mTORC1/2双重抑制剂OSI-027抑制高氧诱导的肺成纤维细胞增殖和分化

目的：分析哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin, mTOR）复合物1/2(mTOR complex 1/2, mTORC1/2)双重抑制剂OSI-027对高体积分数氧（高氧）所致人胚肺成纤维细胞增殖和分化的抑制作用。方法：高氧（95%O——2）处理人胚肺成纤维细胞MRC-5建立增殖分化模型,分为对照组、高氧组、高氧+OSI-027组和高氧+雷帕霉素组。Western blot检测α-平滑肌肌动蛋白（α-smooth muscle actin, α-SMA）、I型胶原蛋白（collagen type I, Col I）、增殖细胞核抗原（proliferating cell nuclear antigen, PCNA）、细胞周期蛋白D1(cyclin D1)、RhoA、Rho相关含卷曲螺旋蛋白激酶1(Rho-associated coiled-coil-containing protein kinase 1, ROCK1)、蛋白激酶B(protein kinase B, PKB/AKT)、p-AKT和mTOR的表达;CCK-8实验检测细胞活力;...     

mTORC2通路在苯丙胺致大鼠纹状体损伤中的变化

目的:探讨哺乳动物雷帕霉素靶蛋白复合物2(m TORC2)通路在苯丙胺致中枢神经损伤过程中的变化。方法:通过注射苯丙胺建立大鼠动物模型,采用open field方法测试大鼠的自主行为活动,用透射电镜观察大鼠纹状体的超微结构,用Western blot方法检测m TORC2信号通路的变化,用免疫组化方法观察m TORC2阳性神经元的变化。结果:苯丙胺大鼠在行为学上出现刻板行为,纹状体细胞和神经纤维的超微结构受损,磷酸化的m TORC2和蛋白激酶B(Akt)表达减少,并且磷酸化的m TORC2阳性细胞也减少。结论:m TORC2通路的抑制可能在苯丙胺致纹状体结构的损伤中起重要作用。     

基于MMP7/mTORC1信号通路探讨小鼠脓毒症急性肾损伤的分子机制

目的：探讨基质金属蛋白酶7(MMP7)在小鼠脓毒症相关急性肾损伤模型中的表达及作用。方法：通过盲肠结扎穿孔（CLP）手术在具有C57BL/6J遗传背景的MMP7敲除（MMP7-KO）小鼠和野生型（WT）C57BL/6J小鼠中诱导脓毒症。采用外源性MMP7重组蛋白对MMP7-KO小鼠进行预处理。通过脂多糖（LPS）刺激正常人近端肾小管上皮细胞系HKC-8建立体外模型。采用Western blot检测MMP7和哺乳动物雷帕霉素靶蛋白复合体1(mTORC1)表达。HE和TUNEL染色评估小鼠的肾损伤。Hoechst 33342染色评估细胞凋亡。结果：与假手术组相比,CLP组在CLP后6 h肾脏组织中MMP7蛋白表达降低,这种降低趋势持续到48 h。MMP7-KO的CLP组小鼠肾小管损伤病理评分和TUNEL阳性肾小管细胞显著高于WT的CLP组（P<0.01）。MMP7重组蛋白孵育可很大程度上降低LPS诱导的HKC-8细胞凋亡。LPS诱导了HKC-8细胞的mTORC1表达,而MMP7可以抑制mTORC1表达。MMP7能够明显促进mTORC1降解,产生分子量为18 kD的较小片段。此外,MM...     

mTOR信号通路与调节

哺乳动物雷帕霉素靶蛋白mTOR是一种非典型丝氨酸/苏氨酸蛋白激酶,可整合细胞外信号,磷酸化下游靶蛋白核糖体p70S6激酶,如S6K1及4E-BP1,影响转录与翻译,从而参与调控细胞生长、增殖等过程.近年来研究发现,调控mTOR通路可以干预某些疾病的病理过程.mTOR研究的新发现,可望为今后相关疾病的治疗提供新的靶点.     

mTOR信号通路与细胞凋亡

哺乳类动物雷帕霉素靶蛋白(mTOR)主要通过上游信号转导通路磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(PKB/Akt)/mTOR信号通路及下游信号通路mTOR/ eIF4E结合蛋白1(4EBP1)、mTOR/p70S6激酶(p70S6K)在细胞生长、增值与分化和在血管再生、蛋白合成与降解中发挥作用.细胞凋亡是细胞的一种程序性死亡,在机体发育、组织代谢中有着重要作用,而细胞凋亡的异常调节与许多疾病的发生和发展紧密相连.近年研究发现,mTOR信号通路在细胞凋亡过程中扮演了重要角色,并已被作为新的药物治疗靶点.     

LKB1/AMPK/mTOR信号通路与血液肿瘤

血液肿瘤已成为严重威胁儿童生命的主要疾病之一,其发病率逐年升高.随着研究的不断深入,LKB1/AMPK/mTOR信号转导通路在多种常见肿瘤如乳腺癌、前列腺癌、肝癌、肺癌及淋巴瘤等的研究中凸现出日益重要的作用,并为肿瘤的治疗提供了新的靶点,成为目前研究的热点.现就LKB1/AMPK/mTOR信号通路的组成及其与血液肿瘤的关系以及分子机制作一综述.     

mTORC1信号通路对能量代谢调控作用及其机制

哺乳动物雷帕霉素靶蛋白（mTORC1）通路是细胞内最主要的能量感受器,能感受营养和激素、与能量需求相关的多个细胞功能的调节相关的上游调节信号。调节下游信号从而影响不同的细胞代谢,从蛋白质和脂质合成到线粒体活性等不同方面调节细胞代谢。作为对细胞代谢过程的调节,mTORC1的活性在外周激活有利于脂肪细胞活化,脂肪生成,葡萄糖摄取和β细胞数量的增加。本文综述了现有的知识对mTORC1的作用在能量平衡和能量代谢的调节,特别是旨在提供有关mTORC1在功能细胞中心能够整合不同激素的作用的背景下的研究进展进行了综述。     

CIRBP对小鼠血管平滑肌细胞增殖及迁移的调控及机制研究

目的：探讨冷诱导RNA结合蛋白(CIRBP)对小鼠血管平滑肌细胞(VSMCs)增殖及迁移过程的调控及机制。方法：将雄性C57BL/6J小鼠分为假手术对照组、假手术干扰组、颈总动脉损伤对照组和颈总动脉损伤干扰组,每组5只。造模后按照组别分别用阴性对照腺病毒(AD-NC)和沉默CIRBP腺病毒(AD-CIRBPI)转染,28 d后观察CIRBP表达及血管内膜的增生情况。将小鼠VSMCs分为对照组和腺病毒沉默组,分别用AD-NC和AD-CIRBPI转染细胞48 h,然后加入激活雷帕霉素靶蛋白复合物1(mTORC1)活性的胰岛素。通过RT-qPCR检测CIRBP的mRNA表达,Western blot检测CIRBP、磷酸化核糖体蛋白S6(p-S6^Ser235/236)和磷酸化4E结合蛋白1(p-4EBP1^Thr37/46)蛋白水平变化,Ki67免疫荧光染色和CCK-8实验检测细胞增殖,划痕实验检测细胞迁移,HE染色检测颈动脉内膜增生程度。结果：沉默CIRBP后,VSMCs的活力下降,Ki67阳性细胞比率降低,细胞迁移速度减慢,同时mTORC1活性下降...     

Ghrelin活化人T细胞翻译起始分子通过mTOR信号转导通路

目的:探讨Ghrelin活化人T细胞翻译起始分子的信号转导机理。方法:采用微柱法分离人外周血T细胞,采用Western blot方法检测mTOR信号通路分子和蛋白质翻译调控分子的磷酸化状态和含量。采用mTOR抑制剂:雷帕霉素,PI3KⅠ抑制剂:LY294002,PI3KⅢ抑制剂:3-甲基腺嘌呤,Ghrelin拮抗剂:Des-Lys-3-GHRP6和AKT抑制剂:A443654和Ghrelin研究相应信号通路。结果:①人T细胞表面有Ghrelin受体(GHSR1a)表达。②Ghrelin与GHSR1a结合后可活化mTOR。③Ghrelin磷酸化两个mTOR下游靶分子4E-BP-1和P70 S6K,P70 S6K进一步磷酸化核糖体S6蛋白。④Ghrelin磷酸化蛋白质翻译起始帽子结构的两个重要分子eIF4E和eIF4G。结论:Ghrelin促进人T细胞mRNA翻译起始的机理是活化mTOR信号转导通路。     

激活mTORC2/Akt信号通路对6-羟基多巴胺模型小鼠多巴胺能神经元和行为学的影响

目的 探讨激活哺乳动物雷帕霉素靶蛋白复合物2(mTORC2)/Akt信号通路对6-羟基多巴胺(6-OHDA)模型小鼠多巴胺能神经元和行为学的影响及可能的机制。方法 将36只体重20～25 g 3月龄Nestin-CreER^TM::ROSA26-LacZ雄性C57BL/6J小鼠分为NS+玉米油组、6-OHDA+玉米油组、6-OHDA+PP242组、6-OHDA+A-443654组,并在小鼠右侧纹状体注射6-OHDA制备帕金森病(PD)小鼠模型以及每日腹腔注射mTORC2/Akt信号通路激动剂A-443654或抑制剂PP242。通过ELISA测定血清肿瘤坏死因子α(TNF-α)和白细胞介素1β(IL-1β)的水平;免疫组织化学和免疫荧光染色考察黑质(SN)-纹状体小胶质细胞、脑室周围神经前体细胞(NPCs)和多巴胺能神经元数目,Western blotting检测中脑水管mTORC2/Akt信号通路各相关蛋白Rictor, p-Akt和DNA损伤反应调节1(REDD1)的表达并通过免疫共沉淀验证它们之间的相互作用,最后观察各组小鼠行为学的变化。结果 6-OHDA模型小...     

LATSl-YAP信号通路对人皮肤成纤维细胞增殖及细胞外基质合成的调控

目的探讨LATSl-YAP信号通路对人皮肤成纤维细胞活力及细胞外基质合成的调控作用。方法实验分为未干预组、LATSlsiRNA干预组和YAPsiRNA干预组。构建LATSlsiRNA和YAPsiRNA,LATSlsiRNA干预组利用LATSlsiRNA转染人皮肤成纤维细胞株HS27,YAPsiRNA干预组利用YAPsiRNA转染HS27。转染后48h利用Western．blot观察LATSl、YAP和c01．1agenI的表达情况,应用MTY检测HS27细胞株的细胞活力情况。结果LATSlsiRNA转染48h后LATSl蛋白表达显著降低,YAP蛋白和collagenI蛋白表达升高,细胞活力显著增高;YAPsiRNA转染48h后LATSl蛋白表达无变化,YAP蛋白和collagenI蛋白表达降低,细胞活力显著降低。结论LATSl．YAP信号通路可调控人皮肤成纤维细胞活力和细胞外基质的合成,可能成为皮肤创伤后修复以及阻止创伤后瘢痕形成提供潜在治疗靶点。     

氨基酸调节mTORC1信号通路的研究进展

氨基酸是合成蛋白质的底物,也可作为信号分子激活哺乳动物雷帕霉素靶蛋白复合体1(mTORC1)。氨基酸调控mTORC1活性的关键步骤是将mTORC1定位于溶酶体表面,Rag GTPase、Ragulator、v-ATPase和SLC38A9等溶酶体相关蛋白及GATOR、Sestrins等胞质蛋白在其中发挥重要的作用。     

外基质激活ERK信号通路促进成纤维细胞增殖及膝关节术后的纤维化

背景:关节纤维化是关节手术的严重并发症之一,目前认为外基质对纤维化的发生至关重要,然而其发生机制尚不明确.目的:探究外基质在膝关节术后纤维化中的作用及体外对成纤维细胞增殖能力的影响.方法:①体内实验:取12只新西兰大白兔构建下肢膝关节粘连模型,术后2,4周取材,分别进行苏木精-伊红染色、马松染色、天狼猩红染色、增殖细胞...     

Intermedin1-53对大鼠心肌成纤维细胞增殖的影响

目的: 研究intermedin_1-53(IMD_1-53)对醛固酮(ALD)促SD乳鼠心肌成纤维细胞(CFBs)增殖的影响及细胞外信号调节激酶(ERK)信号转导途径的作用。方法: 分离培养SD乳鼠CFBs,将细胞分成对照组、不同浓度ALD组、ALD+不同浓度IMD_1-53组,用MTT比色法测定细胞活性。通过Western blotting方法观察IMD_1-53对ALD诱导的p-ERK蛋白表达的影响。结果: (1) IMD_1-53对基础状态下的CFBs增殖无明显抑制作用,但能呈浓度(10^-9~10^-7mol/L)依赖性地抑制ALD刺激CFBs的增殖。(2) IMD_1-53具有浓度(10^-9~10^-7mol/L)依赖性地抑制ALD诱导的p-ERK蛋白表达的作用。结论: IMD_1-53通过ERK信号途径抑制ALD促CFBs增殖的效应。     

miR-146a-5p regulates autophagy and NLRP3 inflammasome activation in epithelial barrier damage in the in vitro cell model of ulcerative colitis through the RNF8/Notch1/mTORC1 pathway

Ulcerative colitis (UC) is a chronic inflammatory disease affecting the colon that can be influenced by microRNAs (miRNAs). This study aims to investigate the impact of miR-146a-5p on lipopolysaccharide (LPS)-induced Caco-2/HT-29 cell autophagy and NLRP3 inflammasome activation and the underlying mechanism, with the aim of identifying potential therapeutic targets. We used LPS to establish Caco-2/HT-29 cell models and measured cell viability by CCK-8. The levels of miR-146a-5p, RNF8, markers of NLRP3 inflammasome activation and autophagy, proteins involved in the Notch1/mTORC1 pathway, and inflammatory factors were assessed by RT-qPCR, Western blot, and ELISA. Intestinal epithelial barrier function was evaluated by measuring transepithelial electrical resistance. Autophagic flux was measured using tandem fluorescent-labeled LC3. miR-146a-5p was highly-expressed in LPS-induced Caco-2/HT-29 cells, and autophagy flux was blocked at the autolysosomal stage after LPS induction. Inhibition of miR-146a-5p suppressed NLRP3 inflammasome activation, reduced intestinal epithelial barrier damage, and facilitated autophagy inhibition in LPS-induced Caco-2/HT-29 cells. The autophagy inhibitor NH4Cl partially nullified the inhibitory effects of miR-146a-5p inhibition on NLRP3 inflammation activation. miR-146a-5p targeted RNF8, and silencing RNF8 partly abrogated the action of miR-146a-5p inhibition on promoting autophagy and inhibiting NLRP3 inflammasome activation. miR-146a-5p inhibition suppressed the Notch1/mTORC1 pathway activation by upregulating RNF8. Inhibition of the Notch1/mTORC1 pathway partially nullified the function of silencing RNF8 on inhibiting autophagy and bolstering NLRP3 inflammasome activation. In conclusion, miR-146a-5p inhibition may be a potential therapeutic approach for UC, as it facilitates autophagy of LPS-stimulated Caco-2/HT-29 cells, inhibits NLRP3 inflammasome activation, and reduces intestinal epithelial barrier damage by upregulating RNF8 and suppressing the Notch1/mTORC1 pathway.     

Pregnancy restores the regenerative capacity of the aged liver via activation of an mTORC1-controlled hyperplasia/hypertrophy switch

Regenerative capacity is progressively lost with age. Here we show that pregnancy markedly improved liver regeneration in aged mice concomitantly with inducing a switch from proliferation-based liver regeneration to a regenerative process mediated by cell growth. We found that the key mediator of this switch was the Akt/mTORC1 pathway; its inhibition blocked hypertrophy, while increasing proliferation. Moreover, pharmacological activation of this pathway sufficed to induce the hypertrophy module, mimicking pregnancy. This treatment dramatically improved hepatic regenerative capacity and survival of old mice. Thus, cell growth-mediated mass reconstitution, which is relatively resistant to the detrimental effects of aging, is employed in a physiological situation and holds potential as a therapeutic strategy for ameliorating age-related functional deterioration.     

晚期糖基化终产物通过mTORC1/uPAR途径促进足细胞移动

 目的:足细胞哺乳动物雷帕霉素靶蛋白复合体1（mTORC1）活化是糖尿病肾病的重要发病机制。我们前期在db/db小鼠中观察到调控足细胞活动度的尿激酶型纤溶酶原激活物受体（uPAR）表达增加。本研究旨在研究晚期糖基化处理的牛血清白蛋白（AGE-BSA）对mTORC1、uPAR和足细胞活动度的影响并初步探索其可能的分子联系。方法: 体外培养小鼠肾小球足细胞,MTT法和免疫荧光分析各刺激物及干预剂对足细胞存活率及细胞骨架的影响。Western blotting检测正常对照组、对照BSA组及AGE-BSA处理组mTORC1的活性及uPAR的表达水平,划痕实验检测足细胞的活动度。进一步采用雷帕霉素抑制AGE-BSA组mTORC1的活性,观察uPAR和细胞活动性的改变。结果: 在预设浓度及干预时间下各刺激物及干预剂对细胞存活率及细胞骨架无明显影响。AGE-BSA 可上调足细胞mTORC1的活性和uPAR的水平,并促进足细胞移动。雷帕霉素能抑制AGE-BSA引起的uPAR表达水平的上调和细胞活动性的增强。结论: AGE-BSA 可能通过mTORC1/uPAR途径导致足细胞移动性增强。     

PDTC对高糖培养大鼠肾成纤维细胞中MCP-1表达的影响

目的:探讨核因子-κB(NF-κB)抑制剂吡咯烷二硫氨基甲酸(PDTC)对高糖培养大鼠肾成纤维细胞(NRK)中单核细胞趋化蛋白-1(MCP-1)表达的影响。方法:将NRK分4组进行体外培养:(1)正常组:5.6mmol/L的葡萄糖;(2)高糖组:30mmol/L葡萄糖;(3)高糖+PDTC1组:30mmo/L葡萄糖+5μmol/LPDTC;(4)高糖+PDTC2组:30mmo/L葡萄糖+10μmol/LPDTC,分别于培养24h、48h取各组NRK采用RT-PCR检测其MCP-1mRNA表达水平,采用WesternBlot检测MCP-1蛋白表达水平。结果:与正常组相比,高糖组MCP-1mRNA和蛋白表达水平显著升高(P<0.05),不同浓度PDTC干预后,MCP-1mRNA和蛋白表达水平显著下降(P<0.05),且随PDTC浓度增大,下降更明显。结论:高糖可使NRK中MCP-1表达增高,PDTC能抑制NRK中MCP-1表达。     

Roles of PINK1, mTORC2, and mitochondria in preserving brain tumor-forming stem cells in a noncanonical Notch signaling pathway

The self-renewal versus differentiation choice of Drosophila and mammalian neural stem cells (NSCs) requires Notch (N) signaling. How N regulates NSC behavior is not well understood. Here we show that canonical N signaling cooperates with a noncanonical N signaling pathway to mediate N-directed NSC regulation. In the noncanonical pathway, N interacts with PTEN-induced kinase 1 (PINK1) to influence mitochondrial function, activating mechanistic target of rapamycin complex 2 (mTORC2)/AKT signaling. Importantly, attenuating noncanonical N signaling preferentially impaired the maintenance of Drosophila and human cancer stem cell-like tumor-forming cells. Our results emphasize the importance of mitochondria to N and NSC biology, with important implications for diseases associated with aberrant N signaling.     

FBP1 is highly expressed in human hypertrophic scars and increases fibroblast proliferation,apoptosis, and collagen expression

Purpose: FBP1, one of the far-upstream element binding proteins(FBPs), is a distal upstream binding protein of c-myc, which is highly expressed in tumor tissues. This study aimed to investigate FBP1 expression in human hypertrophic scars and to determine the effects of FBP1 on fibroblasts. Materials and methods: Human normal skin and scar specimens were collected during clinical surgery. One portion of each tissue specimen was embedded in paraffin and sliced to observe differences in histological features and FBP1 expression by immunohistochemistry and western blotting. The other portion of each tissue specimen was cultured to obtain fibroblasts. Fibroblasts from the second to the sixth passage were used for the experiments, which were divided into the following two groups: an experimental group, whose cells were transfected with an siRNA targeting FBP1, and a control group, whose cells where not transfected. MTT and TUNEL assays were performed, respectively, to assess fibroblast proliferation and apoptosis, and western blotting was performed to assess protein expression. Results: We obtained fibroblasts by primary tissue culture and found that FBP1 was highly expressed in hypertrophic scars. MTT assay showed that an siRNA targeting FBP1 significantly reduced fibroblast proliferation in siRNA-treated cells compared to control cells. TUNEL assay showed that there was no difference in apoptosis between the two groups; however, western blotting showed that collagen I, collagen III, c-myc, caspase-3, and caspase-9 expression levels were all decreased in the experimental group. Conclusion: FBP1 is highly expressed in human hypertrophic scars and increases fibroblast proliferation, apoptosis and collagen expression.