臭椿酮诱导人黑色素瘤A375细胞凋亡及其机制研究

为研究臭椿酮(Ailanthone,AIL)诱导人黑色素瘤A375细胞凋亡的作用及作用机制,以人黑色素瘤A375细胞为研究对象,采用MTT法测定AIL对人黑色素瘤A375细胞生长增殖的抑制作用。用倒置相差显微镜观察AIL对A375细胞形态的影响,用荧光倒置显微镜观察Hoechst33258染色后AIL对A375细胞核的影响,用AnnexinV-FITC/PI双染法检测AIL诱导A375细胞凋亡的作用,用分光光度法检测caspase-3和caspase-9的活性,Westernblot检测p-PI3Kβ(Ser1070),PI3Kβ,p-Akt(Ser473)和Akt蛋白表达水平的变化,接着用PI3K抑制剂LY294002进行干预,进一步验证AIL对PI3K/Akt信号通路及细胞凋亡的影响。实验结果表明,AIL能够明显抑制A375细胞增殖,使A375细胞数目变少、附着力和透光性减弱,AIL能够诱导A375细胞凋亡,使其细胞核染色质发生固缩并呈现高亮,且使A375细胞早期及晚期凋亡率均增加,AIL作用后能够使caspase-3和caspase-9活性增加,AIL能够抑制PI3K和Akt蛋白磷酸化,从而使PI3K/Akt信号通路失活。较AIL单独作用,AIL和LY294002共同作用后对PI3K和Akt蛋白磷酸化的抑制作用增强且诱导凋亡作用增加,进一步说明AIL通过失活PI3K/Akt信号通路来诱导A375细胞凋亡。     

紫草素对肝癌细胞SMMC-7721凋亡的影响及其机制

目的:探讨紫草素对肝癌SMMC-772细胞的作用及分子机制.方法:SMMC-7721细胞分别经0、5、20、80、320 ng/ml的紫草素作用0h、24 h、48 h和72 h后,适时采用CCK8法观察该细胞增殖的活性,hoechst染色分析细胞的核型变化,流式细胞仪检测细胞凋亡水平,Western blot证实细胞...     

杨芽黄素对前列腺癌细胞凋亡的影响及其机制

目的:探讨杨芽黄素对前列腺癌细胞22Rv.1的作用及机制。方法:将0~20μg/ml杨芽黄素作用于22Rv.1细胞和正常前列腺细胞RWPE-1,适时采用MTS法检测细胞的增殖活性,采用流式细胞仪、hoechst染色、LDH释放实验分别检测22Rv.1细胞凋亡、死亡、周期、核型变化和药物的细胞毒作用,利用qPCR和Westernblot分析22Rv.1细胞内基因转录和蛋白表达的改变,并通过抑瘤实验证实该药的抑癌作用。结果:杨芽黄素可显著抑制22Rv.1细胞增殖、诱导其凋亡,促进22Rv.1细胞凋亡相关基因dr4、dr5、trail、p53、caspase-3、caspase-8、caspase-9、bid、bax、foxo3的表达,并抑制抗凋亡基因akt、pi3k和bcl-2的表达。结论:杨芽黄素可通过影响TRAIL和PI3K/AKT信号通路诱导前列腺癌细胞凋亡,具有抗前列腺癌的作用。     

PI3K/Akt信号通路相关的生物学调控机制研究进展

PI3K/Akt信号通路是由酶联受体介导的信号转导通路,该通路不仅参与多种生长因子、细胞因子和细胞外基质等的信号转导,同时还参与细胞增殖、分化、凋亡和葡萄糖转运等多种细胞功能的调节,特别是在细胞凋亡、细胞存活以及调控细胞糖代谢等方面具有重要作用。本研究综述了PI3K-Akt信号通路的结构组成、通路活化、通信过程、调控机制及其生物学功能等方面的研究进展,为进一步研究PI3K/Akt信号通路的生物学调控作用机制提供启示。     

RHCG基因在非小细胞肺癌中的表达及对细胞增殖的影响

为了探讨Rh type C glycoprotein (RHCG)对非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞增殖的影响及可能的作用机制,本研究使用荧光定量PCR法检测12对NSCLC及癌旁组织样本中RHCG mRNA的表达水平及pcDNA3.1-RHCG质粒对A549细胞RHCG m RNA的表达;采用CCK-8法检测细胞增殖能力;运用PI染色法检测细胞周期;使用免疫印迹法检p-PI3K、PI3K、p-AKT以及AKT蛋白表达水平。本研究发现,与癌旁组织比较,NSCLC中RHCG m RNA表达水平明显降低。RHCG过表达能抑制NSCLC细胞系A549细胞增殖能力。此外,RHCG过表达使A549细胞周期G1/S期转化发生阻滞。本研究还发现,RHCG过表达可下调A549细胞p-PI3K/PI3K和p-AKT/AKT水平。本研究表明,RHCG抑制NSCLC细胞增殖的作用与其抑制PI3K/AKT信号通路有关。     

miR-27a-3p通过靶向Sema7A调控病毒性心肌炎心肌细胞损伤的机制研究

目的 探讨微小RNA-27a-3p(miR-27a-3p)过表达对病毒性心肌炎(VMC)细胞损伤的影响及其可能的作用机制.方法 原代培养大鼠心肌细胞,柯萨奇B3病毒(CVB3)感染心肌细胞建立VMC模型(CVB3组).正常心肌细胞作为对照组,分别将miR-NC、miR-27a-3pmimics、si-NC、si-Sem...     

基于PI3K/AKT/mTOR通路的三百棒促进脂多糖诱导的RAW 264.7细胞自噬并抑制炎症CSCD

三百棒来源于芸香科植物飞龙掌血Toddalia asiatica(L.)Lam的根,是一种天然土家族中草药,具有抗炎、抗风湿、抗肿瘤、抗微生物等药理活性。其毒副作用小,疗效显著的特点使之成为当前研究热点。许多天然产物已被证明可通过靶向PI3K/AKT/mTOR介导的自噬来抑制炎症及自身免疫性疾病,本项研究通过调节PI3K/AKT/mTOR信号通路来研究三百棒醇提物(Toddalia asiatica alcohol extract,TAAE)对自噬的影响,用脂多糖(LPS)诱导单核巨噬细胞(RAW 264.7)建立炎症模型,通过细胞毒性检测试剂盒检测TAAE对细胞活力的影响,并筛选出药物的浓度及干预时间,透射电镜和单丹磺酰尸胺染色检测巨噬细胞的生物学功能,酶联免疫吸附法检测上清液中相关炎症因子水平,Western blot检测自噬和通路相关蛋白的表达水平;并采用自噬早期抑制剂(3-MA)和通路PI3K激动剂(740Y-P)进一步验证自噬对炎症和信号通路的影响。实验结果表明TAAE可能通过抑制PI3K/AKT/mTOR信号通路,促进自噬泡的形成、自噬体溶酶体融合和降解,降低LPS处理的RAW 264.7细胞中炎性细胞因子的表达和分泌。总体而言,本研究结果为三百棒的抗炎机制的研究提供了新的线索,并为临床更好的应用三百棒治疗炎症性疾病提供理论依据。     

过表达核糖核酸酶抑制因子通过调节ILK/PI3K/AKT信号途径抑制小鼠黑色素瘤B16-F10细胞体内外生长

核糖核酸酶抑制因子（ribonuclease inhibitor,RI）是胞浆内的一种酸性蛋白质.已有研究证明,RI与核糖核酸酶A（RNaseA）和血管生成素（angiogenin,ANG）结合可抑制其活性.本室前期实验证实,RI可有效抑制某些肿瘤的生长和转移. 然而,RI抑制肿瘤的分子机制尚不清楚. 本研究探讨RI对小鼠黑色素瘤B16-F10细胞生长和凋亡的影响及其机制. MTT法结合流式细胞术分析结果证明,RI基因稳定转染导致B16+F10黑色素瘤细胞S期阻滞,抑制B16-F10黑色素瘤细胞增殖. Annexin V/PI结合流式细胞术结果显示,RI过表达引起细胞凋亡.与此相一致,蛋白质印迹分析显示,过表达RI引起抗凋亡分子Bcl-2表达下调,而Bax上调,同时伴有Pro-casepase 3激活. C57BL/ 6小鼠移植成瘤实验显示,与对照相比,转染RI的B16-F10细胞形成的肿瘤重量显著减少,同时伴有肿瘤组织微血管密度降低.提示RI过表达能抑制微血管生成. 此外,体内外组织/细胞免疫化学和蛋白质印迹结果揭示,过表达RI可显著抑制整合素连接激酶(integrin-linked kinase,ILK)下游靶分子Akt和GSK-3β的磷酸化,并降低β-联蛋白的表达.研究结果证明,过表达RI可通过抑制ILK/ PI3K/AKT信号通路,促进细胞凋亡,引起S期阻滞,并抑制血管生成,从而显著抑制小鼠黑色素瘤B16-F10细胞在体内、外的生长.上述结果提示,RI可能是治疗黑色素瘤的有效分子靶点.     

尼古丁通过PI3K/Akt/mTOR通路抑制自噬诱导BEAS-2B细胞凋亡

吸烟是慢性呼吸系统炎症疾病最主要危险因素.尼古丁是烟草烟雾中最主要成分,与尼古丁相关的慢性呼吸系统疾病的发病率与日俱增.因此,寻找与尼古丁相关的慢性呼吸系统炎症疾病的潜在靶点是急需解决的问题.本研究旨在探究尼古丁对BEAS-2B细胞凋亡的影响及其潜在作用机制.采用蛋白质印迹法检测各组中的细胞内凋亡、自噬和PI3K/Ak...     

过表达MicroRNA-21通过PTEN/PI3K/AKT信号通路调控人退变髓核细胞自噬的研究

目的:探讨过表达mi R-21通过PTEN/PI3K/AKT通路对人退变髓核细胞自噬的影响。方法:构建稳定过表达mi R-21 mimic人退变髓核细胞,转染无意义序列作为mi R-21 mimic control组,采用RT-qPCR检测转染效率;利用MDC荧光染色法观察细胞自噬泡;Western-Blot检测细胞自噬相关蛋白LC3和P62的表达以及PTEN/PI3K/AKT信号通路中关键蛋白PTEN、PI3K及AKT的表达水平。结果:RT-qPCR结果表明mi R-21 mimic转染成功且效率较高,与mi R-21 mimic control组及空白细胞对照组相比,差异显著(P0.05)。荧光显微镜观察MDC染色情况,mi R-21 mimic组的细胞中几乎没有发现自噬体,而mi R-21 mimic control组以及空白对照组细胞中自噬体均较多,与前者相比差异均明显,具有统计学意义(P0.05)。mi R-21 mimic组细胞中LC3-II/LC3-I表达量的比值均显著低于mi R-21 mimic control组及空白对照组细胞(P0.05);而P62在mi R-21 mimic组细胞中表达量显著高于mi R-21 mimic control组及空白细胞对照组,具有统计学意义(P0.05)。mi R-21 mimic组中PTEN蛋白的表达水平较低,与另外两组相比具有统计学意义(P0.05);磷酸化的PI3K(p-PI3K)和AKT(p-Akt)在mi R-21 mimic组中均明显高于mi R-21 mimic control组和空白细胞对照组,差异具有统计学意义(P0.05)。结论:mi R-21可以通过靶向沉默PTEN,促进PI3K和AKT发生磷酸化,进而使PTEN/PI3K/AKT信号通路被激活,最终抑制人椎间盘退变髓核细胞的自噬。     

Overexpression of microRNA-23a-5p induces myocardial infarction by promoting cardiomyocyte apoptosis through inhibited of PI3K/AKT signalling pathway

Myocardial infarction (MI) leads to cardiac remodelling and heart failure. Cardiomyocyte apoptosis is considered a critical pathological phenomenon accompanying MI, but the pathogenesis mechanism remains to be explored. MicroRNAs (miRs), with the identity of negative regulator of gene expression, exist as an important contributor to apoptosis. During the experiment of this study, MI mice models were successfully established and sequencing data showed that the expression of miR-23a-5p was significantly enhanced during MI progression. Further steps were taken and it showed that apoptosis of cardiac cells weakened as miR-23a-5p was downregulated and on the contrary that apoptosis strengthened with the overexpression of miR-23a-5p. To explore its working mechanisms, bioinformatics analysis was conducted by referring to multi-databases to predict the targets of miR-23a-5p. Further analysis suggested that those downstream genes enriched in several pathways, especially in the PI3K/Akt singling pathway. Furthermore, it demonstrated that miR-23a-5p was negatively related to the phosphorylation of PI3K/Akt, which plays a critical role in triggering cell apoptosis during MI. Recilisib-activated PI3K/Akt singling pathway could restrain apoptosis from inducing miR-23a-5p overexpression, and Miltefosine-blocked PI3K/Akt singling pathway could restrict apoptosis from inhibiting miR-23a-5p reduction. In conclusion, these findings revealed the pivotal role of miR-23a-5p-PI3K/Akt axis in regulating apoptosis during MI, introducing this novel axis as a potential indicator to detect ischemic heart disease and it could be used for therapeutic intervention.     

Ropivacaine inhibits proliferation and invasion and promotes apoptosis and autophagy in bladder cancer cells via inhibiting PI3K/AKT pathway

Application of a certain concentration of local anesthetics during tumor resection inhibits the progression of tumor. The effects of ropivacaine in bladder cancer (BC) have never been explored. We explored the effects of ropivacaine on the progression of BC in vitro and in vivo. CCK8 assay and EDU staining was conducted to examine cell proliferation. Flow cytometry and transwell assay were performed to evaluate apoptosis and invasion, respectively. Expression of light chain 3 (LC3) was observed through immunofluorescence. Furthermore, the xenograft tumor model of BC was built to detect the effects of ropivacaine in vivo. IHC and TUNEL assay were conducted to detect cell proliferation and apoptosis in vivo. Ropivacaine inhibited the proliferation of T24 and 5639 cells with the 50% inhibitory concentration (IC50) of 20.08 and 31.86 µM, respectively. Ropivacaine suppressed the invasion ability and induces the apoptosis of cells. Besides, ropivacaine triggers obvious autophagy in BC cells. Moreover, ropivacaine blocks the PI3K/AKT signal pathway in BC cells. The impact of ropivacaine on cell viability, motility, and autophagy was reversed by 740 Y-P, the activator of PI3K/AKT signal pathway. The in vivo experiments demonstrated that ropivacaine inhibited the proliferation and mobility of BC. Ropivacaine has anti-carcinoma effects in BC via inactivating PI3K/AKT pathway, providing a new theoretical reference for the use of local anesthetics in the treatment of BC.     

miR-130a-3p调控SOX4对骨关节炎软骨细胞增殖、分化和炎症因子释放的影响

目的:探讨miR-130a-3p对骨关节炎(osteoarthritis, OA)软骨细胞增殖、分化和炎症因子释放的影响及作用机制。方法:收集我院住院的40例半月板损伤患者和40例OA患者,采用RT-PCR检测半月板损伤患者和OA患者膝关节软骨组织中miR-130a-3p的表达。在OA软骨细胞中分别转染miR-NC、miR-130a-3p mimics、miR-130a-3p inhibitors、si RNA-SOX4(si-SOX4)或过表达SOX4的慢病毒载体(LV-SOX4),采用CCK8法检测细胞增殖情况;RT-PCR和western blot检测细胞分化相关分子BMP2和BMP4;RT-PCR检测炎症因子IFN-γ和TNF-α的表达。结果:半月板损伤患者软骨组织和软骨细胞中miR-130a-3p的表达明显高于OA患者(P均0.05),而SOX4的表达水平明显低于OA患者(P0.05)。OA患者膝关节软骨组织中miR-130a-3p和SOX4的表达呈显著负相关(P0.05)。miR-130a-3p mimics能够明显促进OA软骨细胞增殖(P0.05)、增加分化相关分子BMP2和BMP4的表达(P均0.05)以及抑制炎症因子IFN-γ和TNF-α的表达(P均0.05)。miR-130a-3p inhibitors能够明显抑制OA软骨细胞增殖(P0.05)、分化相关分子BMP2和BMP4的表达(P均0.05)以及促进炎症因子IFN-γ和TNF-α的表达(P均0.05)。OA软骨细胞在转染miR-130a-3p mimics后,SOX4 mRNA和蛋白的表达水平明显降低(P均0.05),在转染miR-130a-3p inhibitors后,SOX4 mRNA和蛋白的表达水平明显升高(P均0.05)。双荧光素酶结果显示miR-130a-3p能够靶向结合SOX4。在OA软骨细胞中,采用si RNA低表达SOX4后,明显促进细胞增殖和分化以及抑制炎症因子的释放。共转染miR-130a-3p mimics和LV-SOX4能够逆转过表达miR-130a-3p对OA软骨细胞增殖、分化和炎症因子释放的影响(P均0.05),而共转染miR-130a-3p inhibitors和LV-SOX4能够进一步加强低表达miR-130a-3p对OA软骨细胞增殖、分化和炎症因子的影响(P均0.05)。结论:miR-130a-3p在OA中明显低表达,并能够通过靶向抑制SOX4促进OA软骨细胞增殖、分化和炎症因子释放。     

MiR-27a-3p enhances the cisplatin sensitivity in hepatocellular carcinoma cells through inhibiting PI3K/Akt pathway

MicroRNAs (miRNAs) play an important role in drug resistance, and it is reported that miR-27a-3p regulated the sensitivity of cisplatin in breast cancer, lung cancer and ovarian cancer. However, the relationship between miR-27a-3p and chemosensitivity of cisplatin in hepatocellular carcinoma (HCC) was unclear, especially the underlying mechanism was unknown. In the present study, we analyzed miR-27a-3p expression levels in 372 tumor tissues and 49 adjacent tissues in HCC samples from TCGA database, and found that the miR-27a-3p was down-regulated in HCC tissues. The level of miR-27a-3p was associated with metastasis, Child–Pugh grade and race. MiR-27a-3p was regarded as a favorable prognosis indicator for HCC patients. Then, miR-27a-3p was overexpressed in HepG2 cell, and was knocked down in PLC cell. Next, we conducted a series of in vitro assays, including MTT, apoptosis and cell cycle assays to observe the biological changes. Further, inhibitor rate and apoptosis rate were detected with pre- and post-cisplatin treatment in HCC. The results showed that overexpression of miR-27a-3p repressed the cell viability, promoted apoptosis and increased the percentage of cells in G₀/G₁ phase. Importantly, overexpression of miR-27a-3p significantly increased the inhibitor rate and apoptosis rate with cisplatin intervention. Besides, we found that miR-27a-3p added cisplatin sensitivity potentially through regulating PI3K/Akt signaling pathway. Taken together, miR-27a-3p acted as a tumor suppressor gene in HCC cells, and it could be useful for modulating cisplatin sensitivity in chemotherapy.     

ROS accumulation contributes to abamectin‐induced apoptosis and autophagy via the inactivation of PI3K/AKT/mTOR pathway in TM3 Leydig cells

Abamectin (ABA) as one of the worldwide used compounds in agriculture has raised safety concerns on nontarget organism toxicity. However, the study of male reproductive system damage caused by ABA remains unclear. Our aim is to investigate the effect of ABA‐induced cytotoxicity in TM3 Leydig cells and their underlying mechanisms. ABA inhibits TM3 cell viability and proliferation via cell cycle arrested in the G0/G1 phase. In addition, ABA‐induced mitochondrial depolarization leads to an imbalance in Bcl‐2 family expression, causing caspase‐dependent apoptosis in TM3 cells. The increased ratio of cells expression LC3 protein and LC3‐II to LC3‐I indicated the activation of autophagy potentially. Further experiments revealed ABA treatment reduced phosphatidylinositol 3‐kinase (PI3K), protein kinase B (AKT) phosphorylation, and mammalian target of rapamycin (mTOR) phosphorylation. Pretreatment with a PI3K/AKT inhibitor, LY294002, mimicked the ABA‐mediated effects on cytotoxicity. Pretreatment with a PI3K/AKT agonist, insulin‐like growth factor‐1, reversed the effects of ABA. ABA caused the accumulation of intracellular reactive oxygen species (ROS) by increased intensity of the ROS indicator. However, N‐acetylcysteine as ROS scavengers inhibited ABA‐induced apoptosis and autophagy and reversed these ABA‐mediated effects on PI3K/AKT/mTOR pathway. On the basis of the above results, it is suggested that ABA exposure induces apoptosis and autophagy in TM3 cells by ROS accumulation to mediate PI3K/AKT/mTOR signaling pathway suppression.     

PI3K/AKT/mTOR信号通路及其在卵巢癌治疗中的应用

卵巢癌是女性生殖系统常见的恶性肿瘤,发病率居于妇科恶性肿瘤第三位,死亡率居于妇科恶性肿瘤之首。目前对卵巢癌的标准治疗包括肿瘤细胞减灭术及卡铂和紫杉醇的联合化疗。PI3K/AKT/mTOR信号通路在卵巢癌的细胞增殖、侵袭、细胞周期进展、血管生成及耐药中发挥重要作用,是卵巢癌中最常发生改变的细胞内途径。本文对PI3K/AKT/mTOR信号通路及其在卵巢癌增殖和进展中的影响、PI3K/AKT/mTOR信号通路抑制剂在卵巢癌中的治疗应用做简要综述。     

MicroRNA-411a-3p过表达抑制Ca2+信号转导与羊驼黑色素细胞的增殖和迁移

黑色素细胞中产生的黑色素转移及黑色素细胞的增殖和迁移均与色素沉积有关。黑色素细胞的增殖需要有丝分裂原协同进行。黑色素细胞的增殖和分化受组织环境以及多种毛色基因的调控。miRNA-411a-3p在不同毛色羊驼皮肤中呈差异表达,且通过靶向IGF1R调控黑色素生成。但miRNA-411a-3p是否与黑色素颗粒迁移、黑色素细胞的增殖和迁移相关未见报道。本研究通过miRNA-411a-3p转染羊驼黑色素细胞后发现,与对照组相比,钙离子信号转导水平下降了（91.73±1.53）% (P<0.01),与黑色素转移有关的Rab27a和肌球蛋白Va在蛋白质水平的表达均被下调,同时与细胞增殖有关的整联蛋白β1和β5相关基因在转录水平分别下降了（44.67 ± 13.67）%（P<0.01）和（30.72 ± 6.23）% (P<0.01),在蛋白质水平表达下降了（45.18 ± 1.96）% (P<0.001)和（11.52 ± 1.09）% (P<0.001)。综上所述,miRNA-411a-3p过表达后会抑制钙离子信号转导,以及羊驼黑色素细胞的增殖和迁移。     

MicroRNA-411a-3p过表达抑制Ca2+信号转导与羊驼黑色素细胞的增殖和迁移

黑色素细胞中产生的黑色素转移及黑色素细胞的增殖和迁移均与色素沉积有关。黑色素细胞的增殖需要有丝分裂原协同进行。黑色素细胞的增殖和分化受组织环境以及多种毛色基因的调控。miRNA-411a-3p在不同毛色羊驼皮肤中呈差异表达,且通过靶向IGF1R调控黑色素生成。但miRNA-411a-3p是否与黑色素颗粒迁移、黑色素细胞的增殖和迁移相关未见报道。本研究通过miRNA-411a-3p转染羊驼黑色素细胞后发现,与对照组相比,钙离子信号转导水平下降了（91.73±1.53）% (P<0.01),与黑色素转移有关的Rab27a和肌球蛋白Va在蛋白质水平的表达均被下调,同时与细胞增殖有关的整联蛋白β1和β5相关基因在转录水平分别下降了（44.67 ± 13.67）%（P<0.01）和（30.72 ± 6.23）% (P<0.01),在蛋白质水平表达下降了（45.18 ± 1.96）% (P<0.001)和（11.52 ± 1.09）% (P<0.001)。综上所述,miRNA-411a-3p过表达后会抑制钙离子信号转导,以及羊驼黑色素细胞的增殖和迁移。