Let-7a下调k-Ras和c-Myc癌基因的表达抑制肾癌细胞增殖

目的：MicroRNA 是近年发现的一类单链小分子RNA,对它的研究已成为一个新的热点。最近的研究发现,1et-7a 在细胞内影响着基因的表达调控,在疾病发生中起着及极重要的作用,尤其是在肿瘤的发展过程中,let-7a扮演着不可替代的角色。本文主要研究let-7a 在肾癌细胞株中的表达情况及其调控的靶基因、抑制细胞增殖的机制,对探索肾癌的致病基因,寻求肾癌新的治疗途径有重要意义。方法：应用化学合成的let-7a 模拟物（mimics）用脂质体Lipofectamine 2000 在体外瞬时转染786-O 和Caki-1肾癌细胞株,转染48 小时后采用荧光定量RT-PCR的方法检测let-7a 及c-Myc、k-Ras mRNA的表达情况,Western blot 检测这两株肾癌细胞转染了let-7a mimics 后c-Myc 及k-Ras 蛋白的表达变化;转染let-7a mimics 后分别在24、48、72 小时三个时间点用CCK-8 试剂盒检测对肾癌细胞株增殖的影响。结果：786-O 和Caki-1 肾癌细胞株中let-7a 的表达量明显低于正常肾小管上皮细胞株HK-2（P〈0.05）; 转染了let-7amimics的786-O 和Caki-1 肾癌细胞株,RT-PCR 及Western blot 结果显示c-Myc、k-Ras 在基因及蛋白的表达水平明显下调（P〈0.05）;CCK-8 检测结果显示转染了let-7a mimics的肾癌细胞株细胞增殖能力明显明显受到抑制,与阴性对照组比较差异有统计学意义（P〈0.05）。结论：Let-7a 在在肿瘤细胞与正常细胞中存在明显差异,let-7a 通过调控c-Myc、k-Ras的表达能抑制肾癌细胞增殖。Let-7a mimics 可以抑制肾癌细胞的增殖,因此上调Let-7a 的表达有可能成为肾癌基因治疗的一种有效治疗手段。     

MicroRNA-34a通过多向调控促癌基因抑制肾癌细胞生长

目的:探讨microRNA-34a(miR-34a)在肾癌细胞中的生物学作用及调控机制.方法:应用miR-34amimics在体外转染769P,786-O和Caki-1细胞;运用qRT-PCR检测miR-34a在三个细胞株的相对表达情况,以及转染后癌基因mRNA的表达情况;观察miR-34a对细胞生长的影响.结果:769P,786-O和Caki-1细胞中miR-34a在786-O中表达最低,769P次之,Caki-1表达最高;利用miR-34a mimics升高769P,786-O和Caki-1细胞miR-34a,发现三个细胞株多个癌基因mRNA表达不同程度的降低(P＜0.05)及生长和集聚能力的降低.结论:miR-34a可能通过调控多个癌基因表达在肾癌中起抑癌作用.miR-34a mimics可抑制肾癌细胞的生长,因此miR-34a有可能作为肾癌基因治疗的新靶点.     

MicroRNA-145在肾癌中的临床意义及其分子机制探讨

目的:探讨miRNA-145在肾癌中的临床意义及其分子机制。方法:通过实时定量PCR检测和比较16例肾癌患者的肿瘤组织及癌旁正常肾组织中miRNA-145的表达,并分析miRNA-145的表达水平与肾癌患者病理特征及临床分级的相关性。进一步采用实时定量PCR检测和比较肾癌细胞株786-O、769P及正常肾细胞株HK2中miRNA-145表达量,通过转染miRNA-145 mimics和阴性对照miRNA至769P、786-O肾癌细胞株后观察癌基因(bcl-2、e2f3、cdk6、ccnd1)mRNA的表达。结果:肾癌组织中miRNA-145的表达显著低于癌旁正常肾组织(P0.05),且与肾癌的直径、病理核分级及临床分级呈显著负相关(P0.05)。肾癌细胞株769P、786-O中miRNA-145的表达显著低于正常HK-2肾细胞(P0.05),而转染miRNA-145 mimics的769P、786-O细胞中多个癌基因(bcl-2、e2f3、cdk6、ccnd1)的mRNA表达均较阴性对照组显著降低(P0.05)。结论:MiRNA-145在肾癌中呈低表达,可能通过调控多个癌基因的表达在肾癌的发生发展过程中发挥重要的作用。     

Spautin-1通过抑制自噬可增强舒尼替尼诱导的肾癌细胞凋亡

目的:研究spautin-1(一种自噬抑制剂)是否能抑制舒尼替尼诱导的肾癌细胞的自噬以及对舒尼替尼诱导的肾癌细胞凋亡的影响。方法:以肾癌细胞系786-O细胞为模型,Western Blot检测spautin-1对舒尼替尼诱导786-O细胞自噬的影响;Cell Counting Kit-8(CCK-8)检测spautin-1和舒尼替尼对786-O细胞增殖的影响;应用流式细胞术,检测spautin-1对舒尼替尼诱导的786-O细胞凋亡的影响;Western Blot检测spautin-1的促凋亡作用与PI3K/AKT/GSK3β信号通路及抗凋亡蛋白Bcl-2和Mcl-1的关系。结果:与舒尼替尼单独处理组相比,spautin-1能通过降低Beclin-1的表达显著抑制舒尼替尼在786-O细胞中诱导的自噬;Spautin-1和舒尼替尼联合作用明显增强舒尼替尼对786-O细胞增殖的抑制作用;Spautin-1能进一步增强舒尼替尼诱导的786-O细胞的凋亡;Spautin-1和舒尼替尼联合处理786-O细胞时,可以显著降低p-AKTSer473和p-GSK3βSer9的蛋白表达水平,增强GSK3β的活性,进而下调Bcl-2、Mcl-1的表达。结论:Spautin-1能通过抑制AKT活性并活化GSK3β,进一步降低抗凋亡蛋白Bcl-2、Mcl-1的表达,增强舒尼替尼诱导的肾癌细胞凋亡。     

RNA干扰Ezrin体外对肾癌细胞株786-0增殖、凋亡的影响(英文)

目的:构建膜-细胞骨架联接蛋白Ezrin基因特异性短发卡RAN表达载体(small hairpin RNA,shRNA),探讨其对肾癌细胞株786-0细胞凋亡、增殖的影响。方法:以Ezrin为靶基因,以Pgenesil-1质粒为载体,设计和构建重组体,设计2条发夹式RNA(shRNA),合成后克隆入载体Pgenesil-1,扩增并中量提取质粒,应用脂质体Lipofectamine 2000转染进786-0细胞,重组质粒转染786-0肾癌细胞株,用运实时荧光定量PCR进行筛选鉴定,筛选出抑制率较高的重组质粒载体shRNA-Ezrin1,用shRNA-Ezrin1转染786-0细胞,采用MTT、流式细胞仪、电镜检测,观察RNA干扰Ezrin后肾癌细胞株786-0细胞增殖能力的改变。结果:shRNA干扰后786-0细胞增殖活性减弱,G0/G1时段明显延长(P0.01),PI缩短(P0.01),细胞凋亡率增加(P0.01)。结论:Ezrin与肾癌细胞凋亡、增殖有关,有望成为肾癌基因治疗的一个新靶点。     

miR-184通过抑制EPB41L5调控大鼠肾癌细胞增殖及迁移

为了探讨miR-184对肾癌细胞的影响及机制,本研究选取肾癌细胞株786-0细胞,随机分为对照组、空白转染组和miR-184转染组,其中miR-184转染组转染miR-184 mimic,空白转染组转染空白mimic,采用CCK-8细胞增殖实验检测各组细胞增殖,流式细胞仪检测各组细胞凋亡,划痕实验检测各组细胞迁移,Western blotting检测各组EPB41L5蛋白表达。研究结果表明miR-184转染组培养48 h和培养72 h时OD值分别为(0.964±0.103)和(1.011±0.121),明显低于对照组和空白转染组(p<0.05);miR-184转染组培养72 h后细胞凋亡率为(18.22±2.26)%,明显高于对照组和空白转染组(p<0.05);miR-184转染组培养24 h后细胞迁移数为(17.21±3.06)个,明显低于对照组和空白转染组(p<0.05);miR-184转染组细胞EPB41L5蛋白相对表达量为(0.241±0.061),明显低于对照组和空白转染组(p<0.05)。本研究初步表明:miR-184可抑制肾癌786-0细胞增殖和迁移,促进细胞凋亡,其可能与其抑制EPB41L5蛋白表达有关。     

miR-21对肾癌细胞凋亡和侵袭力的影响

目的:探讨miR-21对肾癌细胞凋亡和侵袭力的影响.方法:人类肾癌细胞株Caki-1和OS-RC-2细胞,分别以As-miR-21转染沉默miR-21,转染无关乱码寡核苷酸序列作阴性对照,以及不转染寡核苷酸的作空白对照,以MTT法测细胞生长,以AnnexinVFITC凋亡检测试剂盒检测凋亡,采用Transwell肿瘤浸润实验测细胞增殖力.结果:转染无关乱序miRNA的阴性对照组其细胞生存率与空白对照无差异,而转染As-miR-21的Caki-1和OS-RC-2细胞,其生存率均显著下降(P＜0.05),且在72 h时生存率下降最明显;转染AS-miR-21的细胞凋亡率(Caki-1 16.7％; OS-RC-2 14.5％)相对于转染无关乱序miR-21的阴性对照细胞(Caki-12.3％; OS-RC-2 3.3％)和未转染的空白对照细胞(Caki-1 2.5％; OS-RC-2 3.7％)显著增加(P＜0.05).转染AS-miR-21的细胞跨过Transwell膜的相对于转染无关乱序miR-21的阴性对照细胞和未转染的空白对照细胞显著减少(P＜0.05).结论:miR-21能增加肾癌细胞的抗凋亡力和侵袭力,沉默miR-21能抑制肾癌恶性程度.     

抑癌基因NPRL2对肾癌786-O细胞增殖和凋亡的影响

抑癌基因NPRL2(nitrogen permease regulator-like 2)在人类许多正常组织中均有明显的表达,而在人类多种肿瘤组织中的表达明显降低。该实验通过构建重组质粒pEGFP-N1-NPRL2并转染肾癌786-O细胞株,应用倒置荧光显微镜、RT-PCR和Western blot检测NPRL2基因的表达情况;MTT法检测786-O细胞增殖的情况;流式细胞仪分析细胞周期和细胞凋亡情况。结果显示:肾癌786-O细胞株转染重组质粒pEGFP-N1-NPRL2后,通过荧光显微镜可以观察到绿色荧光;RT-PCR和Western blot检测到NPRL2基因的转录和蛋白质表达水平明显增加(P<0.05);MTT法检测发现72 h时pEGFP-N1-NPRL2组、pEGFP-N1组和空白对照组的细胞D490值分别为0.654±0.030、1.528±0.022和1.572±0.036,pEGFP-N1-NPRL2组细胞的增殖较其他两组受到明显的抑制(P<0.05);流式细胞仪检测显示pEGFP-N1-NPRL2组、pEGFP-N1组和空白对照组的凋亡率分别为18.82%±0.40%、5.65%±0.12%和5.85%±0.07%,而处于G0/G1期的细胞比例分别为69.80%±1.40%、46.24%±1.30%和47.03%±0.45%,与其他两组相比,pEGFP-N1-NPRL2组的凋亡率显著增高而且G0/G1期细胞明显增多,表现出G0/G1期阻滞。提示抑癌基因NPRL2的转染可以抑制786-O细胞的增殖,并诱导其凋亡将细胞周期阻滞于G0/G1期。     

Let-7a在哮喘T淋巴细胞失衡中的作用研究

目的:探讨let-7a在哮喘中的表达水平及调节T淋巴细胞亚群失衡的功能作用。方法:收集39例哮喘患者及19例非哮喘对照,Real-time PCR检测let-7a的表达。从哮喘患者外周血单核细胞(Peripheral blood mononuclear cell,PBMC)中分选Th1,Th2和Th17细胞,检测let-7a在T淋巴细胞亚群中的表达。磁珠分选naive T细胞,分别诱导分化成Th1、Th2和Th17细胞,分析let-7a在T淋巴细胞亚群中的表达。Let-7a mimic分别转染Th2和Th17细胞,ELISA检测IL-13和IL-17的表达。结果:与对照组比较,let-7a在哮喘患者肺组织和血清中低表达,并随着哮喘病程进展,let-7a的表达水平也越低。哮喘患者来源的CD4~+T细胞中,let-7a表达明显低于非哮喘对照组。Let-7a在哮喘来源和体外刺激分化的哮喘炎性Th2和Th17细胞中的表达明显下调,而在Th1细胞中的表达没有变化。Let-7a mimic改变了Th2和Th17相关细胞因子IL-13和IL-17的表达。结论:Let-7a在哮喘中低表达,调节哮喘T淋巴细胞亚群失衡,改变了Th2和Th17细胞的功能,是哮喘理想的诊断和治疗靶点。     

白藜芦醇经PI3K/Akt/mTOR 信号通路诱导人肾癌786-O细胞自噬

白藜芦醇(resveratrol)可抑制人肾癌786-O细胞增殖,并诱导其凋亡,但是白藜芦醇对786-O细胞自噬(autophagy)的影响及机制尚不清楚。为探究其机制,体外培养786-O细胞,采用CCK-8检测786-O细胞活力;TUNEL染色检测786-O细胞凋亡;透射电子显微镜观察786-O细胞自噬体;吖啶橙染色观察786-O细胞自噬小泡;GFP-LC3质粒转染分析观察786-O细胞自噬体;Western印迹检测LC3、beclin-1、PI3K、p-PI3K、Akt、p-Akt、mTOR和p-mTOR的表达。结果显示,白藜芦醇以浓度和时间依赖性的方式抑制786-O细胞活力,并诱导细胞凋亡;与对照组相比,白藜芦醇使786-O细胞自噬增强;Western印迹结果显示,与对照组相比,白藜芦醇组LC3-II/LC3-I和Beclin-1显著增高(P0.01),表明白藜芦醇导致786-O细胞自噬体积累。与对照组相比,白藜芦醇使786-O细胞的p-PI3K/PI3K,p-Akt/Akt和p-mTOR/mTOR显著降低(P0.01),表明白藜芦醇可通过PI3K/Akt/mTOR信号通路增强自噬。综上所述,白藜芦醇通过抑制PI3K/Akt/mTOR信号通路从而诱导786-O细胞自噬。     

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。     

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.     

Tuning Cell Motility via Cell Tension with a Mechanochemical Cell Migration Model

Cell migration is orchestrated by a complicated mechanochemical system. However, few cell migration models take into account the coupling between the biochemical network and mechanical factors. Here, we construct a mechanochemical cell migration model to study the cell tension effect on cell migration. Our model incorporates the interactions between Rac-GTP, Rac-GDP, F-actin, myosin, and cell tension, and it is very convenient in capturing the change of cell shape by taking the phase field approach. This model captures the characteristic features of cell polarization, cell shape change, and cell migration modes. It shows that cell tension inhibits migration ability monotonically when cells are applied with persistent external stimuli. On the other hand, if random internal noise is significant, the regulation of cell tension exerts a nonmonotonic effect on cell migration. Because the increase of cell tension hinders the formation of multiple protrusions, migration ability could be maximized at intermediate cell tension under random internal noise. These model predictions are consistent with our single-cell experiments and other experimental results.