两株子宫内膜癌细胞中mTOR信号通路激活的研究

目的：探讨PTEN缺失Ishikawa和PTEN完整HEC-1A细胞中mTOR信号通路的激活。方法：激光共聚焦显微镜检测Ishikawa和HEC-1A细胞中PTEN蛋白表达;RT-PCR和western blot法从mRNA和蛋白水平分别检测mTOR mRNA和下游磷酸话靶蛋白S6K1和4E-BP1表达。结果：Ishikawa细胞中PTEN蛋白表达缺失,HEC-1A细胞PTEN蛋白表达显著 (P<0.01);两株细胞中均存在mTOR mRNA表达,而Ishikawa细胞中表达水平要高于HEC-1A细胞(P<0.05);Ishikawa细胞中S6K1和4E-BP1蛋白的磷酸化要明显高于HEC-1A细胞(P<0.05)。 结论：2种子宫内膜癌细胞中均存在mTOR信号通路的激活,这种激活水平与PTEN相关,PTEN缺失Ishikawa细胞的激活水平较高。 【关键词】子宫内膜癌;雷帕霉素靶蛋白;PTEN;Ishikawa细胞;HEC-1A细胞     

胃肠间质瘤危险性分级与哺乳动物雷帕霉素靶蛋白和磷酸化哺乳 动物雷帕霉素靶蛋白表达的关系

目的研究胃肠间质瘤危险性分级与磷脂酰肌醇3激酶(PI3K)/蛋白激酶(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号传导通路中mTOR和磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)表达的关系。方法用免疫组织化学法检测40例胃肠间质瘤中PI3K/Akt/mTOR信号传导通路相关蛋白mTOR和p-mTOR的阳性表达率,分析其在不同危险程度组之间的差异。结果 mTOR、p-mTOR蛋白在部分胃肠间质瘤呈阳性表达,定位于细胞质。mTOR、p-mTOR在极低和低度危险组的阳性表达率均低于中高度危险组,差异均有统计学意义(P<0.05)。结论胃肠间质瘤PI3K/Akt/mTOR信号传导通路中mTOR和p-mTOR与其侵袭危险性有关,提示该信号传导通路在介导胃肠间质瘤发生、发展过程中起重要作用。     

哺乳动物雷帕霉素靶蛋白对肝癌细胞系Hep3B中侧群细胞的影响

目的探讨哺乳动物雷帕霉素靶蛋白(mTOR)分子对肝癌细胞系Hep3B细胞中侧群(SP)细胞的影响。方法雷帕霉素抑制mTOR,流式细胞术分析Hep3B细胞系中SP细胞的比例,软琼脂克隆形成实验检测Hep3B细胞中SP细胞和非侧群(non-SP)细胞成克隆能力。结果 Hep3B细胞中存在SP细胞,经过雷帕霉素处理后,SP比例明显降低。雷帕霉素能够明显降低SP细胞的软琼脂成克隆能力,对non-SP细胞的软琼脂成克隆能力影响不显著。结论 mTOR的活性对Hep3B细胞中SP细胞的维持起重要作用,抑制mTOR的活性使SP细胞比例明显减低,并能显著抑制SP细胞的软琼脂成克隆能力。     

Inactivation of mammalian target of rapamycin (mTOR) by rapamycin in a murine model of lipopolysaccharide-induced acute lung injury

Background  The mammalian target of rapamycin (mTOR) pathway, a key cellular signaling pathway associated with various cellular functions, has distinct roles in the inflammatory process. In this study, the mTOR inhibitor rapamycin (Rapa) was used to test whether inhibition of mTOR activation attenuates lipopolysaccharide (LPS)-induced acute lung injury (ALI) in a murine model.

Methods  Mice pretreated with Rapa or vehicle were given LPS intratracheally. Local cell numbers and inflammatory cytokines present in the bronchoalveolar lavage fluid (BAL), wet-to-dry weight ratio, histopathology of the lungs, and survival were evaluated.

Results  The phosphorylation of S6, a major downstream target of mTOR, had a 3-fold increase in lung tissue after LPS stimulation, but the increase was blocked by Rapa. Rapa reduced the levels of TNF-α (LPS vs. LPS + Rapa, (1672.74±193.73) vs. (539.17±140.48) pg/ml, respectively; P <0.01) and IL-6 (LPS vs. LPS + Rapa: (7790.88±1170.54) vs. (1968.57±474.62) pg/ml, respectively; P <0.01) in the BAL fluid. However, Rapa had limited effects on the overall severity of ALI, as determined by the wet-to-dry weight ratio of the lungs, number of neutrophils in the BAL fluid, and changes in histopathology. In addition, Rapa failed to reduce mortality in the LPS-induced ALI model.

Conclusions  We confirmed that mTOR was activated during LPS-induced ALI and strongly inhibited by Rapa. Although Rapa reduced the levels of the mediators of inflammation, the overall severity and survival of the ALI murine model were unchanged.

     

Targeting the mammalian target of rapamycin pathway in osteosarcoma using combinative chemotherapy

Osteosarcoma is the most common primary malignant bone sarcoma,and occurs predominantly in children and young adults.The overall survival of patients with osteosarcoma remains low at approximately 60％.1 The prognosis of osteosarcoma patients is highly associated with response to chemotherapy.Although multiple chemotherapy regimens have improved the outcome of patients with osteosarcoma,resistance to current regimens has been reported in more than 30％ of patients,2highlighting the need for novel,targeted therapies.     

miR-27a低表达对子宫内膜癌HEC-1-A细胞增殖的影响

目的研究miR-27a低表达对子宫内膜癌HEC-1-A细胞增殖的影响。 方法构建miR-27a低表达HEC-1-A细胞株。细胞实验分为空白对照组(HEC-1-A不做任何处理)、NC-miR组(转染miR-27a空载体)及miR-27a inhibitor组(转染miR-27a抑制剂)。qRT-PCR法检测各组细胞miR-27a表达情况;MTT比色法和平板克隆技术检测各组细胞增殖能力。 结果成功构建miR-27a低表达HEC-1-A细胞株。miR-27a inhibitor组miR-27a水平及细胞增殖能力均低于NC-miR组及空白对照组(P＜0.05)。 结论miR-27a低表达抑制子宫内膜癌HEC-1-A细胞增殖能力。     

醋酸曲谱瑞林对人子宫内膜癌细胞株HEC-1-B的作用及对PCNA表达的影响

目的研究醋酸曲谱瑞林对人子宫内膜癌细胞株HEC-1-B的作用及其分子作用机制。方法以不同浓度醋酸曲谱瑞林（0，10^-9，10^-8，10^-7，10^-6，10^-5mol／L）体外作用于中分化人子宫内膜腺癌细胞HEC-1-B（0mol/L组作为对照组，其余各组作为实验组），用MTT法检测细胞抑制率；免疫细胞化学检测HEC-1-B细胞PCNA蛋白表达，并用病理图像分析软件进行半定量分析。RT—PCR法检测HEC-1-B细胞PCNAmRNA的表达。结果①当醋酸曲谱瑞林浓度为10^-9mol／g时，HEC-1-B细胞即受到抑制，抑制率为36．8％；随着浓度的增大，抑制率渐高，到浓度为10^-5mol／g时抑制率上升至57．4％。与对照组比较，实验组抑制率差异有统计学意义（P〈0．05）。②浓度从10^-9 - 10^-5mol／L的醋酸曲谱瑞林作用72h后，PC—NA蛋白和PCNAmRNA表达均有不同程度的下降，与对照组相比差异有统计学意义（P〈0．05）。结论醋酸曲谱瑞林在体外对HEC-1-B细胞可能有直接的抑制作用，且呈剂量依赖关系。醋酸曲谱瑞林抑制HEC-1-B细胞增殖的分子机制可能与下调PCNA蛋白表达有关。     

EC9706和Eca109细胞中雷帕霉素靶蛋白信号通路激活状态观察

目的:观察低分化的食管鳞癌细胞EC9706和高分化的食管鳞癌细胞Eca109中雷帕霉素靶蛋白(mTOR)信号转导通路的激活状态.方法:采用细胞免疫组织化学方法检测EC9706和Eca109细胞中mTOR及其下游靶分子p70S6K的表达和定位,并采用RT-PCR和Western blot方法分别从mRNA及蛋白水平检测此通路的活性.结果:在2种细胞中mTOR均有表达,且在EC9706细胞中的表达水平高于Eca109(P＜0.05);EC9706细胞中mTOR下游靶点的磷酸化水平高于Eca109细胞(P＜0.05).结论:在2种食管鳞癌细胞中,mTOR信号通路均特异性激活;但激活状态与细胞的分化程度有关,细胞分化程度低者通路的激活水平较高.     

Mammalian target of rapamycin inhibitor abrogates abnormal osteoclastogenesis in neurofibromatosis type 1

Background Neurofibromatosis type 1 (NF1) is the most common genetic syndrome predisposing patients to various tumors due to dysregulation of the Ras signaling pathway.Recent research has shown NF1 pat...     

n-6／n-3多不饱和脂肪酸对子宫内膜癌Ishikawa细胞增殖的影响

目的探讨n-6／n-3多不饱和脂肪酸（PUFAs）对子宫内膜癌Ishikawa细胞增殖及细胞中哺乳动物雷帕霉素靶蛋白（mammMiantargetofrapamycin,mTOR）、血管内皮生长因子（vascularendothelialgrowthfactor,VEGF）表达的影响。方法用不同比例的n-6／n-3PUFAs处理细胞,MTr法检测细胞增殖能力变化;RT—PCR检测细胞mTOR、VEGF的mRNA表达情况。结果单纯n-6PUFAs和10：1n-6／n-3PUFAs能够明显促进Ishikawa细胞增殖,mTOR、VEGF的mRNA表达增加（P〈0．05）;单纯n-3PUFAs和1：1n-6／n-3PUFAs则显著抑制细胞增殖,同时使VEGF的mRNA表达下降（P〈0．05）;mTOR与VEGFmRNA的表达呈正相关（r=0．94,P〈0．05）。结论n-6／n-3PUFAs可能通过影响mTOR信号调节VEGF表达,从而对Ishikawa细胞增殖产生影响。     

WYE-354调控小鼠同种异体皮肤移植抗排斥反应中的mTOR信号传导通路

目的探讨mTOR激酶ATP竞争抑制剂WYE-354对小鼠同种异体皮肤移植抗排斥反应的作用及机制。方法以C57BL/6为受者,BALB/c为供者建立同种异体皮肤移植模型,C57BL/6小鼠行同种同体皮肤移植为自体移植组。移植-2d给药,共21d,阳性对照组为同种异体皮肤移植,雷帕霉素组口服给药0.2ml、剂量1mg/(kg.d),WYE-354药物组腹腔注射0.2ml:低剂量1mg/(kg.d)、中剂量1.5mg/(kg.d)、高剂量50mg/(kg.d)。术后3、7、11、21d时,应用免疫印记法留样检测每组脾细胞4E-BP1磷酸化水平,11d行皮肤移植物组织病理染色。结果阳性对照组皮肤移植物存活时间明显少于雷帕霉素组、WYE-354低、中、高剂量组,差异有统计学意义(P<0.01);雷帕霉素组皮肤移植物存活时间明显少于WYE-354中、高剂量组,差异有统计学意义(P<0.01)。雷帕霉素组及WYE-354低剂量组皮肤移植物局部淋巴细胞浸润较阳性对照组减少,WYE-354中、高剂量组皮肤移植物局部淋巴细胞浸润较阳性对照组、雷帕霉素组、WYE-354低剂量组均减少。雷帕霉素组、WYE-354低、中、高剂量组均可下调脾细胞中4E-BP1蛋白的磷酸化水平,以WYE-354中、高剂量组作用最为明显。结论 WYE-354可以抑制mTOR信号转导通路相关蛋白磷酸化水平的调控,并阻滞其细胞周期,减少淋巴细胞向皮肤移植物浸润、显著延长小鼠皮肤移植物存活时间。     

小檗碱体外抗子宫内膜癌HEC-1A细胞的作用研究

目的研究小檗碱体外对非激素依赖型人子宫内膜癌HEC-1A细胞增殖、迁移、周期、凋亡的影响。方法 MTT法检测小檗碱对人子宫内膜癌HEC-1A[中分化腺癌,雌激素受体(ER)弱阳性,孕激素受体(PR)阴性]细胞株的抑制作用;倒置显微镜观察细胞形态变化;将实验组分成50、25、12.5 mg/L高、中、低剂量组。Transwell法测定细胞体外迁移能力,流式细胞仪观察细胞周期分布,Annexin V-FITC/PI双染检测HEC-1A细胞凋亡。结果小檗碱可显著抑制HEC-1A细胞生长,在100、50、25、12.5、6.25 mg/L浓度范围内呈剂量依赖性(P0.05)。小檗碱能明显改变细胞形态,小檗碱作用于子宫内膜癌HEC-1A细胞后,倒置显微镜下可见部分细胞体积改变,细胞内出现空泡,结构破坏,裂解成碎片,贴壁细胞减少,并散在排列。抑制子宫内膜癌HEC-1A细胞体外迁移的能力,高剂量组效果更佳,迁移细胞数明显减少。小檗碱低剂量组可以将HEC-1A细胞阻滞于G0/G1期。小檗碱作用24 h能提高HEC-1A细胞总凋亡率、早期凋亡率及晚期凋亡率,早期凋亡率更明显。结论小檗碱能够抑制非激素依赖型子宫内膜癌HEC-1A细胞株的增殖、迁移,并且促进HEC-1A细胞凋亡,低剂量小檗碱能将细胞阻滞在G0/G1期。     

Chronic intermittent hypoxia increases B cell mass and activates the mammalian target of rapamycin/hypoxia inducible factor 1/vascular endothelial growth factor A pathway in mice pancreatic islet

Background Growing evidence from population and clinic based studies showed that obstructive sleep apnea (OSA) and its characterizing chronic intermittent hypoxia (IH) were independently associated wit...     

干扰组蛋白去甲基化酶基因表达对Ishikawa细胞增殖凋亡的影响

目的研究SiRNA干扰组蛋白去甲基化酶（JARID1A）基因表达对人子宫内膜癌Ishikawa细胞增殖凋亡的影响。方法 Ishi-kawa细胞转染JARID1A特异性的SiRNA;将Ishikawa细胞分成sicon组、sicon＋e2组、siJARID1A组和siJARID1A＋e2组,RT-PCR检测四组细胞孕激素受体（progesterone receptor,PR）mRNA表达,Western Blot检测四组细胞PR蛋白,MTT法观察细胞增殖,流式细胞术检测细胞周期。结果干扰JARID1A能显著上调PR表达,E2可进一步上调PR表达（P〈0.01）;E2能促进Ishikawa细胞增殖,转染SiRNA 48 h后细胞增殖受到抑制;转染SiRNA抑制细胞G1、G2/M期（P〈0.05）。结论 JARID1ASiRNA能有效抑制Ishikawa细胞增殖。     

Effect of artesunate on human endometrial carcinoma HEC-1B cells

Objective： To observe the effect of the artesunate （ART） on cellular proliferation in vitro, to search for the possible anti-tumor mechanism of ART on endometrial carcinoma at the molecular level and to provide the experimental and theoretical foundations for the clinical applications of ART. Methods： The cell proliferation was observed by microscope; MTT was used to examine the effects of ART on proliferation of HEC-1B cells, and flow cytometric analysis was used to detect cell cycle and apoptosis. The human endometrial carcinoma HEC-1B cells were conventionally cultured; ART was administered with a concentration of 40 μg/ml before the total RNA were extracted, mRNA expression of Survivin, Caspase-3, N-Cadherin, E-Cadherin, Fibronectinl and Cox-2 were detected using RT-PCR. Results： ART reduced proliferation in human endometrial carcinoma cell line HEC-1B in a dose- and time-dependent effect. The cells of G0/G1 stage were significantly increased （P〈0.05）, but the cells of G2/M stages were significantly decreased （P〈0.05）, so it has shown that the cell cycle was probably blocked in G0/G1 stage. After intervention with ART at 20 and 80 μg/ml for 48 h, cellular apoptosis rate respectively was （36.42±0.77）% and （11.77±0.58）%, and the difference was statistically significant compared with the control （[6.64±0.191%, P〈0.01）. The expression of Cox-2 mRNA in the ART group was lower than those of control group, yet the expression of Caspase-3 and E-Cadherin mRNA in the ART group was higher than those of control group. Conclusion： ART can inhibit HEC-1B cell growth and proliferation in a dose- and time-dependent manner. Furthermore, ART can induce apoptosis in a dose-dependent manner. ART is able to downregulate Cox-2 mRNA expression and to upregulate E-Cadherin and Caspase-3 mRNA expression. So we can conclude that ART could induce the endometrial carcinoma HEC-1B cell apoptosis and inhibit tumor cell proliferation.     

子宫内膜癌组织中PTEN的表达及其与mTOR磷酸化的相关性研究

目的探讨子宫内膜癌组织中磷酸酶与张力蛋白同源物（phosphataseandtensinhomolog,PTEN）的表达及其对哺乳动物雷帕霉素靶蛋白（mammaliantargetofrapamycin,mTOR）磷酸化的影响。方法利用SP免疫组化法对19例正常子宫内膜组织、28例不典型增生子宫内膜组织、53例子宫内膜癌组织中PTEN、磷酸化mTOR（P—mTOR）、真核细胞翻译起始因子4E（eukaryoticinitiationfactor4E,eIF-4E）的表达情况进行检测。结果PTEN在正常子宫内膜组织、不典型增生子宫内膜组织、子宫内膜癌组织中阳性程度逐渐降低（P〈0．05）,P—mTOR和eIF-4E在子宫内膜癌组织中阳性程度最高,在不典型增生子宫内膜组织和正常子宫内膜组织中阳性程度逐渐降低（P〈0．05）;PTEN与P—roTOR在子宫内膜癌组织中的表达呈负相关（r=-0．347,P=0．011）,P—mTOR和elF-4E在子宫内膜癌组织中的表达呈正相关（r：0．806,P=0．000）。结论子宫内膜癌组织中存在mTOR通路蛋白异常高表达,PTEN的表达减少与mTOR的磷酸化及其下游因子eIF-4E的活化有关。     

联合阻断哺乳动物雷帕霉素靶蛋白信号通路及其旁路激活通路对胶质瘤细胞生长的抑制机制

目的 筛选敲减哺乳动物雷帕霉素靶蛋白(mTOR)基因后胶质瘤细胞表达上调的基因及信号通路,探讨联合阻断mTOR信号通路及其旁路激活通路抑制胶质瘤细胞生长的有效性.方法 选取5种人胶质瘤细胞系(U87、U251、U373、T98、LN229),采用蛋白质印迹法验证mTOR蛋白表达.构建靶向mTOR基因的短发夹RNA稳定转...     

食管鳞状细胞癌组织中Pin1和mTOR蛋白的表达

目的:了解食管鳞状细胞癌组织中肽基脯氨酰顺反异构酶(Pin1)与哺乳动物雷帕霉素靶蛋白(mTOR)的表达情况。方法:采用免疫组化SP法检测食管鳞状细胞癌组织和癌旁正常食管黏膜组织中Pin1和mTOR的表达,分析食管鳞状细胞癌组织中二者的表达与临床病理特征的关系以及二者的关联性。结果:食管鳞状细胞癌组织中Pin1蛋白、mTOR蛋白的阳性表达率(60.0%、62.5%)均高于癌旁正常食管黏膜组织(32.5%、27.5%),差异有统计学意义(χ2Pin1=4.762,P=0.027;χ2mTOR=6.036,P=0.014)。Pin1蛋白阳性表达与淋巴结转移有关(P<0.05),与分化程度、年龄、性别及TNM分期等病理学参数无关;mTOR与其他病理参数无关。食管鳞状细胞癌组织中Pin1与mTOR的蛋白表达呈正关联(rP=0.389,P=0.008)。结论:Pin1和mTOR在食管鳞状细胞癌中的表达明显增高,Pin1可能与mTOR相互作用调节细胞周期,从而在食管鳞状细胞癌的发生过程中起着重要作用。     

雷帕霉素联合顺铂对卵巢癌SKOV3细胞株凋亡和周期的影响

目的:探讨雷帕霉素单独及联合顺铂对卵巢癌SKOV3细胞凋亡、周期的影响及可能的分子机制。方法:体外培养卵巢癌SKOV3细胞,流式细胞术检测雷帕霉素单独及联合顺铂对细胞凋亡及周期分布的影响;实时RT-PCR检测对哺乳动物雷帕霉素靶蛋白(mTOR)mRNA表达的影响。结果:雷帕霉素单独作用可抑制细胞生长增殖,引起G1期阻滞,S期及G2-M期细胞明显减少,使得大部分细胞处于有丝分裂间期,进而促进细胞凋亡,呈现明显的时间依从性,72 h效果最明显;联合顺铂凋亡率明显高于单独用药组(P0.01)。实时RT-PCR结果显示,雷帕霉素作用24 h可引起mTOR mRNA表达下调,72 h抑制表达低于24 h(P0.05)。结论:雷帕霉素作用于PI3K/AKT/mTOR信号传导通路的mTOR位点,阻滞细胞周期,诱导细胞凋亡,与传统化疗药顺铂有协同效应,可增强肿瘤细胞对于顺铂的化疗敏感性。     

雷帕霉素联合多西紫杉醇对肺癌细胞增殖和凋亡的影响

目的探讨哺乳动物雷帕霉素靶蛋白(mTOR)信号通路抑制剂雷帕霉素(RAPA)联合多西紫杉醇(DOC)对肺癌细胞系A549、SPC-A-1、95D和NCI-H446增殖抑制及凋亡的影响。方法甲基噻唑基四唑(MTT)法检测不同浓度RAPA组、DOC组及DOC联合20.0 nmol.L-1 RAPA组对肺癌细胞系增殖抑制作用;流式细胞仪法检测RAPA组、DOC组及DOC联合20.0 nmol.L-1 RAPA组对肺癌95D细胞凋亡的影响。结果 RAPA能抑制肺癌细胞的增殖,其作用呈剂量依赖性;单独应用DOC及DOC联合20.0 nmol.L-1 RAPA均可显著抑制肺癌细胞的增殖,且联合应用抑制率显著升高(P<0.05);流式细胞仪检测显示RAPA及DOC均可增加肺癌95D细胞的凋亡率(P<0.05),联合应用明显增加细胞的凋亡率(P<0.05)。结论 mTOR信号通路参与肺癌细胞的增殖与凋亡,mTOR信号通路抑制剂RAPA可增强DOC的抗肿瘤效应。