牙本质基质蛋白1和肿瘤坏死因子-α对汉滩病毒感染的脐静脉内皮细胞的作用

目的  探讨肿瘤坏死因子-α和牙本质基质蛋白1对汉滩病毒感染的脐静脉内皮细胞通透性的影响,为阐明肾综合征出血热发病过程中通透性增高的机制提供实验基础。方法  首先用免疫细胞化学方法检测牙本质基质蛋白1在脐静脉内皮细胞的表达情况,然后通过Transwell单层细胞模型系统检测外源性肿瘤坏死因子-α和牙本质基质蛋白1对汉滩病毒感染的血管内皮细胞通透性的影响。结果  牙本质基质蛋白1可以在血管内皮细胞表达,汉滩病毒感染对牙本质基质蛋白1表达量无显著性影响,汉滩病毒感染后加入肿瘤坏死因子-α使牙本质基质蛋白1表达量显著性增加（P <0.05）。单纯汉滩病毒感染后,通透性无显著性变化（P > 0.05）,而感染细胞加入外源性肿瘤坏死因子-α或牙本质基质蛋白1后,内皮细胞通透性显著增加（P <0.05）,两者联合作用能促进通透性进一步增加（P <0.05）。结论  牙本质基质蛋白1广泛表达在血管内皮细胞,肿瘤坏死因子-α和牙本质基质蛋白1均可使汉滩病毒感染的脐静脉内皮细胞通透性显著增高,且两者存在联合作用。

     

复方苦参注射液联合热疗抗血管生成作用的实验研究

目的探讨复方苦参注射液联合热疗对体内外血管生成的抑制作用。方法采用MTT法观察复方苦参注射液联合热疗对人脐静脉内皮细胞(HUVEC)、人结肠癌LOVO细胞增殖的影响;采用transwell板,观察复方苦参注射液对HUVEC迁移的影响;采用鸡胚绒毛尿囊膜(CAM)模型,观察复方苦参注射液对鸡胚绒毛尿囊膜新生血管的抑制作用。结果复方苦参注射液在6.25~200μL/mL时具有抑制HUVEC增殖的作用(细胞存活率82.2%~32.5%),与浓度呈负相关(相关系数r及P值分别为-0.972、0.001),此浓度范围内对人结肠癌LOVO细胞增殖的抑制明显低于对HUVEC的抑制,具有明显的抗血管生成作用。12.5μL/mL和25μL/mL复方苦参注射液联合热疗在体外具有抗血管生成的协同效应,6.25μL/mL、50μL/mL、100μL/mL和200μL/mL复方苦参注射液联合热疗在体外具有抗血管生成的次加效应。复方苦参注射液在3.125~25μL/mL时具有抑制HUVEC迁移的作用,在12.5~50μL/mL时对鸡胚绒毛尿囊膜新生血管具有明显的抑制作用。结论小剂量复方苦参注射液在体内外具有抑制血管生成作用,联合热疗具有协同或次加效应。     

复方苦参注射液联合热疗抗血管生成作用的实验研究

目的探讨复方苦参注射液联合热疗对体内外血管生成的抑制作用。方法采用MTT法观察复方苦参注射液联合热疗对人脐静脉内皮细胞（讯舰C）、人结肠癌LOVO细胞增殖的影响；采用transwell板，观察复方苦参注射液对HUVEC迁移的影响；采用鸡胚绒毛尿囊膜（CAM）模型，观察复方苦参注射液对鸡胚绒毛尿囊膜新生血管的抑制作用。结果复方苦参注射液在6．25～200μL/L时具有抑制HUVEC增殖的作用（细胞存活率82．2％-32．5％），与浓度呈负相关（相关系数r及P值分别为一0．972、0．001），此浓度范围内对人结肠癌LOVO细胞增殖的抑制明显低于对HUVEC的抑制，具有明显的抗血管生成作用。12．5μL／mL和25μL／mL复方苦参注射液联合热疗在体外具有抗血管生成的协同效应，6．25μL／mL、50μL／mL、100μL／mL和200μL／mL复方苦参注射液联合热疗在体外具有抗血管生成的次加效应。复方苦参注射液在3．125～25μL／mL时具有抑制HUVEC迁移的作用，在12．5-50μL／mL时对鸡胚绒毛尿囊膜新生血管具有明显的抑制作用。结论小剂量复方苦参注射液在体内外具有抑制血管生成作用，联合热疗具有协同或次加效应。     

Metabolic characterization of invaded cells of the pancreatic cancer cell line,PANC‐1

We previously reported that about 0.4% of cells in the cultured human pancreatic cancer cell line, PANC‐1, can invade matrigel during the transwell invasion assay, suggesting that these invaded PANC‐1 cells may have specific characteristics to keep their invasive potential. To identify the metabolic characterization specific in the invaded PANC‐1 cells, metabolome analysis of the invaded PANC‐1 compared with the whole cultured PANC‐1 was performed using CE‐TOFMS, and concentrations of 110 metabolites were measured. In contrast to the whole cultured cells, the invaded PANC‐1 was characterized as a population with reduced levels of amino acids and TCA cycle intermediates, and decreased and increased intermediates in glycolysis and nucleic acid metabolism. In particular, the ratio of both adenosine and guanosine energy charge was reduced in the invaded cells, revealing that the consumption of ATP and GTP was high in the invaded cells, and thus suggesting that ATP‐ or GTP‐generating pathways are stimulated. In addition, the GSH/GSSG ratio was low in the invaded cells, but these cells had a higher surviving fraction after exposure to hydrogen peroxide. Thus, the invaded cells were the population resistant to oxidative stress. Furthermore, reduction in intracellular GSH content inhibited PANC‐1 invasiveness, indicated that GSH has an important role in PANC‐1 invasiveness. Overall, we propose the invaded cells have several unique metabolic profiles.     

胰腺癌细胞与其基质成纤维细胞相互影响的研究

目的：观察胰腺癌细胞系BXPC-3、SW1990能否促进其基质中正常成纤维细胞( NFs)活化成癌相关成纤维细胞( CAFs)及其活化的可能机制,以及活化后的CAFs对BXPC-3、SW1990细胞迁移能力的影响。方法采用差异贴壁法分选出野生型小鼠C57胰腺组织中的NFs,后运用非接触式共培养方法将BXPC-3、SW1990与NFs共培养,共培养后采用免疫荧光方法检测共培养前后NFs中CAFs标志性蛋白α-平滑肌肌动蛋白(α-SMA)、成纤维细胞活化蛋白( FAP)的表达变化,以确定其是否活化,并且运用qRT-PCR法检测目前较公认的胰腺癌中升高的12种 miRNAs 在共培养前后的NFs中含量变化；采用 transwell 法观察活化前后的 NFs 对BXPC-3、SW1990迁移能力的影响。结果免疫荧光检测表明,与BXPC-3、SW1990共培养后, NFs中α-SMA、FAP表达均显著升高；同时,在所选取的12种miRNAs中,与BXPC-3、SW1990共培养后,NFs中miR-155含量均有明显升高。 BX-PC-3、SW1990在CAFs基质环境中迁移能力大大提高。结论胰腺癌细胞能够促进其周围NFs活化成CAFs,其活化可能与胰腺癌中某些特定的 miRNA分泌进入到 NFs中有关,活化后的CAFs又可以反过来促进胰腺癌细胞的迁移。     

Investigation of the effects of soluble fibers on the absorption of resveratrol and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PHIP) in the Caco-2 cellular model of intestinal absorption

Abstract

Soluble fibers are known to modulate intestinal absorption of non-polar compounds in the small intestine. Little is known about the modulation of absorption of more polar compounds. In the present study, we applied the Caco-2-transwell-system in order to investigate the modulation of intestinal bioavailability by soluble fibers. The system was tested using pectin and carrageenan as model soluble fibers at a concentration of 0.1% (w/v), which did not compromise the integrity of the cell monolayer. Modulation of absorption was evaluated for the heterocyclic amine aromatic 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PHIP) and the polyphenol resveratrol. Neither pectin nor carrageenan reduced the high flux of PHIP, apparent permeability coefficient (P_app) of 16?×?10^?6 cm?s^?1. The low P_app of resveratrol was reduced by both soluble fibers, particularly by pectin. These results suggest that the low bioavailability of polyphenols could be further reduced by soluble fibers. Because of their co-occurrence in several fruits, these findings warrant further research.     

富血小板血浆对人牙龈成纤维细胞增殖和迁移的影响

目的:体外研究不同浓度的机采富血小板血浆(PRP)对人牙龈成纤维细胞(HGFs)的增殖和迁移的影响?方法:不同浓度PRP(1%?5%?10%?20%?30%?50%),加入到原代培养的人牙龈成纤维细胞,无血清培养液为阴性对照,10%新生小牛血清为阳性对照,培养时间为3?5?7天,四甲基偶氮唑盐(MTT)法测定细胞增殖效果,Transwell系统测定细胞迁移效果?结果:PRP有明显促进增殖作用,不同浓度PRP与阴性对照组均有明显差异(P < 0.01),且随着时间递增,增殖效果也明显增强(P < 0.01)?在细胞迁移效果中,各浓度组均比阴性对照组效果好?结论:PRP对HGFs功能有明显促进效果,在一定浓度范围有浓度依赖性?     

PCDH8基因表达对鼻咽癌细胞生物学效应的影响

目的 探讨PCDH8基因表达对鼻咽癌细胞株CNE-1生物学行为的影响.方法 采用脂质体介导质粒pcDNA3.1-PCDH8(实验组)与质粒pcDNA3.1(对照组)转染至鼻咽癌细胞株CNE-1,RT-PCR、Western blot检测转染后CNE-1细胞的PCDH8 mRNA及蛋白表达;通过CCK-8细胞生长曲线、流式细胞技术、Transwell侵袭实验及CCK-8细胞药物敏感实验,进一步对转染PCDH8基因质粒的细胞株CNE-1的增殖能力、生长周期、细胞侵袭能力及对化疗药物的敏感性进行了分析,并与转染对照质粒的细胞株进行比较.结果 RT-PCR、Western blot检测结果表明转染质粒pcDNA3.1-PCDH8的实验组细胞株中PCDH8 mRNA及蛋白成功表达并明显高于转染质粒pcDNA3.1的对照组细胞株.CCK-8细胞生长曲线结果表明实验组细胞株的生长速度明显低于对照组.流式细胞仪检测结果表明实验组细胞株G0/G1期较对照组增加,S期细胞较对照组减少;Transwell侵袭实验结果表明实验组细胞株侵袭能力明显低于对照组;CCK-8细胞药物敏感性实验结果表明PCDH8基因的高表达能显著提高鼻咽癌细胞株CNE-1对顺铂化疗药物的敏感性.结论 PCDH8基因的表达能降低鼻咽癌细胞的增殖、侵袭能力,延缓细胞分裂周期,提高对顺铂化疗药物的敏感性.     

Cyclin E表达对于原代乳腺癌细胞侵袭性的影响

目的：从细胞学角度探讨Cycli E表达水平与原代乳腺癌细胞侵袭之间的关系。方法：手术中无菌条件下切取乳腺癌组织,制备原代乳腺癌细胞悬液,运用Transwell小室进行原代乳腺癌细胞侵袭力实验;获取具有不同侵袭力的原代乳腺癌细胞,再用免疫荧光技术检测不同侵袭能力的原代乳腺癌细胞cyclin E的表达水平,研究两者相关性。结果：具有不同侵袭力的乳腺癌细胞表达不同强度的Cyclin E蛋白,分别计数5个高倍视野,其均数分别为81.11±17.25及31.06±4.43,P〈0.05。侵袭性试验结果显示：Cyclin E阳性表达的原代乳腺癌细胞经24小时培养以后浸润到Transwell聚碳酸盐微孔滤膜下的细胞数的均值为：76.32±11.40/高倍视野;而Cyclin E阴性表达的原代乳腺癌细胞的均值为21.56±13.22/高倍视野;P〈0.05。结论：Cyclin E的表达差异与原代乳腺癌细胞的侵袭性有关,Cyclin E高表达者其侵袭性显著强于Cyclin E低表达者。     

Differences in the expression of endogenous efflux transporters in MDR1-transfected versus wildtype cell lines affect P-glycoprotein mediated drug transport

Background and purpose:

P-glycoprotein (Pgp) efflux assays are widely used to identify Pgp substrates. The kidney cell lines Madin-Darby canine kidney (MDCK)-II and LLC-PK1, transfected with human MDR1 (ABCB1) are used to provide recombinant models of drug transport. Endogenous transporters in these cells may contribute to the activities of recombinant transporters, so that drug transport in MDR1-transfected cells is often corrected for the transport obtained in parental (wildtype) cells. However, expression of endogenous transporters may vary between transfected and wildtype cells, so that this correction may cause erroneous data. Here, we have measured the expression of endogenous efflux transporters in transfected and wildtype MDCK-II or LLC cells and the consequences for Pgp-mediated drug transport.

Experimental approach:

Using quantitative real-time RT-PCR, we determined the expression of endogenous Mdr1 mRNA and other efflux transporters in wildtype and MDR1-transfected MDCK-II and LLC cells. Transcellular transport was measured with the test substrate vinblastine.

Key results:

In MDR1-transfected MDCK cells, expression of endogenous (canine) Mdr1 and Mrp2 (Abcc2) mRNA was markedly lower than in wildtype cells, whereas MDR1-transfected LLC cells exhibited comparable Mdr1 but strikingly higher Mrp2 mRNA levels than wildtype cells. As a consequence, transport of vinblastine by human Pgp in efflux experiments was markedly underestimated when transport in MDR1-transfected MDCK cells was corrected for transport obtained in wildtype cells. This problem did not occur in LLC cells.

Conclusions and implications:

Differences in the expression of endogenous efflux transporters between transfected and wildtype MDCK cells provide a potential bias for in vitro studies on Pgp-mediated drug transport.