mTOR、S6K1在双胍类降糖药物改善2型糖尿病中的作用研究

目的 探讨mTOR、S6K1在双胍类降糖药物改善2型糖尿病中的作用和意义.方法 选取本院2015年3月至2017年3月收治的100例糖尿病患者,随机分为观察一组和观察二组,观察一组患者给予常规胰岛素治疗,观察二组患者在此基础上接受二甲双胍治疗3个月.选取同期健康体检者50例为对照组.治疗前后分别检测受试者血清中mTOR、S6K1信号通路的水平变化.结果 与对照组相比,观察一组和观察二组患者体内mTOR和S6K1水平显著升高,IL-6、TNF-α和IL-Iβ水平显著上升,差异具有统计学意义(P＜0.05).观察一组和观察二组患者与治疗前相比,治疗后mTOR、S6K1、IL-6、TNF-α和IL-Iβ水平显著降低,差异具有统计学意义(P＜0.05).与观察一组患者治疗后相比,观察二组患者二甲双胍治疗后,血清mTOR、S6K1、IL-6、TNF-α和IL-Iβ水平显著降低,差异具有统计学意义(P＜0.05).患者血液mTOR和S6K1与其使用二甲双胍治疗的疗效存在显著的负相关关系,Person相关系数为负值,P＜0.01.结论 双胍类降糖药物可以明显改善糖尿病患者的临床症状;mTOR、S6K1信号通路在糖尿病患者的疾病发展和临床治疗中发挥重要的作用.     

mTOR/P70S6K/HIF-1α信号通路在脂多糖诱导Caco-2细胞屏障损伤中的作用及机制

目的:探讨缺氧诱导因子1α(hypoxia-inducible factor-1α,HIF-1α)在脂多糖(lipopolysaccharide,LPS)诱导的Caco-2细胞屏障损伤中的作用,并探究哺乳动物雷帕霉素靶蛋白(mammalian rapamycin target protein,mTOR)/P70核糖体蛋...     

乙醇抑制大鼠原代肝细胞中P70S6K和ERK1/2活性

 目的 研究乙醇对原代肝细胞中P70S6K和ERK1/2活性的影响。方法 分离成年雄性SD大鼠的肝细胞,分别以0、50、100 和200mmol/L的乙醇处理4h,或以200 mmol/L乙醇处理0、1、2和4h,用Western blot法检测P70S6K和ERK1/2的活性。在细胞中加入100 nmol/L的mTOR特异抑制剂雷帕霉素或10μmol/L 的MEK1/2特异抑制剂U0126,培养 24 h后收集细胞,用Western blot法检测P70S6K、ERK1/2及PPARγ的表达;用EMSA检测PPARγ的活性。结果 随着乙醇浓度的增加或处理时间的延长,P70S6K的表达逐渐降低;乙醇也可抑制ERK1/2的活性;U0126同时抑制ERK1/2和P70S6K的活性,而雷帕霉素虽抑制P70S6K的活性,但增强ERK1/2的活性;乙醇不影响PPARγ的表达和活性。 结论 在大鼠原代肝细胞,乙醇对P70S6K的活性有抑制作用,且存在量效关系和时效关系。P70S6K的活性受ERK1/2的控制,但P70S6K也可反向调节ERK1/2。     

GSK-3及P70S6K在胰岛素抵抗大鼠肌肉中的表达及意义

目的:观察饮食诱导的胰岛素抵抗(IR)大鼠骨骼肌中糖原合成酶-3(GSK-3)及核糖体S6蛋白激酶(P70S6K)的表达及变化情况,并探讨GSK-3和P70S6K在IR发生中的作用及意义.方法:将40只4周龄Wistar大鼠随机分为正常对照组、高糖组、高脂组、高脂高糖饲养组,喂养8周后用高胰岛素-正葡萄糖钳夹技术(钳夹试验)对大鼠进行胰岛素敏感性的评估,采用Western blot法检测各组大鼠骨骼肌中GSK-3及P70S6K的含量,同时测定各组大鼠的附睾脂肪垫重量、血糖(BG)、胰岛素(INS)、甘油三酯(TG)、胆固醇(TC)及游离脂肪酸(FFA)水平、超敏C反应蛋白(hsCRP).结果:高脂高糖组、高脂组大鼠产生明显IR,体重增加(P＜0.01),以附睾脂肪垫的重量增加更为显著(P＜0.01),TG、TC及FFA水平、hsCRP增加(P＜0.01),GSK-3、P70S6K在IR大鼠肌肉中的表达明显升高.结论:高脂高糖饮食可诱导大鼠产生IR,IR大鼠的hsCRP、TG、TC及FFA水平明显增高,大鼠产生IR与炎症反应有关,GSK-3、P70S6K在IR大鼠中的表达明显升高,在胰岛素信号传导中起负相调节作用.     

P70S6K在Xp11.2易位/TFE3基因融合相关性肾癌中的表达及临床意义

目的检测P70S6K在Xp11.2易位/TFE3基因融合相关性肾癌(renal cell carcinoma associated with Xp11.2 translocation/TFE3 gene fusion,TFE3 RCC)中的表达,探讨其在TFE3 RCC发生、发展中的作用及临床意义。方法采用免疫组化法检测23例确诊的TFE3 RCC、14例肾透明细胞癌(clear cell RCC,CCRCC)和6例乳头状肾细胞癌(papillary RCC,PRCC)组织中P70S6K的表达,观察其表达与三种RCC的关系,同时观察P70S6K表达与TFE3 RCC发生、发展和相关临床病理参数的关系。结果 P70S6K在TFE3 RCC组织中的表达(91.3%)明显高于其在CCRCC组织(64.2%)和PRCC组织中的表达(66.6%),差异有显著性(P0.05)。P70S6K在TFE3 RCC组织中阳性表达水平的H-score评分值(88.2±9.8)亦显著高于其在CCRCC组织(54.4±7.6)和PRCC组织(43.7±6.2)中阳性表达水平的H-score评分值,差异具有显著性(P0.05)。P70S6K表达与TFE3 RCC患者年龄、有无淋巴结转移和脉管瘤栓有关,与患者性别、肿瘤直径等临床病理参数无关。结论与CCRCC和PRCC组织相比,TFE3 RCC组织中P70S6K表达显著增高,有助于TFE3 RCC的鉴别诊断。P70S6K在TFE3 RCC组织中高表达,同时提示mTOR信号通道在TFE3 RCC的发生、发展中可能起重要作用。为TFE3 RCC的靶向治疗寻找潜在药物靶点提供一定的理论基础。     

鲍曼不动杆菌外膜蛋白A通过Akt/mTOR/p70S6K信号通路引起RAW264.7细胞自噬

目的:分析鲍曼不动杆菌标准株ATCC 19606(Acinetobacter baumannii ATCC 19606)外膜蛋白A(outer membrane protein A,Omp A)对RAW264. 7细胞的自噬诱导作用。方法:建立Omp A刺激RAW264. 7细胞的模型,通过细胞免疫荧光染色、Western blot和透射电子显微镜检测Omp A对RAW264. 7细胞自噬的影响。结果:Omp A可以引起自噬蛋白LC3B-II表达升高,并抑制Akt/mTOR/p70S6K的磷酸化水平;雷帕霉素可以进一步降低mTOR和p70S6K磷酸化,并提高Omp A引起的LC3B-II表达升高。结论:鲍曼不动杆菌Omp A通过Akt/mTOR/p70S6K信号通路引起RAW264. 7细胞自噬。这为将来进一步研究鲍曼不动杆菌引起自噬的分子机制及找到对抗其感染的新方法提供理论依据。     

观察炎症状态和高脂状态对mTOR/S6K/IRS1-Ser和IRS1-Tyr信号通路表达的影响

目的观察在体内外炎症状态下哺乳动物雷帕霉素靶蛋白(mTOR)-70S核糖体蛋白S6激酶(S6K)-胰岛素受体底物1(IRS1)信号传导通路异常磷酸化表达对胰岛素抵抗的影响。方法体外实验,将HepG2分为4组:对照组、高脂组[给予100 mg/L低密度脂蛋白(LDL)处理]、炎症介入组[给予20μg/L肿瘤坏死因子-α(TNF-α)处理]、联合干预组(给予20μg/L TNF-α+100 mg/L LDL处理)。采用实时定量PCR和Western blotting方法检测mTOR、S6K和IRS1 mRNA及蛋白表达水平;体内实验,8周龄c57BL/6J小鼠随机分为4组(正常饮食组、正常饮食加炎症组、高脂饮食组和高脂饮食加炎症组),分别给予隔日皮下注射生理盐水和酪蛋白建立炎症模型,8周后将实验小鼠于深度麻醉下处死并检测其肝脏组织中mTOR、S6K和IRS1 mRNA及蛋白表达水平。结果体内外实验均显示与对照组相比,炎症组与高脂组mTOR、S6K和IRS1-Ser mRNA及蛋白表达增加,而联合干预组表达明显增高,IRS1-Tyr mRNA及蛋白表达下降,联合干预组表达明显降低。结论炎症及高脂状态激活mTOR/s6k/IRS1-Ser信号通路,而抑制IRS1-Tyr信号通路,从而加重胰岛素抵抗。     

Oleanolic acid suppresses the proliferation of human bladder cancer by Akt/mTOR/S6K and ERK1/2 signaling

Oleanolic acid has significant pharmacological activities, such as anti-tumor, regulating blood sugar level and liver protection, which are more effective compared with free aglyconeoleanolic acid. However, it is still unknown if oleanolic acid affects the proliferation of human bladder cancer. We utilized T24 cells to study the effect of oleanolic acid on the proliferation and apoptosis of human bladder cancer. In this study, we found that the anti-cancer effect of oleanolic acid significantly suppressed cell proliferation and increased apoptosis and caspase-3 activity of T24 cells. Furthermore, Akt, mTOR and S6K protein expression was greatly inhibited in T24 cells under oleanolic acid treatment. Meanwhile, ERK1/2 of phosphorylation protein expression was significantly promoted by oleanolic acid treatment. Taken together, we provided evidences that oleanolic acid was Akt/mTOR/S6K and ERK1/2 signaling-targeting anti-tumor agent. These findings represent new evidences that oleanolic acid suppresses the proliferation of human bladder cancer by Akt/mTOR/S6K and ERK1/2 signaling, and oleanolic acid may be used to prevent human bladder cancer.     

mTOR_P70S6K信号通路在小儿脑胶质瘤中的表达及其临床意义

目的:探讨mTOR_P70S6K信号通路在小儿脑胶质瘤组织中的表达及其临床意义。方法:随机选取脑胶质瘤患儿88例,采用SP免疫组化方法,检测脑胶质瘤组织和正常脑组织中mTOR蛋白及P70S6K蛋白的表达。并分析其与脑胶质瘤组织临床分级之间的关系。结果:mTOR蛋白及P70S6K蛋白在脑胶质瘤组织中的阳性表达率分别为81.58%(31/38)和78.95%(30/38),明显高于其在正常脑组织中的阳性表达率[分别为15%(3/20)和10%(2/20)](P<0.01)。mTOR蛋白和P70S6K蛋白在脑胶质瘤组织中的表达呈正相关(r=0.68,P<0.05),且两者在正常脑组织中的表达亦正相关性(r=0.89,P<0.01)mTOR蛋白及P70S6K蛋白阳性表达率在脑胶质瘤高级别组(Ⅲ~Ⅳ级)中的表达均明显高于低级别组(Ⅰ~Ⅱ级),说明随着肿瘤级别的升高二者的表达也增强。结论:mTOR_P70S6K信号通路在小儿脑胶质瘤中发生了特异性的激活。该通路可能与小儿脑胶质瘤的发生、发展密切相关。     

Activation of the PI3K/Akt/mTOR/p70S6K Pathway is Involved in S100A4-induced Viability and Migration in Colorectal Cancer Cells

The S100 protein family member S100A4 regulates various cellular functions. Previous studies have shown that elevated expression of S100A4 is associated with progression and metastasis of colorectal cancer (CRC). However, little is known about whether and how S100A4 contributes to CRC development. In our present study, the elevated expression of S100A4 in CRC tissues compared to matched adjacent normal tissues was confirmed by immunohistochemistry, semi-quantitative RT-PCR and Western blot. Adenovirus-mediated S100A4 overexpression obviously enhanced viability and migration of CRC cells, which was detected by MTT assay and transwell assay, respectively. Additionally, S100A4 overexpression increased the phosphorylation levels of Akt, mTOR and p70S6K. These effects of S100A4 were abolished by treatment with either the specific PI3K/Akt inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, or the specific mTOR/p70S6K inhibitor rapamycin. Furthermore, overexpression of S100A4 resulted in upregulation of VEGF and downregulation of E-cadherin, which were strongly reversed by either {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or rapamycin. Altogether, our results demonstrate that activation of the PI3K/Akt/mTOR/p70S6K signaling pathway is involved in S100A4-induced viability, migration, upregulation of VEGF and downregulation of E-cadherin in CRC cells.     

S6K1 shRNA基因重组腺病毒的构建及对基因沉默效果的研究

目的 构建S6K1 shRNA基因重组腺病毒(S6K1Ax)并在细胞及小鼠肝脏验证对S6K1基因的沉默效果.方法 设计3种S6K1 shRNA序列,通过在pcPUR质粒与pcDNA3.1质粒、cosmid质粒之间的拼接将S6K1 shRNA转染进腺病毒,筛选沉默效果最佳的S6K1Ax并在293细胞扩增纯化得到高效价的S6K1Ax.感染来源于小鼠的肝脏、肌肉、脂肪细胞系,在Western blot水平评价其对S6K1蛋白表达的抑制效果.将S6K1Ax注射进C57BL/6J小鼠尾静脉,6 d后处死小鼠取肝脏,逆转录-聚合酶链反应和Western blot观察小鼠肝脏S6K1在mRNA和蛋白水平的表达.检测注射S6K1Ax前后小鼠血清丙氨酸氨基转移酶(ALT)变化.结果 Western blot证实制备的S6K1 Ax可抑制来源于小鼠的肝脏、肌肉、脂肪3种细胞系和小鼠C57BL/6J肝脏S6K1的蛋白表达.小鼠肝脏S6K1 mRNA结果显示:对照组为1.39±0.21,实验组为0.63±0.09,t=6.132,P<0.01,差异有统计学意义.S6K1Ax注射小鼠,ALT水平注射前为(15.15±4.43)U/L,注射后为(17.32±4.22)U/L,t=1.451,P>0.05,差异没有统计学意义.结论 构建的S6K1Ax可将来源于小鼠的细胞系及C57BL/6J小鼠肝脏S6K1基因沉默,为研究S6K1的基因功能提供了适合的实验工具.     

Immunohistochemical and molecular analysis of PI3K/AKT/mTOR pathway in esophageal carcinoma

Among the numerous signaling pathways involved in tumorigenesis, PI3K‐AKT‐mTOR is a key one that regulates diverse cellular functions. However, its prognostic value in esophageal carcinoma remains unclear. In our study, we examined the immunohistochemical expression of phosphorylated (p‐) AKT, mTOR, p70S6K and 4E‐BP1 along with the mutational status of PIK3CA and AKT1 genes by High Resolution Melting Analysis and Pyrosequencing in 44 esophageal carcinomas. The results were correlated with the clinicopathological characteristics of the patients in an effort to define their possible prognostic significance. Total p‐mTOR cytoplasmic expression, assessed in 10 random areas, was positively correlated with tumor stage (Kruskal–Wallis ANOVA, I/II vs III/IV, p = 0.0500). Μoreover, maximum p‐mTOR cytoplasmic immunoexpression, estimated in hot spot areas, was positively associated with tumor grade (Mann–Whitney U test, I/II vs III, p = 0.0565). Interestingly, p‐4E‐BP1 immunoreactivity was negatively correlated with tumor histological grade (Mann–Whitney U test, I/II vs III, p = 0.0427). No mutation was observed in exons 9 and 20 of PIK3CA gene and in exon 4 of AKT1 gene. In conclusion, our findings depict the presence of activated PI3K/AKT/mTOR pathway in esophageal cancer bringing forward p‐mTOR and p‐4E‐BP1 for their potential role in esophageal carcinogenesis. Additional studies are warranted to validate our findings.     

Characterization of p70 S6 kinase 1 in early development of mouse embryos

The mTOR kinase controls cell growth, proliferation, and survival through two distinct multiprotein complexes mTORC1 and mTORC2. p70 S6 Kinase 1 (S6K1) is characterized as downstream effector of mTOR. Until recently, the connection between S6K1 and mTORC1 /mTORC2 during the early development of mouse embryos has not been well elucidated. Here, the expression level of total S6K1 and its phosphorylation at Thr389 was determined in four phases of one‐cell embryos. S6K1 was active throughout the cell cycle especially with higher activity in G2 and M phases. Rapamycin decreased the activity of M‐phase promoting factor (MPF) and delayed the first mitotic cleavage. Down‐regulating mTOR and raptor reduced S6K1 phosphorylation at Thr389 in one‐cell embryos. Furthermore, rapamycin and microinjection of raptor shRNA decreased the immunofluorescent staining of Thr389 phospho‐S6K1. It is proposed that mTORC1 may be involved in the control of MPF by regulating S6K1 during the early development of mouse embryos. Developmental Dynamics 238:3025–3034, 2009. © 2009 Wiley‐Liss, Inc.     

Analysis of mTOR pathway expression in lymphatic malformation and related diseases

The mammalian target of rapamycin (mTOR) inhibitor sirolimus is an effective treatment for difficult‐to‐treat lymphatic anomalies. However, little is known about the expression of mTOR pathway components in lymphatic anomalies. Here we investigated the expression pattern of mTOR pathway components and their phosphorylated forms (mTOR, p‐mTOR, 4EBP1, p‐4EBP1, S6K1 and p‐S6K1) in normal lymphatic vessels and lymphatic anomalies using immunohistochemistry. We studied 18 patients of lymphatic anomalies, including lymphatic malformation (LM, n = 14), Kaposiform lymphangiomatosis (KLA, n = 2) and Kaposiform hemangioendothelioma (KHE, n = 2). Normal lymphatic vessels expressed 4EBP1, S6K1 and p‐S6K1, but not p‐4EBP1, mTOR or p‐mTOR. The mTOR was detected in all lymphatic anomalies, whereas its activation form p‐mTOR was detected in half cases of KLA and KHE but not in LM. All lymphatic anomalies expressed S6K1 and its activated form p‐S6K1. The expression of 4EBP1 was also found in all lymphatic anomalies, but its activation was detected in approximately half of them. The activation of mTOR was seen in tumor (KLA and KHE) but not in malformation (LM), whereas the activation of S6K1 and 4EBP1 was seen in all and half of lymphatic anomalies, respectively.