水飞蓟宾诱导肺腺癌Anip973 细胞凋亡的分子机制研究

目的:探讨水飞蓟宾诱导肺腺癌Anip973细胞系细胞凋亡的分子机制。方法:采用MTT法、倒置显微镜和电子显微镜等形态学检测以及流式细胞仪(FCM)技术检测、DNALadder分析、凋亡分子PARP的表达检测细胞凋亡,同时进行凋亡相关蛋白Bax、Bcl-2、caspase-3和caspase-9表达活性分析。结果:(1)水飞蓟宾对人肺腺癌Anip973细胞系细胞的增殖有显著抑制作用;(2)水飞蓟宾作用Anip973细胞48h后,随着浓度的增加,倒置显微镜下可见细胞数目减少,胞体变小、变圆,到高浓度时出现较多的死亡细胞;(3)扫描电镜观察发现,随着水飞蓟宾作用浓度的增加,Anip973细胞中出现增多的凋亡细胞,凋亡细胞表现出典型的超微结构特征;(4)流式细胞仪检测的结果发现,随着药物作用时间的延长,Anip973细胞的G1期细胞比例增多,S期细胞明显减少,G2期细胞略有减少,并出现明显的凋亡峰。(5)水飞蓟宾作用后的Anip973细胞出现明显的DNALadder和PARP降解增加等凋亡特征;(6)水飞蓟宾作用后,Anip973细胞中的凋亡相关蛋白Bax表达增加、caspase-3和caspase-9酶活性增加,而Bcl-2表达降低。结论:水飞蓟宾在体外有抑制人肺腺癌细胞Anip973的增殖作用,并通过激活线粒体依赖的caspase凋亡通路,诱导其凋亡。     

水飞蓟宾诱导肺腺癌Anip973细胞凋亡的分子机制研究

目的：探讨水飞蓟宾诱导肺腺癌Anip973细胞系细胞凋亡的分子机制。方法：采用MTT法、倒置显微镜和电子显微镜等形态学检测以及流式细胞仪（FCM）技术检测、DNALadder分析、凋亡分子PARP的表达检测细胞凋亡,同时进行凋亡相关蛋白Bax、Bcl-2、caspase-3和caspase-9表达活性分析。结果：（1）水飞蓟宾对人肺腺癌Anip973细胞系细胞的增殖有显著抑制作用;（2）水飞蓟宾作用Anip973细胞48h后,随着浓度的增加,倒置显微镜下可见细胞数目减少,胞体变小、变圆,到高浓度时出现较多的死亡细胞;（3）扫描电镜观察发现,随着水飞蓟宾作用浓度的增加,Anip973细胞中出现增多的凋亡细胞,凋亡细胞表现出典型的超微结构特征;（4）流式细胞仪检测的结果发现,随着药物作用时间的延长,Anip973细胞的G1期细胞比例增多,S期细胞明显减少,G2期细胞略有减少,并出现明显的凋亡峰。（5）水飞蓟宾作用后的Anip973细胞出现明显的DNALadder和PARP降解增加等凋亡特征;（6）水飞蓟宾作用后,Anip973细胞中的凋亡相关蛋白Bax表达增加、caspase-3和caspase-9酶活性增加,而Bcl-2表达降低。结论：水飞蓟宾在体外有抑制人肺腺癌细胞Anip973的增殖作用,并通过激活线粒体依赖的caspase凋亡通路,诱导其凋亡。     

一个与非小细胞肺癌转移相关的基因――RAB5A基因

采用mRNA差异展示技术（mRNA DD）研究具有相同细胞来源,但转移能力高低不同的人肺腺癌细胞系AGZY83-a（低转移）和Anip973(高转移),分析在两个细胞系中基因差异表达的情况,发现在高转移细胞系中有RAB5A基因的表达。该基因为蛋白质入胞信号的调控者,为RAS超家族成员。为进一步证实其转录表达的调控改变情况,以及RAB5A高表达的临床意义,进一步采用RT-PCR和免疫组织化学的方法检测了50例临床非小细胞肺癌的手术标本,结果表明,RAB5A的表达有随转移发生而增强的趋势,而RAB5A的蛋白表达程度在有转移的病例中明显增强(P<0.05)。 Abstract：　Using mRNA differential display (mRNA DD)techniques, we analyzed the differences of gene expression between two human lung adenocarcinoma cell lines,AGZY83-a and Anip973. Anip973 was isolated from AGZY83-a, but manifested much higher metastatic potential than the parent line. The results showed that there were significant differences on gene expression between the two cell lines and that there was over-expression of RAB5Agene in the Anip973 cell line. The product of RAB5Agene was recognized as signal regulators of endocytotic pathway and protein trafficking at the cell surface, and belong to a member of the RAS superfamily. Furthemore, we compared to the expression of RAB5Agene and RAB5Aprotein in clinical samples of 50 cases non-small lung carcinoma and nearby lymph node by means of RT-PCR and immunohistochemistry method. The results indicated that the high expression of RAB5Ain metastatic tumor and the enhancement level of RAB5Ain metastatic tumor and the enhancement level of expression were corresponded with the increase of metastatic degree. And there were significance of difference on the expression degree of RAB5Aprotein between non-small lung carcinoma with metastasis and non- metastasis (P<0.05).     

鸟氨酸脱羧酶基因反义RNA对淋巴瘤细胞Jurkat生长的影响

探讨鸟氨酸脱羧酶(ornithine decarboxylase,ODC)反义RNA是否对人淋巴瘤细胞Jurkat的生长具有抑制作用。含反义RNA的真核表达载体pcDNA3.1(+)/Rodc用脂质体转染Jurkat细胞,G418筛选ODC表达抑制的细胞株,MTS法分析细胞增殖,Western blotting检测细胞中ODC蛋白表达水平,半定量RT-PCR检测细胞中ODC mRNA含量,流式细胞术检测细胞周期的变化,DNA片段化分析细胞凋亡。结果显示,成功获得ODC表达抑制的淋巴瘤细胞株J/o,ODC反义RNA转染细胞后,引起Jurkat细胞生长缓慢和S/G2细胞周期停滞,细胞对抗癌药物DFMO敏感性显著增加。由此证明,ODC反义RNA能抑制人T淋巴瘤Jurkat细胞的生长,具有治疗人白血病的潜在应用价值。     

一个与非小细胞肺癌转移相关的基因--RAB5A基因

采用mRNA差异展示技术(mRNA DD)研究具有相同细胞来源,但转移能力高低不同的人肺腺癌细胞系AGZY83-a(低转移)和Anip973(高转移),分析在两个细胞系中基因差异表达的情况,发现在高转移细胞系中有RAB5A基因的表达.该基因为蛋白质入胞信号的调控者,为RAS超家族成员.为进一步证实其转录表达的调控改变情况,以及RAB5A高表达的临床意义,进一步采用RT-PCR和免疫组织化学的方法检测了50例临床非小细胞肺癌的手术标本,结果表明,RAB5A的表达有随转移发生而增强的趋势,而RAB5A的蛋白表达程度在有转移的病例中明显增强(P<0.05).     

鼠疫耶尔森菌毒力蛋白YopE新功能的初步研究

鼠疫耶尔森菌外部蛋白E（YopE）是鼠疫耶尔森菌的6种分泌蛋白之一,主要通过其144位的”精氨酸手指”结构与细胞膜耦联蛋白RhoGTP酶相互作用发挥功能.本文构建YopE及其144位突变体YopE(R144A)的可诱导表达系统,并优化了诱导条件. 用该系统结合流式细胞技术检测YopE和YopE(R144A)对细胞凋亡、细胞周期和细胞活性氧(ROS)水平的影响.结果显示：YopE(R144A)促进HeLa细胞凋亡|使G0/G1期细胞比例上升,G2/M期细胞比例下降;随着YopE(R144A)表达量增加,p21蛋白的表达量也增加| YopE(R144A)也能抑制细胞ROS的产生.研究结果提示,YopE在细胞内可能存在新的致病靶点.     

STAT3反义核酸对HL-60细胞周期及c-myc mRNA表达的影响

实验分反义组、无义组、脂质体组、空白组,转染后,应用四唑盐（MTT）比色法分析细胞增殖率,流式细胞仪检测细胞周期,RT-PCR检测细胞中STAT3 mRNA和c-myc mRNA的表达。探讨STAT3反义寡核苷酸对白血病细胞HL-60细胞周期及c-myc的影响。STAT3 ASODN抑制HL-60细胞增殖呈时间和浓度依赖性;反义寡核苷酸作用后,G0/G1期细胞明显减少,S期细胞增多,细胞周期进程受到明显阻滞;反义寡核苷酸作用48h后细胞内STAT3mRNA及c-myc mRNA的表达水平下降,与各对照组比较有显著性差异（P〈0.05）。STAT3 ASODN能够明显抑制HL-60细胞增殖,并能阻滞HL-60细胞于G0/G1期、并下调c-myc mRNA的表达。     

RAB5A基因表达改变对人肺腺癌细胞系GLC-82和SPC-a1的分化和侵袭特性影响

为探讨RAB5A基因对两种人肺腺癌细胞系GLC－82和SPC－al分化及侵袭特性的影响。利用细胞转染技术将构建的RAB5A反义RNA重组质粒（pcDNA3-AntiRAB5A)和RAB5A正义真核表达载体分别转染入低分化人肺腺癌细胞系GLC－82和低转移人肺腺癌细胞系SPC－al中,在稳定筛选后,通过裸鼠体内实验和体外人工基底膜侵袭和细胞趋化运动实验,观察观察转染后细胞分化和转移特性的改变,观察转染前后细胞,发现转染后GLC－82细胞体外侵袭重组基底膜能力及趋化运动能力降低（t检验P＜0．02）;裸鼠体内成瘤实验,瘤块切片病理观察转染后GLC－82细胞出现腺腔样及基底模样结构,分化程度增高,转染后SPC－al细胞体外趋化运动能力,侵袭重组基底膜能力均增强(t检验P＜0．02）。RAB5A基因通过影响细胞的体外趋化运动能力,侵袭重组基底膜能力等对GLC－82和SPC－al细胞的侵袭表型形成及GLC－82细胞的分性发挥重要作用。     

反义细胞周期蛋白E真核表达载体的构建及其对体外人乳腺癌细胞的抑制作用

 为了探讨细胞周期蛋白 E(cyclin E)与人乳腺癌细胞恶性特征间的相关性 ,利用反义 RNA抑制基因表达的技术 ,构建了细胞周期蛋白 E反义 RNA的真核表达载体并转入人乳腺癌细胞中 .通过 G41 8筛选出阳性克隆 ,经 PCR和 Western印迹检测 ,确定细胞中含有重组质粒 ,并且细胞周期蛋白 E蛋白的水平明显降低 ,由此获得了反义 RNA表达载体导致的细胞周期蛋白 E表达受抑制的细胞 .细胞模型建立后 ,观察分析了细胞形态 ,细胞生长的血清依赖性以及软琼脂成集落能力 ,与对照细胞相比所发生的变化 .结果显示 ,细胞周期蛋白 E受抑制后 ,乳腺癌细胞体积变大 ,细胞生长对血清依赖性增加 ,低血清培养到第 6d时 ,细胞密度约为对照细胞的五分之一 ,细胞成集落能力也显著下降 ,软琼脂中克隆形成率下降 57% .这些变化都表明乳腺癌细胞恶性程度由于细胞周期蛋白 E表达受抑制而减弱 ,可以推测 cyclin E与乳腺癌细胞的恶性增殖及非锚定依赖性生长有着明显的关系 .     

LMO3过表达对胶质细胞增殖的作用

构建重组质粒pcDNA4/HisA-LMO3,转染C8-D1A胶质细胞,对照组为pcDNA4/HisA空载体质粒转染组和未转染组,MTT观察各组细胞的体外生长情况,流式细胞术(FCM)测定各组细胞的细胞周期和凋亡细胞百分比,Western-blot检测LMO3转染后凋亡相关蛋白的表达变化,从而观察LMO3基因对C8-D1A胶质细胞体外生长的影响.RT-PCR、Western印迹显示pcDNA4/HisA-LMO3转染组LMO3mRNA及LMO3蛋白表达水平明显高于对照组;与对照组细胞相比,转染组细胞的增殖能力明显高于空载体对照组及C8细胞(P0.01),S期细胞增加,G0/G1期细胞减少,C8细胞转染LMO3后可以促进C8细胞从G0/G1期进入S期,从而促进细胞的增殖.     

A RAB5/RAB4 recycling circuitry induces a proteolytic invasive program and promotes tumor dissemination

The mechanisms by which tumor cells metastasize and the role of endocytic proteins in this process are not well understood. We report that overexpression of the GTPase RAB5A, a master regulator of endocytosis, is predictive of aggressive behavior and metastatic ability in human breast cancers. RAB5A is necessary and sufficient to promote local invasion and distant dissemination of various mammary and nonmammary tumor cell lines, and this prometastatic behavior is associated with increased intratumoral cell motility. Specifically, RAB5A is necessary for the formation of invadosomes, membrane protrusions specialized in extracellular matrix (ECM) degradation. RAB5A promotes RAB4- and RABENOSYN-5–dependent endo/exocytic cycles (EECs) of critical cargos (membrane-type 1 matrix metalloprotease [MT1-MMP] and β3 integrin) required for invadosome formation in response to motogenic stimuli. This trafficking circuitry is necessary for spatially localized hepatocyte growth factor (HGF)/MET signaling that drives invasive, proteolysis-dependent chemotaxis in vitro and for conversion of ductal carcinoma in situ to invasive ductal carcinoma in vivo. Thus, RAB5A/RAB4 EECs promote tumor dissemination by controlling a proteolytic, mesenchymal invasive program.     

Vacuolin-1 potently and reversibly inhibits autophagosome-lysosome fusion by activating RAB5A

Autophagy is a catabolic lysosomal degradation process essential for cellular homeostasis and cell survival. Dysfunctional autophagy has been associated with a wide range of human diseases, e.g., cancer and neurodegenerative diseases. A large number of small molecules that modulate autophagy have been widely used to dissect this process and some of them, e.g., chloroquine (CQ), might be ultimately applied to treat a variety of autophagy-associated human diseases. Here we found that vacuolin-1 potently and reversibly inhibited the fusion between autophagosomes and lysosomes in mammalian cells, thereby inducing the accumulation of autophagosomes. Interestingly, vacuolin-1 was less toxic but at least 10-fold more potent in inhibiting autophagy compared with CQ. Vacuolin-1 treatment also blocked the fusion between endosomes and lysosomes, resulting in a defect in general endosomal-lysosomal degradation. Treatment of cells with vacuolin-1 alkalinized lysosomal pH and decreased lysosomal Ca²⁺ content. Besides marginally inhibiting vacuolar ATPase activity, vacuolin-1 treatment markedly activated RAB5A GTPase activity. Expression of a dominant negative mutant of RAB5A or RAB5A knockdown significantly inhibited vacuolin-1-induced autophagosome-lysosome fusion blockage, whereas expression of a constitutive active form of RAB5A suppressed autophagosome-lysosome fusion. These data suggest that vacuolin-1 activates RAB5A to block autophagosome-lysosome fusion. Vacuolin-1 and its analogs present a novel class of drug that can potently and reversibly modulate autophagy.     

Vacuolin-1 potently and reversibly inhibits autophagosome-lysosome fusion by activating RAB5A

Autophagy is a catabolic lysosomal degradation process essential for cellular homeostasis and cell survival. Dysfunctional autophagy has been associated with a wide range of human diseases, e.g., cancer and neurodegenerative diseases. A large number of small molecules that modulate autophagy have been widely used to dissect this process and some of them, e.g., chloroquine (CQ), might be ultimately applied to treat a variety of autophagy-associated human diseases. Here we found that vacuolin-1 potently and reversibly inhibited the fusion between autophagosomes and lysosomes in mammalian cells, thereby inducing the accumulation of autophagosomes. Interestingly, vacuolin-1 was less toxic but at least 10-fold more potent in inhibiting autophagy compared with CQ. Vacuolin-1 treatment also blocked the fusion between endosomes and lysosomes, resulting in a defect in general endosomal-lysosomal degradation. Treatment of cells with vacuolin-1 alkalinized lysosomal pH and decreased lysosomal Ca²⁺ content. Besides marginally inhibiting vacuolar ATPase activity, vacuolin-1 treatment markedly activated RAB5A GTPase activity. Expression of a dominant negative mutant of RAB5A or RAB5A knockdown significantly inhibited vacuolin-1-induced autophagosome-lysosome fusion blockage, whereas expression of a constitutive active form of RAB5A suppressed autophagosome-lysosome fusion. These data suggest that vacuolin-1 activates RAB5A to block autophagosome-lysosome fusion. Vacuolin-1 and its analogs present a novel class of drug that can potently and reversibly modulate autophagy.     

Receptor-mediated internalization promotes increased endosome size and number in a RAB4- and RAB5-dependent manner

Despite their significance in receptor-mediated internalization and continued signal transduction in cells, early/sorting endosomes (EE/SE) remain incompletely characterized, with many outstanding questions that surround the dynamics of their size and number. While several studies have reported increases in EE/SE size and number resulting from endocytic events, few studies have addressed such dynamics in a methodological and quantitative manner. Herein we apply quantitative fluorescence microscopy to measure the size and number of EE/SE upon internalization of two different ligands: transferrin and epidermal growth factor. Additionally, we used siRNA knock-down to determine the involvement of 5 different endosomal RAB proteins (RAB4, RAB5, RAB8A, RAB10 and RAB11A) in EE/SE dynamics. Our study provides new information on the dynamics of endosomes during endocytosis, an important reference for researchers studying receptor-mediated internalization and endocytic events.     

RAB5A基因对肺腺癌细胞微丝的影响

为研究RAB5A基因对肺腺癌细胞中微丝的影响,通过FITC标记的鬼笔环肽对AGZY83-a细胞骨架中的微丝特异染色,利用共聚焦激光扫描显微镜发现RAB5A过表达后微丝束变密。经Superarray肿瘤转移相关基因微芯片分析RAB5A对肿瘤转移相关基因的表达影响．发现了3个与细胞骨架调节相关基因表达发生变化,NM23H1与Rac1的表达受到抑制．同时S100A4的表达增加。以前有研究认为S100A4基因可抑制NM23H1基因表达,为验证NM23H1基因的表达降低是否由于S100肖4表达增高所致,利用RNAi沉默AGZY83-a细胞中S100A4基因的表达,发现NM23H1基因表达增高,由此推断RAB5A基因可能通过上调S100A4基因表达来抑制NM23H1基因表达。     

A novel ER-localized transmembrane protein,EMC6, interacts with RAB5A and regulates cell autophagy

Autophagy is mediated by a unique organelle, the autophagosome, which encloses a portion of the cytoplasm for delivery to the lysosome. Phosphatidylinositol 3-phosphate (PtdIns3P) produced by the class III phosphatidylinositol 3-kinase (PtdIns3K) complex is essential for canonical autophagosome formation. RAB5A, a small GTPase localized to early endosomes, has been shown to associate with the class III PtdIns3K complex, regulate its activity and promote autophagosome formation. However, little is known about how endosome-localized RAB5A functions with the class III PtdIns3K complex. Here we identified a novel endoplasmic reticulum (ER)-localized transmembrane protein, ER membrane protein complex subunit 6 (EMC6), which interacted with both RAB5A and BECN1/Beclin 1 and colocalized with the omegasome marker ZFYVE1/DFCP1. It was shown to regulate autophagosome formation, and its deficiency caused the accumulation of autophagosomal precursor structures and impaired autophagy. Our study showed for the first time that EMC6 is a novel regulator involved in autophagy.     

克氏原螯虾Rab5、Rab6蛋白及其抗体对血淋巴细胞吞噬活性的影响

为研究克氏原螯虾(Procambarus clarkii)Rab5(PcRab5)和Rab6(PcRab6)的生物学功能, 利用同源重组技术构建了克氏原螯虾pET-B2m-PcRab5和pET-B2m-PcRab6原核表达载体, 并进行了诱导表达和多克隆抗体的制备, 采用ELISA和Western Blot技术检测了抗体效价和特异性。将PcRab5和PcRab6的原核表达蛋白和多克隆抗体注射到健康克氏原螯虾体内, 研究了其对血淋巴细胞吞噬活性的影响。实验结果表明构建的pET-B2m-PcRab5和pET-B2m-PcRab6原核表达载体经诱导后可表达目的蛋白, 分子量均为67 kD, 纯化后蛋白条带单一, 纯度较高。重组表达纯化后的PcRab5和PcRab6蛋白免疫日本大耳兔分别获得效价为1:2048 K和1:512 K的兔抗血清, 制备的抗体可分别特异性识别PcRab5和PcRab6蛋白。将PcRab5和PcRab6蛋白注射健康克氏原螯虾后, 其血淋巴细胞中可吞噬荧光微球的细胞比例分别显著上升至38%和30%(P＜0.01)。而将纯化的PcRab5和PcRab6多克隆抗体分别注射至螯虾体内后, 其血淋巴细胞的吞噬细胞比例均出现显著下降(P＜0.05), PcRab5和PcRab6蛋白参与了血淋巴细胞吞噬功能。实验为进一步研究克氏原螯虾PcRab5和PcRab6分子功能奠定基础, 也可为理解甲壳动物Rab5和Rab6在血淋巴细胞吞噬功能中的作用提供帮助。     

Control of RAB7 activity and localization through the retromer‐TBC1D5 complex enables RAB7‐dependent mitophagy

Retromer is an endosomal multi‐protein complex that organizes the endocytic recycling of a vast range of integral membrane proteins. Here, we establish an additional retromer function in controlling the activity and localization of the late endosomal small GTPase RAB7. Surprisingly, we found that RAB7 not only decorates late endosomes or lysosomes, but is also present on the endoplasmic reticulum, trans‐Golgi network, and mitochondrial membranes, a localization that is maintained by retromer and the retromer‐associated RAB7‐specific GAP TBC1D5. In the absence of either TBC1D5 or retromer, RAB7 activity state and localization are no longer controlled and hyperactivated RAB7 expands over the entire lysosomal domain. This lysosomal accumulation of hyperactivated RAB7 results in a striking loss of RAB7 mobility and overall depletion of the inactive RAB7 pool on endomembranes. Functionally, we establish that this control of RAB7 activity is not required for the recycling of retromer‐dependent cargoes, but instead enables the correct sorting of the autophagy related transmembrane protein ATG9a and autophagosome formation around damaged mitochondria during Parkin‐mediated mitophagy.     

Evidence that Griscelli syndrome with neurological involvement is caused by mutations in RAB27A,not MYO5A

Griscelli syndrome (GS), a rare autosomal recessive disorder, is characterized by partial albinism, along with immunologic abnormalities or severe neurological impairment or both. Mutations in one of two different genes on chromosome 15q can cause the different subtypes of GS. Most patients with GS display the hemophagocytic syndrome and have mutations in RAB27A, which codes for a small GTPase. Two patients with neurological involvement have mutations in MYO5A, which codes for an actin-based molecular motor. The RAB27A and MYO5A gene products interact with each other and function in vesicle trafficking. We report the molecular basis of GS in a Muslim Arab kindred whose members have extremely variable neurological involvement, along with the hemophagocytic syndrome and immunologic abnormalities. The patients have normal MYO5A genes but exhibit a homozygous 67.5-kb deletion that eliminates RAB27A mRNA and immunocytofluorescence-detectable protein. We also describe the molecular organization of RAB27A and a multiplex polymerase chain reaction assay for the founder deletion in this kindred. Finally, we propose that all patients with GS have RAB27A mutations and immunologic abnormalities that sometimes result in secondary neurological involvement. The two patients described elsewhere who have MYO5A mutations and neurological complications but no immunologic defects may not have GS but instead may have Elejalde syndrome, a condition characterized by mild hypopigmentation and severe, primary neurological abnormalities.