纳米氧化锌对人肺腺癌细胞A549的毒性

探讨纳米氧化锌（ZnO）对体外培养的人肺腺癌细胞A549的生物学效应。使用原子力显微镜（AFM）、透射电镜（TEM）和X射线衍射仪（XRD）研究纳米ZnO颗粒物的理化性质。然后,使用0mmol/L、0.1mmol/L、0.5mmol/L、1mmol/L、5mmol/L、10mmol/L的纳米ZnO处理体外培养的人肺腺癌A549细胞,MTT 法测定细胞生长活性。并且测定1mmol/L 纳米ZnO染毒24h后细胞培养液上清中,乳酸脱氢酶（LDH）和胞内的超氧化物歧化酶（SOD）、过氧化氢酶（CAT）的活性以及丙二醛（MDA）的含量。使用透射电镜和流式细胞仪检测细胞凋亡的情况。荧光染色检测细胞产生活性氧（ROS）的生成。实验所用的ZnO纳米颗粒为长75 nm、直径20nm的针状纤锌矿晶体。细胞实验结果表明,纳米ZnO颗粒对A549细胞的生长活性具有明显的抑制作用,存在剂量－效应关系。培养液上清中LDH活性显著升高（P < 0.05）,胞内CAT活性显著下降、MDA含量显著升高（P < 0.01）,但SOD活性下降不明显。荧光染色检测发现染毒后A549细胞出现细胞凋亡,而且细胞内ROS的生成与纳米ZnO存在剂量－效应关系。结论是纳米氧化锌诱导人肺腺癌A549细胞产生活性氧,引发细胞凋亡,并产生细胞毒性。     

丙酮酸乙酯联合顺铂作用裸鼠肝移植瘤的研究

目的:研究丙酮酸乙酯(EP)与顺铂(CDDP)联合处理裸鼠对裸鼠肝癌HepG2移植瘤的作用效果,及对个体的副作用;方法:为裸鼠接种p-Gluc荧光标记的HepG2细胞,分为PBS处理组、CDDP(9.6mM)与EP(60μg)单独注射组、CDDP与EP联合注射组;对各组裸鼠的个体体重、移植瘤体积进行观察测量;结果:药物联合组的肿瘤体积显著小于对照组(*P=0.0146),而CDDP处理组无显著变化。药物联合组与CDDP组中肿瘤抑制率分别为36.8%和62.9%;药物联合组体重平均为21g,CDDP组为19g,EP组与对照组约为25g;结论:EP是CDDP增效剂,药物联合处理有望为肝癌治疗方案提出新的思路,并可能缓解化疗药物单独使用带来的副作用。     

MIF通过PI3K/AKT通路对肺腺癌A549细胞增殖、凋亡、迁移的影响研究

目的:研究巨噬细胞移动抑制因子(MIF)是否通过PI3K/AKT通路对肺腺癌A549细胞的增殖、凋亡、迁移产生影响。方法:免疫组织化学技术检测MIF抗体在肺腺癌癌组织和癌旁组织中的表达情况,DEME完全培养基培养肺腺癌A549细胞株作为对照组,加入不同浓度人重组巨噬细胞移动抑制因子(rh MIF)和PI3K/AKT特异性抑制剂LY294002预处理作为实验组,应用MTT(噻唑蓝比色法)观察A549细胞株的增殖情况,(2) FCM(流式细胞仪技术)检测A549细胞株凋亡情况(3) Western-Blot测定A549细胞株PI3K、p AKT及相关抗凋亡抗体p53、Bcl-2蛋白表达情况。结果:(1)免疫组化发现MIF在肺腺癌癌组织中高表达,且高表达率与癌旁组织有显著差异(2)经rh MIF刺激后PI3K、p AKT、p53、Bcl-2表达更明显,迁移更显著;(3) rh MIF对A549细胞株作用均呈浓度-时间依赖性,rh MIF在50 ng/ml 24 h对A549细胞增殖率最高,LY294002在25μmol/L 24 h对A549细胞抑制作用最显著。结论:MIF在肺腺癌中高表达,肺腺癌细胞中加入rh MIF,rh MIF刺激A549细胞株增殖、迁移,抑制凋亡,与LY294002共同作用下,A549细胞株增殖率和迁移力下降、凋亡率升高,表明MIF有可能通过PI3K/AKT通路对肺腺癌细胞的增殖、凋亡和迁移产生影响。     

冬季和夏季PM10和PM2.5对人肺上皮细胞A549毒性的比较

采集并检测北京市玉泉路地区2008年冬季和夏季大气颗粒物PM10和PM2.5中过渡金属元素的含量,测定A549细胞暴露于颗粒物后的细胞活力及活性氧(ROS)生成.用线性回归统计分析颗粒物中过渡金属元素含量与其细胞毒性的相关性.结果表明,冬季和夏季PM10和PM2.5均明显抑制A549细胞的细胞活力,导致ROS生成升高.颗粒物细胞毒性夏季大于冬季;除Cr、Ni外,各金属元素与颗粒物导致的细胞毒性正相关.冬季和夏季颗粒物中金属元素含量的差异是导致其细胞毒性不同的原因之一.     

“葵龙草”注射液体内外抗肿瘤机制研究

目的:初步探讨"葵龙草"注射液的体内外抗肿瘤作用。方法:以人肿瘤细胞株为模型,采用SRB方法检测观察"葵龙草"注射液对多种肿瘤细胞的增殖抑制作用;以H22、S180、HepA、BGC-823为模型,以不同剂量的"葵龙草"注射液作用于肿瘤裸小鼠模型,观察"葵龙草"注射液的体内抗肿瘤作用。结果:"葵龙草"注射液对人肺癌细胞A549、人乳腺癌细胞MDA-MB-231、人肝癌细胞HepG2、人结肠癌细胞HCT116、人皮肤鳞状细胞癌细胞A431的增殖有明显的抑制作用,对裸鼠移植S180、H22、HepA、BGC-823细胞的增殖也有一定的抑制作用。结论:"葵龙草"注射液能够有效抑制培养的肿瘤细胞生长,注射给药后对多种细胞肿瘤移植的模型小鼠肿瘤增殖有明显的抑制作用,具有较广阔的研究前景。     

激光扫描共聚焦显微镜的使用及其在医学研究领域中的应用

共聚焦显微镜是显微镜制作技术、光电技术、计算机技术相结合的产物,是现代化的光学显微镜,它时普通光镜做了多方面的技术改进:用激光做光源并采用了共聚焦技术消除了透镜的色差和球差;运用点扫描技术,使标本上每一点的图像避免了相邻点的衍射光和散射光的干扰;结果用计算机处理;因而其图像高度清晰且数字化.激光共聚焦显微镜不仅可以观察活细胞、活组织的动态代谢过程,而且可以获得三维图像,与其他的生物学技术相配合,几乎可定性、定量定位地检测组织细胞内的任何一种生化成分.     

一次性使用无菌注射器细胞毒性检测

本文研究一次性使用无菌注射器的细胞毒性.方法:参考GB/T14233.2-2005和ISO10993-5的试验原则,将一次性使用无菌注射器浸提液与L929细胞培养接触,通过倒置显微镜观察其形态,采用MTT(四唑盐)法量化细胞毒性,计算相对增殖率(RGR),对一次性使用无菌注射器进行了细胞毒性试验.结果:部分一次性使用注射器对L929细胞有一定细胞毒性.结论:为客观评价一次性使用无菌注射器的产品质量,保证产品安全提供参考.     

异鼠李素对人鼻咽癌CNE-1细胞增殖的影响

目的:观察异鼠李素对鼻咽癌CNE-1细胞生长及增殖的抑制作用。方法:用不同浓度(10、30、50、70、90μg/m L)的异鼠李素处理人鼻咽癌CNE-1细胞,在不同时间点分别采用CCk-8法检测测细胞生长抑制率;台盼蓝染色测细胞活力;平皿培养测细胞集落形成能力。结果:与正常组相比,异鼠李素处理CNE-1细胞48h后,细胞生长抑制率明升高(p0.05或p0.01);用药物处理细胞6d,随着时间的延长,细胞生长活力逐渐降低;药物处理7天后,药物处理组细胞集落形成能力明显低于正常组(p0.05或p0.01)且以上方法都显示在一定范围内,异鼠李素对CNE-1细胞的生长及增殖抑制作用呈剂量依赖性。结论:异鼠李素可明显抑制鼻咽癌CNE-1细胞的生长和增殖。     

紫草素对淋巴瘤治疗作用研究

目的研究新疆紫草的提取物紫草素抑制B细胞非霍奇金淋巴瘤生长能力。方法对体外培养的Raji细胞PI染色,并进行荧光显微镜观察,同时,采用中性红染色的方法检测细胞在不同浓度紫草素作用下的存活率。结果紫草素对Raji细胞生长有抑制作用,能够诱导Raji细胞死亡,并呈浓度依赖。结论紫草素可以抑制Raji生长,对B细胞非霍奇金淋巴瘤的治疗有重要作用。     

大蒜素对人肝癌HepG2细胞生长的抑制作用研究

目的:研究大蒜素对人肝癌HepG2细胞生长的抑制作用。方法:体外培养人肝癌HepG2细胞,形态学观察大蒜素作用下HepG2细胞的生长变化,四甲基偶氮唑蓝(MTT)比色法检测实验各组HepG2细胞的生长增殖情况。结果:形态学观察显示,在大蒜素作用下,HepG2细胞失去原有"铺路石"样排列,细胞间连接减少或消失,细胞黏附性下降,出现细胞凋亡特征;MTT比色法检测发现,不同浓度的大蒜素均能抑制肝癌细胞的生长(P<0.05),且呈剂量依赖性。结论:大蒜素对人肝癌HepG2细胞的生长起一定的抑制作用。     

Electrotaxis of lung cancer cells in ordered three-dimensional scaffolds

In this paper, we report a new method to incorporate 3D scaffold with electrotaxis measurement in the microfluidic device. The electrotactic response of lung cancer cells in the 3D foam scaffolds which resemble the in vivo pulmonary alveoli may give more insight on cellular behaviors in vivo. The 3D scaffold consists of ordered arrays of uniform spherical pores in gelatin. We found that cell morphology in the 3D scaffold was different from that in 2D substrate. Next, we applied a direct current electric field (EF) of 338 mV/mm through the scaffold for the study of cells’ migration within. We measured the migration directedness and speed of different lung cancer cell lines, CL1-0, CL1-5, and A549, and compared with those examined in 2D gelatin-coated and bare substrates. The migration direction is the same for all conditions but there are clear differences in cell morphology, directedness, and migration speed under EF. Our results demonstrate cell migration under EF is different in 2D and 3D environments and possibly due to different cell morphology and/or substrate stiffness.     

流式细胞术分析五味子乙素促进阿霉素内化的实验研究

[目的]建立流式细胞术分析肿瘤细胞内阿霉素分布的方法,并研究低浓度五味子乙素对K562细胞内化阿霉素的影响。[方法]体外培养猪内皮细胞(pEC),以20μmol/L阿霉素处理2、4和6小时;或加入不同浓度阿霉素(0、10、20、40、80和100μmol/L)处理4小时。培养K562细胞,以5μmol/L阿霉素处理2、4和6小时;或加入不同浓度阿霉素(0、2.5、5、10和20μmol/L)处理4小时。采用不同浓度(0、25、50和100μmol/L)五味子乙素(Sch B)与阿霉素联合处理K562细胞4小时。收集细胞,采用流式细胞术分析阿霉素的特异荧光。[结果]固定浓度处理pEC(20μmol/L)和K562(5μmol/L)后检测,发现细胞内荧光强度随时间延长而增加,两种细胞4小时组荧光强度分别达到6小时组的96.93%和95.23%/。在直方图上,阴性和阳性细胞群界限分明。阿霉素(2.5、5和10μmol/L)单独处理细胞的荧光强度分别为26.78±3.34、64.70±6.24和118.35±9.67;添加25μmol/L Sch B实验后荧光强度增加到43.45±4.25、103.74±7.36和146.69±8.32,Sch B显著增加了K562细胞内阿霉素的分布(p<0.05)。[结论]建立的方案可快速测定细胞内的阿霉素分布;低浓度Sch B促进肿瘤细胞内化阿霉素。     

An optical counting technique with vertical hydrodynamic focusing for biological cells

A barrier in scaling laboratory processes into automated microfluidic devices has been the transfer of laboratory based assays: Where engineering meets biological protocol. One basic requirement is to reliably and accurately know the distribution and number of biological cells being dispensed. In this study, a novel optical counting technique to efficiently quantify the number of cells flowing into a microtube is presented. REH, B-lymphoid precursor leukemia, are stained with a fluorescent dye and frames of moving cells are recorded using a charge coupled device (CCD) camera. The basic principle is to calculate the total fluorescence intensity of the image and to divide it by the average intensity of a single cell. This method allows counting the number of cells with an uncertainty ±5%, which compares favorably to the standard biological methodology, based on the manual Trypan Blue assay, which is destructive to the cells and presents an uncertainty in the order of 20%. The use of a microdevice for vertical hydrodynamic focusing, which can reduce the background noise of out of focus cells by concentrating the cells in a thin layer, has further improved the technique. Computational fluid dynamics (CFD) simulation and confocal laser scanning microscopy images have shown an 82% reduction in the vertical displacement of the cells. For the flow rates imposed during this study, a throughput of 100–200 cells∕s is achieved.     

四味润肺中药对小鼠被动吸烟的保护作用

应用耐缺氧试验、光镜及扫描电镜等方法探讨了四味润肺中药对小鼠被动吸烟的保护作用．结果表明，被动吸烟可导致小鼠耐缺氧时间缩短，气管内膜上皮可见光镜及扫描电镜下病理性改变．太子参及川贝对吸烟所致损害的保护作用较强，玉竹的作用较差．     

乳腺癌耐受蛋白介导5-氟脲嘧啶的耐受及机制探讨

AIM To filtrate breast cancer resistance protein(BCRP)-mediated resistance agents and investigate the mechanism,so as to provide valuable datum for optimization clinical chemotherapy scheme to tumor with evaluation marker of BCRP expression. METHODS MTT assay was used to filtrate BCRP-mediated resistance agents with PA317/Tet-on/TRE-BCRP cell of different expression levels of BCRP after treated with different concentration anticancer agents. High performance liquid chromatography(HPLC) was applied to measure relative dose of intracellular retention resistance agents. Nuclear DNA fluorescence dye,Hochest 33258, staining and flow cytometry were adopted to detect apoptotic cells after treated with drugs. RESULTS There were shown increasing durg-resistance to 5-fluorouracil,methotrexate, doxirubicin, pirarubicin,etoposide and mitoxantrone followed with increasing expression of BCRP on PA317/Tet-on/TRE-BCRP cells(P＜0.05, n=3),but shown sensitive to paclitaxel, cisplatin, vincristine, mitomycin and vindesine. There also was shown significant negative correlation between the intracellular retention dose of 5-fluorouracil with different expression of BCRP(r=-0.885, P＜0.05, n=3).There were shown parallel results of that decreasing cellular apoptotic rate with increasing cellular expression of BCRP after treated with 5-fluorouracil by fluorescence dye staining and flow cytometry(P＜0.05, n=3),and also shown significate rise of the apoptotic rate of BCRP expression cells after treated with Ko143 (P＜0.05, n=3). Every group of cells could be different extently blocked in phase of G0/G1 treated with 5-fluorouracil. CONCLUSION Resistance of 5-fluorouracil could be especially mediated by conjugated with BCRP and acted as drug exclude-pump substrate. Cellular ability resistant to 5-fluorouracil-induced apoptosis could be reinforced by BCRP expression.     

Enhanced fluorescence emitted by microdroplets containing organic dye emulsions

In this paper, laser beam resonant interaction with pendant microdroplets that are seeded with a laser dye (Rhodamine 6G (Rh6G)) water solution or oily Vitamin A emulsion with Rhodamine 6G solution in water is investigated through fluorescence spectra analysis. The excitation is made with the second harmonic generated beam emitted by a pulsed Nd:YAG laser system at 532 nm. The pendant microdroplets containing emulsion exhibit an enhanced fluorescence signal. This effect can be explained as being due to the scattering of light by the sub-micrometric drops of oily Vitamin A in emulsion and by the spherical geometry of the pendant droplet. The droplet acts as an optical resonator amplifying the fluorescence signal with the possibility of producing lasing effect. Here, we also investigate how Rhodamine 6G concentration, pumping laser beam energies and number of pumping laser pulses influence the fluorescence behavior. The results can be useful in optical imaging, since they can lead to the use of smaller quantities of fluorescent dyes to obtain results with the same quality.     

五种重组人源IL-18突变子表达质粒的构建及其在BxPC-3细胞中的表达

目的:设计获得5段hIL-18异构体,定向插入pEGFP-C1质粒形成融合基因,转染胰腺癌Bx-PC-3细胞并表达,为进一步研究改建的hIL-18蛋白功能奠定实验基础.方法:设计引物从pGEM-T-hIL-18质粒中PCR扩增得到5段不同的hIL-18片段,分别连接入pEGFP-C1载体构建不同的突变子(Mu0、Mu1、Mu2、Mu3和Mu4),转染原位胰腺癌BxPC-3细胞,以荧光显微镜观察绿色荧光蛋白的表达,RT-PCR法检测转染细胞中hIL-18表达水平.结果:(1)五种重组质粒经酶切与测序证实构建成功;(2)BxPC-3细胞转染重组质粒后可观察到绿色荧光蛋白的表达.结论:(1)成功构建了hIL-18五种异构体的绿色荧光蛋白表达载体;(2)改建的hIL-18蛋白可与绿色荧光蛋白在BxPC-3细胞融合表达.     

离体黄瓜下胚轴不定根发生的细胞组织学研究

在0.01mg/L NAA诱导下，离体黄瓜下胚轴切段显示了较强的发根能力，发根率达88％以上，切片观察显示，在NAA诱导24h时，下胚轴切段维管柱中靠近外韧皮部外则的薄壁细胞发生变化，细胞质变浓，细胞核变大，部分细胞分裂并形成分生组织结节；48h时，分裂的细胞团增大，逐渐形成根原基，96h时，不定根中分化出许多网纹导管分子并与下胚轴切段的导管分子相连，而且大量不定根穿破表皮达到肉眼可见的程度，下胚轴切段正插时，不定根在形态学下端发生；反插时，不定根在形态学上，下两端发生，切片观察显示，正插和反插时不定根发生部位相同，均位于外韧皮部外侧的薄壁细胞处，但插时不定根发生在时间上比正插要快1天左右。