Caco-2细胞模型用于乳源ACE抑制肽LL、LPEW的小肠吸收研究

通过建立并验证Caco-2细胞模型,分析血管紧张素转化酶(angiotensin-I converting enzyme,ACE)抑制肽LL、LPEW在小肠中的转运量,研究LL、LPEW的小肠吸收机制。从细胞形态、跨膜电阻和碱性磷酸酶活性3个方面验证Caco-2细胞模型可用性。分析ACE抑制肽LL、LPEW的Caco-2细胞转运量,LL的表观渗透系数(P_(app))为(275.17±8.28)×10~(-7 )cm/s,肠道吸收良好;LPEW的P_(app)为(5.13±1.49)×10~(-7) cm/s,相比于LL,肠道吸收量较低。加入旁路转运促进剂去氧胆酸钠、内吞抑制剂渥曼青霉素(Wortmannin)、肽转运载体竞争性抑制剂Gly-Pro,对比无抑制剂时LL的转运量,分析得到LL的跨膜转运机制可能为内吞途径。加入ATP能量生成抑制剂叠氮化钠、多药耐药蛋白抑制剂MK-571、P-糖蛋白抑制剂维拉帕米,对比无抑制剂时肽LL的转运量,得出LL没有外排作用,所以LL肠道吸收较好。     

白杨素及其衍生物在Caco-2细胞模型中的转运规律

目的 研究白杨素（chrysin,ChR）及其衍生物6,8-二-三氟甲基-7-乙酰氧基白杨素（dFMAChR）在人源结肠腺癌细胞系Caco-2细胞单层模型的转运规律.方法 建立精确灵敏、重复性好的ChR及dFMAChR的高效液相色谱（HPLC）检测方法;MTT（四甲基偶氮唑盐）法测定ChR和dFMAChR对Caco-2细胞的毒性作用.体外培养Caco-2细胞,用Transwell建立单层细胞模型;确定ChR和dFMAChR转运的最佳pH条件.评价时间、浓度对dFMAChR和ChR的双向转运的影响,计算转运速率及表观渗透系数（Papp）,并与阳性对照药普萘洛尔进行比较.结果 ChR和dFMAChR的转运最佳介质pH分别为6.0和6.5,两者的转运无论是Apical侧（A侧）到Basolateral侧（B侧）还是B侧到A侧,均具有时间依赖性和浓度依赖性.ChR和dFMAChR的平均Papp （A-B）分别是（1.60±0.15）×10-6 cm/s和（10.63±0.35）×10-6 cm/s,平均Papp （B-A）分别是（1.22±0.17）×10-6 cm/s和（10.43±0.28）×10-6 cm/s,两者Papp （A-B） ＞Papp （B-A）,且Papp （A-B） /Papp（B-A） ＜2.结论 在Caco-2模型中,dFMAChR和ChR均由被动扩散方式转运,dFMAChR的转运速率明显高于ChR,吸收接近普萘洛尔,属于吸收良好的化合物.     

转运葡萄糖苷类化合物的Caco-2细胞模型建立

建立转运葡萄糖苷类化合物的Caco-2细胞模型,为葡萄糖及葡萄糖苷类化合物肠道吸收研究提供基础.通过对Caco-2细胞最适培养基的选择、单层致密性的测定、单层通透性和细胞两侧碱性磷酸酶活性比的测定、单层亚显微结构的观察、细胞葡萄糖转运蛋白SGLT1和GLUT2的mRNA及蛋白表达水平的分析,建立可靠的细胞模型.研究表明:Caco-2细胞的最适培养基为添加体积分数15％血清的DMEM低糖培养基;Caco-2细胞在14～17 d的跨膜电阻趋于稳定,苯酚红的表观渗透系数达到0.49×10-6～0.51×10-6 cm·s-1,微绒毛分化成熟,细胞两侧碱性磷酸酶比值达到2.0以上.葡萄糖转运载体SGLT1的mRNA表达在14 d时显著高于17 d和21 d,葡萄糖转运载体SGLT1和GLUT2的蛋白表达在14、17、21 d无显著差异(P<0.05).Caco-2细胞培养14～17 d即可建立可靠的肠细胞吸收模型,用于葡萄糖及葡萄糖苷类化合物吸收的体外研究.     

猕猴桃籽油微胶囊在Caco-2单细胞层的吸收特性

以玉米肽为壁材,猕猴桃籽油为芯材,吐温-20为乳化剂,采用喷雾干燥法制备猕猴桃籽油微胶囊。MTT试验结果表明,当微胶囊浓度≤400μg/mL时,其对Caco-2细胞生长无明显抑制作用,且质量浓度为100~400μg/mL的微胶囊对细胞的生长有促进作用。对微胶囊在Caco-2单细胞层的吸收特性进行研究,结果表明,微胶囊在高、中、低3个浓度时,微胶囊的生物利用率均高于25%。当质量浓度为100μg/mL时,微胶囊的生物利用率达到最高,为41.70%±1.65%。微胶囊溶液从AP侧到BL侧的表观渗透系数Papp在1×10^-7~1×10^-6cm·s^-1之间,吸收水平较好。     

基于Caco-2单层细胞模型的兜唇石斛多糖的转运机制研究

以兜唇石斛多糖(DAP)为研究对象,借助人小肠上皮细胞Caco-2(the human colon carcinoma cell line)单层细胞模型,体外模拟DAP在小肠中的转运情况。在前期得到的DAP为基础,探究DAP的粘度对其转运情况的影响,同时建立Caco-2细胞的模型,采用MTT法测定DAP对Caco-2细胞存活率的影响,并考察了DAP在小肠的吸收转运的特征。实验表明:DAP的粘度与其在Caco-2细胞中的转运能力呈负相关趋势,粘度越大,转运速率越慢;在测定TEER值及荧光素钠渗透情况,观察Caco-2细胞的形态,建立了良好的细胞模型;DAP的存在下,Caco-2细胞的存活率为91.8%以上,因此DAP对Caco-2细胞存活无影响;DAP在Caco-2不同单边层的转移率分别为0.35%±0.02%和0.32%±0.02%,表观渗透率分别为(0.42±0.02)×10-6 cm/s和(0.19±0.18)×10-6 cm/s,表明DAP在小肠中不易被吸收。本研究所得结果为进一步研究DAP在肠道中的吸收机制奠定基础。     

Caco-2细胞模型快速培养法的建立与评价

Caco-2细胞模型可用于预测各种途径的食品营养物质吸收,被普遍用于营养物质开发的早期快速筛选过程中。传统Caco-2单层细胞模型的建立通常需要培养21 d,本研究建立一种快速培养Caco-2单细胞层的方法以缩短实验周期、降低成本。采用向标准培养基中添加生长因子的方法培养Caco-2细胞,分别研究细胞模型跨膜电阻值(TEER)、亲水标志物的渗透性、关键蛋白基因的表达、细胞膜特征形态等评价模型的完整性。表明快速培养法(9 d)获得的Caco-2模型跨膜电阻值(TEER)220Ω·cm2;漏出标志物荧光黄表观分配系数为(0.39±0.15)×10-6 cm/s;肠腔侧(apical,AP)/基底侧(Basolateral,BL)碱性磷酸酶活力比值为5.481±0.5304,单细胞层极性分化明显;q RT-PCR检测细胞单层关键蛋白质(P-gp、MRP2)的m RNA表达量快速法同标准培养法无显著差异。此法仅需9 d即可形成完整Caco-2细胞模型。结论是向标准培养基中添加生长因子的Caco-2细胞模型快速培养法可推广。     

基于Caco-2细胞单层与大鼠小肠模型的大豆皂苷Ⅰ和Ⅱ经上皮传递的变化研究

利用Caco-2细胞单层与大鼠小肠模型研究大豆皂苷Ⅰ和Ⅱ的吸收变化与机制。在Caco-2细胞单层中,大豆皂苷Ⅰ和Ⅱ从肠腔侧到基底侧的表观渗透系数（apparent permeability coefficients,Papp）随时间的延长趋向平稳,前120 min近似线性,且随浓度增大,斜率减小,Papp值分别为（1.02×10－6～3.41×10－6）cm/s和（0.9×10－6～3.05×10－6） cm/s;传递的饱和性、双侧Papp比率＞1.5以及线粒体呼吸链抑制剂叠氮化钠的抑制作用表明了两者的主动转运机制。抑制剂维拉帕米没有提高大豆皂苷Ⅰ和Ⅱ的吸收,排除了p-糖蛋白介导的外排;吸收促进剂按照冰片＞脱氧胆酸钠＞卡波姆934P＞聚山梨酯80的强弱提高两者的吸收,壳聚糖则未能加强渗透。跨膜转运也表现出组织差异性：两者在大鼠空肠的Papp是十二指肠和回肠的2 倍多。因此,控制的传递应能提高大豆皂苷Ⅰ和Ⅱ的小肠吸收以便两者实施它们的生理功能。     

胶束化对Caco-2上皮细胞叶黄素吸收和转运的影响

本试验为研究胶束化促进叶黄素肠上皮细胞转运的特性,采用体外消化模型探究胶束化处理对叶黄素生物利用度的影响以及Caco-2细胞模型测定胶束化对叶黄素肠细胞摄取、表观渗透系数和细胞内吞的影响。结果显示:随浓度升高胶束化叶黄素生物可给率先增大后减小,浓度为6×10^-5 mol/L时胶束化叶黄素的生物可给率最高,是叶黄素单体的1.42倍;叶黄素胶束化显著促进了其细胞吸收量,细胞内积累量是单体的2.6倍。表观渗透系数(P_app)测定表明胶束化叶黄素累积转运分数大于1.5%,且被动扩散为其跨膜输送主要途径;胶束化后,P_app(B→A)与P_app(A→B)比值明显降低。进一步通过细胞内吞抑制实验发现制霉菌素(Nystain)、3-羟基-2-萘甲酸[(3,4-二羟基苯基)亚甲基]酰肼(Dynasore)均能抑制胶束化叶黄素的转运(P<0.05),而5-(N-乙基-N-异丙基)阿米洛利(EIPA)无显著抑制作用(P>0.05)。以上研究结果表明,胶束化处理显著促进了叶黄素的生物利用度,其在肠细胞中的跨膜吸收途径以被动扩散为主,兼具网格蛋白介导和小窝/脂筏蛋白介导的细胞内吞途径。     

鳕鱼皮胶原蛋白肽在Caco-2细胞单层模型中的吸收机制

目的：鳕鱼皮胶原蛋白肽（cod skin collagen peptide,CSCP）对肝损伤具有较好的保护作用,但其吸收机制尚不明确,本实验拟采用人结肠腺癌Caco-2细胞单层模型对CSCP在肠道中的吸收机制进行研究,为CSCP在动物肠道内的吸收机制提供依据。方法：在进行转运实验前,先对CSCP在人工胃、肠液、不同pH值条件及在Caco-2细胞单层中的稳定性进行评价;采用CCK-8（cell counting kit-8）法确定CSCP在转运实验中的最高质量浓度,然后建立Caco-2细胞单层模型并测定跨膜电阻值和碱性磷酸酶活力以检验细胞单层的致密性、完整性和极化现象;考察转运时间、CSCP质量浓度、维拉帕米、MK-571、氧化苯砷和去氧胆酸钠对转运的影响,利用高效液相色谱法检测CSCP质量浓度并计算累积转运量和表观渗透系数。结果：在3 h内,CSCP在人工胃、肠液及接近胃肠道pH值环境中基本保持稳定,转运过程中未见多肽发生水解。CSCP的转运具有时间和浓度依赖性,不受维拉帕米和氧化苯砷的影响,去氧胆酸钠和MK-571对CSCP的转运具有非常显著的促进作用（P＜0.05）。结论：CSCP跨Caco-2细胞单层转运的机制为细胞旁路途径,其外排受到多药耐药蛋白的介导。     

食用酸枣仁多糖的小鼠肠道菌群培养上清液对Caco-2细胞吸收相关蛋白表达的影响

为了探究食用酸枣仁多糖(ZSSP)的小鼠肠道菌群培养上清液是否会调节Caco-2细胞中外排转运蛋白、紧密连接蛋白和Ⅱ相代谢酶的表达水平。将20只C57BL/6小鼠随机分为对照组和ZSSP组,分别灌胃超纯水和酸枣仁多糖水溶液(100 mg/kg·d^-1)14 d,收集小鼠粪便制备肠道菌群培养上清液。采用噻唑蓝(methyl thiazolyl tetrazolium,MTT)法检测肠道菌群培养上清液对Caco-2细胞活力的影响,采用免疫印迹(western blot,WB)法检测Caco-2细胞中P-糖蛋白(P-glycoprotein,P-gp)、多药耐药相关蛋白2(multidrug resistance-associated protein 2,MRP2)、咬合蛋白(Occludin)和闭合蛋白1(Claudin-1)的表达水平,用酶联免疫检测(enzyme linked immunosorbent assay,ELISA)试剂盒测定磺酸基转移酶(sulfotransferase,SULT)和尿苷二磷酸葡萄糖醛酸基转移酶(UDP-glucuronosyltransferase,UGT)的含量。结果表明,ZSSP组的肠道菌群培养上清液能显著降低Caco-2细胞中外排转运蛋白(P-gp和MRP2)的表达水平(P<0.05),显著升高UGT的表达水平(P<0.05),但对紧密连接蛋白(Occludin和Claudin-1)和SULT没有显著影响(P>0.05)。由此可知,食用酸枣仁多糖的小鼠肠道菌群培养上清液能调节Caco-2细胞中的外排转运蛋白和Ⅱ相代谢酶的表达水平。     

Molecular mechanisms of the naringin low uptake by intestinal Caco-2 cells

Naringin, the main flavanone of grapefruit, was reported to display numerous biological effects: antioxidant, hypocholesteremic, anti-atherogenic and favoring drug absorption. Naringin absorption mechanisms were studied in Caco-2 cells (TC7 clone). We investigated the possible involvement of several membrane transporters implicated in polyphenolic compounds intestinal transport (sodium-dependent glucose transporter 1, monocarboxylate transporter, multidrug-associated resistance proteins 1 and 2, and P-glycoprotein). Naringin was poorly absorbed by Caco-2 cells, according to its low value of apparent permeability coefficient (P(app) = 8.1 +/- 0.9 x 10(-8) cm/s). In the presence of verapamil, a specific inhibitor of P-glycoprotein, cellular uptake was increased by almost threefold after 5 min, and P(app) was doubled after 30 min. Our results indicated the involvement of P-glycoprotein, an ATP-driven efflux pump, capable of transporting naringin from the Caco-2 cell to the apical side. This phenomenon could explain, at least in part, the low absorption of this flavanone at the upper intestinal level.     

Esterification of Quercetin Increases Its Transport Across Human Caco‐2 Cells

Plant polyphenols showed useful biochemical characteristics in vitro; however, the assessments of their clinical applications in vivo are restricted by their limited bioavailability due to their strong resistance to 1st‐pass effects during absorption. In order to improve the bioavailability of quercetin (QU), the ester derivative of QU (3,3′,4′,5,7‐pentahydroxy flavones, TAQU) was synthesized, followed by examining the oil–water partition coefficient as well as the transport mechanisms of QU and its ester derivative (TAQU) using human Caco‐2 cells. The transport characteristics of QU and TAQU transport under different conditions (different concentrations, time, pH, temperature, tight junctions, and potential transporters) were systematically investigated. Results showed that QU had a lower permeability coefficient (2.82 × 10^?6 cm/s) for apical‐to‐basolateral (AP‐BL) transport over 5 to 50 μM, whereas the transport rate for AP to BL flux of TAQU (5.23 × 10^?6 cm/s) was significantly greater than that of QU. Paracellular pathways were not involved during the transport of both QU and TAQU. QU was poorly absorbed by active transport, whereas TAQU was mostly absorbed by passive diffusion. Efflux transporters, P‐glycoproteins, multidrug resistance proteins were proven to participate in the transport process of QU, but not in that of TAQU. These results suggested that improving the lipophicity of QU by esterification could increase the transport of QU across Caco‐2 cells.     

静态超高压处理对抗菌复合蛋白薄膜的改性研究

本研究以乳清分离蛋白和酪蛋白酸钠为成膜基材,甘油为增塑剂,山梨酸钾为抗菌剂制备复合蛋白成膜溶液,在其流延成薄膜之前对成膜溶液分组并进行了静态超高压处理:压力分别为200、300、400 MPa,处理时间分别为5、10、20、30 min,未经超高压处理的为对照组。通过对13组薄膜样品的机械性能、光学性能、水溶性、微观结构、阻隔性能等参数的分析,结果表明,超高压处理的薄膜表面更光滑和均匀,有较少的孔洞。超高压处理对薄膜的机械性能、阻隔性能均有显著性(p<0.05)影响,超高压压力300 MPa,处理20 min后,薄膜抗拉强度达到最大值4.86 MPa;C₄组薄膜的水蒸气透过系数降低到1.177×10^-9 g·cm/cm²·s·Pa;超高压压力200 MPa,处理10 min后氧气渗透系数降低到0.93×10^-9 cm³·cm/cm²·s·cmHg。超高压处理对组B₁、C₁、D₄水溶性均有显著性(p<0.05)影响,能够使薄膜水溶性降低。不同超高压处理后的薄膜可适应不同包装食品的货架期要求。     

食品胶对甘薯淀粉膜机械及渗透性能的影响

本实验以抗张强度、断裂拉伸应变、透湿性和透氧性为指标,研究食品胶对甘薯淀粉膜性能的影响。结果表明,添加食品胶能够显著增加甘薯淀粉膜的抗张强度,当甘薯淀粉膜中羟丙基羧甲基纤维素(HPCMC)含量为5g/100g 淀粉时,其抗张强度达36.22MPa,是对照膜(25.93MPa)的1.4 倍。添加羧甲基纤维素钠(CMC-Na)和魔芋葡甘露聚糖(KGM)可显著增加甘薯淀粉膜的透湿性,其他食品胶对甘薯淀粉膜的透湿性影响不显著,同时不同浓度的HPCMC 对甘薯淀粉膜的透湿性的影响也不显著。添加HPCMC、黄原胶和壳聚糖(5g 胶体/100g 淀粉)能够显著增加甘薯淀粉膜的透氧性,添加KGM 能够显著降低淀粉膜的透氧性,CMC-Na 和甲基纤维素(MC)对透氧性影响不显著。高浓度HPCMC(＞ 7g/100g 淀粉)能够降低甘薯淀粉膜的透氧性。当HPCMC 含量为10g/100g 淀粉时,淀粉膜的透氧性降低至0.749 × 10-6cm3·m/(m2·d·kPa),比对照膜降低了67%。     

Transport of methyl tert-butyl ether through alfalfa plants

Concentrations measured in alfalfa plant stem segments indicated that plants grown in methyl tert-butyl ether (MTBE)-contaminated soil took up the chemical through their roots. Assuming a cylindrical shape for the plant stem, a mathematical model was developed to describe the transport of MTBE through the stems. Simulation results from uniform and nonuniform initial concentration distributions across the stem radius were compared with steady-state experimental data. With known values of plant stem radius, water usage, water content, and the distance over which the concentration decreased by 50%, the diffusion coefficient of MTBE radial transport across the plant stem was estimated with 95% confidence to be in the range of 8.43-16.2 x 10(-8) cm2/s with a mean of 1.23 x 10(-7) cm2/s. When the diffusion coefficient was calculated based on transient experimental data, the values with 95% confidence interval ranged from 4.14 x 10(-7) to 8.00 x 10(-7) cm2/s with a mean value of 6.07 x 10(-7) cm2/s. The difference between these two results can be reduced by more accurate estimation of the water flow velocity through plant stems. The model is applicable to other species including sunflowers and poplars upon substitution of appropriate parameters.     

Evaluation of the permeability of agricultural films to various fumigants

A variety of agricultural films are commercially available for managing emissions and enhancing pest control during soil fumigation. These films are manufactured using different materials and processes which can ultimately result in different permeability to fumigants. A systematic laboratory study of the permeability of the agricultural films to nine fumigants was conducted to evaluate the performance of commonly used film products, including polyethylene, metalized, and high-barrier films. The permeability, as expressed by mass transfer coefficient (cm/h), of 27 different films from 13 manufacturers ranged from below 1 × 10(-4) cm/h to above 10 cm/h at 25 °C under ambient relative humidity test conditions. The wide range in permeability of commercially available films demonstrates the need to use films which are appropriate for the fumigation application. The effects of environmental factors, such as temperature and humidity, on the film permeability were also investigated. It was found that high relative humidity could drastically increase the permeability of the high-barrier films. The permeability of some high-barrier films was increased by 2-3 orders of magnitude when the films were tested at high relative humidity. Increasing the temperature from 25 to 40 °C increased the permeability for some high-barrier films up to 10 times more than the permeability at 25 °C, although the effect was minimal for several of these films. Analysis of the distribution of the permeability of the films under ambient humidity conditions to nine fumigants indicated that the 27 films largely followed the material type, although the permeability varied considerably among the films of similar material.