乳杆菌对致病性大肠杆菌感染小鼠肠黏膜屏障功能的影响

目的:研究乳杆菌对致病性大肠杆菌感染小鼠肠黏膜屏障的保护作用.方法:将BALB/c小鼠随机分为对照组、致病性大肠杆菌感染组、乳杆菌JCM1081灌胃组、乳杆菌治疗组等,实验进行14 d.观察各组小鼠肠黏膜形态变化、肠道菌群变化、细菌移位以及肠黏膜钙黏蛋白表达的差异.结果:致病性大肠杆菌感染组小鼠与对照组相比,肠黏膜形态有明显改变,肠道内厌氧菌数量显著下降,肠杆菌和肠球菌数量明显增加,在肝、脾、肾以及肠系膜淋巴结中有细菌存在,但肠黏膜钙黏蛋白无明显变化.乳杆菌灌胃组小鼠各项指标无显著变化.乳杆菌治疗组小鼠各项指标变化程度较致病性大肠杆菌感染组显著减轻.结论:乳杆菌能黏附定植于肠黏膜并形成生物屏障,起着保护肠黏膜免受损伤的作用.     

乳酸菌对感染肠上皮细胞通透性及紧密连接蛋白表达的影响

目的:研究乳酸菌对侵袭性大肠杆菌(EIEC) 感染肠上皮细胞(Caco-2)后,对其通透性和紧密连接(TJ)蛋白表达的影响. 方法:建立Caco-2 单层细胞感染模型.实验分为正常组、感染组、乳酸菌组和庆大霉素组.用电阻仪测定单层细胞的跨膜电阻值(TER),用高效液相法(HPLC)测定甘露醇透过率的变化,采用免疫组化法观察TJ相关蛋白,如Claudin-1蛋白,Occludin蛋白, JAM-1蛋白,ZO-1蛋白的分布和结构变化. 结果:EIEC感染Caco-2细胞后,单层细胞的TER值和甘露醇的透过率随时间延长而升高,而经乳酸菌处理后,TER值升高趋缓(P＜0.05),甘露醇的透过率降低(P＜0.05);EIEC感染后,相邻Caco-2细胞间TJ结构遭到破坏,TJ相关蛋白的表达亦减少,而乳酸菌处理后可使EIEC引起的TJ结构受损减轻、相关蛋白的表达增多. 结论:乳酸菌黏附于肠上皮细胞Caco-2后,可抑制EIEC破坏单层细胞的完整性,并改善TJ的结构变化和相关蛋白的表达分布.     

结直肠癌患者术后肠道菌群变化及微生态制剂治疗效果研究

目的:探讨结直肠手术对结直肠癌患者肠道菌群的影响及微生态制剂治疗肠道菌群失调的效果.方法:选择50例结直肠癌择期手术患者随机分为微生态组和对照组各25例,术后对照组常规处理,微生态组给予三联活菌制剂口服治疗,采用细菌DNA PCR分析定量肠道细菌量,检测血浆D-乳酸和尿L/M水平,对比手术前后肠道菌群变化和肠道屏障功能.结果:术后10天双歧杆菌、乳酸杆菌显著降低,而微生态组显著高于对照组,大肠杆菌、粪肠球菌显著升高,微生态组显著低于对照组,B/E呈显著倒置,对照组倒置更为显著(P值＜0.05);术后60天,微生态组双歧杆菌、乳酸杆菌、大肠杆菌、粪肠球菌量及B/E基本恢复术前水平,而对照组仍显著低于术前(P值＜0.05).术后1天血浆D-乳酸和尿L/M水平显著升高(P值＜0.05),术后10天两组血浆D-乳酸和尿L/M水平均回落,微生态组回落较对照组显著且显著低于对照组(P值＜0.05).结论:结直肠癌术后患者肠道内双歧杆菌、乳酸杆菌等益生菌显著减少,大肠杆菌和粪肠球菌显著增加,肠道菌群严重失调,肠道屏障功能受损,微生态制剂治疗有助重建肠道菌群平衡,恢复肠道屏障功能.     

铜对Caco-2细胞单层屏障功能的影响

目的研究铜对Caco2细胞单层细胞间通透性、P糖蛋白(Pgp)活性的影响。方法应用Caco2细胞单层模型,通过测定铜暴露后细胞单层的跨上皮细胞电阻(TEER)来反映通透性的改变;通过免疫荧光和荧光染色观察铜对紧密连接蛋白ZO1和F微丝的影响;通过测定细胞单层对罗丹明123的跨膜转运和细胞内罗丹明123的蓄积来反映P糖蛋白活性的改变。结果细胞单层顶面铜暴露(30～100μmol/L,Hanks缓冲溶液,0～3h)可导致TEER值呈浓度和时间依赖性降低,同时伴随着F微丝的解聚,但对紧密连接蛋白ZO1无显著影响;在不影响细胞活性和细胞单层通透性的剂量下,顶面铜暴露(300μmol/L,完全培养基,24h)后细胞单层对罗丹明123的表观通透系数(Papp)BL→AP从对照组的(737±020)10-6cm/s降低为(643±027)×10-6cm/s,PappAP→BL从对照组(123±005)×10-7cm/s增加为(341±008)×10-7cm/s,同时细胞内罗丹明-123的蓄积量从对照组的每膜(031±001)nmol增加为每膜(050±003)nmol。结论铜可显著增加Caco2细胞单层的通透性和抑制Pgp的活性,从而推测可能影响肠道上皮细胞的正常屏障功能。     

0.4mT工频磁场对CHL细胞微丝装配的影响

目的 探讨0.4mT工频磁场对中国仓鼠肺成纤维细胞(CHL)微丝(F-actin)装配的影响及作用机制。方法 用免疫荧光染色法标记F-actin,激光共聚焦荧光显微镜观察F-actin形态并拍照,用Western-blotting方法检测了与去垢剂不可溶的细胞骨架相连的表皮生长因子受体(EGFR)蛋白的含量。结果 0.4 mT工频磁场辐照CHL细胞30min导致细胞F-actin应力纤维减少,在细胞周边出现丝状伪足,磁场对CHL细胞F-actin形态的影响与细胞经50nmol/L的表皮生长因子(EGF)作用30min类似,且磁场和EGF都使与去垢剂不可溶的细胞骨架相连的EGFR增多。结论 0.4mT工频磁场辐照导致CHL细胞F-actin装配发生变化,磁场对F-actin的影响可能与磁场诱导表皮生长因子受体聚集,并引起信号向下传递有关。     

0.2 mT工频磁场引起人羊膜成纤维细胞微丝骨架重组

目的探讨50Hz工频磁场对人羊膜成纤维细胞微丝装配的影响,并分析磁场对肌动蛋白和表皮生长因子受体蛋白表达的影响。方法将人羊膜成纤维细胞分别用0．1、0．2．0．3、0．4、0．5mT,50Hz工频磁场辐照30min,以异硫氰四甲基罗丹明一鬼笔环肽标记细胞微丝并在显微镜下观察分析微丝形态的变化。用荧光光谱及紫外光谱检测法测定细胞内微丝总含量,用激光共聚焦显微镜逐层扫描的方法观测细胞微丝骨架的高度,用扫描电镜观测细胞外部形态等方法进一步分析磁场对细胞微丝骨架及细胞形态的影响。用Western blotting检测去垢剂不可溶的蛋白的表达量以分析磁场作用的可能机制。结果人羊膜成纤维细胞经不同强度工频磁场辐照30min后,只有0．2mT工频磁场辐照显著导致细胞中平行排列的微丝应力纤维减少,诱导细胞周边出现致密微丝外周带并伸出丝状伪足,停止辐照2h后微丝形态恢复,伪足消失。光谱检测发现胞内微丝总含量无显著变化。激光共聚焦显微镜逐层扫描观测到0．2mT磁场辐照使细胞微丝骨架平均高度由（12．37±1．28）μm降低到（9．97±0．38）μm（t=6．96,P〉0．05）。扫描电镜图像显示磁场辐照使细胞更为扁平,有足状伪足出现。Western blotting检测到磁场辐照后细胞骨架中去垢剂不可溶部分的肌动蛋白和表皮生长因子受体含量与对照组相比,分别升高（16．8±2．3）％（t=16．68,P〈0．05）和（31．2±4．1）％（t=17．10,P〈0．05）。结论0．2mT工频磁场短时辐照影响了人羊膜成纤维细胞微丝骨架的形态,此效应可随磁场撤销而解除,且可能与磁场诱导了细胞膜上表皮生长因子受体聚簇有关。     

益生菌对急性胰腺炎大鼠肠道微生态及紧密连接的影响

目的 探讨经肠道补充益生菌对急性胰腺炎（AP）大鼠肠上皮细胞紧密连接、屏障功能及微生态的影响.方法 经胰管注射3%牛磺胆酸钠造成大鼠AP后,分别给予肠外营养（PN组）和PN＋益生菌（益生菌组）,持续5天;第6天处死大鼠,取腔静脉血、肺及肝组织和肠系膜淋巴结匀浆作细菌培养测细菌易位率;取末段回肠和结肠,采用免疫组织化学法测定其跨膜结合蛋白表达,电镜观察肠上皮细胞紧密连接超微结构;取盲肠内粪便作厌氧培养及细菌种群DNA指纹图谱分析.结果 益生菌组大鼠肠道内乳杆菌和双歧杆菌数量高于PN组（P＜0.05）,而潜在致病菌产气荚膜梭菌低于PN组（P＜0.05）,DNA指纹图谱也显示益生菌组优势菌群的基因条带和正常大鼠具有较高一致性,与PN组相比则有明显差异;益生菌组小肠和大肠跨膜结合蛋白的表达明显优于PN组（P=0.001,P=0.036）,肠上皮细胞紧密连接、微绒毛较完整;益生菌组大鼠的血、肺、肝、肠系膜淋巴组织的细菌易位率低于PN组（P=0.0413）.结论 益生菌能纠正AP大鼠PN时肠道菌群紊乱,增加肠上皮跨膜结合蛋白表达,维持肠上皮紧密连接,减少细菌易位。     

CPP-ACP对发酵乳杆菌生长、黏附影响的实验研究

[目的]研究酪蛋白磷酸肽钙磷复合体(Casein phosphopeptide-Amorphic Calcium Phosphate,CPP-ACP)对发酵乳杆菌生长、黏附的影响,进一步探讨CPP-ACP的防龋机制。[方法]将发酵乳杆菌接种到BHI培养基,实验组中加入不同浓度(0.5%～5.0%(W/V))的CPP-ACP溶液,厌氧培养48h后,采用噻唑蓝(MTT)比色分析法测定细菌浓度的吸光度A值(λ=550nm)。采用唾液包被的羟磷灰石(Saliva-coated hydroxyapatite,S-HA)形成实验性膜,用不同浓度(0.5～5.0%(W/V))CPP-ACP处理S-HA,定量观察发酵乳杆菌在S-HA上的黏附情况。[结果]随CPP-ACP浓度的升高,发酵乳杆菌甲臜产物的二甲亚砜溶液的吸光度值降低,即发酵乳杆菌的活菌数减少(P﹤0.01)。发酵乳杆菌对经各实验浓度的CPP-ACP处理后的S-HA黏附能力明显下降,黏附量(cpm)随CPP-ACP浓度的升高而降低,黏附抑制率达100%。[结论]CPP-ACP对发酵乳杆菌的生长以及在S-HA上的黏附具有抑制作用,随CPP-ACP浓度的升高抑制作用增强。     

碱性鞘磷脂酶对Caco-2细胞胆固醇吸收的影响及机制研究

目的探讨碱性鞘磷脂酶(Alk-SMase)对Caco-2细胞胆固醇吸收的影响以及可能机制。方法建立Caco-2细胞单层模型,用100 units/L Alk-SMase孵育细胞24 h后,再用[14C]-胆固醇微胶溶液孵育细胞2 h。用液闪计数仪(LSC)检测细胞胆固醇吸收量,薄层层析法分离膜磷脂、LSC检测细胞膜[14C]-鞘磷脂含量。结果 [14C]-胆固醇在Caco-2细胞中的吸收呈时间依赖性增加。Alk-SMase能够明显降低Caco-2细胞胆固醇吸收,并减少Caco-2细胞膜鞘磷脂含量,100 mu/ml Alk-SMase能够引起80%[14C]-标记的膜鞘磷脂水解。结论 Alk-SMase能够抑制Caco-2细胞胆固醇吸收,这可能与其水解膜鞘磷脂有关。     

肠上皮细胞的紧密连接与肠道疾病

紧密连接(TJ)是肠上皮细胞间的主要连接方式,在维持肠上皮细胞极性和调节肠黏膜屏障的通透性中发挥着重要的作用.TJ受损与多种疾病的发生发展相关.现主要就肠上皮细胞TJ的结构、功能、调节途径及其与相关疾病的关系进行综述.     

Effect of lactobacilli on paracellular permeability in the gut

Paracellular permeability is determined by the complex structures of junctions that are located between the epithelial cells. Already in 1996, it was shown that the human probiotic strain Lactobacillus plantarum 299v and the rat-originating strain Lactobacillus reuteri R2LC could reduce this permeability in a methotrexate-induced colitis model in the rat. Subsequently, many animal models and cell culture systems have shown indications that lactobacilli are able to counteract increased paracellular permeability evoked by cytokines, chemicals, infections, or stress. There have been few human studies focusing on the effect of lactobacilli on intestinal paracellular permeability but recently it has been shown that they could influence the tight junctions. More precisely, short-term administration of L. plantarum WCSF1 to healthy volunteers increased the relocation of occludin and ZO-1 into the tight junction area between duodenal epithelial cells.     

Enteropathogenic E. coli, Salmonella, and Shigella: masters of host cell cytoskeletal exploitation

Bacterial pathogens have evolved numerous strategies to exploit their host's cellular processes so that they can survive and persist. Often, a bacterium must adhere very tightly to the cells and mediate its effects extracellularly, or it must find a way to invade the host's cells and survive intracellularly. In either case, the pathogen hijacks the host's cytoskeleton. The cytoskeleton provides a flexible framework for the cell and is involved in mediating numerous cellular functions, from cell shape and structure to programmed cell death. Altering the host cytoskeleton is crucial for mediating pathogen adherence, invasion, and intracellular locomotion. We highlight recent advances in the pathogenesis of enteropathogenic Escherichia coli, Salmonella Typhimurium, and Shigella flexneri. Each illustrates how bacterial pathogens can exert dramatic effects on the host cytoskeleton.     

Glutamine reduces phorbol-12,13-dibutyrate-induced macromolecular hyperpermeability in HT-29Cl.19A intestinal cells.

BACKGROUND: Loss of mucosal integrity is associated with intestinal hyperpermeability, which may be inhibited by glutamine. The nature of this effect is unknown. The effect of glutamine on protein kinase C (PKC)-mediated hyperpermeability in HT-29Cl.19A intestinal cells was studied. METHODS: Confluent monolayers of HT-29C1.19A cells were cultured on permeable filters and mounted in Ussing chambers for permeability studies. Apical to basolateral transepithelial permeability for intact horseradish peroxidase (HRP) was determined. Phorbol-12,13-dibutyrate (PDB) was used to activate PKC-mediated hyperpermeability, and the effect of glutamine (0.6 mmol/L) was studied. RESULTS: Two hours of PDB stimulation increased the HRP flux, reaching five times control values after 4 hours. Bilateral exposure to glutamine for 4 hours reduced PDB-induced hyperpermeability (37%). Preincubation with glutamine 2 hours before PDB stimulation showed an earlier and greater effect (3 hours, 43%; 4 hours, 50%). This bilateral effect of glutamine was mimicked by separate apical exposure. Basolateral exposure alone had no effect. CONCLUSIONS: Glutamine rapidly reduced the PKC-mediated hyperpermeability for HRP in HT-29Cl.19A intestinal cells. The dependency on apical exposure suggests that glutamine may be more effective when delivered by the enteral route.     

P fimbriae and other adhesins enhance intestinal persistence of Escherichia coli in early infancy

Resident and transient Escherichia coli strains were identified in the rectal flora of 22 Pakistani infants followed from birth to 6 months of age. All strains were tested for O-antigen expression, adhesin specificity (P fimbriae, other mannose-resistant adhesins or type 1 fimbriae) and adherence to the colonic cell line HT-29. Resident strains displayed higher mannose-resistant adherence to HT-29 cells, and expressed P fimbriae (P = 0.0036) as well as other mannose-resistant adhesins (P = 0.012) more often than transient strains. In strains acquired during the first month of life, P fimbriae were 12 times more frequent in resident than in transient strains (P = 0.0006). The O-antigen distribution did not differ between resident and transient strains, and none of the resident P-fimbriated strains belonged to previously recognized uropathogenic clones. The results suggest that adhesins mediating adherence to intestinal epithelial cells, especially P fimbriae, enhance the persistence of E. coli in the large intestine of infants.     

Competition for adhesion between probiotics and human gastrointestinal pathogens in the presence of carbohydrate

The adhesion of Lactobacillus rhamnosus GG to human enterocyte-like Caco-2 cells was not inhibited by eight carbohydrates tested, namely N-acetyl-glucosamine, galactose, glucose, fructose, fucose, mannose, methyl-alpha-D-mannopyranoside and sucrose. The degree of hydrophobicity predicted the adhesion of L. rhamnosus GG to Caco-2 cells. L. rhamnosus GG, however, was able to compete with Escherichia coli and Salmonella spp. of low hydrophobicity and high adhesin-receptor interaction for adhesion to Caco-2 cells. The interference of adhesion of these gastrointestinal (GI) bacteria by L. rhamnosus GG was probably through steric hindrance, and the degree of inhibition was related to the distribution of the adhesin receptors and hydrophobins on the Caco-2 surface. A Carbohydrate Index for Adhesion (CIA) was used to depict the binding property of adhesins on bacteria surfaces. CIA was defined as the sum of the fraction of adhesion in the presence of carbohydrates, with reference to the adhesion measured in the absence of any carbohydrate. The degree of competition for receptor sites between Lactobacillus casei Shirota and GI bacteria is a function of their CIA distance. There were at least two types of adhesins on the surface of L. casei Shirota. The study provides a scientific basis for the screening and selection of probiotics that compete with selective groups of pathogens for adhesion to intestinal surfaces. It also provides a model for the characterisation of adhesins and adhesin-receptor interactions.     

Induction of apoptosis in HT-29 colon cancer cells by phloretin

Phloretin, which is present in apples and pears, has been found to inhibit the growth of several cancer cells and induce apoptosis of B16 melanoma and HL60 human leukemia cells. The present study examined whether and how phloretin induces apoptosis of HT-29 human colon cancer cells. Phloretin (0-100 micromol/L) substantially decreased viable cell number and induced apoptosis of HT-29 cells in a dose-dependent manner. Western blot analysis of total cell lysates revealed that phloretin increased the protein levels of Bax but had no effect on Bcl-2. In addition, phloretin induced cleavage of caspase-8, -9, -7, and -3 and poly(ADP-ribose) polymerase. Furthermore, phloretin increased the levels of cytochrome c and Smac/Diablo in the cytosol. The present results indicate that phloretin inhibits HT-29 cell growth by inducing apoptosis, which may be mediated through changes in mitochondrial membrane permeability and activation of the caspase pathways.     

Survival of Campylobacter jejuni and pathogenic Escherichia coli in mahewu, a fermented cereal gruel

The survival of strains of Campylobacter jejuni and enteropathogenic and enterotoxigenic (LT) Escherichia coli was investigated in mahewu, a traditional fermented cereal gruel. The mahewu was inoculated with 10(6) to 10(7) colony forming units of the bacterial cells per ml of the cereal gruel. None of the strains of C. jejuni were detected 30 min after inoculation. All the strains of E. coli were detected after 24 h in mahewu but did not show any increase in numbers of surviving cells. The enterotoxigenic E. coli strains showed a more marked decrease in numbers than did enteropathogenic E. coli strains during the 24 h period after inoculation in the mahewu. This suggests that mahewu has some antimicrobial qualities and is unlikely to play a major role in the transmission of these 2 enteric pathogens.     

Induction of apoptosis by the aqueous extract of Rubus coreanum in HT-29 human colon cancer cells

OBJECTIVES: The incompletely ripened fruit of Rubus coreanum (IRFRC) has been used in traditional herbal medicine to manage various diseases. To explore the possibility that IRFRC has chemopreventive effects, we examined whether or not extracts of IRFRC inhibits HT-29 cell growth and explored the mechanism for this effect. METHODS: We cultured HT-29 cells in the presence of the aqueous or ethanol extract of IRFRC. DNA synthesis was estimated by 5-bromo-2'-deoxyuridine incorporation. We measured apoptosis using a DNA fragmentation assay and Annexin V staining. We used western blot analyses to determine the cleavage of caspases and poly (adenosine diphosphate-ribose) polymerase. RESULTS: Aqueous extract of IRFRC substantially inhibited viable HT-29 cell number in a dose-dependent manner, whereas ethanol extract had only a minimal effect. Aqueous extract inhibited DNA synthesis and induced apoptosis of HT-29 cells in a dose-dependent manner. Aqueous extract induced cleavage of caspase-3, -7, and -9 and induced the activity of caspase-3 and cleavage of poly (adenosine diphosphate-ribose) polymerase. CONCLUSIONS: We have shown that aqueous extract of IRFRC inhibits cell proliferation and stimulates apoptosis in HT-29 cells, and that this may be mediated by its ability to activate the caspase-3 pathway. It remains to be determined whether the aqueous extract of IRFRC has chemopreventive activities in animal models.