Transepithelial transport of putrescine across monolayers of the human intestinal epithelial cell line, Caco-2

AIM: To study the transepithelial transport characteristics of the polyamine putrescine in human intestinal Caco-2 cell monolayers to elucidate the mechanisms of the putrescine intestinal absorption. METHODS: The transepithelial transport and the cellular accumulation of putrescine was measured using Caco-2 cell monolayers grown on permeable filters. RESULTS: Transepithelial transport of putrescine in physiological concentrations (】 0.5 mM) from the apical to basolateral side was linear. Intracellular accumulation of putrescine was higher in confluent than in fully differentiated Caco-2 cells, but still negligible (less than 0.5%) of the overall transport across the monolayers in apical to basolateral direction.EGF enhanced putrescine accumulation in Caco-2 cells by four fold, as well as putrescine conversion to spermidine and spermine by enhancing the activity of S adenosylmethionine decarboxylase. However, EGF did not have any significant influence on putrescine flux across the Caco-2 cell monolayers. Excretion of putrescine from Caco-2 cells into the basolateral medium did not exceed 50 picomoles, while putrescine passive flux from the apical to the basolateral chamber, contributed hundreds of micromoles polyamines to the basolateral chamber. CONCLUSION :Transepithelial transport of putrescine across Caco2 cell monolayers occurs in passive diffusion, and is not influenced when epithelial cells are stimulated to proliferate by a potent mitogen such as EGF.

Quantitative Comparison of Bile Acid Distribution and Intestinal Transport from Native Cow-bezoar and Artificial and in vitro Cultured Substitutes using Caco-2 Cell Monolayer Model

Objective This study was conducted to examine the absorption and translocation of conjugated bile acids(BAs)in Calculus bovis and its substitutes to detect differences in these materials.Methods A Caco-2 monolayer cell model was used to compare the apparent permeability coefficient(Papp)value and efflux ratio(ER)of BAs in natural cow-bezoar(NCB),artificial cow-bezoar(ACB),and in vitro cultured cow-bezoar(Ivt-CCB).Papp and ER values were determined by liquid chromatography-mass spectrometry.Samples were separated on an analytical column.Results The distribution of BAs in NCB was significantly different from that in ACB and Ivt-CCB.The percentages of conjugated BAs were significantly higher in NCB than in the two substitutes.The distribution differences of conjugated and unconjugated BAs can be used to distinguish costly NCB from relatively inexpensive substitutes.Conclusion The transport characteristics of BAs in Ivt-CCB were more consistent with NCB than with ACB,even when the proportions of BAs in Ivt-CCB were closer to those of ACB.

基于自噬角度探究A2AR对内毒素诱导的Caco-2肠上皮细胞炎性损伤的影响

目的探究腺苷A2A受体(A2AR)对内毒素脂多糖(LPS)诱导的Caco-2肠上皮细胞炎性损伤的影响及机制。方法首先将Caco-2细胞分为对照组、LPS组(10μg/ml LPS处理12 h)、A2AR激动剂(CGS21680)组(10μmol/L CGS21680预处理10 min)、CGS21680+LPS组(10μmol/L CGS21680预处理10 min、10μg/ml LPS处理12 h),CCK-8法测定各组细胞活力,ELISA法测定各组细胞上清液中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)的分泌水平,实时荧光定量PCR检测各组细胞中TNF-α、IL-1β、IL-6的mRNA表达水平,Western blot分析各组细胞中自噬相关蛋白微管相关轻链蛋白3(LC3)-Ⅱ/LC3-Ⅰ、自噬相关蛋白(Beclin1)的蛋白表达水平;再将Caco-2细胞分为对照组、LPS组(10μg/ml LPS处理12 h)、CGS21680+LPS组(10μmol/L CGS21680预处理10 min、10μg/ml LPS处理12 h)、CGS21680+LPS+雷帕霉素(Rapa)组(10μmol/L CGS21680预处理10 min、10μg/ml LPS与5μmol/L Rapa处理12 h),CCK-8法测定各组细胞活力,ELISA法测定各组细胞上清液中TNF-α、IL-1β、IL-6的分泌水平。结果与对照组比较,LPS组Caco-2细胞活力显著降低(P<0.05),上清液中TNF-α、IL-1β、IL-6的水平显著升高(P<0.05),细胞中TNF-α、IL-1β、IL-6的mRNA相对表达量均显著上调(P<0.05),并检测到LC3-Ⅱ/LC3-Ⅰ比值与Beclin1蛋白相对表达量显著上调(P<0.05);与LPS组比较,CGS21680+LPS组Caco-2细胞活力显著升高(P<0.05),上清液中TNF-α、IL-1β、IL-6的水平显著降低(P<0.05),细胞中TNF-α、IL-1β、IL-6的mRNA相对表达量均显著下调(P<0.05),且LC3-Ⅱ/LC3-Ⅰ比值与Beclin1蛋白相对表达量显著下调(P<0.05);此外,与CGS21680+LPS组比较,CGS21680+LPS+Rapa组Caco-2细胞活力又显著降低(P<0.05),上清液中TNF-α、IL-1β、IL-6的水平也显著升高(P<0.05)。结论使用A2AR激动剂能够减轻内毒素LPS诱导的Caco-2肠上皮细胞炎性损伤,提高细胞活力,这可能与其抑制自噬水平有关。

Separate basolateral and apical phosphatidylcholine secretion routes in intestinally differentiated tumor cells

AIM:To investigate whether the secretion of phospha-tidylcholine(PC)in intestinal mucus occurs by apical secretion or via basolateral excretion and to determine its subsequent passage across the tight junctions to the apical mucus.METHODS:We addressed this question using the po-larized intestinally differentiated tumor cell line CaCo-2 grown on filters to confluence in Transwell culture chambers.The released PC and sphingomyelin(Sph)from apical and basolateral media were analyzed by mass spectrometry.RESULTS:The secreted PC species were identical in both compartments indicating the same intracellular origin of PC.However,PC secretion into the basolateral compart-ment was more effective,and the PC:Sph ratio in the ba-solateral compartment was signif icantly higher than that in the apical compartment(8.18 ± 1.84 vs 4.31 ± 1.22,P = 0.01).Both pathways were temperature sensitive and were unaltered in the presence of cyclosporine.CONCLUSION:The data demonstrate the PC secre-tion capacity of CaCo-2 cells and indicate two sepa-rated apical and basolateral release mechanisms.

Interaction between enteric epithelial cells and Peyer's patch lymphocytes in response to Shigella lipopolysaccharide: Effect on nitric oxide and IL-6 release

AIM： TO investigate the effect of interaction between enteric epithelial cells and lymphocytes of Peyer＇s patch on the release of nitric oxide （NO） and IL-6 in response to Shigella lipopolysaccharide （LPS）. METHODS： Human colonic epithelial cells （Caco-2） were mixed cocultured with lymphocytes of Peyer＇s patch from wild-type （C57 mice） and inducible NO synthase knockout mice, and challenged with Shigella F2a-12 LPS. Release of NO and raiL-6 was measured by Griess colorimetric assay and enzyme-linked immunosorbent assay （ELISA）, respectively. RESULTS： In the absence of LPS challenge, NO was detected in the culture medium of Caco-2 epithelial cells but not in lymphocytes of Peyer＇s patch, and the NO release was further up-regulated in both cocultures with lymphocytes from either the wild-type or iNOS knockout mice, with a significantly higher level observed in the coculture with iNOS knockout lymphocytes. After Shigella F2a-12 LPS challenge for 24-h, NO production was significantly increased in both Caco-2 alone and the coculture with lymphocytes of Peyer＇s patch from the wild-type mice but not from iNOS knockout mice. LPS was found to stimulate the release of mIL-6 from lymphocytes, which was suppressed by coculture with Caco-2 epithelial cells. The LPS-induced mIL-6 production in lymphocytes from iNOS knockout mice was significantly greater than that from the wild-type mice. CONCLUSION： Lymphocytes of Peyer＇s patch maintain a constitutive basal level of NO production from the enteric epithelial cell Caco-2. LPS-induced mIL-6 release from lymphocytes of Peyer＇s patch is suppressed by the cocultured epithelial cells. While no changes are detectable in NO production in lymphocytes from both wild-type and iNOS knockout mice before and after LPS challenge, NO from lymphocytes appears to play an inhibitory role in epithelial NO release and their own mIL-6 release in response to LPS.

Differential activation of mitogen-activated protein kinases by γ-irradi-ation in IEC-6 cells: Role of intracellular Ca^(2+)

Objective: To explore the effects of γ-irradiation on mitogen-activated protein kinases (MAPKs) and role of intracellular calcium in this event in intestinal epithelial cell line 6 (IEC-6 cells). Methods: After cultured rat IIEC-6 cells with or without the pretreatment of intracellular Ca2+ chelator were exposed to Y-ir-radiation of 6 Gy, the total and phosphorylated MAPKs in the cells were determined with Western blotting and apoptosis was examined with flow cytometry. Activities of Extracellular signal-regulated protein kinase (ERK) and p38 MAPK were determined by using immuoprecipitation followed by Western blotting. Results: In response to γ-irradiation, phosphorylation of ERK was not significantly observed, while the levels of phosphorylated c-Jun NH2-terminal kinase (JNK) and p38 MAPK were increased in 30 min and reached the peak 2 h after exposure to 6 Gy γ-irradiation, though the cell viability was significantly lowered 12 h. On the other hand, no obvious changes were seen in the total protein levels of ERK, JNK and p38 MAPK. Chelation of intracellular Ca2+ almost completely suppressed the JNK and p38 MAPK phosphorylation induced by γ-irradia-tion, but removal of external Ca2+ had no such effect. Activation of p38 MAPK, but not of ERK, was seen to have a correlation with γ-irradiation induced apoptosis. Conclusion: The results suggest that γ-irradiation is a potent activator for JNK and p38 MAPK, and Ca2+ mobilized from intracellular stores plays an important role in the activation of MAPKs and the induction of apoptosis in IEC-6 cells.

3D （Three-Dimensional） Caco-2 Spheroids： Optimized in vitro Protocols to Favor Their Differentiation Process and to Analyze Their Cell Growth Behavior

3D （Three-dimensional） Caco-2 spheroids closely recapitulating in vivo physiological organization of intestinal epithelial cells, provide an excellent in vitro model system to study their pathophysiology and their response to stressful stimuli. The objective of this technical note is to provide optimized in vitro experimental protocols for culturing 3D Caco-2 spheroids and for analyzing their cell growth features. An optimized 3D Caco-2 spheroid culturing technique based on a new configuration of the culture medium is provided A methodological approach to determine the distribution of the cell cycle phases in disaggregated Caco-2 spheroids by using cytofluorimetric analysis is also described. The optimized culturing protocol favors 3D Caco-2 spheroid differentiation process, as evaluated by the number of well-differentiated spheroids with a single hollow lumen. The cytofluorimetric analysis allows rapid collection of cell cycle phase data from high numbers of spheroid samples, thus, permitting to estimate their growth dynamics in a relatively short time. The optimized technical approaches described here can be applied in systematic manner to a variety of research activities utilizing 3D Caco-2 spheroids. Ease of use, time and economic saving advantages deriving from these protocols further highlight their potential.

Rho激酶在氢气保护LPS诱导的Caco-2细胞上皮屏障功能障碍中的机制研究

目的探讨Rho激酶(ROCK)在氢气改善离体脓毒症肠屏障功能中的作用。方法常规培养人结肠上皮细胞Caco-2,分为6组(n=3):对照组(C组)、富氢培养基组(H组)、脂多糖(LPS)处理组(L组)、富氢培养基+LPS组(HL组)、Rho激酶抑制剂Y-27632组(Y组)、Y-27632+LPS组(YL组)。H组给予0.6 mmol/L富氢培养基;LPS和Y-27632的处理浓度分别为50 mg/L、25μmol/L。建立Transwell小室模型,定期检测跨上皮电阻值(TEER值),当TEER值达到800Ω·cm^2后给予处理,于6、12、24 h检测TEER值,24 h时检测FITC-右旋糖酐通过率。细胞接种于6孔板,融合达80%~90%后给予处理,实时聚合酶链式反应技术检测闭锁小带蛋白1(ZO-1)mRNA和ROCK mRNA表达情况;蛋白免疫印迹技术检测ZO-1蛋白和ROCK蛋白表达水平。结果与C组比较,H组12、24 h TEER值升高(P<0.05),FITC-右旋糖酐通过率、ZO-1蛋白和ROCK蛋白表达水平差异无统计学意义;Y组6、12、24 h TEER值升高(P<0.05),FITC-右旋糖酐通过率差异无统计学意义,ZO-1 mRNA表达增加,ROCK mRNA表达减少(均P<0.05);L组6、12、24 h TEER值降低,FITC-右旋糖酐通过率增高,ZO-1 mRNA和蛋白表达均下降,ROCK mRNA和蛋白表达均增加(P<0.05)。与L组比较,6、12、24 h YL组TEER值增高,FITC-右旋糖酐通过率降低,ZO-1 mRNA表达增加,ROCK mRNA表达降低(均P<0.05)。与L组比较,HL组6、12、24 h TEER值增高,FITC-右旋糖酐通过率降低,各时间点ZO-1蛋白表达上升,ROCK蛋白表达下降(均P<0.05)。结论氢气可保护脓毒症肠屏障功能,改善肠上皮屏障完整性和通透性,增加肠细胞间紧密连接蛋白表达,这些保护机制可能与氢气抑制LPS诱导的ROCK过度表达有关。

应用Caco-2细胞模型评价真菌毒素毒性研究进展

真菌毒素是一些病原真菌在侵染作物过程中产生的次级代谢产物,许多食品和饲料在生产、加工、贮存和流通过程中都有可能受到真菌毒素的污染。人和动物摄入真菌毒素后,体内能够引发多种生理毒性反应,如肝肾毒性、致癌性、肠道损伤及炎症、中枢神经系统异常、生殖紊乱等。肠上皮细胞是分隔机体内部环境与外界的屏障,在真菌毒素的毒性评价中,对真菌毒素引起肠上皮细胞损伤的评价是一个重要方面。人结肠癌Caco-2细胞系常用于建立体外肠道屏障模型,该模型可应用于体外评价药物或毒素在小肠粘膜或上皮的吸收转运效率,以及对肠道屏障功能的影响。本文综述了Caco-2细胞单层模型的建立和评价指标,应用该模型评价几种常见真菌毒素对肠上皮细胞的运输、屏障功能的影响,以及肠上皮细胞毒性等研究进展,为进一步研究多种真菌毒素对肠上皮损伤的机制提供支持。

Intestinal Transport and Biotransformation of Resibufogenin and Cinobufagin in Chan Su via HPLC/APCI-MS^n

In vitro models of human colon carcinoma cell line(Caco-2 cell monolayer) and human intestinal bacteria were used to investigate the intestinal transport and biotransformation of resibufogenin and cinobufagin in Chan Su by HPLC/APCI-MSn. The experimental results of Caco-2 cell monolayer demonstrate that the apparent permeability coefficients(Papp) of resibufogenin and cinobufagin are higher than 10–6 cm/s, which indicates that both resibufogenin and cinobufagin have a good absorption in the small intestine. And the biotransformation result of human intestinal bacteria shows that resibufogenin has been transformed to 3-epiresibufogenin and cinobufagin has been transformed to 3-epicinobufagin, deacetylcinobufagin and 3-epideacetycinobufagin, respectively.

柚叶总黄酮对LPS所致Caco-2结肠上皮细胞间高通透性的保护作用

探讨柚叶总黄酮(PLTFE)对脂多糖(LPS,2μg/m L)诱发Caco-2高通透性细胞模型的保护作用。Caco-2模型细胞以不同浓度的PLTFE(0、10、50、100、150μg/m L)处理培养24 h进行后续实验。MTT法测定细胞生存率,细胞乳酸脱氢酶(lactate dehydrogenase,LDH)水平依说明书使用试剂盒测定。酶联法(enzyme linked immunosorbent assay,ELISA)测定白介素(interlukin-1β,IL-1β)、IL-8和肿瘤坏死因子(tumor necrosis factor-α,TNF-α)分泌水平。跨上皮细胞电阻(trans epithelial electrical resistance,TEER)值和异硫氰酸荧光素-右旋糖酐(FD40)透过度用于评估细胞通透性水平。实时定量PCR(Quantitative real-time PCR,q RT-PCR)检测细胞IL-1β、IL-8、TNF-α、闭锁蛋白(Occludin)、紧密连接蛋白-1(claudin-1)、封闭小带蛋白(ZO-1)和肌球蛋白轻链激酶(myosin light chain kinase,MLCK)的m RNA表达。结果表明,柚叶总黄酮能显著提高受损细胞生存率至86.1%,抑制受损细胞中LDH的溢出(p <0.05)。同时还能有效抑制受损细胞中炎性细胞因子(IL-1β、IL-8、TNF-α)的分泌及m RNA转录。此外,柚叶总黄酮可增强细胞紧密连接因子(Occludin、claudin-1、ZO-1)的m RNA转录,抑制MLCK的m RNA转录,改善细胞间高通透性q RT-PCR法检测细胞中相关因子的m RNA转录水平。结果提示,柚叶总黄酮具有较强的抗炎活性,能通过上调细胞内细胞紧密连接相关因子的m RNA转录显著改善LPS造成的Caco-2细胞间高通透性的发生(p <0.05)。

黄芪多糖通过影响Ca^(2+)调节蛋白及其复合体促进大鼠小肠隐窝上皮细胞迁移

目的观察黄芪多糖对大鼠小肠隐窝上皮IEC-6细胞迁移过程中多胺信号通路Ca^(2+)调节指标的影响,并探讨黄芪促进胃肠黏膜损伤修复的作用机制。方法采用划痕法建造细胞迁移模型并观察黄芪多糖在无钙培养时对细胞迁移的影响;采用qPCR法检测经典瞬时受体电位通道1(TRPC1)、基质交感分子(STIM)1、STIM2 mRNA表达;采用免疫荧光法检测STIM1蛋白的分布及表达;采用蛋白质印迹法检测TRPC1、STIM1和STIM2蛋白表达;采用免疫沉淀法检测STIM1/TRPC1、STIM1/STIM2蛋白复合体的表达。结果无钙培养(去除胞外Ca^(2+)内流来源)减弱了黄芪多糖促进细胞迁移的作用(P<0.01)。黄芪多糖能提高TRPC1 mRNA和蛋白的表达(P<0.05或P<0.01)、逆转二氟甲基鸟氨酸(DFMO)所致的TRPC1 mRNA和蛋白表达降低(P<0.01);黄芪多糖能促进STIM1向胞膜移位并提高其表达水平,改善DFMO所致的STIM1向胞膜移位延迟及表达抑制;黄芪多糖能提高STIM1 mRNA和蛋白表达、逆转DFMO对STIM1 mRNA和蛋白表达的抑制(P<0.05或P<0.01);黄芪多糖能降低STIM2 mRNA和蛋白表达,逆转DFMO对STIM2 mRNA和蛋白表达的提高(P<0.05或P<0.01)。黄芪多糖能提高STIM1/TRPC1的表达水平,逆转DFMO对STIM1/TRPC1表达的抑制,还可通过提高复合体中STIM1表达水平、降低STIM2表达水平调节STIM1/STIM2表达,并能拮抗DFMO对STIM1/STIM2表达的影响。结论黄芪多糖促进IEC-6细胞迁移的作用与其影响Ca^(2+)调节蛋白及蛋白复合体表达有关。

Intestinal permeability of liquiritin and isoliquiritin in the Caco-2 cell monolayer model

The intestinal permeability of two flavonoid compounds liquiritin （LQ） and isoliquiritin （ILQ） was investigated using the Caco-2 cell monolayer model. In order to evaluate the permeability and predict the absorption mechanism of the two compounds, the study on bidirectional permeability from the apical （AP） side to the basolateral （BL） side as well as from the BL side to the AP side was carried out. The determination was performed by HPLC-UV method. And the permeability parameters, especially the apparent permeability coefficients （Papp）, were then calculated. The Papp values of LQ and ILQ are （5.40±0.16）× 10^-7 and （8.69±0.15）× 10^-7 cm/s, respectively. The results of time- and concentration-dependent transport experiments indicate that both LQ and ILQ are poor absorbed mainly through passive diffusion.

Intestinal permeability of atractylenolides across the human Caco-2 cell monolayer model

The intestinal permeability of three sesquiterpene lactones, atractylenolide Ⅰ, Ⅱ, and Ⅲ, was investigated using the human Caco-2 cell monolayer model. The bidirectional permeability of the three compounds from the apical （AP） to the basolateral （BL） side and in the reserved direction was studied. The three compounds were assayed using HPLC. The Papp values of atractylenolide Ⅰ, Ⅱ, and Ⅲ were all at the level of 10^-5 cm/s, suggesting high intestinal permeability and good absorption. The bidirectional transport of the three compounds was time- and concentration-dependent, and indicated the main mechanism of the passive diffusion of the three compounds across the intestinal epithelium membrane. Moreover, atractylenolide Ⅰ might be partly actively transported.

植物乳杆菌对肠道吸收短链脂肪酸的调控作用

短链脂肪酸(short-chain fatty acids,SCFAs)是肠道微生物发挥益生作用的重要物质基础,但当肠道微生态发生紊乱时,会缺失或过多地吸收SCFAs,对机体健康产生不利的影响。该文利用肠上皮Caco-2细胞建立肠道吸收混合SCFAs模型,探究植物乳杆菌对肠道吸收SCFAs的调控作用及可能的作用机制。结果表明,培养至第21天时,肠上皮Caco-2细胞单层表面被微绒毛覆盖,具有良好的致密性和完整性;当模型中混合SCFAs(乙酸∶丙酸∶丁酸=3∶1∶1,摩尔比)为5.0 mmol/L时,模型中的细胞存活率较高。植物乳杆菌f28、f2及f5均显著促进了细胞对乙酸、丙酸和丁酸的吸收(P<0.05);菌株f5促细胞吸收乙酸的作用显著高于其他菌株(P<0.05),而菌株f16则起到显著抑制作用(P<0.05),但促细胞吸收丙酸和丁酸的作用均显著高于其他菌株(P<0.05);菌株f19显著抑制细胞对丁酸的吸收(P<0.05)。菌株f2和f19显著上调了细胞中单羧酸转运蛋白(monocarboxylate transporter 1,MCT 1)蛋白和基因的表达水平(P<0.05),而菌株f28和f5则起到显著下调作用(P<0.05);菌株f28、f5、f16和f19显著上调了Na^(+)耦合单羧酸转运蛋白-1(sodium-coupled monocarboxylate transporter,SMCT1)蛋白和基因的表达水平(P<0.05);菌株f16显著上调了Na^(+)/H^(+)交换泵3(Na^(+)-H^(+)exchanger 3,NHE3)蛋白和基因的表达水平(P<0.05),而菌株f2、f5和f19则起到显著下调作用(P<0.05)。试验的植物乳杆菌能够通过上调或下调肠上皮Caco-2细胞SCFAs转运体的表达水平来调控其对SCFAs的吸收,研究为改善机体对SCFAs的生物利用度及相关制品的开发应用提供理论依据。

胰高血糖素样肽-2对28日龄断奶仔猪肠上皮细胞的保护及修复效应研究

本研究旨在探讨胰高血糖素样-肽2(Glucagon-like peptide-2,GLP-2)对28日龄断奶仔猪的肠黏膜上皮细胞(IEC)损伤后增殖、代谢、凋亡的影响及可能的作用机理。试验首先采用单因子设计,分别用1.2和2.4 mg.mL-1的β-伴球蛋白对原代培养的断奶仔猪肠上皮细胞进行攻毒,通过测定细胞增殖、代谢及凋亡的变化而建立细胞损伤模型;然后再采用2×3因子设计,考察添加不同浓度的GLP-2(1×10-9、1×10-8、1×10-7mol.L-1)对致敏的肠上皮细胞的影响。结果表明,使用β-伴球蛋白攻毒,细胞MTT OD值显著降低(P<0.05),细胞蛋白质沉积量和细胞总蛋白含量极显著降低(P<0.01),胞外LDH活力极显著增加(P<0.01),Na+-K+-ATP酶活力显著(P<0.05)或者极显著(P<0.01)降低,半胱氨酸蛋白酶-3(caspase-3)酶活力极显著(P<0.01)升高;使用β-伴球蛋白攻毒的同时添加不同浓度的GLP-2,细胞MTT OD值、细胞蛋白质沉积量、细胞总蛋白含量和Na+-K+-ATP酶活力均显著或极显著(P<0.05或P<0.01)升高,且随着GLP-2浓度的增加而升高,而胞外LDH活力则随着GLP-2浓度的增加而逐渐下降(P<0.05或P<0.01),caspase-3酶活力极显著(P<0.01)降低。提示β-伴球蛋白对断奶仔猪小肠上皮细胞的增殖和细胞完整性有不利影响,而GLP-2能够减轻或者避免β-伴球蛋白对断奶仔猪小肠上皮细胞的不利影响,这种效应可能是通过调节细胞Na+-K+-ATP酶、caspase-3等的活力变化并影响细胞代谢而实现。

胰高血糖素样肽-2对脂多糖应激的IPEC-J2细胞形态和紧密连接相关基因表达的影响

本研究旨在考察胰高血糖素样肽-2(Glucagons-like peptide-2,GLP-2)和脂多糖(Lipopolysaccharide,LPS)对仔猪空肠上皮细胞及其紧密连接(Tight Junctions,TJ)相关蛋白基因表达的影响,并探讨GLP-2调控仔猪肠道TJ蛋白基因表达可能的作用机理。试验一设对照、GLP-2、LPS、LPS+GLP-2共4个组,考查各处理对IPEC-J2细胞形态和紧密连接关键蛋白Occludin、Claudin-1和ZO-1基因表达的影响。结果:添加100nmol·L-1 GLP-2能显著改善IPEC-J2细胞形态,显著提高Occludin、Claudin-1和ZO-1mRNA表达(P<0.01);100μg·mL-1 LPS处理能显著破坏细胞形态、降低IPEC-J2细胞紧密连接蛋白mRNA的表达(P<0.01);在添加LPS基础上添加100nmol·L-1 GLP-2能有效维护细胞形态,显著增加IPEC-J2细胞TJ相关蛋白Occludin、Claudin-1和ZO-1mRNA的表达(P<0.01),增加量分别为46.3%、65.1%和30.3%。试验二考察了添加PI3K特异性抑制剂Wortmannin(Wort)和LY294002(LY)阻断PI3K-Akt-mTOR信号转导途径后Occludin、Claudin-1和ZO-1mRNA表达量的变化,试验共设对照、GLP-2、GLP-2+Wort、GLP-2+LY等4个组。结果:添加抑制剂Wort后可显著降低IPEC-J2细胞Akt、mTOR、Occludin、Claudin-1和ZO-1mRNA的表达(P<0.01),降低量分别为46.9%、50.5%,38.1%、49.6%和18.9%;添加LY后上述mRNA的表达量分别显著降低了67.2%、70.8%,49.6%、60.9%和25.8%(P<0.01)。以上结果表明:添加GLP-2能够有效抑制LPS应激对IPEC-J2细胞形态和TJ相关基因表达的损伤,PI3K-Akt-mTOR信号转导途径可能是GLP-2调控肠道紧密连接蛋白基因表达的重要信号通路之一。

双歧杆菌胞壁蛋白诱导人肠腺上皮细胞β-防御素-2 mRNA的表达

目的 检测双歧杆菌对人肠腺上皮细胞β-防御素 - 2 (h BD- 2 )基因的激活作用及其活性组分。方法 应用逆转录聚合酶链反应 (RT- PCR)法和 Northern杂交检测 HT- 2 9细胞 h BD- 2 m RNA的表达 ;采用超声破碎细胞、超速离心、蛋白萃取等方法分离双歧杆菌胞壁蛋白质成分。结果 RT- PCR和 Northern杂交分析显示 ,在正常培养条件下 HT- 2 9细胞无可见的 h BD- 2 m RNA表达信号 ,但在双歧杆菌菌体、细胞壁和胞壁蛋白刺激下均检测出显著的 h BD- 2 m RNA表达。结论 双歧杆菌能诱导人肠腺上皮细胞 h BD- 2基因的表达 。

血管活性肠肽、表皮生长因子上调支气管上皮细胞bcl-2基因表达

为探索肺内调节肽血管活性肠肽（ＶＩＰ） 和表皮生长因子（ＥＧＦ） 抗氧化保护的基因机制， 用逆转录聚合酶链式反应（ＲＴＰＣＲ） 及Ｓｏｕｔｈｅｒｎ ｂｌｏｔ 杂交等方法检测原代培养的兔支气管上皮细胞（ＢＥＣ） 内ｂｃｌ２ 和ｃｍｙｃ 基因的表达情况， 观察ＶＩＰ、ＥＧＦ及热应激对这两个基因表达的影响。结果显示：（１） 基础情况下ＢＥＣ内有ｂｃｌ２ 和ｃｍｙｃ 基因的低水平表达；（２） ＥＧＦ和ＶＩＰ均明显增强ｂｃｌ２ 和ｃｍｙｃ 的转录，ＥＧＦ的作用更强， 而热应激无明显效应；（３） ｂｃｌ２ 和ｃｍｙｃ 两者的转录有显著相关性。上述结果提示，ＶＩＰ和ＥＧＦ等肺内调节肽可通过上调ｂｃｌ２ 基因表达增强支气管上皮细胞的抗氧化能力；ｃｍｙｃ 基因的编码产物可能是ｂｃｌ２ 基因转录的上游调节因子。

胰高血糖素样肽-2对体外培养的断奶仔猪小肠黏膜上皮细胞形态、增殖及其酶活力的影响

本试验旨在研究不同浓度的胰高血糖素样肽-2(GLP-2)对断奶仔猪小肠黏膜上皮细胞形态、增殖及其酶活力的影响。试验采用单因子试验设计,以体外培养的28日龄断奶仔猪回肠上皮细胞作为模型,细胞培养液中的人胰高血糖素样肽-2(hGLP-2)浓度分别为0、1×10-11、1×10-10、1×10-9、1×10-8和1×10-7mol/L,培养时间为144 h。结果表明,加入hGLP-2培养细胞96 h后,28日龄断奶仔猪回肠上皮细胞已具备单层柱状上皮细胞的典型特征,各试验组细胞数量均显著多于对照组(P<0.05),相对应的MTT OD值均极显著高于对照组(P<0.01);各试验组培养液乳酸浓度、总蛋白含量和蛋白质沉积量都显著高于对照组(P<0.05);各试验组的胞外碱性磷酸酶、乳酸脱氢酶和肌酸激酶活力都极显著低于对照组(P<0.01),各试验组的Na+-K+-ATP酶活力都显著高于对照组(P<0.05)。由此可知,GLP-2可以促进体外培养的28日龄断奶仔猪小肠黏膜上皮细胞增殖,抑制细胞凋亡,维持细胞形态的完整性。