microRNA对细胞自噬调节

细胞自噬是细胞内高度保守的细胞自我消化和分解代谢过程,细胞内变性蛋白、衰老和受损的细胞器被转运到溶酶体降解. 自噬过程失调引起多种疾病,包括感染、衰老、神经退行性疾病、癌症和心脏疾病等,因此,自噬过程需要非常精确的调控. MicroRNA是一类在基因转录后水平调控目的基因的功能性小RNA分子.研究发现,microRNA可以通过RNA干扰（RNA interference, RNAi）途径调控某些自噬相关基因(autophagy related gene, ATG)及其调节因子.这些microRNA表达异常足以影响自噬水平,使得microRNA成为自噬研究的新视角,同时也使microRNA成为治疗自噬失调引起的疾病的潜在靶点.本文将对有关microRNA参与细胞自噬调控的最新研究动态进行综述.     

细胞间隙连接通讯与肿瘤

由连接蛋白构成的细胞间隙连接通讯(GJIC)广泛存在于脊椎动物中,可以直接介导细胞间小分子物质的传递,而不需要通过细胞间质。GJIC与肿瘤密切相关,多数肿瘤GJIC能力降低或丧失,连接蛋白不表达或胞内定位,而恢复GJIC可以抑制肿瘤, GJIC已成为肿瘤预防与治疗研究的潜在靶点之一。     

细菌细胞间通讯的群感效应

自然界中的细菌大多数以生物膜的形式存在,这种存在方式增强了细菌对环境的适应性和病原菌的致病性。近年来研究表明,细菌群感效应(quorum—sensing)是调控生物膜形成和其它生物学功能的机制。细菌能够分泌特定的信号分子并感应它的浓度,当信号分子浓度达到阈值时,细菌就能够引发包括致病基因在内的相关基因的表达以适应环境的变化。由于生物膜的形成是病原菌致病性和其它要求一定细胞密度才能产生功能的基础,所以细菌群感效应的发现为防止病原菌的毒害作用提供了新的思路。     

胃癌细胞与正常细胞的间隙连接通讯和促癌剂效应的研究

本文介绍用一种新的快速、简便SLDT’技术研究培养细胞的间隙连接通讯功能。细胞划痕后,标记两种荧光染料——Lucifer’Yellow(LY, MW.457.2)和Rhodamine Dextran(RD,MW.10,000)。RD停留原位,LY小分子传输到邻近细胞,表示间隙连接通讯现象。用SLDT方法观察到人胃腺癌MGC一803细胞无连接通讯功能。钙调素抑制剂T"FP有使胃癌细胞恢复连接通讯的效应。中国地鼠V,。细胞,wB大鼠肝细胞和鸡胚成肌细胞有通讯功能。促癌变剂TPA阻断正常细胞的间隙连接通讯,并阻断由。rFP丐导的胃癌细胞连接通讯。     

肾小球系膜细胞间隙连接的通讯功能及其影响因素

本文利用激光扫描共聚焦显微镜及荧光漂白后荧光回复技术对人肾小球系膜细胞间隙连接的通讯功能及其影响因素进行了观察。结果发现,体外培养的肾小球系膜细胞在融合生长状况下,相邻两细胞有间隙连接结构形成。不同浓度的血管紧张素Ⅱ对肾小球系膜细胞间隙连接的通讯功能具有促进作用,而甲基强的松龙则具有抑制作用。     

培养的内皮细胞和平滑肌细胞之间间隙连接形成及连接通讯的研究

本实验以离子电渗法将荧光黄注入细胞内,观察培养的EC 与SMC 同类及异类细胞间连接通讯现象,证实SMC 与EC 在培养中可形成同类或异类细胞GJ 结构,两种细胞间有接触介导的物质交流。EC-EC 之间的细胞通讯比SMC-SMC 和SMC-EC 之间为强。对SMC 有促增殖作用的LDL(100 μg LDL-蛋白质/ml)及胰岛素(15 mu/ml)对这种连接通讯有抑制作用,促癌剂TPA 几乎完全抑制此种连接通讯。结果提示高浓度LDL 及胰岛素等可能通过抑制SMC 与EC 之间的连接通讯而使SMC 脱离正常控制而大量增殖,促进AS发生、发展。故推论促进细胞连接通讯的因子,可能对AS 有防治作用,值得进一步研究。     

应用激光扫描共聚焦显微镜FRAP技术研究兔早期胚胎发育中细胞间隙连接介导通讯

本研究应用激光扫描共聚焦显微镜的光漂白恢复技术(FRAP)分析兔早期胚胎卵裂球之间通过间隙连接介导的细胞通讯(GJIC)。研究结果发现,用强激光分别将4-细胞期胚胎、异裂胚胎和8-细胞期胚胎的一个卵裂球荧光光漂白后,经过15分钟的荧光恢复,4-细胞期胚胎的光漂白恢复率为17.8％,异裂胚胎的光漂白恢复率为23.7％,二者之间没有明显的差异;8-细胞期胚胎的光漂白恢复率为78.2％,与前二者之间存在明显的差异。推测兔早期胚胎卵裂球细胞间隙连接建立的时间在8-细胞阶段,胚胎卵裂球间隙连接通讯可能是兔胚胎正常发育的重要条件。     

间隙连接细胞间通讯与胸苷激酶系统 "旁观者效应"的关系及其调控

增强“旁观者效应”是提高单纯疱疹胸苷激酶／更昔洛韦(HSV-TK／GCV)基因系统治疗效果的关键手段,旨在探讨间隙连接细胞间通讯(GJIC)在HSV-TK／GCV系统“旁观者效应”中的作用及其化学调控。通过采用划痕标记染料示踪技术(SLDT)比较ACHN、HeLa、NIH-3T3、Cos-7及L-02等五种不同细胞系GJIC功能状况,四甲基偶氮唑盐酶反应比色法(MTT法)检测HSV-TK／GCV对其杀伤效应和“旁观者效应”,并比较GJIC上调剂apigenin和抑制剂18-α-glycyrrhetinic acid(AGA)对GJIC和“旁观者效应”的影响。结果表明,GJIC功能强大的NIH-3T3、Cos-7及L-02细胞对HSV-TK／GCV敏感性及“旁观者效应”程度远较GJIC功能低下的ACHN和HeLa细胞高(P＜0．001)。Apigenin可显著提高ACHN细胞GJIC功能,对HeLa细胞则无影响;相反,AGA可显著抑制NIH-3T3、Cos-7和L-02等三种靶细胞的GJIC功能。相应地,apigenin可明显提高ACHN细胞的“旁观者效应”而对HeLa细胞的“旁观者效应”则无影响,AGA则可明显降低上述三种细胞的“旁观者效应”。因此认为,细胞内在的GJIC功能与靶细胞对HSV-TK／GCV的敏感性和“旁观者效应”程度有正向关系,人为调控细胞GJIC后,“旁观者效应”程度亦发生相应改变。     

一种用FRAP测定细胞间隙连接介导通讯的方法

本文探讨了利用荧光漂白恢复技术(FRAP)测定体外培养小鼠大脑皮层神经胶质细胞间隙连接通讯的方法。经细胞培养、荧光染色、荧光激发、荧光漂白、激光扫描及计算 机分析等处理,结果各所选神经胶质细胞内荧光均得到不同程度的恢复,即经扫描15.3分钟后,1—5号细胞内荧光相对强度分别上升了14％、28％、43％、17.5%、12.5％,三次平均上升了18.57±10.06％,荧光恢复速率平均为1.223±0.785％/min(n=14)。表明该方法可用来测定细胞间隙连接通讯。     

Intercellular communication of notochord cells during their differentiation in Cynops orientalis

Intercellular communication of notochord cells during their differentiation was studied by microinjection of a fluorescent dye.Lucifer Yellow,Close correlation existed between the incidences of dye coupling and quantitative evaluation of gap junctions.high incidences of dye coupling and of gap junctions occurred at a stage when notochord cells were active in the change of cell shape and cell arrangement.With the subsidence of cell movements,both dye coupling and gap junctions were reduced to lower levels.It was,therefore,Suggested that intercellular communication via gap junctions played an important role in the coordination of notochord cell movements.Gap Junctions of altered configuration occurred in notochord cells in late taibud stage.The comparison of incidences of dye coupling at this stage with those at other stages strongly suggested that the gap junctions of altered configuration functioned just as those of generalized type.     

多途径介导的小胶质细胞间钙波传递

利用微局域机械力刺激,快速实时观察机械力引起的细胞间钙波传递,系统地研究了BV-2小胶质细胞间钙通讯机制.结果表明,在细胞种植密度较小且彼此未接触的情况下,旁分泌途径可介导BV-2小胶质细胞间钙波传递.在细胞密度较大且相互接触的情况下,旁分泌和间隙连接两种途径可共同介导胞间钙波传递.更为有趣的是,在体外发现BV-2小胶质细胞间存在通道纳米管类似物连接,也可介导小胶质细胞间钙波传递.综上所述,小胶质细胞间钙波传递可通过旁分泌、间隙连接和通道纳米管类似物连接三种途径介导.     

Quantitative Determination of Gap Junction Intercellular Communication by Scrape Loading and Image Analysis

Gap junction intercellular communication (GJIC) consists of intercellular exchange of low molecular weight molecules. Chemically induced alterations of this communication have been suggested to result in abnormal cell growth and tumour promotion. Several in vitro assays have been developed to determine the effect of chemicals on gap junction communication in cultured cells. The scrape loading dye transfer technique is based on studying the transfer of the fluorescent dye Lucifer Yellow in cells where the dye is loaded through a cut in the cell monolayer. This technique is rapid and relatively uncomplicated, but has only been used to qualitatively demonstrate communication, due to lack of an appropriate method for quantification of the dye spreading. We show here that analysis of digital fluorescence images of cells scrape loaded with Lucifer Yellow can be used for quantitative determination of GJIC. We have analysed the images both by means of distance of diffusion of the dye in the cell monolayer, as well as by area of dye-coupled cells. The results are consistent with that obtained using microinjection of Lucifer Yellow and the method offers a simple way for quantitative determination of GJIC.     

Quantitative Determination of Gap Junction Intercellular Communication Using Flow Cytometric Measurement of Fluorescent Dye Transfer

Gap junction intercellular communication (GJIC) is involved in several aspects of normal cell behaviour, and disturbances in this type of communication have been associated with many pathological conditions. Reliable and accurate methods for the determination of GJIC are therefore important in studies of cell biology. (Tomasetto, C., Neveu, M.J., Daley, J., Horan, P.K. and Sager, R.(1993) Journal of Cell Biology, 122, 157–167) reported some years ago the use of flow cytometer to determine transfer between cells of a mobile dye, calcein, as a measure of cell communication through gap junctions. In spite of this being a method with potential for quantitative and reliable determination of GJIC, it has been modestly used, possibly due to technical difficulties. In the present work we have illustrated several ways to use flow cytometric data to express cell communication through gap junctions. The recipient cells were pre-stained with the permanent lipophilic dye PKH26, and the donor cell population were loaded with the gap junction permeable dye, calcein. We show that the method may be used to measure the effect of chemicals on GJIC, and that the information is reliable, objective and reproducible due to the large number of cells studied. The data may give additional information to that obtained with other methods, since the effect observed will be on the establishment of cell communication as compared to what is observed for microinjection or scrape loading, where the effect is on already established communication. This is probably the reason for the more potent effects of DMSO on GJIC measured by the present method than on already existing GJIC measured by microinjection or quantitative scrape loading. We also show that the problem related to the mobile dye calcein not being fixable with aldehydes will not affect the results as long as the cells are kept on ice in the dark and analysed by flow cytometer within the first hours after formalin cell fixation.     

Liver tumor potency indicators for technical toxaphene and congeners simulating weathered toxaphene

Technical toxaphene (TT) is a liver tumor promoter in B6C3F1 mice but not in F344 rats. To further evaluate dose-response relationships for weathered toxaphene, B6C3F1 mouse hepatocytes were treated with TT alone, five selected persistent congeners (p-26, p-50, p-62, Hx-Sed, and Hp-Sed), or two selected congener mixtures (simulating weathered toxaphene) and dose-response relationships were characterized for cytotoxicity and gap junction intercellular communication (GJIC) inhibition. Phenobarbital was included as a positive control for mouse liver tumor promotion and GJIC inhibition and dose ranges were calibrated to define benchmark dose concentrations. Each treatment group exhibited significant cytotoxicity and GJIC inhibition for at least one sex (M/F) after 3 and/or 24 h of treatment. Maximum GJIC inhibition was observed at certain noncytotoxic concentrations with sex-specific differences in relative potency estimated as the effective concentration at 20% inhibition (EC20); however, no significant EC20 differences were observed between the treatment groups. Analysis of mixture interactions at the EC20 showed that GJIC inhibition of the two weathered toxaphene mixtures was significantly less than additive compared to that for the component congeners. These findings suggest that the persistent toxaphene congener mixtures tested are not more tumorigenic than the parent insecticide mixture.     

过表达微小RNA miR-125b对人胚胎干细胞增殖的影响

目的:通过在人胚胎干细胞(hESC)中有效转染微小RNA miR-125b的真核表达载体,研究过表达miR-125b对hESC增殖的影响。方法:将在无饲养层上培养至第3 d,克隆融合达70%的hESC用Accutase酶消化为单细胞,然后用LipofectAMINE2000对hESC单细胞转染pHRS-1cla-miR125b-CMV-EGFP载体及其对照pHRS-1cla-CMV-EGFP载体,通过实时定量PCR对转染后细胞中成熟miR-125b的表达进行检测;进一步进行细胞计数和克隆计数,对miR-125b表达上调的hESC的增殖情况进行分析。结果:实时定量PCR检测结果表明,细胞转染后72 h,miR-125b的表达上调1.45倍,说明hESC转染成功;克隆计数及细胞计数结果显示过表达miR-125b的hESC增殖受到明显抑制(P<001)。结论:转染miR-125b真核表达载体的hESC能够上调成熟miR-125b的表达,hESC中miR-125b的表达上调能明显抑制hESC的增殖。     

Inhibition of Gap Junction Intercellular Communications in Cell Culture by Polycyclic Aromatic Hydrocarbons (PAH) in the Absence of PAH Metabolism

We have studied the effect of polycyclic aromatic hydrocarbons (PAH) on gap junction intercellular communications (GJIC) in culture of hepatoma cells Hep G2 and G27. Carcinogenic PAH inhibited GJIC in both cultures in contrast to non-carcinogenic PAH. We showed that both constitutive and inducible expressions of mRNAs of Ah receptor and cytochrome P4501A1 (the main isoform involved in PAH metabolism) were absent in hepatoma G27 cells. We concluded that the initial, non-metabolized molecules of carcinogenic PAH are responsible for changes in GJIC through interaction with an unknown factor in the cellular membrane.     

Effects of tumor promoters,genotoxic carcinogens and hepatocytotoxins on mouse hepatocyte intercellular communication

Intercellular communication via gap junctions may be an important mechanism of cellular growth control. Tumor promoters can inhibit intercellular communication between cultured cells, while genotoxic carcinogens apparently lack this capability. The inhibition of intercellular communication by tumor promoters may be an essential mechanism by which tumor promotion occurs in vivo. In this study, the liver tumor promoters phenobarbital, lindane (1,2,3,4,5,6-hexachlorocyclohexane, -isomer), DDT (1,1-Bis[4-chlorophenyl],-2,2,2-trichloroethane), Aroclor 1254 (a polychlorinated biphenyl mixture) and dieldrin inhibited intercellular communication between male B6C3F1 mouse hepatocytes in primary culture. Intercellular communication was detected as the passage of [5-³H]uridine nucleotides from pre-labelled donor hepatocytes to non-labelled recipient heptocytes. Mouse hepatocyte intercellular communication was also inhibited by the skin tumor promoter TPA (12-0-tetradecanoyl phorbol-13-acetate), but not by the bladder tumor promoter saccharin. The genotoxic hepatocarcinogens dimethylnitrosamine, diethylnitrosamine, benzo[a]pyrene and 2-acetylaminofluorene, and the hepatocytotoxins bromobenzene, acetaminophen, carbon tetrachloride, chloroform and methotrexate had no effect on mouse hepatocyte intercellular communication at non-cytotoxic levels. These results suggest that the ability to inhibit mouse hepatocyte intercellular communication is an effect specific to tumor promoters.Abbreviations DDT 1,1-Bis[4-chlorophenyl]-2,2,2-trichloroethane - FBS fetal bovine serum - LDH lactate dehydrogenase - TCA trichloroacetic acid - TPA 12-0-tetradecanoyl-phorbol-13-acetate