐influenza virus (IFV) potential as a probiotic, partly through enhancement of innate immunity by modulation of the intestinal immune system. Bifidobacterium longum MM‐2 (MM‐2), a very safe bacterium in humans, was isolated from healthy humans and its protective effect against IFV infection in a murine model shown. In mice that were intranasally inoculated with IFV, oral administration of MM‐2 for 17 consecutive days improved clinical symptoms, reduced mortality, suppressed inflammation in the lower respiratory tract, and decreased virus titers, cell death, and pro‐inflammatory cytokines such as IL‐6 and TNF‐α in bronchoalveolar lavage fluid. The anti‐IFV mechanism of MM‐2 involves innate immunity through significant increases in NK cell activities in the lungs and spleen and a significant increase in pulmonary gene expression of NK cell activators such as IFN‐γ, IL‐2, IL‐12 and IL‐18. Even in non‐infected mice, MM‐2 administration also induced significant enhancement of both IFN‐γ production by Peyer's patch cells (PPs) and splenetic NK cell activity. Oral administration of MM‐2 for 17 days activates systemic immunoreactivity in PPs, which contributes to innate immunity, including NK cell activation, resulting in an anti‐IFV effect. MM‐2 as a probiotic may function as a prophylactic agent in the management of an IFV epidemic.     

富硒双歧杆菌缓解5-FU导致的化疗性肠粘膜炎的药效学研究

通过腹腔注射5-FU建立小鼠肠黏膜炎模型,探讨富硒长双歧杆菌(Selenium-enriched Bifidobacterium longum,Se-B.longum)能否改善5-FU所致的小鼠肠黏膜炎。将健康BALB/c小鼠随机分为对照组、5-FU组和Se-B.longum/5-FU组,分别灌胃生理盐水、生理盐水和Se-B.longum(Se0.3 mg/kg BW,1×106bacteria/只)6 d,然后5-FU组和Se-B.longum/5-FU组小鼠均腹腔注射5-FU(250 mg/kg),观察小鼠腹泻及死亡情况,5 d后处死小鼠,计算体重变化、脏器指数及考察肠道组织变化;将健康BALB/c小鼠随机分为对照组、5-FU组和Se-B.longum/5-FU组,分别灌胃生理盐水、生理盐水和Se-B.longum(Se 0.3 mg/kg BW,1×106bacteria/只)6 d,然后5-FU组和Se-B.longum/5-FU组小鼠均腹腔注射5-FU(300 mg/kg),观察小鼠死亡情况,绘制生存曲线。Se-B.longum能缓解5-FU导致的正常小鼠的肠粘膜炎、降低小鼠死亡率。     

Molecular docking analysis of vascular endothelial growth factor receptor with bioactive molecules from Piper longum as potential anti-cancer agents

It is known that vascular endothelial growth factor receptor (VGFR) is linked with cancer. Therefore, it is of interest to document the molecular binding features of bioactive molecules from Piper longum as potential anti-cancer agents with VGFR2 for further consideration. Thus, we document the binding features of four compounds (sesamin, fargesin, longamide and piperlonguminine) with VGFR2 for further consideration in drug discovery.     

辣椒优良自交系间遗传差异的分子分析

作物自交系间遗传差异的分析与评价是杂种优势育种和杂交育种成功的基础。鲜食尖椒类(Capsicum an-nuumvar.longum)品种是我国辣椒生产的主要品种类型之一。针对我国鲜食尖椒的育种目标,以国内外10份尖椒优良自交系为材料,利用相关序列扩增多态性(SRAP)和简单序列重复(SSR)标记技术对其进行了遗传差异分析。结果显示:SRAP技术具有较高的位点和多态性检测能力,平均每次检测的位点数和多态性位点数分别为34个和10个,是SSR的10倍和5倍;辣椒自交系间基于SRAP标记的遗传距离和基于SSR标记的遗传距离之间的相关程度较低(r=0.144);基于SRAP标记和SSR标记联合数据计算的遗传距离,10个尖椒自交系被分为3大类,这种分类结果与辣椒杂种优势育种实践相一致。本研究结果表明,SRAP具有较高的遗传分析效力;基于不同分子标记遗传分析结果的差异与标记间共享位点的多少有关;10个尖椒自交系的分类结果可用于指导育种实践。     

双歧杆菌对新生大鼠坏死性小肠结肠炎肠损伤及肠细胞凋亡影响

目的探讨添加双歧杆菌对新生鼠坏死性小肠结肠炎(necrotizing enterocolitis,NEC)模型肠损伤的保护作用。方法 32只新生SD大鼠按析因设计随机分成4组,每组动物8只。A1B1组为NEC模型组并添加双歧杆菌(109CFU/d),A1B2组为NEC模型组,但未添加双歧杆菌;A2B1组为对照组并添加双歧杆菌(109CFU/d),A2B2组为对照组,且未添加双歧杆菌。在出生48 h开始给予鼠配方奶人工喂养,100%氮气缺氧90 s,4℃冷刺激10 min,每天2次,连续3 d,建立新生大鼠NEC模型;在最后1次缺氧、冷刺激后24 h空腹断头处死小鼠,解剖留取十二指肠下端至直肠上端肠道组织,其中,回盲部近端肠管进行病理学检查及肠损伤评分,组织学评分≥2为NEC,其余肠管进行肠细胞凋亡率检测及电镜观察。采用流式细胞仪检测肠细胞凋亡率。SPSS 11.0统计学软件进行统计分析,α=0.05为显著性检验标准。结果造模后,A1B1组、A1B2组相继出现腹泻、腹胀、生长发育减慢和活动度减少,显微镜下可见肠黏膜坏死、黏膜下层出血以及肌肉层坏死等肠损伤表现,透射电镜显示肠黏膜出现大量凋亡细胞,形成凋亡小体,但A1B1组程度较轻。A1B1、A1B2、A2B1和A2B24组肠损伤组织病理评分(x±s)分别为2.04±0.52、3.38±0.55、0.33±0.36和0.38±0.33,肠细胞凋亡率分别为(23.97±10.48)%、(47.28±21.98)%、(11.42±4.75)%和(12.16±4.95)%;各组间肠损伤组织评分、肠细胞凋亡率差异有显著统计学意义(H分别为26.657、20.916,P均0.01);与A1B2组相比,A1B1组新生鼠肠损伤组织评分、肠细胞凋亡率均明显降低,但仍高于A2B1、A2B22个对照组(P均0.01);肠组织损伤评分值、肠细胞凋亡率均受到NEC造模和添加双歧杆菌两个因素的影响,NEC造模与补充双歧杆菌之间均存在交互作用。结论添加双歧杆菌可以降低新生鼠NEC肠损伤程度,可能通过抑制肠上皮细胞凋亡,从而降低新生大鼠发生NEC危险性。     

Interaction between Polyoxin and Active Center of Chitin Synthetase

Various derivatives of polyoxin C, other polyoxins and several uridine analogues have been known as competitive inhibitors of chitin-UDP acetylglucosaminyitransferase (EC 2.4.1.16, chitin synthetase). Their inhibitory activities were more or less dependent on pH. The variation of inhibitor constants Ki or Michaelis-Menten parameters Km and V with pH was investigated and the data obtained were plotted according to the method proposed by Dixon et al. From the results of the pKi-pH plots for the above competitive inhibitors, it was concluded that the ionized amino group at C?2″ position acted a very important role for the binding of polyoxins to chitin synthetase. The carbonyl oxygen atoms at C?1″ and of the carbamoyloxy group probably participated in the hydrogen bond formation with the enzyme. And pH scarcely influenced on the interaction between the carboxyl group at C?5″ and the enzyme. The results of the Dixon plots for variations of Km and V with pH suggested that an unionized imidazole group (pK_a = 6.3) and an ionized amino group (pK_a = 7.7) of chitin synthetase were concerned in the enzyme reaction.     

Effect of production conditions on the stability of a human bifidobacterial species Bifidobacterium longum in yogurt

Aims:  Human bifidobacteria are more sensitive to external environmental factors than animal bifidobacteria, and it is difficult to ensure their stable survival in yogurt. The purpose of this investigation was to observe the survival of human bifidobacteria in yogurts produced under various production conditions.
Methods:  Frozen or lyophilized bifidobacteria starters containing Bifidobacterium longum BB536 originally isolated from an infant, and commercial lyophilized yogurt starters were used for yogurt preparation. After producing yogurts under various conditions, the survival of bifidobacteria in these yogurts over various storage periods was observed.
Results:  Although there were some differences in bifidobacterial survival in yogurt between various production conditions, more than 1·0 × 10⁷ CFU g⁻¹ of Bif. longum survived in yogurt after 35 days' storage at 5°C. Lower fermentation temperature (37°C) and inclusion of Lactococcus lactis in the starter significantly ( P  < 0·05) improved survival of Bif. longum in the yogurt.
Conclusion:  In this investigation, the human bifidobacterial strain Bif. longum survived adequately in yogurt, although the fermentation temperature and starter composition affect bifidobacterial survival.
Significance and Impact of the Study:  This investigation indicates that stable probiotic yogurt using human bifidobacteria can be produced by choosing optimal production conditions.