胶质母细胞瘤中P16和cyclinD1基因表达异常的研究

目的：探讨胶质母细胞瘤（glioblastomas,CBMs)中P16,cylinD1基因表达的异常及其与细胞增殖活性之间的关系。方法：应用流式细胞仪检测32例胶质母细胞瘤标本中P16、cyclinD1基因蛋白表达的相对含量。结果;胶质母细胞瘤中P16基因蛋白表达量较正常脑组织降低,cyclinD1基因蛋白表达量较正常脑组织升高,二者的异常可使细胞发生过度增殖。结论：P16基因蛋白表达量降低和cyclinD1基因蛋白表达量升高是胶质母细胞瘤发生发展的重要因素之一。     

Smad7对脑胶质母细胞瘤U251细胞生物学行为的影响

目的 观测Smad7对脑胶质母细胞瘤U251细胞增殖及浸润的影响.方法 应用CCK-8和RT-PCR分别检测Smad7稳定转染U251细胞与空载体转染U251细胞及U251细胞在增殖和N-cadherin 、E-cadherin、 vimentin、 twist基因表达的差异.结果 Smad7转染后,上皮细胞间质化相关基因表达未见改变(P＞0.05),但相对于空载体转染U251细胞及U251细胞其增殖速度减慢.结论 Smad7稳定转染后未见胶质母细胞瘤上皮细胞间质化改变,但Smad7高表达可抑制胶质母细胞瘤细胞增殖.     

蛋白酶体抑制剂乳胞素对胶质母细胞瘤U87MG的抑制作用

目的目的探讨蛋白酶体抑制剂乳胞素对人胶质母细胞瘤U87MG的抑制作用。方法培养人胶质母细胞瘤U87MGM,分别以5、10、15μmol/L作用于细胞株。MTT法检测细胞存活率;bcl-2、Bax及caspase-3 mRNA水平用RT-PCR法检测;Rh123法检测线粒体膜电势;Hoechst33342检测细胞凋亡情况;U87MG细胞Bax及bcl-2的蛋白表达用Western印迹法检测。结果与未处理组相比对,U87MG细胞在实验组的存活率下降,bcl-2、Bax mRNA及蛋白表达和线粒体膜电势均下降,caspase-3 mRNA表达水平上升。结论在体外条件下,乳胞素对胶质母细胞瘤U87MG细胞具有较强的抑制效应。     

miR-138通过促进PDK1和激活AKT信号通路促进胶质母细胞瘤细胞增殖

目的 检测miR-138在胶质母细胞瘤中的表达水平并研究其对肿瘤细胞增殖的影响.方法 收集胶质母细胞瘤患者的临床标本,RT-聚合酶链反应(PCR)检测miR-138和PDK1的表达,并分析二者表达水平的相关性.培养T98G、H4、U251和U87细胞,并分别转染miR-control、miR-138 mimic或inh...     

survivin在胶质母细胞瘤中的表达及临床意义

目的探讨survivin在胶质母细胞瘤中的表达及与其凋亡、增殖和微血管密度（MVD）的相关性。方法应用免疫组织化学（SP）法检测42例份胶质母细胞瘤石蜡切片中survivin、Ki-67和第8因子相关抗原（FVIRAg）的表达情况,并用末端转移标记法测定凋亡指数（AI）。结果survivin在胶质母细胞瘤中的表达阳性率为81．0％,而正常对照组中无1例阳性表达;survivin的表达与Ki67及MVD呈正相关,而与AI负相关。结论survivin在胶质母细胞瘤的发生和发展中可能通过调控细胞的凋亡和增殖起着重要作用,并参与肿瘤血管的生成。可望成为诊断治疗的新靶点。     

褪黑素对胰腺癌细胞株SW1990体外增殖及凋亡的影响

目的 探讨褪黑素(MT)体外抑制胰腺癌细胞株SW1990增殖及诱导其凋亡的作用.方法 以不同浓度的MT(0.1、0.5、1.0、2.5及5.0 mmol/L)处理体外培养的胰腺癌细胞株SW1990细胞24、48、72 h.用MTT法测定细胞增殖,以Annexin V/PI检测细胞凋亡,流式细胞仪分析细胞周期及Western blotting检测细胞Bcl-2、Bax蛋白表达.结果 MT呈浓度和时间依赖性抑制SW1990细胞的增殖.0.1～5.0 mmol/L MT作用48 h后,细胞的增殖抑制率为7.4%～85.8%.1.0～5.0 mmoL/L MT作用48 h后,G0/G1期比例为72.6%～85.3%,细胞凋亡率为21.5%～41.7%,同时Bcl-2蛋白表达下调,Bcl-2/Bax比值下降.结论 MT可以抑制SW1990细胞增殖,其机制可能与上调Bax表达,下调Bcl-2表达,促进细胞凋亡,将细胞周期阻止于G0/G1期有关.     

YM155对人胃癌细胞株MKN28的作用及其机制研究

背景:研究发现生存素(survivin)在胃癌组织中高表达,YM155是survivin的特异性抑制剂。目的:探讨YM155对人胃癌细胞株MKN28的作用及其机制。方法:以不同浓度YM155作用于人胃癌细胞株MKN28。采用甲基噻唑基四唑(MTT)法检测细胞增殖抑制率;以原位末端标记(TUNEL法)检测细胞凋亡率;以逆转录聚合酶链反应(RTPCR)、蛋白质印迹法分别检测survivin mRNA和survivin、多聚ADP核糖聚合酶(PARP)、caspase-3蛋白表达。结果:YM155作用后,MKN28细胞增殖抑制,凋亡增加。随着YM155浓度升高,survivin mRNA和蛋白表达水平明显降低,并伴随PARP、caspase-3蛋白裂解。结论:YM155可抑制人胃癌细胞株MKN28增殖,并诱导其凋亡,此机制可能与抑制survivin表达,继而激活caspase凋亡信号通路有关。     

亚砷酸对肝癌BEL-7402细胞增殖、凋亡及其Bcl-2表达的影响

目的探讨亚砷酸（AA）对人肝癌BEL-7402细胞增殖、凋亡及其Bcl-2表达的影响。方法采用MTT比色法检测从作用后的BEL-7402细胞增殖抑制率,流式细胞术检测BEL-7402细胞周期及凋亡细胞,HE染色法观察凋亡细胞的形态,RT-PCR检测BEL．7402细胞的Bcl-2 mRNA,免疫组化法检测细胞的Bcl-2蛋白。结果1．0—8．0μmol／L的AA可使BEL-7402细胞增殖抑制率上升,能诱导BEL-7402细胞凋亡并阻滞细胞周期于S、G2／M期,呈剂量依赖性;8．0μmol／L的AA作用BEL-7402细胞48h后,细胞呈现明显的凋亡形态改变,其Bcl-2 mRNA及蛋白表达明显减弱。结论AA体外有抑制BEL-7402细胞增殖及诱导凋亡的作用,且呈时间、剂量依赖性,其作用机制可能与降低其Bcl-2表达有关。     

沉默SIRT1基因表达对胰腺癌细胞PANC1增殖和凋亡的影响

目的 应用RNA干扰技术沉默胰腺癌PANC1细胞的SIRT1基因表达,观察其对细胞增殖和凋亡的影响.方法 构建靶向SIRT1基因表达的短发夹RNA(shRNA)真核表达质粒pGC-shRNA,转染胰腺癌细胞PANC1.设对照shRNA(shRNA-C)转染组和未转染对照组.实时定量PCR和免疫细胞化学法检测转染后细胞SIRT1 mRNA及蛋白的表达;MTT法检测细胞的增殖率;ELASA法检测细胞caspase-3和caspase-9活性;Western bloting检测细胞Bax、Bcl-2蛋白表达.结果 与未转染组相比,shRNA组转染后48 h,PANC1细胞SIRT1 mRNA及蛋白表达的抑制率分别为(76.2%±10.4)%和(80.1±11.6)%;细胞增殖抑制率为(45.1±6.5)%;caspase-3和caspase-9酶活性显著增高;Bax蛋白表达上调,Bcl-2蛋白表达下调.结论 应用RNA干扰技术能有效沉默PANC1细胞SIRT1基因的表达,其机制可能与caspasa酶活性升高及Bax表达上调、Bcl-2表达下调有关.     

幽门螺杆菌对胃上皮细胞及胃癌细胞增殖凋亡的影响

目的 探讨幽门螺杆菌(Hp)对正常胃上皮细胞、胃癌细胞的形态、增殖、细胞周期以及细胞凋亡的影响.方法 倒置显微镜下观察经Hp作用24小时、48小时后,人胃上皮细胞GES-1、人胃腺癌细胞株SGC-7901、人低分化胃腺癌细胞株MGC-803、人胃癌细胞株BGC-823的形态学变化,并应用噻唑蓝比色法(MTT)检测Hp对上述细胞增殖活性的影响.采用流式细胞仪检测4类细胞增殖周期及细胞凋亡情况,Western-blot方法检测凋亡相关蛋白Caspase-3和Caspase-9的表达以及增殖周期相关蛋白P53、P21及E2F-1表达情况.结果 4种细胞与Hp共同培育后细胞正常形态发生改变,且随着细菌数目增加,细胞形态失常更加明显.MTT细胞增殖活性检测显示4种细胞株经Hp处理后,细胞增殖活性受到不同程度抑制.流式细胞术(FCM)检测4种细胞株呈不同比例的S期细胞增多,G1期细胞减少现象.Western-blot检测显示经Hp细菌处理后4种细胞株凋亡蛋白及增殖蛋白呈不同程度变化.结论 Hp对正常胃上皮细胞及不同类型的胃癌细胞在细胞增殖和细胞凋亡上存在不同程度的影响.     

乙酰肝素酶反义寡核苷酸对肺癌A-549细胞侵袭力的抑制作用

目的 探讨乙酰肝素酶(heparanase,HPSE )反义寡核苷酸(antisense oligodeoxynucleotide,ASODN)对人肺癌A-549细胞侵袭力的抑制作用.方法 设计合成互补于HPSE mRNA起始密码区的HPSE ASODN,以阳离子脂质体Lipofectin包埋后转染人肺癌A-549细胞进行培养,采用流式细胞分析技术和逆转录-聚合酶链反应(RT-PCR)方法检测肺癌A-549细胞HPSE蛋白及其mRNA表达的变化;以Matrigel细胞侵袭试验对HPSE AS-ODN对肺癌A-549细胞侵袭的抑制进行检测.结果 ASODN各组与正常对照组和脂质体组比较及ASODN各组间比较HPSE mRNA和蛋白的表达及细胞侵袭力均受到明显抑制 (P<0.01);ASODN在终浓度为100、200、400 nmol/L时对肺癌A-549细胞侵袭力的抑制率分别为55.6%、82.3%和91.2%.结论 HPSE ASODN通过下调HPSE mRNA和蛋白的表达可显著抑制肺癌A-549细胞的侵袭力,并呈剂量依赖性.     

Bcl-x(L) and Myeloid cell leukaemia-1 contribute to apoptosis resistance of colorectal cancer cells

AIM： TO explore the role of Bcl-XL and Myeloid cell leukaemia （Mcl）-I for the apoptosis resistance of colorectal carcinoma （CRC） cells towards current treatment modalities. METHODS： Bcl-XL and Mcl-1 mRNA and protein expression were analyzed in CRC cell lines as well as human CRC tissue by Western blot, quantitative PCRand immunohistochemistry. Bcl-XL and Mcl-1 protein expression was knocked down or increased in CRC cell lines by applying specific siRNAs or expression plasmids, respectively. After modulation of protein expression, CRC cells were treated with chemotherapeutic agents, an antagonistic epidermal growth factor receptor （EGFR1） antibody, an EGFR1 tyrosine kinase inhibitor, or with the death receptor ligand TRAIL. Apoptosis induction and cell viability were analyzed. RESULTS： Here we show that in human CRC tissue and various CRC cell lines both Bcl-xL and Mcl-1 are expressed. Bcl-xL expression was higher in CRC tissue than in surrounding non-malignant tissue, both on protein and mRNA level. Mcl-1 mRNA expression was significantly lower in malignant tissues. However, protein expression was slightly higher. Viability rates of CRC cells were significantly decreased after knock down of Bcl-xL expression, and, to a lower extent, after knock down of Mcl-1 expression. Furthermore, cells with reduced Bcl-xL or Mcl-1 expression was more sensitive towards oxaliplatinand irinotecan-induced apoptosis, and in the case of Bcl-xL also towards 5-FU-induced apoptosis. On the other hand, upregulation of Bcl-xL by transfection of an expression plasmid decreased chemotherapeutic drug-induced apoptosis. EGF treatment clearly induced Bcl-xL and Mcl-1 expression in CRC cells. Apoptosis induction upon EGFR1 blockage by cetuximab or PD168393 was increased by inhibiting Mcl-1 and Bcl-xL expression. More strikingly, CD95- and TRAIL-induced apoptosis was increased by Bcl-xL knock down. CONCLUSION： Our data suggest that Bcl-xL and, to a lower extent, Mcl-1, are important anti-apoptotic factors in CRC. Specific do     

辛二酰苯胺异羟肟酸对鼻咽癌细胞回复引导半胱氨酸丰富蛋白含kazal基元以及基质金属蛋白酶9表达与活性的影响

目的探讨辛二酰苯胺异羟肟酸(suberoylanilide hydroxamic acid,SAHA)对鼻咽癌细胞回复引导半胱氨酸丰富蛋白含kazal基元(reversion-inducing-cysteine-rich protein with kazal motifs,RECK)以及基质金属蛋白酶9(matrix metalloproteinase-9,MMP-9)表达与活性的影响。方法体外培养鼻咽癌细胞系CNE-1,用0、0.1、1、5μmol/L SAHA处理后,采用蛋白质印迹法(Western blot)和Real-Time PCR(RT-PCR)分别检测RECK,MMP-9蛋白和mRNA表达;MTT法检测CNE-1细胞的增殖。同时采用明胶酶谱实验观察SAHA处理后MMP-9酶活性变化。结果随着SAHA浓度的增加,RECK蛋白和mRNA的表达显著增高(P0.05);随着SAHA浓度的增加,MMP-9蛋白和mRNA的表达显著降低(P0.05);CNE-1细胞随着SAHA浓度的增加,存活率逐渐降低(P0.05)。明胶酶谱实验显示,随着SAHA浓度的增加,MMP-9酶活性逐渐降低。结论 SAHA可能通过上调RECK基因的表达,抑制MMP-9的表达与活性而发挥对CNE-1细胞生长抑制作用。     

In leukaemic CD5+ B cells the expression of BCL-2 gene family is shifted toward protection from apoptosis

This study further investigated the mechanisms that control apoptosis in leukaemic CD5⁺ B cells, and focused on the Bcl-2 gene family. The pattern of expression of Bcl-2, Bcl-xL, Bcl-xS and Bax genes, selected because of their interrelated role in the control of apoptosis, was analysed in a series of CD5⁺ B-cell chronic lymphoid leukaemias.
Cells from 34 patients with chronic lymphoid leukaemia of B-cell type (23 B-chronic lymphocytic leukaemia (B-CLL) and 11 mantle cell lymphoma (MCL) in leukaemic phase) were investigated. High levels of Bcl-2 mRNA were observed by Northern blot and high levels of Bcl-2 protein were detected by cytofluorograph analysis with a specific monoclonal antibody (MAb) in all cases. Strong Bax expression was detected by RT-PCR in 20/23 B-CLL cases; Bax was also observed in 8/11 MCL in leukaemic phase with variable degree of intensity. In both B-CLL and MCL samples the presence of Bax protein was confirmed by cytofluorograph analysis. RT-PCR detected high levels of Bcl-xL in 16/23 B-CLL and in 8/11 MCL in leukaemic phase, whereas Bcl-xS was detectable in low to trace amounts respectively in 13/23 B-CLL and in 6/11 MCL in leukaemic phase.
According to the functional role of Bcl-2, Bcl-xL, Bcl-xS and Bax, these data indicate that the pattern of Bcl-2 family genes expression in leukaemic CD5⁺ B cells is skewed toward prevention of apoptosis and may thus favour the relentless accumulation of CD5⁺ leukaemic B cells.     

Follicle-stimulating hormone inhibits adenosine 5'-monophosphate-activated protein kinase activation and promotes cell proliferation of primary granulosa cells in culture through an Akt-dependent pathway

FSH, acting through multiple signaling pathways, regulates the proliferation and growth of granulosa cells, which are critical for ovulation. The present study investigated whether AMP-activated protein kinase (AMPK), which controls the energy balance of the cell, plays a role in FSH-mediated increase in granulosa cell proliferation. Cells isolated from immature rat ovaries were grown in serum-free, phenol red free DMEM-F12 and were treated with FSH (50 ng/ml) for 0, 5, and 15 min. Western blot analysis showed a significant reduction in AMPK activation as observed by a reduction of phosphorylation at thr 172 in response to FSH treatment at all time points tested. FSH also reduced AMPK phosphorylation in a dose-dependent manner with maximum inhibition at 100 ng/ml. The chemical activator of AMPK (5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside, 0.5 mm) increased the cell cycle inhibitor p27 kip expression significantly, whereas the AMPK inhibitor (compound C, 20 microm) and FSH reduced p27kip expression significantly compared with control. FSH treatment resulted in an increase in the phosphorylation of AMPK at ser 485/491 and a reduction in thr 172 phosphorylation. Inhibition of Akt phosphorylation using Akt inhibitor VIII reversed the inhibitory effect of FSH on thr 172 phosphorylation of AMPK, whereas ERK inhibitor U0126 had no effect. These results show that FSH, through an Akt-dependent pathway, phosphorylates AMPK at ser 481/495 and inhibits its activation by reducing thr 172 phosphorylation. AMPK activation by 5-amino-imidazole-4-carboxamide-1-beta-D-ribofuranoside treatment resulted in a reduction of cell cycle regulatory protein cyclin D2 mRNA expression, whereas FSH increased the expression by 2-fold. These results suggest that FSH promotes granulosa cell proliferation by increasing cyclin D2 mRNA expression and by reducing p27 kip expression by inhibiting AMPK activation through an Akt-dependent pathway.     

弓形虫ROP16蛋白对人乳腺癌MCF-7细胞增殖、周期和凋亡的影响

目的 探讨弓形虫Ⅰ型(RH)ROP16蛋白在人乳腺癌MCF-7细胞增殖、周期及凋亡方面的作用。方法 以空载体(MCF-7-HBLV)和过表达ROP16蛋白的慢病毒(HBLV-RH ROP16)分别感染MCF-7细胞,嘌呤霉素筛选出稳定表达ROP16的细胞株,Real time PCR、Western blot法检测MCF-7细胞中ROP16 mRNA和蛋白表达水平。CCK-8和流式细胞术检测细胞增殖、周期和凋亡。Western blot法检测细胞周期及凋亡相关蛋白表达。结果 相比MCF-7-HBLV(空载体组)和MCF-7细胞组(对照组),MCF-7-RH ROP16细胞组中ROP16 mRNA和蛋白表达升高;细胞增殖率降低(P<0.01);S期细胞比例、细胞凋亡率升高(P<0.01)。促凋亡因子Bax、P53、Caspase-9、Caspase-3及细胞周期蛋白依赖激酶抑制因子P21表达增高(P<0.01);抗凋亡蛋白Bcl-2及细胞周期蛋白A1(CyclinA1)、周期蛋白依赖性激酶2(CDK2)的表达均下降(P<0.01)。结论 弓形虫Ⅰ型(RH)ROP1...     

Lysophosphatidic acid and adenylyl cyclase inhibitor increase proliferation of senescent human diploid fibroblasts by inhibiting adenosine monophosphate-activated protein kinase

Abstract This study was designed to elucidate the molecular mechanism underlying lysophosphatidic acid (LPA) and adenylyl cyclase inhibitor SQ22536 (ACI)-induced senescent human diploid fibroblast (HDF) proliferation. Because adenosine monophosphate (AMP)-activated protein kinase (AMPK) is known to inhibit cell proliferation, we examined the phosphorylation status of AMPK and p53 and the expression level of p21(waf1/cip1) after treating HDFs with LPA and ACI. Phosphorylation of AMPKalpha on threonine-172 (p-Thr172-AMPKalpha) increases its catalytic activity but phosphorylation on serine-485/491 (p-Ser485/491-AMPKalpha) reduces the accessibility of the Thr172 phosphorylation site thereby inhibiting its catalytic activity. LPA increased p-Ser485/491-AMPKalpha, presumably by activating cAMP-dependent protein kinase (PKA). However, ACI reduced p-Thr172-AMPKalpha by inhibiting the LKB signaling. Our data demonstrated that both LPA and ACI inhibit the catalytic activity of AMPKalpha and p53 by differentially regulating phosphorylation of AMPKalpha, causing increased senescent cell proliferation. These findings suggest that the proliferation potential of senescent HDFs can be modulated through the regulation of the AMPK signaling pathway.