干扰SET8在胃癌细胞肿瘤进展和顺铂敏感性中的作用

目的 探究SET8对胃癌的进展以及对顺铂敏感性的影响。方法 采用免疫组织化学和蛋白质免疫印迹分析胃癌癌组织中SET8表达,采用qRT-PCR检测胃癌细胞中SET8 mRNA水平;胃癌BGC-823细胞转染SET8-shRNA后,采用CCK-8法检测细胞增殖,采用细胞集落形成实验检测细胞集落形成能力,采用transwell法检测细胞的迁移和侵袭能力;最后,采用CCK-8法检测干扰SET8表达对胃癌BGC-823细胞顺铂敏感性的影响。结果 胃癌组织及胃癌细胞中SET8表达明显升高;干扰SET8表达可抑制胃癌BGC-823细胞增殖、集落形成、迁移与侵袭,并提高胃癌细胞对顺铂敏感性。结论 干扰SET8表达可抑制胃癌细胞进展并提高顺铂敏感性。     

CDX2基因高表达对胃癌细胞生物学行为的影响

目的探讨尾侧型同源转录因子-2(CDX2)基因过表达对胃癌BGC-823细胞增殖、迁移、凋亡等生物学特征的影响。方法采用脂质体转染法建立CDX2基因过表达的胃癌BGC-823稳定细胞株,分别采用RT-PCR、Western blotting和免疫细胞化学等方法检测转染重组表达载体pEGFP-C1/CDX2后,BGC-823细胞中CDX2基因及其蛋白的表达。MTT法检测CDX2基因过表达对细胞的增殖能力的影响;划痕实验检测CDX2过表达对细胞迁移能力的影响;流式细胞术检测CDX2过表达对细胞的凋亡的影响;应用基因芯片技术检测转染前后相关基因的差异表达。结果 RT-PCR及Western blotting检测结果显示,与对照组相比,转染pEGFP-C1/CDX2后,BGC-823细胞中CDX2基因和蛋白均呈高表达;CDX2过表达能明显降低转然组BGC-823细胞增殖能力和迁移能力;但对细胞凋亡影响不明显;基因芯片结果提示CDX2基因高表达能影响某些基因的表达。结论 CDX2过表达能明显抑制胃癌细胞增殖、降低迁移能力,提示CDX2在胃癌中可能发挥抑癌基因的作用。     

下调lincRNA HOTAIR对胃癌细胞迁移及增殖抑制作用的研究

通过下调人胃癌细胞BGC823中linc RNA HOTAIR基因的表达,该文探讨了linc RNA HOTAIR低表达对胃癌细胞迁移、侵袭及增殖能力的影响。该文构建针对人linc RNA HOTAIR基因的干扰质粒sh HOTAIR,稳定转染入胃癌细胞BGC823、筛选稳转株,q PCR检测linc RNA HOTAIR在胃癌细胞中表达水平。采用划痕试验、侵袭试验、MTT法分别检测转染胃癌细胞迁移、侵袭及增殖能力。结果表明,稳定转染干扰质粒sh HOTAIR后下调linc RNA HOTAIR表达的胃癌细胞株细胞迁移、侵袭及增殖能力较阴性对照组明显减弱。下调胃癌细胞中linc RNA HOTAIR的表达,可降低胃癌细胞的迁移力、侵袭性、抑制其增殖能力,提示linc RNA HOTAIR可作为分子靶点用于胃癌的分子靶向治疗。     

大黄素抑制胃癌细胞糖酵解并促进凋亡

目的:探讨大黄素对人胃癌BGC-823细胞凋亡及糖酵解的影响。方法:采用不同浓度大黄素(30μmol/L、90μmol/L、180μmol/L)、磷脂酰肌醇3-激酶(Phosphatidylinositol 3 kinase,PI3K)抑制剂处理人胃癌BGC-823细胞,通过四甲基偶氮唑盐(MTT)检测细胞活力,采用试剂盒检测细胞葡萄糖消耗及乳酸水平,western blotting检测细胞己糖激酶Ⅱ、Bcl-2相关蛋白(Bcl-2 Associated X Protein,Bax)、PI3K、人低氧诱导因子1α(Human Hypoxia-inducible factor 1α,HIF-α)的表达。结果:大黄素能浓度依赖性的抑制BGC-823细胞增殖、葡萄糖消耗,降低乳酸水平;并降低己糖激酶Ⅱ的表达,促进凋亡蛋白Bax表达。PI3K抑制剂可抑制胃癌细胞糖酵解水平,而将大黄素与PI3K抑制剂联合使用后,与单一抑制剂组比,对细胞糖酵解抑制水平进一步加强,大黄素可下调PI3K下游蛋白及HIF-α的表达。结论:大黄素对人胃癌BGC-823细胞的增殖抑制作用其作用机制与调节PI3K途径及HIF-α,并抑制己糖激酶Ⅱ表达降低胃癌细胞糖酵解水平相关。     

PI3K/AKT/NF-κB轴调控胃癌细胞HGC-27增殖

目的:构建磷酸化AKT1(Ser473)位点突变真核表达载体,并检测PI3K/AKT/NF-κB信号通路轴对胃癌细胞增殖的影响。方法:以带有pcDNA3.0-Flag标签的AKT1质粒为模板,扩增出AKT1(Ser473A)(丝氨酸突变成丙氨酸)位点突变编码序列,将其插入pcDNA3.0-Flag载体中,双酶切和测序验证后瞬时转染人胚肾293T细胞,Western印迹检测其表达情况;将突变质粒与空载体分别转染胃癌细胞HGC-27,通过Western印迹检测其下游基因核转录因子κB(NF-κB)在蛋白水平的变化;通过CCK-8法检测对细胞生长曲线的影响。结果:双酶切和测序结果表明,pcDNA3.0-Flag-AKT1(Ser473A)真核表达质粒构建成功;转染293T细胞后获得表达;转染胃癌细胞HGC-27后,Western印迹验证去磷酸化AKT1(Ser473A)可下调NF-κB的蛋白水平(P0.01);细胞生长曲线结果显示,转染pcDNA3.0-Flag-AKT1(Ser473A)较空载体细胞生长慢(P0.01)。结论:PI3K/AKT/NF-κB信号通路轴在胃癌发生发展过程中发挥重要作用。     

H.pylori-L型对胃癌BGC-823细胞侵袭力影响的研究

目的研究幽门螺杆菌L型(Helicobacter pyloriL-form,H.pylori-L型)感染对胃癌BGC-823细胞侵袭力影响,探讨H.pylori-L型在胃癌发展中的作用和可能机制。方法将胃癌BGC-823细胞与H.pylori-L型按1:50、1:200和1:500的不同比例共培养24 h,进行以下实验:(1)应用具有聚碳酸酯和重建基底膜的Transwell小室细胞侵袭模型,观察与H.pylori-L型作用后胃癌BGC-823细胞的侵袭能力;(2)应用Western-blotting实验测定胃癌BGC-823细胞OPN和MMP2蛋白表达量的变化。结果 (1)Transwell侵袭实验发现随着胃癌BGC-823细胞与H.pylo-ri-L型细菌的浓度比例增大,胃癌BGC-823细胞穿透重建基底膜的数量逐渐增多,穿透重建基底膜的胃癌细胞数量在不同实验组之间的差异有统计学意义(F=24.78,P0.01);(2)Western-blotting实验发现,随着胃癌BGC-823细胞与H.pylori-L型比例增大,胃癌BGC-823细胞中OPN和MMP2的表达量逐渐增加,呈细菌浓度依赖性。结论 H.pylori-L型感染增强胃癌细胞的侵袭能力,具有细菌浓度依赖效应,其机制可能与其上调了胃癌细胞OPN、MMP2表达有关。     

靶向survivin的siRNA对胃癌BGC-823细胞增殖的影响

目的探讨干扰RNA沉默生存素（survivin）基因表达对人胃癌BGC-823细胞增殖和凋亡的影响。方法设计并合成3条靶向survivin的小分子干扰RNA（siRNA）,构建表达性干扰RNA质粒（shRNA）——shRNA-survivin-1、shRNA-survivin-2和shRNA-survivin-3,分别转染胃癌BGC-823细胞,实时定量PCR检测干扰RNA沉默survivin mRNA表达效果,Westernblot观察对胃癌BGC-823细胞survivin蛋白质表达的抑制,MTT（四甲基偶氮唑盐）比色法分析检测细胞生长抑制率,流式细胞计数检测各组细胞周期和凋亡率,探讨干扰RNA对胃癌BGC-823细胞生长的影响。结果在体外,shRNA-survivin-1有效沉默人胃癌BGC-823细胞survivin mRNA的表达,使sur-vivin mRNA相对水平明显降低（P〈0.05）,survivin蛋白质表达抑制,72h细胞生长抑制率达74.92%（P〈0.05）,shRNA-survivin-1使G2/M期细胞百分比明显增加,凋亡率显著增加（P〈0.05）。结论 shRNA-survivin-1可以沉默survivin基因的表达,可以显著抑制胃癌BGC-823细胞的增殖,在一定程度上诱导其自发凋亡。本研究为靶向sur-vivin的RNA干扰在胃癌的基因治疗提供了有力的理论依据和技术储备。     

shRNA沉默HuR抑制肝细胞癌细胞系SMMC-7721的增殖、迁移及侵袭能力

人抗原R (human antigen R,HuR)基因在肺癌、乳腺癌等多种肿瘤组织中高表达。推测HuR基因也参与肝癌的发展过程。为探索HuR对肝细胞癌细胞系SMMC-7721的增殖、迁移和侵袭的作用,本研究通过蛋白质印迹实验,检测HuR在肝细胞癌细胞系和正常肝细胞中蛋白质的表达水平。结果显示,肝细胞癌细胞系SMMC-7721 HuR的表达量显著高于正常肝细胞HL-7702。合成特异性靶向HuR基因的shRNA,转染肝细胞癌细胞系SMMC-7721,检测结果发现,HuR基因表达量下调90%。沉默HuR,实时细胞分析技术(real time cell analysis,RTCA)结果显示,细胞增殖能力降低55%,侵袭能力下降75%;细胞划痕实验结果显示,迁移能力下降80%;克隆形成实验中细胞克隆数减少85%;此外,在HuR敲低稳转肝细胞癌细胞系SMMC-7721细胞中过表达Bcl-2,其细胞学现象部分获得逆转。激光共聚焦扫描系统检测结果显示,Bcl-2主要定位于肝细胞癌细胞系SMMC-7721的核膜及胞质。蛋白质印迹法检测结果显示,沉默HuR,Bcl-2下调92%,Survivin下调55%,Twinst1下调69%,N-钙黏着蛋白(N-cadherin)下调48%,E-钙黏着蛋白(E-cadherin)上调1.5倍。以上结果表明,HuR可能通过调控定位于核膜及胞质上的Bcl-2来参与肝细胞癌细胞系SMMC-7721的增殖、迁移、侵袭及克隆形成过程,HuR基因有望成为临床上治疗肝癌的一个新的潜在靶点。     

B-RAF基因特异的siRNA干扰对胃癌BGC823细胞的影响

为探讨B-RAF基因特异的siRNA干扰对胃癌BGC823细胞的增殖和凋亡的影响, 设计并合成B-RAF小分子干扰RNA(B-RAF-siRNA)和阴性对照siRNA, 用TransMessenger介导转染胃癌BGC823细胞, RT-PCR分析检测胃癌BGC823细胞中B-RAF基因以及Bcl-2基因的表达; MTT检测胃癌BGC823细胞增殖情况; 流式细胞仪检测细胞凋亡情况, 并与对照组进行比较。TransMessenger能够有效介导B-RAF-siRNA和阴性对照siRNA转染胃癌BGC823细胞, TransMessenger介导的B-RAF-siRNA有效地抑制胃癌BGC823细胞B-RAF以及Bcl-2基因的表达, 与对照组相比, 抑制率达90.0%以上, 最高达100%; 同时明显抑制胃癌BGC823细胞增殖; 促进胃癌BGC823细胞的凋亡(P < 0.01)。B-RAF基因特异的siRNA干扰能有效地抑制胃癌BGC823细胞中B-RAF基因以及Bcl-2基因的表达, 同时促进胃癌细胞凋亡和抑制胃癌细胞增殖。     

Sprouty-2对胃癌细胞上皮间质转化及侵袭转移的影响

目的:研究Sprouty2(SPRY2)基因在胃癌肿瘤细胞上皮间质转化(EMT)和侵袭转移的影响。方法:体外培养人胃癌细胞(BGC-823),采用慢病毒介导的sh RNA沉默SPRY2基因,并用实时定量PCR与Western blot检测其SPRY2、E-钙黏蛋白(E-cadherin)、波形蛋白(vimentin)的表达,采用细胞划痕实验、Transwell实验检测SPRY2基因沉默后的胃癌细胞侵袭转移能力变化。结果:在慢病毒介导sh RNA沉默SPRY2基因的人胃癌BGC-823细胞中,SPRY2的m RNA和蛋白表达明显降低(P0.05),SPRY2沉默后人胃癌细胞E-cadherin的蛋白表达增多(P0.05),vimentin的蛋白表达减少(P0.05)。此外,SPRY2沉默后,胃癌细胞迁移能力和侵袭能力明显减弱(P值均P0.05)。结论:Sprouty-2基因通过调节E-cadherin与vimentin的表达参与胃癌细胞的上皮-间质转化,进而促进胃癌细胞的迁移与侵袭。     

颌下腺导管细胞与胶原海绵细胞相容性的实验研究

为探讨胶原海绵对颌下腺 (submandibulargland ,SMG)导管细胞的细胞相容性 ,采用HE染色光镜观察及免疫组化观察SMG导管细胞接种于胶原海绵后 ,细胞的生长情况。光镜下可见接种后第 1d细胞数量较少 ,分散于胶原海绵支架中间 ,第 7d细胞数量明显增加 ,免疫组织化学染色抗IV型胶原抗体染色呈阳性 ,说明细胞与支架材料之间已经有细胞外基质产生。胶原海绵具有良好的细胞相容性 ,是一种理想的支架材料。与胶原海绵复合培养 ,颌下腺导管细胞仍可保持良好的增殖能力。     

Determination of Mammalian Cell Counts,Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls^1-5. A critical breakthrough in cell/particle counting technologies was the development of the Coulter technique by Wallace Coulter over 50 years ago. The technique involves the application of an electric field across a micron-sized aperture and hydrodynamically focusing single particles through the aperture. The resulting occlusion of the aperture by the particles yields a measurable change in electric impedance that can be directly and precisely correlated to cell size/volume. The recognition of the approach as the benchmark in cell/particle counting stems from the extraordinary precision and accuracy of its particle sizing and counts, particularly as compared to manual and imaging based technologies (accuracies on the order of 98% for Coulter counters versus 75-80% for manual and vision-based systems). This can be attributed to the fact that, unlike imaging-based approaches to cell counting, the Coulter Technique makes a true three-dimensional (3-D) measurement of cells/particles which dramatically reduces count interference from debris and clustering by calculating precise volumetric information about the cells/particles. Overall this provides a means for enumerating and sizing cells in a more accurate, less tedious, less time-consuming, and less subjective means than other counting techniques⁶.Despite the prominence of the Coulter technique in cell counting, its widespread use in routine biological studies has been prohibitive due to the cost and size of traditional instruments. Although a less expensive Coulter-based instrument has been produced, it has limitations as compared to its more expensive counterparts in the correction for "coincidence events" in which two or more cells pass through the aperture and are measured simultaneously. Another limitation with existing Coulter technologies is the lack of metrics on the overall health of cell samples. Consequently, additional techniques must often be used in conjunction with Coulter counting to assess cell viability. This extends experimental setup time and cost since the traditional methods of viability assessment require cell staining and/or use of expensive and cumbersome equipment such as a flow cytometer.The Moxi Z mini automated cell counter, described here, is an ultra-small benchtop instrument that combines the accuracy of the Coulter Principle with a thin-film sensor technology to enable precise sizing and counting of particles ranging from 3-25 microns, depending on the cell counting cassette used. The M type cassette can be used to count particles from with average diameters of 4 - 25 microns (dynamic range 2 - 34 microns), and the Type S cassette can be used to count particles with and average diameter of 3 - 20 microns (dynamic range 2 - 26 microns). Since the system uses a volumetric measurement method, the 4-25 microns corresponds to a cell volume range of 34 - 8,180 fL and the 3 - 20 microns corresponds to a cell volume range of 14 - 4200 fL, which is relevant when non-spherical particles are being measured. To perform mammalian cell counts using the Moxi Z, the cells to be counted are first diluted with ORFLO or similar diluent. A cell counting cassette is inserted into the instrument, and the sample is loaded into the port of the cassette. Thousands of cells are pulled, single-file through a "Cell Sensing Zone" (CSZ) in the thin-film membrane over 8-15 seconds. Following the run, the instrument uses proprietary curve-fitting in conjunction with a proprietary software algorithm to provide coincidence event correction along with an assessment of overall culture health by determining the ratio of the number of cells in the population of interest to the total number of particles. The total particle counts include shrunken and broken down dead cells, as well as other debris and contaminants. The results are presented in histogram format with an automatic curve fit, with gates that can be adjusted manually as needed.Ultimately, the Moxi Z enables counting with a precision and accuracy comparable to a Coulter Z2, the current gold standard, while providing additional culture health information. Furthermore it achieves these results in less time, with a smaller footprint, with significantly easier operation and maintenance, and at a fraction of the cost of comparable technologies.     

Transformations of the Cell Center during Cell Differentiation

A question was posed as to how the multicomponent and polyfunctional organelle dynamically changes during metazoan ontogenesis. The centrosome structure is gradually formed and its functions are switched on during early embryogenesis, one of which is the cell center formation. During cell differentiation, the condition of the cell center and surrounding structures may be different: first, the cell center is quite distinct; second, the cell center is absent due to redistribution of the microtubule organizing centers; third, the cell center disappears due to reversible or irreversible inactivation of the centrosome and other centers of microtubule organization. The assembly of the Golgi complex does not depend directly to the cell center presence. In some cell types, the Golgi complex is topologically associated with the cell center, while in others it exists as individual dictyosomes despite the cell center presence. In some other cell types, the common Golgi complex is assembled without the cell center, but in the presence of microtubules that are formed by noncentrosome centers of microtubule organization. In still others, degradation of both the cell center and the common Golgi complex takes place in the case of centrosome inactivation.     

Determining Cell Number During Cell Culture using the Scepter Cell Counter

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and accuracy. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses.Despite this need for speed and accuracy in cell counting, 71% of 400 researchers surveyed¹ who count cells using a hemocytometer. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse, which results in inaccurate counts. Hemocytometers are made of special optical glass on which cell suspensions are loaded in specified volumes and counted under a microscope. Sources of errors in hemocytometry include: uneven cell distribution in the sample, too many or too few cells in the sample, subjective decisions as to whether a given cell falls within the defined counting area, contamination of the hemocytometer, user-to-user variation, and variation of hemocytometer filling rate².To alleviate the tedium associated with manual counting, 29% of researchers count cells using automated cell counting devices; these include vision-based counters, systems that detect cells using the Coulter principle, or flow cytometry¹. For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments¹.The Scepter cell counter is an automated handheld device that offers the automation and accuracy of Coulter counting at a relatively low cost. The system employs the Coulter principle of impedance-based particle detection³ in a miniaturized format using a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip is engineered with a microfabricated, cell- sensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter reports cell population statistics graphically displayed as a histogram.     

细胞提取物重编程研究进展

体细胞重编程是在特定的条件下使已分化的细胞转变成为另一种细胞.体细胞重编程的方式主要有体细胞核移植技术、细胞融合技术、细胞提取物处理技术及特定转录因子转染技术.现有研究表明,细胞提取物重编程技术在体细胞重编程中发挥着一定的作用,为此,就该技术的最新研究进展和可能机制作一综述.     

Cell Physician: Reading Cell Motion

Cell motility is an essential phenomenon in almost all living organisms. It is natural to think that behavioral or shape changes of a cell bear information about the underlying mechanisms that generate these changes. Reading cell motion, namely, understanding the underlying biophysical and mechanochemical processes, is of paramount importance. The mathematical model developed in this paper determines some physical features and material properties of the cells locally through analysis of live cell image sequences and uses this information to make further inferences about the molecular structures, dynamics, and processes within the cells, such as the actin network, microdomains, chemotaxis, adhesion, and retrograde flow. The generality of the principals used in formation of the model ensures its wide applicability to different phenomena at various levels. Based on the model outcomes, we hypothesize a novel biological model for collective biomechanical and molecular mechanism of cell motion.