miR-382-5p通过靶基因PTEN阻滞NB4细胞分化

该文主要研究在急性早幼粒细胞白血病NB4细胞中, miR-382-5p通过调控靶基因PTEN(phosphatase and tensin homologue)抑制了全反式维甲酸(all-trans retinoic acid, ATRA)诱导的急性早幼粒细胞分化。我们运用ATRA(1μmol/L)诱导细胞分化; Western blot检测PTEN及髓系分化标志物CD11b的蛋白质水平;实时荧光定量PCR检测miR-382-5p的表达水平;过表达PTEN的慢病毒载体分别感染NB4细胞和HL-60、THP-1细胞;脂质体转染miR-382-5p的模拟剂(mimics)和特异性抑制剂(inhibitors)NB4细胞。结果显示, PTEN促进ATRA诱导的NB4细胞分化,而在HL-60和THP-1细胞中并无明显促分化效应。NB4细胞中,脂质体转染miR-382-5p mimics在mRNA和蛋白水平均抑制了PTEN的表达,并且抑制了ATRA诱导的分化;转染miR-382-5p inhibitors则恢复了PTEN表达,同时促进了ATRA诱导的急性早幼粒细胞NB4细胞的分化。该文结果提示, miR-382-5p靶向抑制了PTEN的表达从而抑制ATRA诱导的NB4细胞分化。     

SMMC7721肝癌细胞蛋白激酶B活性增高可驱动有功能的上皮钙粘着蛋白到细胞表面(英文)

为了研究蛋白激酶B (PKB)对上皮钙粘着蛋白 (E cadherin)的调节 ,使用了用胰岛素处理的野生型SMMC 772 1细胞及稳定表达持续激活PKB的SMMC 772 1细胞株 (Gag PKB/SMMC 772 1) .用RNA印迹法和蛋白质印迹法检测细胞E cadherin表达 ,发现通过胰岛素刺激或在细胞中表达持续激活PKB从而增加PKB活性 ,不影响E cadherin的转录和蛋白质合成 ,但用流式细胞术和免疫荧光定位E cadherin ,则发现PKB活性增加能明显驱动E cadherin到细胞表面 ,从而导致部分通过E cadherin途径的细胞粘聚增加和细胞调亡的抑制 .因此 ,我们提供新的证据表明 ,增加PKB活性可驱动有功能的E cadherin分子到细胞表面 .     

应用两种方法诱导SH-SY5Y 细胞分化的研究

目的:观察全反式维甲酸(ATRA)处理和ATRA与十四烷酰佛波醇乙酸酯(TPA)序贯处理(ATRA/TPA)对人类神经母细胞瘤细胞系SH-SY5Y细胞增殖抑制和形态分化的影响。方法:应用10μM ATRA处理6天和10μM ATRA处理3天继以80 nMTPA处理3天这两种方法使SH-SY5Y细胞分化;用倒置光学显微镜动态观察SH-SY5Y细胞形态学变化;并用MTT比色法比较两种分化方法对SH-SY5Y细胞的体外抗增殖作用。结果:ATRA处理和ATRA与TPA序贯处理对SH-SY5Y细胞都有抗增值和诱导细胞分化作用,细胞形态发生明显的变化,分化成神经元表型,前者主要表现为两端带有长突起的纺锤体样细胞形态,而后者主要是由细胞体延伸出多个突起的多边形的细胞。ATRA分化6天的细胞的存活率下降为78.7%±2.0%。当去除ATRA后,继续培养1天的细胞存活率上升为89%±0.2%,而继续培养2天的细胞存活率为86.3%±1.4%;ATRA与TPA序贯分化6天细胞存活率下降为75.9±0.4%。当去除TPA后,继续培养一天的细胞存活率为75.5±0.7%,继续培养2天的细胞存活率为74.9±1.0%。结论:维甲酸(ATRA)处理和ATRA与十四烷酰佛波醇乙酸酯(TPA)序贯处理(ATRA/TPA)均能明显诱导SH-SY5Y细胞分化。这两种分化细胞为神经科学的研究提供了优良的体外培养模型细胞,尤其是ATRA与TPA序贯处理能获得分化完全而稳定的神经元样细胞。     

CD11b/DNA双参数流式细胞术检测HL-60细胞分化的细胞周期

目的采用双参数流式细胞术研究全反式维甲酸(alltransretinoidacid,ATRA)诱导人类急性早幼粒白血病细胞HL-60细胞分化的细胞周期。方法HL-60细胞经分化诱导剂ATRA(终浓度为1μmol/L)诱导不同时间点后,利用CD11b/DNA双参数流式细胞术同时检测分化细胞表面抗原CD11b的表达及分化细胞DNA含量。结果HL-60细胞经ATRA诱导后,细胞表面分化抗原CD11b表达明显升高,细胞阻滞于G0/G1期,且CD11b阳性细胞主要位于G0/G1期。结论CD11b/DNA双参数流式细胞术能简便,快速,直观地检测细胞分化的细胞周期。     

TGFβ1参与视黄酸诱导的HL-60细胞分化

为研究内源性TGF-β１对全反式视黄酸（ATRA）作用HL60细胞的影响,应用定量RT-PCR和ELISA方法,研究ATRA诱导HL-60细胞分化过程中TGF-β１ 表达的变化.并构建TGF-β１ RNA干扰表达质粒,抑制HL-60细胞内源性TGF-β１表达,进而研究ATRA诱导内源性TGF-β１表达下降的HL-60细胞分化的情况．结果发现,ATRA诱导HL-60细胞分化过程中,TGF-β１ 表达明显增高．得到4个针对不同靶位点的RNA干扰表达质粒.其中,针对起始编码区的质粒转染48 h后对HL-60细胞的TGF-β１蛋白抑制率为73~2％．内源性TGF-β１表达下降后,ATRA作用的HL-60细胞NBT还原试验的光密度值降低,CD33抗原阳性的细胞比例较对照组升高,CD11b抗原阳性的细胞比例较对照组降低.表明内源性TGF-β１表达下降后,ATRA诱导HL-60细胞分化的作用有所减弱,提示内源性TGF-β１在ATRA诱导HL-60细胞分化中起一定的作用．     

RARβ在胃癌细胞生长调节中的作用

为探讨 RARβ受体介导全反式视黄酸 ( ATRA)抑制胃癌细胞生长的作用机理 ,用 Northern印迹测定 RARβ m RNA表达水平 ,脂质体介导的转染方法将含有 RARβ基因的表达载体转染MKN- 45细胞并稳定表达 ,MTT和软琼脂集落形成等实验测定细胞生长速率和生长状态 ,氯霉素乙酰转移酶活性 ( CAT)测定视黄酸应答元件βRARE的转录活性以及 AP- 1 ( activator protein- 1 )活性 .RARβ在 ATRA敏感细胞株 MGC80 - 3、BGC- 82 3和 SGC- 790 1中表达 ,而在 ATRA抗性细胞株 MKN- 45中不表达 .当 RARβ基因转染 MKN- 45细胞时 ,细胞变为 ATRA敏感 ,由此导致ATRA抑制 MKN- 45细胞生长和软琼脂集落形成 .ATRA可以加强诱导 MGC80 - 3、BGC- 82 3和SGC- 790 1细胞βRARE的转录活性 ,但对 MKN- 45细胞影响不大 ,不能抑制细胞 AP- 1活性 .当RARβ基因转染 MKN- 45细胞后 ,ATRA则能够诱导细胞 βRARE的转录活性 ,并抑制细胞的 AP-1活性 .RARβ表达与 ATRA抑制胃癌细胞生长密切相关 .ATRA诱导 βRARE转录活性和抑制AP- 1活性可能是其调控胃癌细胞生长的机制之一 .     

Wnt信号转导及其生物效应

 Wnt蛋白与其下游效应物构成一组重要的信号转导通路 .信号转导过程包括 :Wnt首先激活细胞表面受体佛力子 (FZ) ,活化的FZ将通过Dvl、CKIε抑制糖原合成酶激酶 3β ,继而拮抗 β 链结素( β cat)催毁器的作用 ,使胞浆中 β cat积聚并进入核内 .β cat在核内与转录因子LEF TCF协作 ,激活控制胚胎发育和细胞命运的靶基因 ;活化的FZ还经激活JNK及Flamingo来影响细胞骨架的聚合 ,以决定细胞的平面极性及纺缍体定位     

NLS-RARα对人白血病细胞NB4分化抑制及其机制的研究

该文旨在探讨带核定位信号的维甲酸受体α(nuclear localization signal retinoic acid receptor alpha,NLS-RARα)对人急性早幼粒白血病(acute promyelocytic leukemia,APL)细胞株NB4分化的影响及其机制。免疫印迹实验检测全反式维甲酸(all-trans retinoic acid,ATRA)诱导的NB4细胞分化标志物C/EBPβ、CD11b和p38α蛋白质水平;利用慢病毒介导的NLS-RARα基因过表达,进一步用免疫印迹实验验证过表达效率并检测NLS-RARα对NB4细胞分化标志物C/EBPβ、CD11b和p38α蛋白质水平的影响;间接免疫荧光实验分析NLS-RARα与p38α的空间共定位;免疫共沉淀实验分析NLS-RARα与p38α的相互作用。结果显示,生理浓度和药理浓度的ATRA促进NB4细胞分化的同时也激活了p38α,且p38α的活性变化与髓系分化标志物C/EBPβ变化一致;髓系分化表面标志物CD11b表达量在药理浓度ATRA(1μmol/L)处理下达到最高;NLS-RARα抑制NB4细胞的分化,且只有在ATRA存在的条件下,NLS-RARα抑制NB4细胞的分化与下调p38α活性相关;NLSRARα与p38α存在空间共定位且NLS-RARα与p38α直接相互作用。该研究结果提示,当存在ATRA诱导时,NLS-RARα与p38α直接相互作用后下调p38α的活性进而抑制NB4细胞的分化。     

肝癌细胞67kD层粘连蛋白受体N-糖链结构与功能的变化

探讨肝癌细胞与正常肝细胞 6 7kD层粘连蛋白受体 (6 7LR)N 糖链结构与功能的差异 .采用流式细胞术检测SMMC 772 1肝癌细胞和L 0 2正常肝细胞膜表面 6 7LR的表达 ,并分别从这 2株细胞分离纯化到高亲和力的 6 7LR ,利用凝集素结合分析其糖链结构 ,并用肽 N 糖苷酶水解N 糖链 ,观察糖链在与层粘连蛋白结合过程中的作用 .结果发现 ,L 0 2细胞膜表面 6 7LR表达的阳性率为5 5 3% ,而SMMC 772 1细胞为 34.7% ,这两株细胞 6 7LR与伴刀豆素 (ConA)的结合能力无显著差异 ,但SMMC 772 1细胞的 6 7LR与麦胚凝集素的结合能力明显高于L 0 2细胞的 6 7LR ,说明 2株细胞 6 7LR的糖链结构存在显著差异 .当N 糖链被切除后 ,SMMC 772 1细胞的 6 7LR与层粘连蛋白的结合能力明显下降 ,而L 0 2细胞则没有变化 .这些资料表明 ,SMMC 772 1肝癌细胞和L 0 2正常肝细胞与层粘连蛋白结合能力的差别 ,以及两株细胞的 6 7LR与层粘连蛋白结合能力的不同 ,很可能是由于这两株细胞的层粘连蛋白受体的N 糖链结构不同所引起     

转谷氨酰胺酶2通过mTOR通路和自噬调控全反式维甲酸诱导的白血病细胞HL60和U937的髓系分化

全反式维甲酸(ATRA)是具有融合基因PML-RARα的急性早幼粒细胞白血病(APL)特异的靶向治疗药物。此外，ATRA在无PML-RARα融合的急性髓系白血病及其它一些肿瘤中也有一定治疗效果。但ATRA治疗也会引起一些并发症或发生愈后复发。因此，对ATRA诱导分化调控机制的研究非常重要。转谷氨酰胺酶2(TGM2)是一种多功能酶，能调控mTOR信号通路和自噬等。ATRA能诱导APL细胞中TGM2表达上调，TGM2敲低抑制ATRA诱导的细胞分化。但其调控机制及涉及的信号通路尚不明确。本研究发现，在HL60和U937细胞中，ATRA能够上调CD11b和TGM2的表达(P<0.05),抑制mTOR信号通路，并增强自噬；与对照相比，敲低TGM2,mTOR信号通路增强，自噬被抑制，而ATRA诱导的CD11b表达被抑制(P<0.05),分化减弱，被ATRA抑制的mTOR信号通路得到部分恢复，而被ATRA增强的自噬适当减弱。这表明ATRA使HL60和U937细胞发生髓系分化，并诱导TGM2表达升高；而TGM2通过mTOR信号通路和自噬途径调控ATRA诱导的髓系分化。该研究将有利于更深入地...     

粘着斑激酶在TNF-α作用下影响SMMC-7721细胞PKB的表达水平

 探讨在肿瘤坏死因子α(TNF-α)作用下 ,粘着斑激酶 (focal adhesion kinase,FAK)对蛋白激酶 B(PKB)蛋白水平的影响 .利用构建、转染 FAK反义质粒来特异性降低 SMMC- 772 1细胞的FAK含量 ,及用 Western杂交的方法来检测 PKB的蛋白含量 .文献报道 TNF- α能够激活磷脂酰肌醇 3-激酶 (PI3K)而使 PKB发生磷酸化 .但是至于 TNF-α对 PKB蛋白水平的影响目前并无报道 .研究发现 ,当用 wortmannin特异性抑制 PI3K活性后可以显著降低 PKB的蛋白含量 .提示PI3K对维持 PKB的基础蛋白水平是必需的 .但是 TNF- α本身对 PKB的蛋白水平无明显影响 .而当用不同浓度的 TNF- α和 wortmannin处理 SMMC- 772 1细胞时 ,发现 PKB的蛋白含量随着TNF-α浓度的增加而降低 .提示 TNF-α可能除了通过 PI3K外 ,还可能通过另一条途径来下调PKB的表达 .而当用 FAK反义质粒转染 SMMC- 772 1细胞后 (FAK下降了 60 % ) ,发现在当用不同浓度的 TNF- α处理的情况下 ,FAK反义质粒转染株 AS- 772 1细胞的 PKB含量降低为对照的70 % ;而在用 TNF-α和 wortmannin处理的情况下 ,下降为对照的 40 %～ 60 % .TNF-α能够通过PI3K及另一未知途径来影响 PKB的蛋白水平 .而 FAK在 TNF- α作用下能够不通过 PI3K来影响PKB的蛋白水平 .     

E‐, P‐, and N‐cadherin are co‐expressed in the nasopharyngeal carcinoma cell line TW‐039

The cadherin/catenin complex plays a key role in the initiation of cell‐cell recognition, and adhesion, and the elaboration of structural and functional organization in multicellular tissues and organs. It is associated with tumor metastasis and also acts as an “invasion suppressor” of cancer cells. Nasopharyngeal carcinoma (NPC) is notorious for its highly metastatic nature. The expression of the E‐cadherin/catenin complex is down‐regulated in NPC tumor specimens. To obtain better insight into the intercellular adhesive property of NPC cells, we used immunofluorescence microscopy, immunoprecipitation, and immunoblot analysis to examine the expression of the classical cadherins and β‐catenin in a NPC cell line, TW‐039. The results demonstrate a change in the distribution of E‐cadherin from cytosolic flakes to cell‐cell contacts with increasing time in culture. Between days 1 and 5 after plating, the detergent‐insoluble fraction of E‐cadherin increased from 20% to 37% of total E‐cadherin, and that for P‐cadherin increased from 33% to 40%. By contrast, the values for β‐catenin remained unchanged (26% and 25%). Both immunofluorescence and immunoblot studies suggested that P‐cadherin may be involved in pioneer contact adhesion of TW‐039 cells. Interestingly, E‐, P‐, and N‐cadherin are co‐expressed in this cell line. Immunoprecipitation studies also showed that other members of the cadherin family may be involved in the contact adhesion of TW‐039 cells. J. Cell. Biochem. 76:161–172, 1999. © 1999 Wiley‐Liss, Inc.     

人U6基因POI Ⅲ启动子表达反义VEGF cDNA抑制SMMC-7721肝癌细胞VEGF表达的观察

 由 RNA聚合酶启动子在细胞内转录高浓度反义 RNA是抑制靶蛋白的一种有效手段 ,有报道 POI 启动子转录小分子 RNA存在效率不高 ,带有较多的非特异性序列等缺点 ,为了克服这些问题 ,表达反义 VEGF RNA的人 U6基因表达盒对人肝癌细胞株 SMMC- 772 1 VEGF表达的抑制作用进行了研究 .首先 PCR扩增 2 0 0 bp VEGF c DNA以正、反向插入人 U6 sn RNA.通过测序证实反向插入的正确性 .采用细胞原位杂交 ,RNA酶保护分析 ,Northern印迹来证实反义 RNA表达的情况 ,利用 RT- PCR方法研究了其对人肝癌细胞株 SMMC- 772 1 VEGF表达的抑制效果 .细胞原位杂交结果显示 U6启动子转录产物主要分布于细胞核内 ,细胞浆内亦有表达 ,RNA酶保护分析显示 U6基因 POI 启动子能高表达所需大小反义 RNA,Northern印迹结果显示脂质体 lipo-fectamine介导含 U6基因 POI 启动子的质粒转染人肝癌细胞株 SMMC- 772 1后 1 2 h即有表达且可持续表达 6 d. RT- PCR证实 U6基因 POI 启动子转录的反义 VEGF RNA能有效抑制SMMC- 772 1细胞 VEGF的 m RNA表达 .已有的研究结果揭示 POI 启动子是反义基因治疗中一种好的选择     

The effect of receptor protein tyrosine phosphatase kappa on the change of cell adhesion and proliferation induced by N‐acetylglucosaminyltransferase V

N‐acetylglucosaminyltransferase V (GnT‐V) has been reported to be positively associated with tumor progression, but its mechanism still remains unknown. In the present study, we found that GnT‐V overexpression not only changed the glycosylation of receptor protein tyrosine phosphatase kappa (RPTPκ) but also decreased its protein level. Moreover, GnT‐V overexpression decreased cell calcium‐independent adhesion and increased the tyrosine phosphorylation level of β‐catenin, in which RPTPκ played an important role. Since RPTPκ has an RXKR motif, which is a favored cleavage site for furin, we used furin inhibitor to further explore the effect of RPTPκ on the change of cell adhesion and β‐catenin signaling induced by GnT‐V. Our results showed that preventing RPTPκ cleavage rescued the above effects of GnT‐V, suggesting that furin cleavage could be one of the factors for RPTPκ to regulate cell adhesion and β‐catenin signaling in GnT‐V overexpression cell lines. In addition, the increased tyrosine phosphorylation level of β‐catenin was associated with the increased nuclear level of β‐catenin and downstream signaling molecules such as c‐myc and cyclin D1 that were associated with cell proliferation. Our results suggest that GnT‐V could decrease human hepatoma SMMC‐7721 cell adhesion and promote cell proliferation partially through RPTPκ. J. Cell. Biochem. 109: 113–123, 2010. © 2009 Wiley‐Liss, Inc.     

紫杉醇对人肝癌SMMC7721细胞NDRG1表达及增值的影响

旨在探索紫杉醇对人肝癌SMMC7721细胞NDRG1表达的影响,及紫杉醇对肝癌SMMC7721细胞系增殖的抑制作用。分别提取紫杉醇处理前后SMMC7721细胞的RNA,进行逆转录-聚合酶链反应(RT-PCR),判断紫杉醇处理前后肝癌细胞中NDRG1表达的情况;采用蛋白印迹技术(Western blotting)分析紫杉醇处理前后肝癌细胞中NDRG1蛋白表达的情况;应用不同浓度紫杉醇处理肝癌细胞,以MTT法检测处理前后肝癌细胞的抑制率,流式细胞术(FCM)观察细胞周期变化的况。结果表明紫杉醇处理后的肝癌SMMC7721细胞中NDRG1表达下降,紫杉醇浓度越高,NDRG1表达水平越低,具有浓度依赖性。以MTT法观察紫杉醇对肝癌细胞的抑制作用,试验结果表明不同浓度的紫杉醇处理肝癌SMMC7721细胞后,癌细胞被明显抑制;以流式细胞术观察紫杉醇作用后肝癌SMMC7721细胞周期的变化,结果显示G2-M期细胞比例升高的程度随浓度增高而升高,细胞越来越多地被阻滞在G2-M期,不能继续分裂增殖。分化相关基因NDRG1的表达可能是肝癌的发病机制之一,紫杉醇可抑制肝癌SMMC 7721细胞中NDRG1的表达;同时紫杉醇能使肝癌SMMC7721细胞的生长阻滞在G2-M期,从而显著抑制SMMC7721细胞的增殖,并且具有剂量、时间依赖效应。     

Analysis of cadherin/catenin complexes in transformed thyroid epithelial cells: modulation by beta 1 integrin subunit

We have analysed the expression of cadherin/catenin complex molecules in PC C13 rat thyroid cells transformed in vitro with different oncogenes. No significant downregulation of either E-cadherin, alpha-, beta- and gamma-catenin was detected following the introduction of activated forms of myc, adenovirus E1A, ras, raf, myc + ras, E1A + raf. However, ras- and raf-transformed PC C13 cells showed altered adherens junctions. An altered distribution of cadherin/catenin complexes characterized by radially oriented membrane spikes perpendicular to cell edges was the most prominent feature evidenced by immunofluorescence. No beta1 integrin localization was observed in areas where this altered pattern of E-cadherin expression was detected. However, beta1 integrin subunit expression was detected at areas of cell-cell contact where E-cadherin showed a normal pattern of expression. Furthermore, ras- and raf-transformed PC C13 cells showed the ability to migrate in collagen gels, in contrast to their normal untransformed counterpart. Overexpression of beta1 integrin was found to restore normal E-cadherin localization at cell-cell contacts and to partially inhibit the ability to migrate in collagen gels. Finally, two cell lines obtained by ras transformation in vivo, and derived from a rat primary thyroid carcinoma (TK6) and its lung metastasis (MPTK6), were found to have lost gamma-catenin expression. TK6 lost also E-cadherin expression and membrane localization of alpha-catenin. These results suggest that: i) in vitro thyroid cell transformation is associated to a change in cadherin/catenin complexes distribution rather than to a decrease in expression; ii) in vivo transformation is associated to the loss of expression of some of these molecules likely due to tumor progression; iii) alterations in beta1 integrin subunit expression can result in changes in cadherin/catenin function thus implying that an integrin-cadherin synergy may exist in thyroid cells.     

An acidic extracellular pH disrupts adherens junctions in HepG2 cells by Src kinases‐dependent modification of E‐cadherin

We have previously shown that culturing HepG2 cells in pH 6.6 culture medium increases the c‐Src‐dependent tyrosine phosphorylation of β‐catenin and induces disassembly of adherens junctions (AJs). Here, we investigated the upstream mechanism leading to this pH 6.6‐induced modification of E‐cadherin. In control cells cultured at pH 7.4, E‐cadherin staining was linear and continuous at cell–cell contact sites. Culturing cells at pH 6.6 was not cytotoxic, and resulted in weak and discontinuous junctional E‐cadherin staining, consistent with the decreased levels of E‐cadherin in membrane fractions. pH 6.6 treatment activated c‐Src and Fyn kinase and induced tyrosine phosphorylation of p120 catenin (p120ctn) and E‐cadherin. Inhibition of Src family kinases by PP2 attenuated the pH 6.6‐induced tyrosine phosphorylation of E‐cadherin and p120ctn, and prevented the loss of these proteins from AJs. In addition, E‐cadherin was bound to Hakai and ubiquitinated. Furthermore, pH 6.6‐induced detachment of E‐cadherin from AJs was blocked by pretreatment with MG132 or NH₄Cl, indicating the involvement of ubiquitin‐proteasomal/lysosomal degradation of E‐cadherin. An early loss of p120ctn prior to E‐cadherin detachment from AJs was noted, concomitant with a decreased association between p120ctn and E‐cadherin at pH 6.6. PP2 pretreatment prevented the dissociation of these two proteins. In conclusion, pH 6.6 activated Src kinases, resulting in tyrosine phosphorylation of E‐cadherin and p120ctn and a weakening of the association of E‐cadherin with p120ctn and contributing to the instability of E‐cadherin at AJs. J. Cell. Biochem. 108: 851–859, 2009. © 2009 Wiley‐Liss, Inc.