PNRC在不同发育时期大鼠组织中的表达及其在人乳腺癌和相应癌旁组织中的表达差异

目的：探索核受体辅活化子（coactivator）PNRC（proline-rich Nuclear receptor coregulatory protein）在不同发育时期正常大鼠各种组织中的表达与其生理作用的关系;PNRC在人乳腺癌和相应癌旁组织中的表达与乳腺癌的关系.方法：以β-actin基因为参照,应用半定量RT-PCR和southern印迹技术,检测不同发育时期正常大鼠各种组织中PNRC的表达水平及其在人乳腺癌和癌旁组织中的表达.结果：PNRC在正常大鼠和胎鼠相应组织中广泛表达且无组织差异性（P＞0.05）;在成年大鼠各种组织中的表达明显高于胎鼠相应组织（P＜0.01）.PNRC在人乳腺癌中的表达水平明显低于相应癌旁组织（P＜0.01）.结论：PNRC是一种必需的、广泛表达的转录调节因子,它可能参与了机体多种正常组织的发育过程;在人乳腺癌和癌旁组织中的表达差异与它在乳腺癌发生中的潜在作用密切相关.     

内质网应激对乳腺癌MCF-7细胞CCL5表达的影响

目的探讨内质网应激水平与人乳腺癌MCF-7细胞CCL5表达之间的关系,明确CCL5与人乳腺癌MCF-7细胞增殖侵袭转移能力之间的关系。方法使用内质网应激诱导剂(Tuniamycin）和内质网应激抑制剂(4-PBA）分别处理人乳腺癌MCF-7细胞,24小时后提取细胞总蛋白。Western blot检测MCF-7细胞内质网应激水平及CCL5表达情况。MTT比色法,Transwell小室法检测细胞增殖和侵袭转移能力。结果内质网应激诱导剂处理组细胞内质网应激处于高水平,同时CCL5表达量增高;抑制剂组细胞内质网应激水平低,CCL5表达量也随之降低。并且诱导剂组肿瘤细胞增殖较抑制剂组活跃。升高的CCL5部分分泌到培养液中,影响细胞侵袭转移能力。结论内质网应激能够诱导人乳腺癌MCF-7细胞CCL5的表达;内源性CCL5能促进MCF-7细胞的增殖;外源性CCL5能促进MCF-7细胞侵袭转移。     

紫杉醇脂质体对乳腺癌MCF-7细胞生长抑制作用的机制

目的 分析紫杉醇脂质体对乳腺癌MCF-7细胞的生长抑制作用,观察药物作用的时间浓度关系并探究其作用机制.方法 应用不同剂量紫杉醇脂质体对MCF-7细胞进行处理,通过MTT法检测细胞存活率,倒置显微镜观察细胞形态,流式细胞分析技术检测细胞凋亡情况及其对细胞周期的影响,Western blot法检测bcl-2蛋白表达情况.结果 紫杉醇脂质体对MCF-7细胞有明显的剂量时间依赖效应.紫杉醇与紫杉醇脂质体均可将MCF-7细胞阻滞在G2/M期,且随药物浓度增加,凋亡细胞所占比例逐渐增加.通过Western blot法检测发现,紫杉醇脂质体作用后MCF-7细胞bcl-2蛋白表达明显下降,bax蛋白表达明显上调,bax/bcl-2比例明显上调.结论 与紫杉醇相比,紫杉醇以脂质体对人乳腺癌MCF 7细胞具有同样的抑制作用,其促凋亡机制之一为调控bcl-2及bax蛋白表达.     

siRNA沉默DNMT1对人乳腺癌细胞MCF-7生长的影响

目的 靶向人DNMT1（DNA methyltransferase 1, DNMT1）构建RNA干扰载体,研究其对乳腺癌细胞周期、增殖及凋亡的影响。方法 靶向DNMT1基因设计三条短发夹状RNA(short hairpin RNA, shRNA) 的寡核苷酸片段,构建重组体pGCsi-DNMT1,转染至乳腺癌细胞株MCF-7,定量PCR 法检测DNMT1 mRNA表达水平;流式细胞技术分析细胞周期的变化;MTT 法检测细胞生长情况;Annexin V/PI双染法观察细胞凋亡情况;定量PCR 法分析沉默DNMT1基因后对RASSF1A、p16、p21、p27及ERβ基因表达的影响。结果 在构建的靶向DNMT1的shRNA重组质粒pGCsi-DNMT1中,成功筛选到pGCsi-T3能显著下调DNMT1的表达。实时定量PCR检测结果证实重组质粒pGCsi-DNMT1对乳腺癌MCF-7细胞中DNMT1基因的转录有明显的抑制作用。MCF-7细胞转染后,pGCsi-DNMT1可明显抑制乳腺癌MCF-7细胞的增殖;大量细胞发生凋亡;细胞周期分析可见S期明显减少,而G₁/G₀期细胞显著增加;定量PCR检测到乳腺癌细胞中RASSF1A、p16、p21及ERβ基因mRNA表达水平明显升高,而p27基因表达水平未见明显变化。结论 重组质粒pGCsi DNMT1能特异有效地抑制MCF-7细胞内DNMT1的表达、 抑制细胞增殖、促进细胞凋亡,并可通过抑制DNMT1的活性来解除相关基因启动子区的高度甲基化状态,从而促进肿瘤相关基因的表达,提示DNMT1可作为乳腺癌基因治疗的新靶标。     

补骨脂素对乳腺癌MCF-7细胞基因表达谱的影响

 目的 观察中草药补骨脂分离提取的有效成分—补骨脂素对人乳腺癌MCF 7(ER阳性)细胞基因表达谱的影响。 方法 用补骨脂素对人乳腺癌细胞株MCF-7(ER阳性)进行体外生长抑制实验,将经过补骨脂素作用的实验组细胞与对照组细胞株分别提取总RNA,用22K人类基因表达谱芯片检测基因表达情况,分析补骨脂素作用后MCF-7细胞出现的差异表达基因。 结果 经补骨脂素作用的实验组细胞样本与对照组细胞样本比较, 呈现差异表达的基因有1 053个(4.78%)。其中表达上调的有456个(43.3%, Radio>2.0),表达下调的有657个(56.7%,Radio<0.5)。差异表达明显的基因主要与细胞凋亡、细胞周期调控、核酸转译调节、转译因子活性、核苷酸转移酶活性、血管收缩调节等有关。 结论 补骨脂素对人乳腺癌MCF-7细胞抑制作用的分子机制是通过对细胞凋亡、细胞周期、核酸转译等多种基因的调控而起作用的。     

米托蒽醌对人乳腺癌细胞MCF-7增殖的影响

目的:观察米托蒽醌对人乳腺癌细胞MCF-7的影响.方法: 台盼蓝拒染观察药物对细胞的不良反应,流式细胞术观察药物对细胞周期的影响.结果: 米托蒽醌对MCF-7细胞具有明显的增殖抑制作用,呈一定的时效性;药物作用MCF-7 48h后能引起其细胞周期发生变化.结论: 米托蒽醌能明显地抑制MCF-7细胞的增殖,并能引起其细胞周期变化.     

米托蒽醌对人乳腺癌细胞MCF-7增殖的影响

目的：观察米托蒽醌对人乳腺癌细胞MCF-7的影响。方法：台盼蓝拒染观察药物对细胞的不良反应,流式细胞术观察药物对细胞周期的影响。结果：米托蒽醌对MCF-7细胞具有明显的增殖抑制作用,呈一定的时效性;药物作用MCF-748h后能引起其细胞周期发生变化。结论：米托葸醌能明显地抑制MCF-7细胞的增殖,并能引起其细胞周期变化。     

毛蚶抗癌蛋白对人乳腺癌细胞MCF-7的生长抑制作用

目的:从毛蚶软体部位中分离出具有一定抗癌活性的毛蚶蛋白组分,研究其对人乳腺癌细胞MCF-7的生长抑制作用。方法:以新鲜毛蚶软体部分为原材料,分离获得毛蚶抗癌蛋白组分(命名为NS);倒置相差显微镜观察不同浓度(50、100、200、400μg/mL)NS组分对MCF-7癌细胞生长的影响,将200μg/mL NS组分作用MCF-7细胞不同时间(12、24、48 h)后,Giemsa染色观察癌细胞及核形态的变化,流式细胞术检测NS组分对MCF-7细胞周期及细胞凋亡的影响,Western blot检测细胞周期及细胞凋亡相关蛋白的表达。结果:与阴性对照组比较,各浓度NS组分对人乳腺癌MCF-7细胞生长均具有明显的抑制作用(P<0.05或P<0.01);倒置相差显微镜下可见200μg/mL NS组分作用MCF-7细胞12 h后,部分细胞收缩变圆、脱落,24、48 h后细胞脱落现象更加明显;Giemsa染色发现,NS组分处理12 h后细胞出现有丝分裂相增加,继而出现多核或核碎裂等现象;流式细胞术检测发现NS组分主要引起细胞G2-M期阻滞;NS处理组凋亡率均较对照组明显升高(P均<0.01),且随着药物作用时间的延长,细胞凋亡比例逐渐增加。Western blot检测发现NS组分作用MCF-7细胞后p53及procaspase-3蛋白表达量均上调。结论:毛蚶抗癌蛋白NS组分体外对人乳腺癌MCF-7细胞的细胞毒作用主要通过引起细胞发生G2-M期阻滞、诱导细胞凋亡实现的,期间伴随p53及procaspase-3蛋白表达上调。     

靶向沉默NOB1基因对乳腺癌细胞MCF-7生长和周期分布的影响

背景与目的:NOB1(NIN1/RPN12 binding protein 1 homolog)是2005年新克隆的一个基因,属于RNA结合蛋白,该类蛋白的功能与恶性肿瘤的发生密切相关。本研究旨在观察利用慢病毒介导的RNAi沉默NOB1基因对人乳腺癌MCF-7细胞增殖及细胞周期的影响。方法:包装表达NOB1短发夹RNA(shRNA)的慢病毒,感染MCF-7细胞,实时定量PCR和蛋白质印迹法(Western blot)验证NOB1的抑制效率;MTT和克隆形成实验检测NOB1对细胞增殖能力的影响;流式细胞术检测细胞周期的变化。结果:NOB1-shRNA慢病毒感染3 d后,可显著下调MCF-7细胞NOB1 mRNA和蛋白的表达水平,显著抑制细胞增殖和体外成瘤能力,并导致细胞周期分布紊乱,G0/G1期及G2/M期细胞增加,S期细胞减少。结论:NOB1基因通过调节细胞周期分布促进乳腺癌细胞恶性增殖,可能是乳腺癌基因治疗的分子靶点。     

Effect of growth on the estrogen receptor levels in MCF-7 cells

MCF-7 cells have been shown to contain estrogen receptor in several cell fractions following homogenization: nuclei, microsomes, and cytosol. The amount of 17 beta-estradiol-binding capacity found in each cellular compartment depended on the inclusion of detergent in homogenization buffers and on the use of 0.25 M sucrose in the nuclear washes. 17 beta-Estradiol receptor (E2R) associated with nuclei (whole nuclei exchange assay, 0.6 M KCl soluble, and that found on membranes sheared from crude nuclear pellets by centrifugation in 0.25 M sucrose buffer) displayed a dissociation constant (Kd) of 0.77 +/- 0.01 (S.D.) nM (n = 7). KdS of the cytoplasmic (microsomes and soluble) receptors were determined to be 0.33 +/- 0.10 nM (n = 9). Exchangeable ligand on partially purified nuclei assumed its highest level in MCF-7 cells during logarithmic growth in serum-containing media (0.8 pmol/micrograms DNA) but declined after the culture reached confluence (0.2 pmol/micrograms DNA). Seventy-five % of the nuclear E2R declined linearly after feeding MCF-7 cells in logarithmic growth phase an estrogen- and serum-free medium (t1/2 3.5 days). Another class of salt-extractable nuclear receptor (0.2 pmol/micrograms DNA) persisted in postconfluent cultures whether fed estrogen (serum-containing media) or not (serum-free media). This residual binding capacity remained in nuclei of MCF-7 cells for an extended period of time. MCF-7 cells demonstrated functionality of E2R throughout their growth phases as evidenced by the replenishment of cytosolic E2R and the induction of progesterone receptor when given 17 beta-estradiol.     

Matrilysin over-expression in MCF-7 cells enhances cellular invasiveness and pro-gelatinase activation

Matrilysin (MMP-7) is over-expressed in various cancers and is thought to play important roles in tumor invasion and metastasis. However, the function of MMP-7 in breast cancer remains unclear. We therefore examined the expression of the MMP-7 gene in breast cancer (MCF-7) cells and the effect of its over-expression on cellular invasion. We transfected human MMP-7 into MCF-7 cells and selected clones that stably over-expressed the MMP-7 gene. The in vitro invasiveness of MCF-7 cells was quantified by use of the Matrigel invasion assay. Expression of MMP-7 mRNA was analyzed by quantitative RT-PCR. MMP secretion and activation were detected by gelatin zymography. We found that MMP-7-expressing clones had significantly increased invasion (P < 0.001), with increased MMP-7 expression and gelatinase activation as compared to the vector controls. We conclude that MMP-7 over-expression correlates with breast cancer in vitro invasiveness and that MMP-7 may promote invasion by increasing the secretion and activation of proMMP-2 and proMMP-9.     

弗林蛋白酶抑制剂对乳腺癌MCF-7细胞生长和迁移的影响

目的 探讨乳腺癌转移的机制,为深入研究乳腺癌发生、发展机制提供理论基础.方法 应用不同浓度的弗林蛋白酶（Furin）抑制剂α1-PDX处理乳腺癌MCF-7细胞.用四甲基偶氮唑蓝（MTT）和克隆形成实验检测Furin抑制剂对MCF-7细胞增殖和克隆形成的影响.单层细胞迁移实验和Transwell实验检测MCF-7细胞迁移和浸润能力.Hoechst 33342/PI双染法检测细胞凋亡.酶联免疫吸附法检测细胞培养液中基质金属蛋白酶2（MMP-2）和MMP-9蛋白水平.Western blot检测细胞迁移相关蛋白MT1-MMP、血管内皮生长因子（VEGF）-C和VEGF-D水平.结果 不同浓度α1-PDX作用MCF-7细胞48 h以上时,细胞的生长受到抑制,集落形成降低,细胞凋亡率升高.但在低浓度情况下对细胞迁移和侵袭起抑制作用.α1-PDX降低了细胞内MT1-MMP、VEGF-C、VEGF-D的表达,同时细胞培养液上清中MMP-2和MMP-9浓度也低于对照组.结论 Furin抑制剂通过抑制乳腺癌MCF-7细胞MMP及VEGF表达抑制肿瘤的迁移能力.     

In vitro modulation of MCF-7 breast cancer cell growth by myoepithelial cells

Growth and differentiation of MCF-7 breast adenocarcinoma cells were studied in mixed cultures of MCF-7 cells and PA 16/23 myoepithelial cells and in isolated cultures of MCF-7 cells grown in the absence and presence of conditioned medium of PA 16/23 cells. In the cocultures, the MCF-7 cells grew in smaller aggregates and showed a more differentiated phenotype than in the isolated cultures. The conditioned medium of PA 16/23 cells enhanced growth and reduced differentiation of the MCF-7 cells. Hence, a direct relationship between MCF-7 cells and PA 16/23 cells seems to play a leading role in influencing the behaviour of the former cells, thus overriding the opposite effects of soluble factors secreted by the PA 16/23 cells.     

PNRC多肽通过干扰核受体途径抑制BT474细胞的增殖

目的:运用核受体辅活化子PNRC的抗Ras PTD融合多肽(PTD-PARP)研究其在乳腺癌细胞BT474中的分布、对核受体信号传导途径的影响及其抗肿瘤细胞增殖活性.方法:用多肽合成仪合成PTD-PARP、单独的PARP和PTD多肽以及FTFC标记的PTD-PARP、PARP.HPLC分析和纯化后,荧光显微镜结合流式细胞仪检测PTD-PARP、PARP在BT474细胞中的分布.虫荧光素酶报告基因检测PTD-PARP对野生型PNRC辅活化ER反式激活功能的影响.MTS、软琼脂克隆形成实验检测PTD-PARP对BT474细胞的抗增殖活性.结果:PTD-PARP能进入BT474细胞,抑制野生型PNRC辅活化ER的反式激活功能并抑制BT474细胞的增殖.结论:PNRC抗RasP DT融合多肽可通过干扰核受体途径抑制乳腺癌细胞的增殖.     

Invasive and metastatic properties of MCF-7 cells and rasH-transfected MCF-7 cell lines.

In vitro invasion and in vivo metastasis assays were performed with a panel of MCF-7 cells transfected with isogenic constructs of mutated rasH genes. Both increased levels of rasH expression and rasH oncogene activation increased activity of derivative cell lines in in vitro invasion assays. In vivo formation of spontaneous metastases was assessed after intradermal inoculation of MCF-7 cells in the vicinity of the mammary fat pads of ovariectomized nude mice. No metastases were seen in the absence of estradiol treatment of the mice. With estradiol supplementation of the mice both the rasH-transfected and control transfected cell lines gave a higher incidence of metastases than parental MCF-7 cells. Prolonged treatment of mice with exogenous estradiol (60 days vs. 21 days) resulted in more frequent metastases to liver and lung at the end of the 90-day observation period. In contrast to activated rasH-gene enhancement of metastatic capacity of rodent fibroblast and epithelial cell lines, there was no correlation of rasH expression with in vivo metastatic capacity of a human mammary carcinoma cell line.     

Differential regulation of growth and invasiveness of MCF-7 breast cancer cells by antiestrogens

Estrogen increases the ability of the estrogen-dependent MCF-7 human breast cancer cell line to both proliferate and invade through an artificial basement membrane. In studying the response of MCF-7 cells to various antiestrogens, we found that 4-hydroxytamoxifen and tamoxifen inhibited cell proliferation but increased their invasiveness. In contrast, the structurally unrelated benzothiophene antiestrogens, LY117018 and LY156758, were potent antiproliferative agents which did not stimulate invasiveness. The differential effects of these antiestrogenic agents on invasion correlated with changes in production of collagenase IV, while no significant change was seen in the chemotactic activity of the cells. Invasiveness was increased by 17 beta-estradiol or 4-hydroxytamoxifen after a few hours of treatment and was rapidly lost when 17 beta-estradiol was withdrawn. Stimulation of invasiveness with 17 beta-estradiol was blocked by the antiestrogen, LY117018. Cells from the MDA-MB-231 line which lacks estrogen receptors were not affected by estrogen or antiestrogen in terms of proliferation or invasion. These studies indicate that the invasiveness of MCF-7 cells is regulated by antiestrogens through the estrogen receptor and may be mediated by collagenase IV activity. Antiestrogens which reduce both the proliferation and invasiveness of these cells may be interesting new candidates for clinical application.     

曲格列酮对人乳腺癌MDA-MB-231和MCF-7细胞株增殖影响的研究

目的:探讨过氧化物酶体增殖物激活受体γ(PPARγ)激动剂曲格列酮(TRG)对体外培养雌激素受体(ER)阴性乳腺癌MDA-MB-231细胞及ER+乳腺癌MCF-7细胞增殖的影响。方法:以不同浓度的TRG(0～50μmol/mL)分别处理体外培养的MDA-MB-231细胞和MCF-7细胞48h,MTT法检测细胞存活率;流式细胞术(FCM)检测细胞周期分布;蛋白质印迹法检测PPARγ和ER-α的表达。结果:MTT结果示TRG浓度≥5μmol/mL时MDA-MB-231细胞(P值分别为0.006、0.006、0.000和0.000)和MCF-7细胞(P值分别为0.007、0.006、0.004和0.001)的存活率降低。FCM检测结果表明,经TRG作用后肿瘤细胞主要集中在G1期。以上2种检测结果显示,MDA-MB-231细胞对TRG的敏感程度较MCF-7细胞高;蛋白质印迹法检测结果表明,TRG处理后的MDA-MB-231细胞中PPARγ蛋白的表达水平随药物浓度增加逐渐升高(相对强度分别为2.045、2.600和3.038),MCF-7细胞的ER-α表达水平则随药物浓度增加逐渐降低(相对强度分别为1.164、1.130和0.680)。结论:TRG对ER+和ER-乳腺癌细胞均有抑制作用,且ER-乳腺癌细胞对TRG更敏感。     

反义c-myc对MCF-7细胞端粒酶活性的影响

目的研究脂质体LipfectAMINE^TM（LR）介导的c-myc反义寡核苷酸（ASODN）对MCF-7细胞端粒酶活性的影响及其机制。方法端粒重复序列扩增聚丙烯酰胺凝胶电泳（TRAP-PAGE）及端粒重复序列扩增酶联免疫吸附测定（TRAP-ELISA）定量检测不同时间段MCF-7细胞的端粒酶活性,流式细胞仪检测c-myc蛋白表达。结果TRAP-PAGE结果显示,c-myc ASODN对MCF-7细胞的作用,随时间的递增（24h、48h、72h）,端粒酶梯度条带明显地减少,端粒酶活性明显降低。TRAP-ELISA显示,2．5μmol／L的c-myc ASODN作用MCF-7细胞48h吸光度A值降为0．486±0．041,作用72hA值降为0．263±0．034,与未经任何处理的MCF-7细胞A值0．684±0．031相比,端粒酶活性明显受到抑制,分别与正义寡核苷酸组、空白对照组相比差异有统计学意义（P〈0．05）。未经任何处理的MCF-7细胞c-myc蛋白阳性率为（99．684±2．937）％,经c-myc ASODN作用48h c-myc蛋白阳性率低至（61．295±2．825）％,c-myc ASODN作用72h c-myc蛋白阳性率低至（29．482±2．726）％。而c-mye正义寡核苷酸（SODN）无上述作用。结论c-myc ASODN能有效降低MCF-7细胞端粒酶活性和c-myc蛋白表达。