G6PD通过细胞周期蛋白D1/D2调控人黑色素瘤细胞周期的进程

葡萄糖-6-磷酸脱氢酶(G6PD)在人皮肤黑色素瘤A375细胞中处于高表达与高活性状态, 但G6PD在黑色素瘤发生发展过程中的作用及其具体机制尚不明确.本文在前期运用 siRNA方法构建G6PD敲减的黑色素瘤A375稳转细胞(A375-G6PDΔ)基础上,构建表达载体pBabe-puro-G6PDWT在A375-G6PDΔ细胞中过表达野生型的G6PD基因,从而构建G6PD表达恢复的稳转细胞(A375-G6PDΔ-G6PDWT).3株细胞A375-WT、A375-G6PDΔ和 A375-G6PDΔ-G6PDWT经G6PD酶活性测定、MTT测定、克隆形成实验、流式细胞仪分析细胞周期和Western 印迹检测.结果显示,A375-G6PDΔ-G6PDWT细胞的G6PD蛋白表达量 (0.847 ± 0.080)及其活性(0.394 ± 0.029)分别是A375-G6PDΔ的3.28倍(P＜0.01) 和7.34倍(P＜0.01),分别是A375-WT细胞的91-57%和2.12倍(P＜0.05).与A375-WT细 胞相比,A375-G6PDΔ细胞G0/G1期细胞数增加,S期细胞数减少,增殖指数PI降低了25-70%（P＜0.05）,细胞周期蛋白D1/D2、细胞周期蛋白E表达分别下降37.4%、54.3% (P＜0.01)和17.3%;而A375-G6PDΔ-G6PDWT细胞呈现G1/S期阻滞解除,细胞周期蛋白D1/D2蛋白分别恢复到A375-WT细胞的89.5%和87.6%,细胞周期蛋白E表达未见 恢复,呈现生长增殖和克隆形成率的恢复并接近于A375-WT细胞. 结果提示,G6PD通 过细胞周期蛋白D1/D2调控人皮肤黑色素瘤A375细胞G1期向S期转换的进程,这为黑色 素瘤发病机制的研究提供了新的思路.     

G6PD缺陷抑制人黑色素瘤细胞裸鼠移植瘤的生长

敲减葡糖6-磷酸脱氢酶(G6PD)表达的人黑色素瘤A375细胞(A375-G6PDΔ) 呈现生长增殖抑制和凋亡率升高. 为明确G6PD缺陷对裸鼠体内成瘤的影响及其可能机制,用A375-WT与A375-G6PDΔ细胞制作裸鼠荷瘤模型,观察体内瘤体生长,real-time PCR、免疫组织化学染色与紫外分光光度法分别检测瘤体组织G6PD mRNA、G6PD蛋白及酶活性,Western 印迹分析凋亡相关蛋白,分光光度法测定NADPH和GSH/GSSG水平. 结果显示,A375-G6PDΔ细胞注射组的裸鼠成瘤时间延长,瘤体生长明显减慢,瘤体的体积与质量显著低于A375 WT细胞注射组（P <0.01）;与A375-WT细胞注射组相比,A375 G6PDΔ细胞注射组的裸鼠瘤体组织中G6PD mRNA表达、G6PD阳性细胞数与G6PD活性分别降低了87.10%、77.20%与75.77%（P<0.01）,G6PD、p53和Bcl-2的表达分别降低了67.92%、65.54%和62.32%（P<0.01）,Fas升高了86.38%（P<0.01）,NADPH和GSH/GSSG分别降低了74.37%和86.02%（P<0.01）. 结果提示,G6PD缺陷可能通过减少核酸等合成的原料、改变细胞内氧化还原状态及凋亡相关蛋白表达抑制裸鼠瘤体生长与增殖,这为黑色素瘤发生和治疗研究提供了新的线索.     

G6PD缺陷通过Caspase-3和PARP-1诱导人黑色素瘤细胞凋亡

葡糖-6-磷酸脱氢酶(G6PD)在许多肿瘤细胞中高表达,但其发生的作用机理目前仍然不明确.以正常人表皮黑色素细胞(HEM)、野生型人黑色素瘤A375细胞(A375-WT)和G6PD缺陷的A375细胞(A375-G6PDΔ)为对象,经real-time PCR、Western印迹和紫外分光光度法分析显示,A375-WT细胞的mRNA、G6PD蛋白和G6PD活性分别是HEM细胞的1.89倍(P0.05)、6.86倍(P0.01)和2.30倍(P0.05).Annexin V/PI流式细胞仪和Western印迹测定表明,A375-G6PDΔ的凋亡率是A375-WT的5.10倍(P0.01),活化半胱氨酸蛋白酶3(caspase-3)增高1.84倍(P0.01)以及89 kD多聚二磷酸腺苷核糖聚合酶-1(PARP-1)生成增加2.87倍(P0.01).分光光度法分析显示,A375-G6PDΔ的NADPH和GSH分别降低了72.30%(P0.01)和27.39%(P0.05),并伴有75.43%的H2O2增高(P0.01).结果提示,G6PD在黑色素瘤细胞中高表达和高活性,而敲减G6PD表达通过caspase-3和PARP-1信号诱发人黑色素瘤细胞凋亡,这为深入揭示黑色素瘤的发生机理提供了新思路。     

敲减NADPH氧化酶4能降低STAT3活性抑制人黑色素瘤A375细胞的增殖

NADPH氧化酶参与细胞活性氧族（ROS）的生成过程,而ROS与肿瘤细胞增殖密切相关. 为了阐明NADPH氧化酶影响黑色素瘤A375细胞增殖的分子机制,本文首先应用荧光定量PCR和Western 印迹证实NOX4为人黑色素瘤A375细胞的NADPH氧化酶功能核心亚基;随后根据NOX4基因设计3条干扰序列和对照序列并连接到pSuper-retro-puro载体,经鉴定后转化E.coli DH5α感受态细胞、筛选有效干扰序列并用于逆转录病毒包装,病毒液感染A375细胞并经嘌呤霉素筛选10 d,构建了NOX4缺陷的A375稳转细胞珠（A375 NOX4Δ）,其NOX4的mRNA和蛋白表达分别下降了66.02%和77.35%,伴随NADPH氧化酶活性和ROS水平分别下降了79.17%和64.16%;MTT、EdU法检测显示,A375-NOX4Δ细胞的增殖能力比A375-WT细胞明显降低、倍增时间延长,增殖细胞数量下降了68.27%（P＜0.01）,呈现G1→S期阻滞;Western blot检测表明A375 NOX4Δ细胞的 cyclin D1、CDK4分别下降了55.7%（P＜0.01）和64.8%（P＜0.01）,而P53、P21分别增加了6.89 倍（P＜0.01）和3.27 倍（P＜0.01）,STAT3、P-STAT3分别下降了51.80%（P＜0.05）和82.58%（P＜0.01）;电泳迁移率变动分析（EMSA）表明,A375 NOX4Δ细胞的STAT3-DNA结合活性明显降低. 上述结果提示,敲减A375细胞的NOX4表达可能通过减少ROS生成使得STAT3磷酸化水平及其结合DNA的活性下降,最终导致A375-NOX4Δ细胞增殖减少、呈现G1→S期阻滞,这为黑色素瘤发病机制研究提供了新思路及可能的药物作用靶点.     

MAT2A基因小干扰RNA诱导人肝癌细胞凋亡的分子机制

为探讨甲硫氨酸腺苷转移酶2A(MAT2A)小干扰RNA对人肝癌细胞生长和细胞凋亡的影响及其机 制,采用脂质体转染法将MAT2A小干扰RNA质粒表达载体转染人肝癌细胞系Bel 7402细胞、HepG 2细胞和 HepG3B细胞.半定量RT PCR检测MAT2A mRNA表达,Western印迹检测MAT2A 蛋白质表达, M TT法观察MAT2A小干扰RNA对肝癌细胞生长的影响,流式细胞仪及DAPI染色检测siRNA对肝癌细 胞凋亡的影响.为探讨其作用机制, 进一步检测转染后肝癌细胞MAT的活性、MAT1A mRNA表 达及SAM、SAH含量.结果发现, MAT2A小干扰RNA特异性抑制人肝癌细胞MAT2A mRNA和蛋白质 的表达, 刺激MAT表达由MAT2A向MAT1A转变, 降低了肝癌细胞中MATⅡ活性（P<005） ,从而诱导肝癌细胞凋亡; MAT2A小干扰RNA诱导Bel－7402细胞、HepG 2细胞、 Hep 3B细胞凋亡 指数分别为19．3%±2．8%、22．8%±3．5%、21．8%±4．2%, 较对照组siRNA(凋亡指数为5 2%±19%)具有明显差异(P<005).DAPI染色显示, MAT2A小干扰RNA转染组可见多个细胞核 浓缩、碎裂成蓝色的小块状,染色质凝聚,形成典型的凋亡小体, 而对照siRNA转染组未发现典型的 凋亡小体.肝癌细胞的生长也受到抑制,MAT2A小干扰RNA转染Bel 7402细胞、HepG 2细胞 、HepG3B细胞72 h后,细胞生长抑制率达高峰,分别为39．62%、41．27%、38．84%.肝癌细胞 中SAM含量明显升高(P<001),而SAH含量改变不明显, SAM/SAH变化伴随SAM含量变化而改 变.提示靶向MAT2A基因的siRNA通过升高肝癌细胞中SAM含量,刺激MAT表达由MAT2A向MAT1A转变, 从而诱导肝癌细胞凋亡,抑制肝癌细胞生长.     

臭椿酮诱导人黑色素瘤A375细胞凋亡及其机制研究

为研究臭椿酮(Ailanthone,AIL)诱导人黑色素瘤A375细胞凋亡的作用及作用机制,以人黑色素瘤A375细胞为研究对象,采用MTT法测定AIL对人黑色素瘤A375细胞生长增殖的抑制作用。用倒置相差显微镜观察AIL对A375细胞形态的影响,用荧光倒置显微镜观察Hoechst33258染色后AIL对A375细胞核的影响,用AnnexinV-FITC/PI双染法检测AIL诱导A375细胞凋亡的作用,用分光光度法检测caspase-3和caspase-9的活性,Westernblot检测p-PI3Kβ(Ser1070),PI3Kβ,p-Akt(Ser473)和Akt蛋白表达水平的变化,接着用PI3K抑制剂LY294002进行干预,进一步验证AIL对PI3K/Akt信号通路及细胞凋亡的影响。实验结果表明,AIL能够明显抑制A375细胞增殖,使A375细胞数目变少、附着力和透光性减弱,AIL能够诱导A375细胞凋亡,使其细胞核染色质发生固缩并呈现高亮,且使A375细胞早期及晚期凋亡率均增加,AIL作用后能够使caspase-3和caspase-9活性增加,AIL能够抑制PI3K和Akt蛋白磷酸化,从而使PI3K/Akt信号通路失活。较AIL单独作用,AIL和LY294002共同作用后对PI3K和Akt蛋白磷酸化的抑制作用增强且诱导凋亡作用增加,进一步说明AIL通过失活PI3K/Akt信号通路来诱导A375细胞凋亡。     

抑制survivin表达增强人肝癌HepG2细胞对高线性能量转移射线的辐射敏感性

探讨了肿瘤细胞中survivin的表达对高线性能量转移(LET)射线辐射敏感性的影响.根据Gen Bank提供的survivin序列,合成特异性survivin-siRNA寡核苷酸,转染人肝癌HepG2细胞,抑制survivin的表达.发现siRNA转染后诱导了HepG2细胞G2/M期阻滞,增加了自发性和辐射诱导的细胞凋亡.在高线性能量转移(LET)碳离子辐照后,siRNA转染细胞的克隆存活率明显下降.这些结果表明survivin表达是HepG2细胞产生对高LET射线辐射抗性的关键因素.     

干扰素α1b对黑色素瘤细胞A375增殖、迁移和凋亡的影响

目的:研究干扰素α1b(IFN-α1b)对人恶性黑色素瘤细胞A375增殖、迁移和凋亡的影响。方法:采用体外培养的黑色素瘤细胞A375,通过MTT法检测和比较IFN-α1b和IFN-α2b对A375细胞的增殖的抑制作用;细胞划痕实验分析IFN-α1b对A375细胞迁移的影响;Annexin V-FITC/PI染色后采用荧光显微镜观察和流式细胞术分析IFN-α1b对A375细胞凋亡的影响。结果:MTT结果显示随着IFN-α1b和IFN-α2b浓度的增加,A375细胞的生长抑制率逐渐升高,药物作用48 h和72 h后,IFN-α1b作用的A375细胞生长抑制率明显高于IFN-α2b,IFN-α1b和IFN-α2b作用48 h的IC50分别为(22.69±1.52)μg/mL和(35.69±1.01)μg/mL(P0.01);IFN-α1b和IFN-α2b作用72 h的IC50分别为(10.00±0.98)μg/mL和(25.02±0.44)μg/mL(P0.01)。细胞划痕实验显示IFN-α1b能够使A375细胞的迁移指数和迁移能力降低,且随着IFN-α1b药物浓度的增加抑制细胞迁移作用增强。随着50μg/mL IFN-α1b作用的A375细胞的凋亡率为(29.31±0.45)%,较对照组(8.21±1.36)%相比明显上升(P0.01)。结论:IFN-α1b对人恶性黑色素瘤细胞A375具有生长抑制作用,与IFN-α2b相比,IFN-α1b的抑制作用更加显著;IFN-α1b能够降低细胞A375的迁移能力并诱导其凋亡。     

早孕因子单克隆抗体对黑色素瘤细胞A-375增殖、凋亡的影响

目的:研究早孕因子单克隆抗体(EPF-McAb)对黑色素瘤细胞A-375增殖、凋亡的影响。方法:体外培养黑色素瘤细胞A-375,通过CCK-8实验检测EPF-McAb对A-375细胞增殖的影响;通过流式细胞术检测EPF-McAb对A-375细胞凋亡的影响;Western Blot检测EPF-McAb对A-375细胞EPF蛋白表达的影响。结果:CCK-8结果显示EPF-McAb可以抑制黑色素瘤细胞A-375的增殖,且随着作用时间延长和药物浓度的增加,对A-375细胞增殖的抑制作用也增强;流式细胞术实验结果显示EPF-McAb可以促进黑色素瘤细胞A-375的凋亡,且调亡率随着药物浓度的增加而升高,0.2、0.4、0.8 mg/m L的EPF单抗作用于A-375细胞24 h后,细胞调亡率分别为:14.68%(P0.01)、19.81%(P0.01)、23.97%(P0.01);Western Blot实验结果显示EPF-McAb可以降低黑色素瘤细胞A-375 EPF蛋白的表达。结论:早孕因子单克隆抗体可以抑制黑色素瘤细胞A-375的增殖,并促进其凋亡。     

siRNA 诱导CyclinB1沉默增加HePG2细胞对柔红霉素的敏感性

探讨了CyclinB1在肝癌药物耐受形成中的作用.用siRNA技术沉默CyclinB1在细胞中的表达,使用流式细胞仪技术分析细胞的凋亡和周期分布,用细胞克隆形成能力分析和细胞毒性实验分析细胞的增殖能力.由siRNA诱导的CyclinB1下调导致40%～50%肝癌细胞阻滞在G2/M期,并显著抑制肝癌细胞的单克隆形成能力;柔红霉素联合CyclinB1 siRNA较单独使用柔红霉素能更加有效地导致肝癌细胞凋亡,而在人正常肝细胞HL-7702中这种现象不明显.实验结果表明针对于CyclinB1的靶向性下调与肝癌药物的联合使用将有可能成为肝癌治疗的新策略.     

微流控芯片技术在细胞生物学研究中的应用进展

20世纪90年代以来,微流控芯片技术得到了快速发展。由于具有小型化、集成化、高通量、低消耗、分析快速等特点,微流控芯片作为一种新型的生物学研究平台,能够提供传统方法不具备的精细和可控制的细胞研究条件,在细胞生物学研究领域中得到了广泛关注。该文主要介绍其在细胞培养、分选、裂解、计数、凋亡检测、迁移、单细胞捕获、细胞间作用等方面的研究进展。     

Efficient and Innocuous Live‐Cell Delivery: Making Membrane Barriers Disappear to Enable Cellular Biochemistry

The confluence of protein engineering techniques and delivery protocols are providing new opportunities in cell biology. In particular, techniques that render the membrane of cells transiently permeable make the introduction of nongenetically encodable macromolecular probes into cells possible. This, in turn, can enable the monitoring of intracellular processes in ways that can be both precise and quantitative, ushering an area that one may envision as cellular biochemistry. Herein, the author reviews pioneering examples of such new cell‐based assays, provides evidence that challenges the paradigm that cell penetration is a necessarily damaging and stressful event for cells, and highlights some of the challenges that should be addressed to fully unlock the potential of this nascent field.     

Ultrastructural evidence for two-cell and three-cell neural pathways in the tentacle epidermis of the sea anemone Aiptasia pallida.

Sensory and ganglion cells in the tentacle epidermis of the sea anemone Aiptasia pallida were traced in serial transmission electron micrographs to their synaptic contacts on other cells. Sensory cell synapses were found on spirocytes, muscle cells, and ganglion cells. Ganglion cells, in turn, synapsed on sensory cells, spirocytes, muscle cells, and other neurons and formed en passant axo-axonal synapses. Axonal synapses on nematocytes and gland cells were not traced to their cells of origin, i.e., identified sensory or ganglion cells. Direct synaptic contacts of sensory cells with spirocytes and sensory cells with muscle cells suggest a local two-cell pathway for spirocyst discharge and muscle cell contraction, whereas interjection of a ganglion cell between the sensory and effector cells creates a local three-cell pathway. The network of ganglion cells and their processes allows for a through-conduction system that is interconnected by chemical synapses. Although the sea anemone nervous system is more complex than that of Hydra, it has similar two-cell and three-cell effector pathways that may function in local responses to tentacle contact with food.     

The role of glutamine, serum and energy factors in growth of enterocyte-like cell lines

Background: Glutamine is routinely added to most cell cultures. Glutamine has been found to be the preferential nutrient to the rapidly replicating intestinal mucosa, but whether this is a metabolic effect or due to other properties of this amino acid is not determined. To study the importance of glutamine on the growth of two enterocyte-like cell lines, the effects of depriving the media or supplementing it with glutamine were assessed in media with different serum and energy supplements. Methods: CaCo-2 and HT-29 cells were grown in serum-free medium, with fetal bovine or synthetic serum, and with or without glucose or galactose. The glutamine content was varied between 0 and 4 mM. All growth assays were performed in triplicate by counting in a hemocytometer. Results: Both cell lines were dependent of serum factors for growth, but displayed distinct requirements on glutamine supplementation. Glutamine was an obligate supplement with dose-dependent correlation to growth (r=0.87, p<0.01) for CaCo-2 cells cultured in synthetic, but not in fetal bovine serum. In HT-29 cells, the correlation between glutamine and growth was significant (r=0,68, p<0,05) only in fetal bovine serum in the absence of galactose. Conclusion: This study shows that glutamine has different growth stimulating effects on two enterocyte-like cell lines studied. This could reflect different modes of action of glutamine on proliferation and differentiation in an enterocyte cell population.     

Of mice, frogs and flies: generation of membrane asymmetries in early development

Embryonic development begins with cleavage of the fertilized egg. Cleavage comprises two major processes: cytokinesis and formation of a polarized epithelial cell layer. The focus of this review is comparison of the generation of membrane polarity during embryonic cleavage in three different developmental model systems. In mammalian embryos, as exemplified by analysis of the mouse, generation of distinct membrane domains is uncoupled from cleavage divisions and is initiated in a specific developmental phase, called compaction. In Xenopus laevis embryos, generation of polarized blastomeres occurs simultaneously with cytokinesis. The origin of specific membrane domains of X. laevis polar blastomeres, however, can be traced back to oogenesis. Finally, in Drosophila melanogaster, generation of polarized cells occurs at cellularization. The relevance of cell adhesion, cell junctions and cytocortical scaffolds will be discussed for each of the model systems. Despite enormous morphologic differences, the three models share many common features; in particular, many important molecular interactions are conserved.     

微囊化K562细胞生长周期及代谢特性的研究

以K562细胞为模型,分别进行微囊化和游离培养,运用流式细胞术考察两种培养体系下细胞周期和生长代谢变化;建立数学模型,模拟了两种培养体系下细胞的生长活性和代谢特性。实验发现：微囊化培养过程中的K562细胞处于DNA合成期（S期）的百分含量显著高于游离培养,并且细胞保持较高的增殖活性。模型计算表明,所建模型动力学参数能够很好地描述微囊化和游离两种培养体系下细胞的代谢情况;对细胞活性的理论计算表明,微囊化的细胞具有较高的增殖和代谢活性,同时细胞能够较长时间保持此活性;模型参数表明,两种培养体系下,葡萄糖对细胞生长的影响无显著差别 (k^Free_L≈k^APA_L),乳酸对游离培养细胞的生长具有明显抑制作用,但对微囊化培养细胞抑制作用较小(kFreeL>≈kAPAL)。     

哺乳动物体细胞核移植中供体细胞的研究进展

在哺乳动物体细胞核移植中,供体细胞是影响其效率的主要因素之一。供体细胞的类型、细胞周期、细胞的培养代数、冷藏与冷冻处理,以及供体动物的性别、年龄等都可能影响核移植胚胎的发育。根据现有资料,简要综述了在哺乳动物体细胞核移植中有关供体细胞的研究进展。     

Engineering Cardiac Cell Junctions In Vitro to Study the Intercalated Disc

Abstract

This review article discusses a recent work using engineered cardiac cells to study the function of the intercalated disc putting emphasis on mechanical and electrical coupling.     

Perivascular cells for regenerative medicine

Mesenchymal stem/stromal cells (MSC) are currently the best candidate therapeutic cells for regenerative medicine related to osteoarticular, muscular, vascular and inflammatory diseases, although these cells remain heterogeneous and necessitate a better biological characterization. We and others recently described that MSC originate from two types of perivascular cells, namely pericytes and adventitial cells and contain the in situ counterpart of MSC in developing and adult human organs, which can be prospectively purified using well defined cell surface markers. Pericytes encircle endothelial cells of capillaries and microvessels and express the adhesion molecule CD146 and the PDGFRβ, but lack endothelial and haematopoietic markers such as CD34, CD31, vWF (von Willebrand factor), the ligand for Ulex europaeus 1 (UEA1) and CD45 respectively. The proteoglycan NG2 is a pericyte marker exclusively associated with the arterial system. Besides its expression in smooth muscle cells, smooth muscle actin (αSMA) is also detected in subsets of pericytes. Adventitial cells surround the largest vessels and, opposite to pericytes, are not closely associated to endothelial cells. Adventitial cells express CD34 and lack αSMA and all endothelial and haematopoietic cell markers, as for pericytes. Altogether, pericytes and adventitial perivascular cells express in situ and in culture markers of MSC and display capacities to differentiate towards osteogenic, adipogenic and chondrogenic cell lineages. Importantly, adventitial cells can differentiate into pericyte‐like cells under inductive conditions in vitro. Altogether, using purified perivascular cells instead of MSC may bring higher benefits to regenerative medicine, including the possibility, for the first time, to use these cells uncultured.     

Mammalian testis: a target of in vivo electroporation

Mammalian spermatogenesis consists of three biologically significant processes: stem cell self-renewal and differentiation, meiosis, and haploid cell morphogenesis. Understanding the molecular mechanisms behind these processes might provide clues to the puzzle of species preservation and evolution, and to treatments for male infertility. However, few useful in vitro systems exist to investigate these processes at present. To elucidate these mechanisms, in vivo electroporation of the testis might be a convenient option. Since DNA solution can be injected into the seminiferous tubule via the rete testis, similar to germ cell transplantation, it is easy to transfect expression vectors into various differentiated germ cells and supporting Sertoli cells with adequate electric shock. Unfortunately, it is difficult to create transgenic animals using this method because of its low efficiency. However, gain- and loss-of-function assays, promoter assays, and tagged-protein behavior assays can be conducted with this technique, as in in vitro culture systems.