表皮生长因子对ER阴性乳腺癌细胞株细胞周期的影响

目的　研究表皮生长因子 (epidermalgrowthfactor,EGF)对雌激素受体 (ER)阴性乳腺癌细胞株MDA MB 4 35S细胞周期的影响。方法　采用Westernblot检测MDA MB 4 35S细胞周期蛋白D1(cyclinD1 )的表达 ,用流式细胞技术检测细胞周期的变化。结果　EGF促cyclinD1 表达作用显著 ,蛋白激酶C抑制剂Go6 .976能抑制核转录因子κB(NF κB)的激活 ,并能抑制EGF的促cyclinD1 表达作用 ;EGF组G0/ G1 期 6 9 .36 %,S期 2 2 . 77%,细胞增殖指数 (PI) 0 . 31 ,与对照组比较差异有统计学意义 (P <0. 0 5 ) ,加用Go6 976后 ,G0 /G1 期明显上升达 91. 5 4 %,S期比例降至 7. 81 %,PI0. 0 9,与EGF组比较差异有统计学意义 (P <0 .0 5 )。结论　EGF促进MDA MB 4 35S的cyclinD1 表达 ,使肿瘤细胞进入DNA合成期。Go6 976可以抑制NF κB的活性。     

乳腺

20060084新辅助化疗对乳腺癌雌孕激素受体表达的影响,20060085核转录因子κB激活对雌激素受体阴性乳腺癌细胞增殖的影响，20060086尼美舒利对二甲基苯并蒽诱导的乳腺癌的影响殛机制的实验研究，20060087乳腺癌根治术中创面使用纤维蛋白胶促进切口愈合的研究，20060088局部进展期及较大乳腺癌的保乳治疗。[编者按]     

乳腺癌细胞中表皮生长因子对雌激素受体表达的影响及机制

目的研究表皮生长因子（EGF）在雌激素受体（ER）阳性及阴性乳腺癌细胞株中对ER表达的影响及其可能的机制。方法以逆转录-聚合酶链反应（RT—PCR）技术分别研究EGF途径以及抑制该途径的信号传导后，对乳腺癌细胞株中ERcxmRNA的影响。结果在ER阳性乳腺癌细胞株中，EGF能显著抑制ERα mRNA的表达（P〈0．05），而通过抑制表皮生长因子受体（EG-FR）、磷脂酰肌醇3激酶（P13K）阻断EGF信号传导可减弱上述抑制作用（P〈0．05）；在ER阴性乳腺癌细胞株中，ERα mRNA无显著变化。结论EGF能够明显抑制ER阳性乳腺癌细胞株中ER的表达，这种抑制作用可能通过EGFR、蛋白激酶B（PKB，又称AKt）信号传导途径完成；这种作用在ER阴性乳腺癌细胞株中并不明显。     

肼苯哒嗪与三苯氧胺协同抗雌激素受体α阴性乳腺癌的研究

目的观察肼苯哒嗪对雌激素受体(ER)α阴性人乳腺癌细胞株MDA-MB-231和MDA-MB-435ERα基因诱导表达作用；肼苯哒嗪联合三苯氧胺(TAM)对ERα阴性乳腺癌细胞的体外抑制作用。方法肼苯哒嗪处理ERα阴性人乳腺癌细胞株MDA-MB-231和MDA-MB-435，逆转录一聚合酶链反应(RT-PCR)检测ERα mRNA表达；肼苯哒嗪、TAM分别或联合作用于MDA-MB-231和MDA-MB-435细胞，噻唑蓝(MTT)比色法分析细胞生长抑制作用，流式细胞仪测定细胞周期分布和凋亡率。透射电镜观察细胞超微结构。结果肼苯哒嗪能诱导MDA-MB-231和MDA-MB-435细胞表达ERα mRNA。当肼苯哒嗪浓度≥10μmol／L可抑制MDA-MB-231和MDA-MB-435细胞生长、阻滞细胞周期于G0／G1期，诱导这两种细胞凋亡，凋亡率分别为11．20％和8．71％；TAM对MDA-MB-231和MDA-MB-435细胞生长、细胞周期无影响；联合用药组显著抑制细胞生长，诱导这两种细胞凋亡，凋亡率分别为48．8％和53．1％。对照组和TAM组细胞形态较好；肼苯哒嗪作用组细胞变性改变多见；联合用药组主要表现为细胞坏死，但未发现有凋亡小体的存在。结论肼苯哒嗪能诱导ERα阴性乳腺癌表达ERα mRNA，恢复ERα阴性乳腺癌细胞对TAM的敏感性，联合TAM能协同抑制ERα阴性乳腺癌细胞生长，诱导细胞凋亡，从而为ERα阴性乳腺癌开辟新的内分泌治疗途径提供实验依据。     

畸胎瘤细胞源性生长因子对雌激素受体阴性乳腺癌细胞雌激素依赖性的影响

目的分析乳腺癌细胞雌激素依赖性与畸胎瘤细胞源性生长因子（PC—cell derived growth factor,PCDGF）表达之间的关系,探讨雌激素受体（ER）阴性乳腺癌患者接受内分泌治疗的可能性。方法荧光定量聚合酶链反应检测乳腺癌细胞系MCF-7、MDA-MB-231、T47D、MDA-MB-435s中PCDGF的表达,CCK-8法检测不同浓度雌激素培养条件下细胞的存活情况。以ER阳性乳腺癌细胞系MCF-7为对照,用RNA干扰技术抑制ER阴性乳腺癌细胞系MDA-MB-231中PCDGF基因的表达,观察抑制前后雌激素依赖性的变化。结果PCDGF在4株乳腺癌细胞系中均有不同程度的表达,其中在MDA-MB-435s中表达量最高,PCDGF高表达的乳腺癌细胞系的雌激素依赖性较高,PCDGFshRNA可以有效抑制乳腺癌细胞系MCF-7和MDA-MB-213中PCDGF的表达（抑制率分别为81．1％和86．7％）。MDA-MB-231细胞系雌激素依赖性增加的程度高于MCF-7细胞系（P〈0．05）。结论PCDGF高表达于ER阴性乳腺癌细胞,抑制其表达可能会更好的逆转内分泌治疗的耐药性。     

核因子-κB与乳腺癌的研究进展（文献综述）

核因子-κB(NF-κB)在乳腺癌中异常激活，同时持续激活的NF-κB和乳腺癌的浸润与转移有关，NF-κB是雌激素受体阴性乳腺癌的一个潜在治疗靶点。     

胃癌细胞株核因子κB组成性激活对细胞增殖的影响

目的探讨胃癌细胞株是否存在核转录因子(NF)-κB组成性激活及对胃癌细胞增殖的影响。方法采用逆转录-聚合酶链反应(RT-PCR法)和蛋白免疫印迹法(Westernblot法)检测4株不同分化程度的胃癌细胞株NF-κB的mRNA和蛋白表达,比较4株胃癌细胞株中NF-κB的转录和蛋白质表达水平的差异,并利用TransAMTMNF-κBP65试剂盒比较不同胃癌细胞株中P65亚基的蛋白活性差异。采用噻唑蓝(MTT)比色法观察NF-κB活性抑制剂PDTC(吡咯烷二硫代氨基甲酸盐)对胃癌细胞增殖的影响。结果4株胃癌细胞株中均存在NF-κB的组成性激活,且存在蛋白表达和活性差异。活化的P65亚基在MKN28、MKN45细胞株中表达较低,在AGS、SGC-7901细胞株中表达较高。经PDTC处理的实验组胃癌细胞的增殖明显受到抑制,对照组呈正常生长。结论胃癌细胞株中存在NF-κB组成性激活及P65蛋白表达差异。抑制NF-κB的活性可明显抑制胃癌细胞的增殖。     

核因子kappa B与体外循环炎性反应

核因子κB(NF-κB)是近年发现的一种重要转录因子，参与多种细胞因子及免疫基因表达的转录、调节。一般情况下，NF-κB与抑制蛋白IκB结合呈非活性状态，受刺激激活后与其抑制蛋白解离，移位到核内，参与细胞因子、粘附分子、生长因子和急性期蛋白等因子的转录、调控，在炎性疾病的启动中起重要作用，并在体外循环中诱发炎性反应。现就NF-κB在体外循环炎性反应发生机制中所起的作用，并对现有NF-κB激活的抑制药物和措施作一综述。     

核因子kappa B与体外循环炎性反应

核因子κB(NF κB)是近年发现的一种重要转录因子 ,参与多种细胞因子及免疫基因表达的转录、调节。一般情况下 ,NF κB与抑制蛋白IκB结合呈非活性状态 ,受刺激激活后与其抑制蛋白解离 ,移位到核内 ,参与细胞因子、粘附分子、生长因子和急性期蛋白等因子的转录、调控 ,在炎性疾病的启动中起重要作用 ,并在体外循环中诱发炎性反应。现就NF κB在体外循环炎性反应发生机制中所起的作用 ,并对现有NF κB激活的抑制药物和措施作一综述。     

核转录因子-κB组成性激活对胃癌细胞增殖的影响及其机制

目的 探讨胃癌细胞株是否存在核转录因子(NF)-κB组成性激活及其对胃癌细胞增殖的影响机制。方法 采用蛋白免疫印迹法(Western blot法)检测4株不同分化程度的胃癌细胞株NF-κB蛋白表达,比较4株胃癌细胞株中NF-κB蛋白质表达水平的差异。通过Trans AM~(TM)NFκBp65试剂盒来比较不同胃癌细胞株中p65亚基的蛋白活性差异。选取活性较高细胞株,观察吡咯烷二硫代氨基甲酸盐(PDTC)对NF-κB活性的影响;利用噻唑蓝(MTT)法,分别观察24、48、72h,PDTC对细胞增殖的影响。并采用流式细胞分析技术(FCM)检测细胞的凋亡情况。结果 4株胃癌细胞株胞核中均存在NF-κB蛋白的表达,即存在NF-κB的组成性激活,且存在表达差异。活化的p50亚基在AGS细胞株中表达较低,在MKN28、MKN45、SGC-7901细胞株中表达较高;活化的p65亚基在MKN28、MKN45细胞株中表达较低,在AGS、SGC-7901细胞株中表达较高。经PDTC处理的实验组胃癌细胞,NF-κB的活性均被抑制(P<0.01);同时细胞表现为不同程度的增长抑制(P<0.01),FCM结果显示PDTC可诱导细胞的凋亡。结论 胃癌细胞株中存在NF-κB的组成性激活,并在不同的细胞株中存在表达差异。抑制NF-κB的活性可明显抑制胃癌细胞的增殖,其机制与PDTC所诱导的细胞凋亡有关。     

Activation of cyclin D1-Cdk4 and Cdk4-directed phosphorylation of RB protein in diabetic mesangial hypertrophy

To determine the role of cell-cycle proteins in regulating pathological renal hypertrophy, diabetes was induced in mice expressing a human retinoblastoma (RB) transgene and in wild-type littermates. Whole-kidney and glomerular hypertrophy caused by hyperglycemia was associated with specific G1 phase cell-cycle events: early and sustained increase in expression of cyclin D1 and activation of cyclin D1-cdk4 complexes, but no change in expression of cyclin E or cdk2 activity. Overexpression of RB alone likewise caused hypertrophy and increased only cyclin D1-cdk4 activity; these effects were not further augmented by high glucose. Identical observations were made when isolated mesangial cells conditionally overexpressing RB from a tetracycline-repressible system hypertrophied in response to high glucose. A mitogenic signal in the same cell-culture system, in contrast, transiently and sequentially activated both cyclin D1-cdk4 and cyclin E-cdk2. In vivo and in cultured mesangial cells, high glucose resulted in persistent partial phosphorylation of RB, an event catalyzed specifically by cyclin D1-cdk4. These data indicate that mesangial hypertrophy caused by hyperglycemia in diabetes results in sustained cyclin D1-cdk4-dependent phosphorylation of RB and maintenance of mesangial cells in the early-to-middle G1 phase of the cell cycle.     

Inhibition of cell proliferation in head and neck squamous cell carcinoma cell lines with antisense cyclin D1

Cyclin D1 and cyclin G are essential regulatory factors in the progression of the cell cycle from G0 through G1 and S phase. Aberrations in expression of these cyclins may lead to dysregulated cellular proliferation that could result in neoplasia. Amplification and overexpression of cyclin D1 have been observed in many human cancers, whereas cyclin G is a new cyclin recently described in osteosarcoma cells. This study was performed to determine whether these cyclins were amplified in head and neck squamous cell carcinoma (HNSCC) tumors. Polymerase chain reaction of DNA extracted from 22 HNSCC primary tumors and three HNSCC cell lines did not reveal amplification of cyclin D1 in any of the tumor samples. Southern blot analysis identified amplification of cyclin D1 in a single tumor. Amplification of cyclin G was not observed in any of the tumors by Southern blot hybridization with a cyclin G probe. HNSCC cell lines transfected with antisense cyclin D1 were tested for cell proliferation by the incorporation of ³ H-thymidine into cells grown in serum-free media. By 72 hours of incubation, there was a greater than 30% reduction in proliferation of cells transfected with antisense cyclin D1 as compared with nontransfected control cells. The results indicate that cyclin D1 may play an important role in the growth and proliferation of HNSCC cells. (Otolaryngol Head Neck Surg 1998;119:593-9.)     

雄激素受体在乳腺癌细胞中的表达及其对细胞增殖的影响

目的 检测雄激素受体(AR)在乳腺癌细胞中的表达,并观察雄激素刺激对乳腺癌细胞增殖的影响.方法 选择雌激素受体(ER)阳性的MCF-7和ER阴性的MDA-MB-453乳腺癌细胞,体外培养,Western blot技术检测两乳腺癌细胞株中AR蛋白的表达,MTT法检测用1×10-7、1×10-8和1×10-9 mol/L不同浓度的雄激素二氢睾酮(DHT)分别干预48、96、144 h后的细胞增殖,并应用流式细胞术检测DHT刺激乳腺癌细胞72 h后细胞周期的变化.结果 两个乳腺癌细胞株经DHT作用后AR蛋白表达均增多,DHT通过AR抑制MCF-7和MDA-MB-453两个乳腺癌细胞株的生长,各时间段不同浓度组比较A值差异无统计学意义(P＞0.05),细胞周期结果显示G1期细胞比例增高,S期细胞比例降低.结论 雄激素受体途径对ER阳性的MCF-7和ER阴性的MDA-MB-453乳腺癌细胞均有抑制生长作用,可能通过抑制细胞由G1期到S期转化来实现的.

Abstract：

Objective To evaluate the expression of androgen receptor (AR) in the breast cancer cell lines and its effect on proliferation of breast cancer cells. Methods The estrogen receptor (ER) -positive MCF-7 and ER-negative MDA-MB-453 cells were involved in this study and cultured in vitro. The expression of AR was detected by using Western blotting. Cell proliferation was determined by methyl thiazol tetrazolium (MTT) assay after the treatment with different concentrations of dihydrotestosterone (DHT) ( 1 x 10 -7, 1 x 10- 8, 1 x 10 -9 mol/L) for 48, 96 and 144 h respectively. Cell cycle was analyzed by flow cytometry following culture for 72 h. Results DHT increased the AR expression in the two breast cancer cell lines. AR pathway could inhibit proliferation of MCF-7 and MDA-MB-453 cells. There was no significant difference in absorbance values among three treatment groups at different time points (P ＞ 0. 05). Cell cycle analysis revealed that the proportion of cells at G1 phase was increased, and that at S phase decreased. Conclusion AR pathway may inhibit proliferation of ER-negative MDA-MB-453 breast cells as well as ER-positive MCF-7 cells, by suppressing the process of G1 to S phase progression.     

Dipyridamole inhibits PDGF-stimulated human peritoneal mesothelial cell proliferation

BACKGROUND: It has been proposed that proliferation of human peritoneal mesothelial cells (HPMCs) accompanied by collagen synthesis may contribute to the development of peritoneal fibrosis (PF) in patients of long-term continuous ambulatory peritoneal dialysis (CAPD). However, the precise molecular mechanism regulating HPMC proliferation has never been reported. Dipyridamole has been reported to have potential as an antiproliferative and antifibrotic agent. We investigated the mechanism and effect of dipyridamole in regulation of HPMC proliferation. METHODS: HPMCs were cultured from human omentum by an enzyme digestion METHOD: Cell proliferation was measured by the methyltetrazolium assay and intracellular cAMP was measured using an enzyme immunoassay kit. Cell-cycle distribution of HPMC was analyzed by flow cytometry. Extracellular signal-regulated protein kinase (p44/p42 ERK) activity and expressions of cell-cycle proteins (cyclin D(1), CDK4, pRB and p27(Kip1)) were determined by Western blotting. RESULTS: The addition of DP suppressed PDGF-stimulated HPMC proliferation by cell-cycle arrest at the G1 phase. The antimitogenic effect of dipyridamole was mediated through the cAMP pathway. PDGF (25 ng/mL) increased the ERK1/2 activity of HPMC within 15 minutes, which maximized at 30 minutes, and the pretreatment with dipyridamole (17 microg/mL) substantially reduced the ERK response to PDGF by approximately 78.5%. PDGF induced elevated protein levels of cyclin D(1), but the CDK4 protein level did not change. Dipyridamole and DBcAMP had no effect on the levels of cyclin D(1) and CDK4 in PDGF-stimulated HPMC. PDGF decreased p27(Kip1) and induced pRB phosphorylation of HPMC. In contrast, dipyridamole prevented PDGF-induced p27(Kip1) degradation and attenuated PDGF-stimulated pRB phosphorylation. CONCLUSION: Dipyridamole appears to inhibit PDGF-stimulated HPMC proliferation through attenuated ERK activity, preservation of p27(Kip1), and decreased pRB phosphorylation. Thus, dipyridamole may have therapeutic efficacy to prevent or alleviate PF.     

Melatonin and prostate cancer cell proliferation: interplay with castration,epidermal growth factor,and androgen sensitivity

BACKGROUND: Potential modulatory effects of melatonin on the proliferation of androgen-sensitive LNCaP and androgen-insensitive PC-3 and DU 145 prostate cancer cells were reported recently. In this study, we investigated the effects of combined melatonin and castration on LNCaP tumor growth in vivo, the interactions between melatonin and epidermal growth factor (EGF) on LNCaP cell proliferation, and melatonin actions on the proliferation of PC-3 and DU 145 cells. METHODS: Tumor development and growth in castrated nude mice inoculated with LNCaP cells or in intact animals inoculated with DU 145 cells, with or without daily melatonin treatment, were monitored by observation and caliper measurement. MT(1) receptor expression in native or transfected prostate cancer cell lines was examined by immunocytochemistry or 2-[(125)I]iodomelatonin binding. Cyclin D1 expression in LNCaP cells was assessed by Western blotting, and cell proliferation was measured by thymidine incorporation and/or cell count. RESULTS: Melatonin treatment was associated with further decreases in LNCaP tumor incidence and growth rate in castrated nude mice. Melatonin and 2-iodomelatonin (a melatonin receptor agonist) attenuated EGF-stimulated increases in LNCaP cell proliferation and cyclin D1 levels. Melatonin had no effect on the proliferation or growth of MT(1) receptor-expressing DU 145 cells, and of PC-3 cells in which MT(1) receptor protein was undetectable. The proliferation of transfected PC-3 cells expressing MT(1) receptor was unaffected by 2-iodomelatonin. CONCLUSION: Together with previous data, the present results indicate synergistic action of melatonin and castration in inhibiting the growth of androgen-sensitive LNCaP tumor. Androgen-sensitive prostate cancer cell proliferation may be modulated by opposite changes in cyclin D1 levels induced by activated MT(1) and EGF receptors. In androgen-insensitive prostate cancer cells, MT(1) receptor-mediated signal transduction may become defective not only through changes in membrane receptor protein expression and/or functions, but also by means of alterations in downstream postreceptor signaling events.     

尿酸活化ERK1/2信号通路刺激大鼠肾小球系膜细胞增殖

目的 探讨尿酸（UA）对大鼠肾小球系膜细胞（GMC）增殖的影响及其可能的机制。 方法 体外培养大鼠GMC,应用不同浓度的UA（50、100、300 μmol/L）刺激或应用细胞外信号调节激酶（ERK1/2）特异性抑制剂U0126（10 μmol/L）、NADPH氧化酶特异性抑制剂夹竹桃麻素（500 μmol/L）、线粒体复合体Ⅰ抑制剂鱼藤酮（10 μmol/L）预处理 30 min 后,再加入UA（300 μmol/L）。于实验终点收集细胞,应用3H-TdR掺入法、细胞计数及流式细胞术测定GMC增殖和细胞周期变化;应用实时定量PCR、Western印迹法检测细胞周期素cyclin D1和cyclin A2的表达及ERK1/2的磷酸化水平;应用荧光探针2,7-二氯二氢荧光素乙酰乙酸（DCFDA）检测细胞内活性氧（ROS）的变化。 结果 （1）与对照组相比,3H-TdR掺入法和细胞计数均显示,UA呈剂量依赖性促进GMC增殖,300 μmol/L UA刺激组其细胞数为对照组的1.5倍以上。（2）流式细胞术显示,UA呈剂量依赖性减少G1/G0期细胞数,增加S期细胞数,300 μmol/L UA刺激组其S期细胞数为对照组的2倍以上。（3）UA呈剂量依赖性促进系膜细胞周期蛋白cyclin D1和cyclin A2的表达。（4）UA呈剂量依赖性促进系膜细胞ERK1/2磷酸化且U0126能够抑制UA诱导的GMC增殖。细胞计数和3H-TdR掺入法分别显示,U0126的抑制率分别是UA 300 μmol/L刺激组的22%和31%（均P < 0.05）。（5）UA呈剂量依赖性促进ROS产生增加,夹竹桃麻素能够明显抑制UA诱导的ROS生成、ERK1/2的磷酸化和系膜细胞增殖（均P < 0.05）,而鱼藤酮对其无明显影响。 结论 UA可促进GMC增殖,其可能的机制为UA诱导NADPH 氧化酶来源的 ROS 产生增加,从而激活ERK1/2信号通路,引起周期蛋白表达增加,促进GMC增殖。     

Cyclin D1 and breast cancer

Cyclin D1 is one of the frequently overexpressed proteins and one of the commonly amplified genes in breast cancer. This article reviews the roles of cyclin D1 in cell-cycle regulation (normal and abnormal), mammary gland development and carcinogenesis and the relationship to oestrogen in breast tissues. It concludes by presenting the clinical, prognostic and therapeutic implications of our current knowledge of cyclin D1 in breast cancer.     

TRIM55对大鼠系膜细胞增殖的调控作用

目的系膜增殖性肾炎是世界范围内高发的肾小球疾病,其发病与系膜细胞异常增殖有关,但调控系膜细胞增殖的内在分子机制尚不明确。本研究旨在探索TRIM55对大鼠系膜细胞(RMCs)增殖的调控作用及机制。 方法向8周龄雄性SD大鼠尾静脉注射2.5 mg/kg抗Thy-1抗体建立抗Thy-1肾炎模型。PAS染色观察肾脏病理表现,qPCR检测大鼠肾小球TRIM55 mRNA表达量;利用质粒及siRNA转染分别得到TRIM55过表达及低表达的RMCs,利用流式细胞仪检测其细胞周期;Western印迹检测p27及Cyclin D1的蛋白表达量。 结果TRIM55在抗Thy-1肾炎模型系膜增殖期高表达(P<0.01,T＝3.625)。体外RMCs中,TRIM55过表达可促进RMCs细胞周期由G1期向G2/S期转化,诱导系膜细胞增殖(P<0.01,T＝13.1);TRIM55低表达可引起RMCs的G1期阻滞,抑制RMCs增殖(P<0.01,T＝5.31)。此外,TRIM55过表达可下调p27表达、上调Cyclin D1;反之,TRIM55低表达可上调p27表达、下调Cyclin D1。 结论TRIM55可通过调节p27及Cyclin D1表达水平影响细胞由G1期到G2/S期的转化,进而调控RMCs的增殖。