锰对神经元细胞内钙稳态影响的研究

目的研究锰对神经细胞内钙稳态的影响,重点观察神经元细胞钙离子浓度、Na+-K+-ATPase和Ca2+-ATPase活性的改变。方法选用原代培养神经元为模型,待细胞生长至最佳状态时,予以分组处理:锰处理组为含不同浓度氯化锰(0,25,100,400μmol/L)的培养液,培养神经细胞12h后,通过倒置相差显微镜观察细胞形态的改变,并检测神经元细胞内钙离子浓度、Na+-K+-ATPase和Ca2+-ATPase活性的变化。结果随着染Mn剂量的加大,神经元形态出现异常,细胞内钙离子浓度逐渐升高;细胞Na+-K+-ATPase和Ca2+-ATPase活性也逐渐下降。结论锰通过影响与维持钙稳态相关的酶(Na+-K+-ATPase和Ca2+-ATPase)活性,干扰神经元细胞内钙稳态,进而造成神经元细胞损伤。     

大鼠血清培养液对食管癌细胞增殖影响

目的筛选食管癌细胞增殖培养液中大鼠血清的最佳浓度和活性状态。方法采用常规饲料喂饲大鼠30d,股动脉采血制备血清,采用不同浓度和活性状态的大鼠血清培养液培养人食管上皮鳞癌细胞株Eca-109,采用四甲基偶氮噻唑蓝(MTT)法,筛选大鼠血清培养液对Eca-109细胞生长增殖的最佳作用条件,以小牛血清培养液及人正常肝上皮细胞株HL7702作为对照进行比较。结果各浓度未灭活大鼠血清培养的人食管癌细胞Eca-109基本上均呈指数增长趋势,72h时各浓度组间差异有统计学意义(F=159.263,P0.05);用5%未灭活大鼠血清培养人食管癌细胞Eca-109和人正常肝上皮细胞HL7702,培养72h时,2株细胞的生长增殖能力均强于5%灭活大鼠血清,差异均有统计学意义(F=37.746,P0.05;F=43.399,P0.05);与小牛血清对2株细胞生长增殖的作用接近(P0.05)。结论 5%相同浓度条件下,未灭活大鼠血清培养液更适合细胞生长。     

微量元素砷、硒、碲、铼、铱对梨形四膜虫生长的作用

本文在先前用锗和稀土元素促进梨形四膜虫(Tetraphymena Pyriformis S1)细胞生长分裂工作的基础上,继续探寻稀有微量元素砷、硒、碲、铼、铱对它的作用。实验发现,当聚蛋白胨培养液中添加的砷浓度为0.03—0.05ppm,硒浓度在0.01—1.0ppm范围时,它们分别显示出对四膜虫生长分裂的促进作用。首次发现,稀有元素铼、铱对四膜虫群同样具有促进繁殖的作用,其浓度分别是:钵0.02—0.08ppm,铱1.0—3.0ppm;而它们抑制该细胞生长分裂的浓度分别为:铼>50ppm,铱>20ppm;从而表明它们是属于低毒或无害元素。另一元素碲未见增益作用,当碲浓度大于10ppm时出现抑制的影响。     

锌对成骨细胞增殖 分化 矿化活性的影响

目的:探讨锌对成骨细胞增殖、分化、矿化活性的影响。方法:采用MTT法研究锌对成骨细胞增殖活性的影响;采用碱性磷酸酶测定法以及大鼠I型胶原蛋白(Col I)酶联免疫分析法研究锌对成骨细胞分化活性的影响;采用细胞钙茜素红染色法观察不同浓度的锌培养液作用成骨细胞后结节的数量,探讨锌对成骨细胞矿化活性的影响。结果:不同浓度的ZnCl_2培养液均具有促进成骨细胞增殖的作用(P0.05;P0.001);ZnCl_2浓度为1μM的培养液对成骨细胞的分化无影响,而ZnCl_2浓度为5μM和25μM的培养液具有促进成骨细胞分化的作用(P0.05;P0.001),各浓度的ZnCl_2培养液均提高了成骨细胞中Col I的水平(P0.001);不同浓度的ZnCl_2培养液作用于成骨细胞后,细胞内桔红色阳性结节的数量明显增多。结论:锌能促进成骨细胞的增殖、分化、矿化,从而促进骨形成过程。     

氮和磷对铜绿微囊藻细胞生长的影响

目的 了解氮、磷浓度及氮磷比值对铜绿微囊藻生长的影响,为阐述微囊藻水华形成的机制提供科学依据.方法 将经过扩大培养3次的铜绿微囊藻细胞(FACHB 936)接种于无氮、无磷的BG-11培养液中饥饿培养3 d,分别接种到含有0、0.5、1.0、5.0、10.0 mg/L磷的BG-11培养液中培养19 d;并分别接种于含磷0.05、5.0 mg/L的BG-11培养液中,分别按照氮∶磷物质量比为5∶1、10∶1、20∶1、50∶1、100∶1加入所需的NaNO3,培养19 d.观察各组铜绿微囊藻细胞计数的变化及培养液中可溶性磷浓度的变化情况.结果 培养的初始阶段(第1～6天),低磷培养液(0、0.5 mg/L组)中铜绿微囊藻细胞的生长稍好于高磷培养液(1.0、5.0、10.0 mg/L组).而在对数生长后期(第7～21天),高磷培养液(除10 mg/L组)中铜绿微囊藻细胞的生物量超过低磷培养液.此后,培养液中的铜绿微囊藻细胞逐渐停止增长,且高磷培养液中铜绿微囊藻细胞的对数生长期长于低磷培养液.对初始磷浓度为0.05 mg/L的各组铜绿微囊藻细胞最大生物量进行比较,差异无统计学意义(P＞0.05).而对初始磷浓度为5.0 mg/L的各组铜绿微囊藻细胞最大生物量进行比较,差异有统计学意义(P＜0.05);且依次为:20∶1＞10∶1＞5∶1＞50∶1＞100∶1.不同初始磷浓度、不同氮磷比值培养液中可溶性磷浓度、铜绿微囊藻细胞增长率均随培养时间的延长呈下降趋势.结论 磷浓度对铜绿微囊藻的生长有较大影响,应综合探讨磷浓度和氮磷比值对铜绿微囊藻细胞生长的影响;通过各种手段控制水体中的磷浓度可能是解决微囊藻水华的有效途径.     

酵母对无机铁的富集及其影响因素研究

目的 :观察不同培养条件对富铁酵母的生长影响 ,研究其实验制备的方法。方法 :实验中选用 4株不同酵母菌株 ,在不同的铁浓度梯度下 ,研究其对铁的富集情况。并就实验筛选所得啤酒酵母细胞生长的影响因素进行了研究 ,观察了啤酒酵母细胞在不同影响因素下的细胞生长情况、菌体收获量及菌体富铁量。双吡啶比色法测定铁浓度。结果 :获得富铁啤酒酵母细胞培养的最佳培养温度为 2 8℃ ,pH值为 5～ 7之间 ,偏酸性 ,培养基添加铁盐为硫酸亚铁 ,搅拌速度为 2 0 0r/min ,接种量以 10 %为最佳。结论 :通过生物发酵技术 ,能用于制备富铁酵母。     

蔗糖铁及右旋糖酐铁对儿科静脉营养液中脂肪乳剂稳定性的影响

目的 分析静脉营养液中分别加入两种不同铁剂（蔗糖铁及右旋糖酐铁）后对脂肪乳剂稳定性的影响。方法 按静脉营养配制操作规范加入两种不同剂量铁剂（分别为蔗糖铁及右旋糖酐铁）的静脉营养液各10袋,肉眼观察含不同当量铁剂（0.25、0.50、0.75和1.00 mg ）的静脉营养液在室温（25 ℃）悬挂静置3 d,扫描电镜观察3 d内的脂肪颗粒平均大小、直径＞0.5 μm百分比、营养混合液的pH值及渗透浓度。结果 含不同铁剂的营养液在不同时间点的脂肪颗粒大小差异无统计学性意义（F=0.32,P=0.7836;F=1.73,P=0.1321）。72 h内各组脂肪乳颗粒平均大小均＜0.5 μm,并且均未见到直径＞5 μm的脂肪颗粒,含不同浓度铁的TNA在不同时间点间脂肪颗粒＞0.5 μm的百分比、pH值及渗透压差异均无统计学意义 （百分比：F=1.47,P=0.3467;F=1.04,P=0.4758。pH值：F=0.63,P=0.5942;F=0.46,P=0.6825。渗透压：F=1.37,P=0.3648;F=0.65,P=0.6023）。结论 浓度小于1%蔗糖铁及右旋糖酐铁分别加入儿科静脉营养液是稳定的。     

铜对人类肠道上皮Caco-2细胞的毒性研究

目的　了解铜对人类肠道上皮Caco 2细胞的毒性影响。方法　应用噻唑蓝 (MTT)实验、P 糖蛋白(P gp)活性检测实验、活性氧检测及克隆形成实验研究铜对肠道上皮Caco 2细胞的毒性及可能作用机制 ,同时利用Caco 2细胞和肠炎沙门氏菌为模型 ,研究铜对Caco 2细胞对细菌侵入和存活易感性的影响。结果　在一定的暴露场景下 ,铜可明显地降低细胞的活力 ,抑制细胞膜表面P gp的活性 ,促进细胞内活性氧自由基的产生 ,降低细胞的克隆形成能力。此外 ,细胞经铜暴露后 ,肠炎沙门氏菌侵入细胞的数量增加 ,但细胞内存活的细菌数量却下降。结论　过量的铜可导致Caco 2细胞氧化损伤 ,从而引起更广泛的细胞毒性效应 ,但其对细胞对细菌侵入和存活易感性的影响有待进一步研究。     

Caco-2细胞单层模型及其在毒理学中的应用

Caco 2细胞来源于人类结肠腺癌细胞 ,当其在生长达到融合后细胞可自发形成极性 ,表达出成熟肠道上皮细胞的某些形态和功能特征 ,其细胞单层模型是目前最为成熟的体外模拟肠道上皮细胞摄取和转运营养物质和外来化学物的模型 ,被广泛地应用于药理学和毒理学研究。本文介绍了Caco 2细胞单层模型的培养条件、细胞特性及常用功能指标 ,以及其在对外源化学物代谢、摄取、转运和消化道毒理学方面的应用。     

缺铁性贫血患儿淋巴细胞体外增殖及细胞因子分泌水平研究

目的 评估铁缺乏对患儿淋巴细胞增殖功能及细胞因子分泌水平的影响,初步探讨缺铁性贫血(IDA)导致儿童免疫功能下降的原因。方法 2018年3月4日-2018年4月28日选取IDA患儿和健康体检患儿各20例,作为IDA组和正常对照组,MTT比色法检测两组儿童T、B淋巴细胞在10、20、50 μmol/L铁浓度培养液下淋巴细胞体外刺激指数(SI),ELISA法检测两组儿童在不同铁浓度培养液下 单个核细胞(PBMC) INF-γ、IL-2、IL-6、IL-10的分泌水平。结果 1)对照组20 μmol/L铁浓度培养液下B、T淋巴细胞SI值高于其他浓度组,且高于同浓度组IDA患儿,IDA组在给予生理浓度铁补充下SI值有所升高;2)IDA组各铁浓度下INF-γ、IL-2分泌水平均显著低于对照组,同组中20 μmol/L浓度组显著高于10 μmol/L浓度组;3)IDA组10、20 μmol/L浓度组IL-6、IL-10分泌水平均显著高于对照组。结论 IDA患儿T、B淋巴细胞增殖功能障碍以及细胞因子分泌水平的改变可能是导致患儿免疫功能失衡,免疫力下降的原因之一。     

Comparison of In Vivo with In Vitro Pharmacokinetics of Mercury Between Methylmercury Chloride and Methylmercury Cysteine Using Rats and Caco2 Cells

The in vivo and in vitro pharmacokinetics of mercury (Hg) were compared between methylmercury chloride (MeHg·Cl) and methylmercury cysteine (MeHg-Cys) using rats and Caco2 cells because humans can be exposed to MeHg compounds through dietary fish. The in vivo pharmacokinetics of Hg immediately after the digestion of MeHg compounds are still obscure. In Caco2 cells, membrane uptake and subcellular distribution of MeHg compounds were examined. When rats received it intravenously, MeHg·Cl showed 20-fold greater plasma and 2-fold greater blood concentrations of Hg than MeHg-Cys, indicating that their pharmacokinetic properties are different. One hour later, however, Hg concentrations in plasma and blood became virtually identical between MeHg·Cl and MeHg-Cys, although blood Hg concentrations were?>100-fold greater than those in plasma. When administered into the closed rat’s jejunum loop, MeHg·Cl and MeHg-Cys were rapidly and efficiently taken up by intestinal membranes, and Hg was retained in intestinal membranes for a relatively long time. When administered orally, no difference was observed in plasma and blood Hg concentrations between MeHg·Cl and MeHg-Cys: plasma and blood Hg concentrations increased gradually and reached steady levels at 8?h after administration. In Caco2 cells, uptake of MeHg-Cys was significantly suppressed by l-leucine, although this was not seen with MeHg·Cl. In Caco2 cells, 81?% of Hg was recovered from cytosol fractions and 13?% of Hg from nuclear fractions (including debris) after a 2-h incubation with MeHg-Cys. In conclusion, the mechanism of membrane uptake and volume of distribution in the initial distribution phase were clearly different between MeHg·Cl and MeHg-Cys. However, such pharmacokinetic differences between them disappeared 1?h after intravenous and after oral routes of administration, possibly due to the metabolism in the body.     

Sodium butyrate inhibits cell growth and stimulates p21WAF1/CIP1 protein in human colonic adenocarcinoma cells independently of p53 status

Butyric acid, one of the short-chain fatty acids produced by microbial fermentation in the colon, exhibits antiproliferative activities in various cancer cell lines. The initial objective of the study was to assess whether the effect of sodium butyrate (NaB) on cell growth differed by p53 status of the cells. Four human colorectal adenocarcinoma cell lines were used: HT29 (p53 point mutation), Caco2 (p53 truncation), LS513 (p53 wild type), and Lovo (p53 wild type). NaB significantly inhibited cell growth in all four cell lines. NaB arrested HT29 and LS513 cells in G0/G1 and Caco2 and Lovo in G2-phase. A second objective was to determine whether NaB similarly affected the cyclin-dependent kinase inhibitor, p21WAF1/CIP1. In all cell lines, p21 mRNA levels were immediately elevated after NaB exposure, and p21 protein levels were increased within 6 h. NaB increased p21 promoter activity in both Caco2 and Lovo, suggesting p53 independence. NaB did not influence p21 mRNA stability. Although three DNase I hypersensitivity sites were identified in the region of the p21 gene, induction of p21 mRNA by NaB was not accompanied by relaxation of the chromatin in the region of the p21 gene.     

锌对Caco2细胞ZIP4 mRNA表达的影响

目的研究锌对Caco2细胞ZIP4mRNA表达的影响及其规律。方法通过锌特异螯合剂TPEN建立低锌Caco2细胞模型,RT-PCR法获得ZIP4cDNA片断,10μmol/LTPEN培养基诱导后,分别检测0、2、4、6、8和10h时点ZIP4mRNA的表达,及0、2·5、5、7·5、10μmol/LTPEN培养基诱导6h,检测各浓度组ZIP4mRNA的表达。结果RT-PCR获得单一条带的片断,大小与设计一致,获得正确的ZIP4cDNA片断,随着低锌时间的增加,ZIP4mRNA表达也升高,6h达到峰值;随着TPEN浓度的升高,ZIP4mRNA表达也随之升高。结论ZIP4mRNA表达受锌的调控,提示可能参与小肠对锌的吸收。     

Genome and transcriptome scale portrait of sigma factors in Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne's disease (JD), a chronic gastroenteritis of ruminants and other animals, including primates. Many evidences suggested association of MAP to Crohn's disease, a chronic granulomatous gastrointestinal disease of humans with strong similarities with JD. The present study attempts to evaluate global gene regulation in MAP, which has not been addressed previously, despite the availability of MAP genome sequence. For this purpose, we investigated: (i) the presence of sigma factors and their relationship to sigma factors of other mycobacteria (M. avium subsp.avium, M. tuberculosis, M. bovis, M. leprae and M. smegmatis), and (ii) their expression during different growth conditions and in vitro infection of intestinal epithelial Caco2 cells. MAP genome contains 19 putative sigma factor, but only 12 belong to gene families common to other mycobacteria. Gene expression was evaluated with Real-Time PCR during growth in 7H9 medium and mycobactin J, in 7H9 medium plus mycobactin J and lisozyme, and during infection of Caco2 cells: very different expression patterns were observed and, on the whole, only 7 sigma factors were found to be expressed. sigJ was upregulated during the infection of Caco2 cells. Even if only few sigma factors were expressed in the three conditions tested, the overall high numbers of MAP sigma factors suggests a noteworthy flexibility of this pathogen. Thus, this first report on expression of MAP sigma factors opens the way to an extensive characterization of global gene regulation, as a key to understand strategies of survival and mechanisms of infections used by this organism.     

维生素D_3对报告载体荧光素酶表达的作用

目的：　研究１,２５－二羟维生素Ｄ３ 对结肠癌细胞系Ｃａｃｏ－２ 细胞中报告基因表达的作用,并探讨在报告载体ｐＧＬ２ 序列中存在潜在的抑制性维生素Ｄ应答元件（ＶＤＲＥ）的可能性。方法：　采用磷酸钙沉淀法将报告载体转染入Ｃａｃｏ－２ 细胞。Ｃａｃｏ－２细胞经不同浓度１,２５－二羟维生素Ｄ３ 处理后测定细胞裂解液中表达的荧光素酶活性。结果：　应用ｐＧＬ２ 报告载体时,当用ｐＳＧ５－ＶＤＲ表达载体共转染后,１,２５－二羟维生素Ｄ３显著地抑制Ｃａｃｏ－２ 细胞荧光素酶的表达（Ｐ＜ ０．０５）;而未使用该表达载体共转染则无抑制作用（Ｐ＞ ０．０５）。应用ｐＧＬ３ 报告载体时,不同浓度的１,２５－二羟维生素Ｄ３ 对ｐＬＧ３转染后Ｃａｃｏ－２ 细胞表达的荧光素酶活性均无显著抑制作用（Ｐ＞ ０．０５）,该作用不依赖是否存在有ｐＳＧ５－ＶＤＲ表达载体共转染。结论：１,２５－二羟维生素Ｄ３ 对报告载体ＰＧＬ２ 荧光素酶表达具有抑制作用,而对ｐＧＬ３ 则否;类似人类ＰＴＨ基因中的潜在抑制性ＶＤＲＥ存在于报告载体ｐＧＬ２,在ｐＧＬ３ 中该ＶＤＲＥ业已改变。     

姜黄素对人宫颈癌SiHa细胞株增殖、凋亡和COX-2表达的影响

目的:体外研究中药姜黄素(Cur)对子宫颈癌SiHa细胞的增殖抑制和诱导凋亡的作用及与环氧合酶(COX-2)表达的关系。方法:以不同浓度的姜黄素(10～30μmol/L),分12～72 h四个时间点处理SiHa细胞,光镜观察细胞形态变化;用四甲基偶氮唑蓝(MTT)法测定细胞增殖抑制率;用DNA梯状电泳(DNA ladder)检测凋亡的发生;Western blot检测COX-2蛋白的表达;用放射免疫法检测细胞PGE2释放水平。结果:姜黄素可抑制SiHa细胞增殖,作用呈明显的时效和量效关系,差异有统计学意义(P<0.01);姜黄素可诱导SiHa细胞凋亡,DNA ladder呈梯状条带;姜黄素可明显抑制COX-2的表达,差异有统计学意义(P<0.01),并呈浓度依赖性;姜黄素明显抑制SiHa细胞PGE2释放水平,差异有统计学意义(P<0.01)。结论:姜黄素体外对SiHa细胞具有增殖抑制作用和促进凋亡作用,其机制可能与抑制COX-2蛋白表达、降低PGE2释放水平有关。     

Iron bioavailability from maize and beans: a comparison of human measurements with Caco-2 cell and algorithm predictions

BACKGROUND: An in vitro digestion and Caco-2 cell model may predict iron bioavailability to humans; however, direct comparisons are lacking. OBJECTIVE: The objective was to test the differences in iron bioavailability between 2 maize varieties and 2 bean varieties (white beans and colored beans) by comparing human, Caco-2, and algorithm results. DESIGN: Two randomized, 2 x 2 factorial experiments compared women's iron absorption from 2 maize varieties (ACR and TZB; n = 26) and 2 bean varieties (great northern and pinto; n = 13), each fed with and without ascorbic acid (AA) from orange juice. Nonheme iron bioavailability was determined from 2-wk retention of extrinsic radioiron tracers and was compared with Caco-2 cell and algorithm results from identical meals. RESULTS: Without AA supplementation, women absorbed only about 2% of the iron from the maize or bean meals. The results were unaffected by the variety of either maize or beans. Adding AA (15-20 molar ratios of AA:iron) roughly tripled the iron absorption (P < 0.0001) from all test meals. Although the Caco-2 model predicted a slightly improved bioavailability of iron from ACR maize than from TZB maize (P < 0.05), it accurately predicted relative iron absorption from the maize meals. However, the Caco-2 model inaccurately predicted both a considerable difference between bean varieties (P < 0.0001) and a strong interaction between bean varieties and enhancement by AA (P < 0.0001). The algorithm method was more qualitatively than quantitatively useful and requires further development to accurately predict the influence of polyphenols on iron absorption. CONCLUSIONS: Caco-2 predictions confirmed human iron absorption results for maize meals but not for bean meals, and algorithm predictions were only qualitatively predictive.     

Terminalia ferdinandiana Exell. Fruit and Leaf Extracts Inhibit Proliferation and Induce Apoptosis in Selected Human Cancer Cell Lines

Terminalia spp. are characterized by their high antioxidant contents and several species have anticancer activity. This study examined T. ferdinandiana fruit and leaf extracts for antiproliferative and apoptotic activities against a panel of human carcinoma cell lines. All extracts inhibited Caco2, HeLa, Jeg-3, JAR, MC3T3-E1, and MG63 proliferation. The leaf ethyl acetate extract was the most potent inhibitor of proliferation (MC3T3-E1 IC₅₀ = of 6 µg/ml; Caco2 IC₅₀ = 102 µg/ml). Furthermore, IC₅₀'s < 500 µg/ml were determined against all cell lines tested against that extract. The methanolic leaf extract was also a potent inhibitor of cell proliferation (Jeg-3 IC₅₀ = 147 µg/ml; MC3T3-E1 IC₅₀ = 40 µg/ml). The fruit extracts were also good inhibitors of carcinoma cell proliferation. Cell imaging studies detected morphological features consistent with apoptosis in Caco2 cells exposed to the ethyl acetate, methanolic, and aqueous extracts. Caspase 3 activity was significantly elevated in Caco2 cells exposed to these extracts, indicating that apoptosis was induced. The leaf ethyl acetate extract contained a high diversity and relative abundance of tannins and flavonoids. All T. ferdinandiana fruit and leaf extracts displayed either no toxic or low toxicity in the Artemia franciscana bioassay and in a HDF viability assay.