磷脂酰肌醇-3-激酶/蛋白激酶B/雷帕霉素靶蛋白信号转导研究进展及与自身免疫病的关系

磷脂酰肌醇-3-激酶/蛋白激酶B/雷帕霉素靶蛋白(PI3K/Akt/mTOR)信号通路介导许多在肿瘤和自身免疫病发生中至关重要的细胞生物学过程,对于细胞增殖、细胞活化、血管生成起到重要作用.PI3 K/Akt/mTOR信号通路作为药物治疗靶点进行抗细胞增殖活化的研究近年来发展迅速.本文从PI3K/Akt/mTOR信号通路的上下游、信号通路抑制剂以及与信号通路相关的自身免疫病等方面作一综述.     

异硫氰酸苯乙酯对结肠癌SW480细胞PI3K/Akt/mTOR信号通路的影响

目的探讨异硫氰酸苯乙酯(PEITC)对结肠癌SW480细胞PI3K/Akt/mTOR信号通路的影响。方法选择6~8周龄的SPF级BALB/c(nu/nu)雄性裸鼠24只,将其随机分为对照组、低剂量组、中剂量组和高剂量组,各6只。体外培养结肠癌细胞系SW480,对照组加入等体积的二甲基亚砜(DMSO),低剂量组、中剂量组和高剂量组分别给予10、30和50μmol/L PEITC作用24 h,Western blot检测PI3K和PTEN的表达和Akt、mTOR磷酸化情况。最后,建立裸鼠异种移植瘤动物模型,观察PEITC对异种移植瘤生长的抑制作用。结果 PEITC能抑制PI3K的表达以及Akt和mTOR磷酸化。肿瘤质量:高剂量组中剂量组低剂量组对照组,抑瘤率:高剂量组中剂量组低剂量组(P0.05)。结论 PEITC可能通过影响PI3K/Akt/mTOR通路发挥抗肿瘤作用。     

抑制PI3K/Akt/mTOR信号通路对兔原代巨噬细胞自体吞噬的影响及机制

目的 探讨抑制PI3K/Akt/mTOR信号通路对兔原代巨噬细胞自体吞噬中的影响。方法 分离培养纯种新西兰兔腹腔原代巨噬细胞并分为4组,加入磷脂酰肌醇3激酶（PI3K）抑制剂LY294002（10 μmol/L）组、哺乳动物雷帕霉素靶蛋白（mTOR）抑制剂雷帕霉素（10 nmol/L）组、蛋白激酶B（Akt）抑制剂曲西立滨组（20 μmol/L）以及空白对照组。共培养4 h、12 h后分别收集细胞,运用透射电镜观察巨噬细胞自噬体的变化,细胞免疫荧光法检测微管相关蛋白轻链3Ⅱ（LC3Ⅱ）分子的表达,Western blot检测 Akt、mTOR、磷酸化Akt（p-Akt）、磷酸化mTOR（p-mTOR）及自噬相关蛋白Beclin-1和自噬蛋白Atg5-Atg12连接体的表达,单丹酰尸胺（MDC）染色法观察自噬溶酶体的变化。结果 与空白对照组相比,透射电镜下LY294002组自噬体、自噬空泡、髓磷脂图像等自噬标记物明显减少,雷帕霉素组、曲西立滨组明显增多;激光共聚焦显微镜下LY294002组LC3Ⅱ表达显著减少,雷帕霉素组、曲西立滨组表达显著增多;Western blot结果显示LY294002组Beclin-1及Atg5-Atg12蛋白表达水平显著下降,p-mTOR、p-Akt蛋白表达显著减少;雷帕霉素组、曲西立滨组Beclin-1及Atg5-Atg12蛋白表达水平明显上调,共培养4 h后p-Akt表达增多,雷帕霉素组p-mTOR表达增多,曲西立滨组减少;共培养12 h后雷帕霉素组、曲西立滨组p-mTOR表达显著减少,雷帕霉素组p-Akt表达显著增多,曲西立滨组显著减少;MDC染色显示LY294002组自噬溶酶体明显减少,雷帕霉素组、曲西立滨组明显增多。结论 抑制PI3K/Akt/mTOR信号通路能促进兔原代巨噬细胞自体吞噬,抑制PI3K能减少兔原代巨噬细胞自体吞噬,可能是不同类型的PI3K分子通过其他通路起作用。     

非小细胞肺癌中PI3K/Akt信号通路通过Cdh1调控Skp2表达的机制研究

目的探讨非小细胞肺癌（NSCLC）中Cdc20同源蛋白1（Cdh1）参与磷脂酰肌醇三羟基激酶（PI3K）/Akt信号通路对S期激酶相关蛋白2（Skp2）表达调控的机制。方法体外培养NSCLC细胞系A549、LK2和H460,LY294002特异性阻断PI3K/Akt信号通路后,Western blot检测Skp2、Cdh1及p-Akt蛋白表达的变化,免疫荧光（IF）检测Cdh1在NSCLC中的定位变化。结果 LY294002处理后,与对照组相比3种细胞中Skp2蛋白表达和Akt磷酸化水平均降低（P〈0.01）,Cdh1在3种细胞的核内表达均增多。结论 NSCLC中PI3K/Akt信号通路抑制剂LY294002使Skp2蛋白表达下调与Cdh1由细胞质向细胞核转位有关。     

黄连温胆汤调控PI3K/Akt/mTOR信号通路干预ApoE-/-小鼠动脉粥样硬化的作用机制

目的观察黄连温胆汤对载脂蛋白E(ApoE)^-/-小鼠动脉粥样硬化的干预效应,从磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物西罗莫司靶蛋白(mTOR)信号通路探讨其机制。方法采用高脂饲料饲养ApoE^-/-小鼠4周构建动脉粥样硬化模型。采用全自动生化分析仪检测小鼠血脂水平;采用苏木精-伊红(HE)染色、油红O染色、Masson染色观察主动脉斑块情况;采用免疫组化法检测小鼠主动脉根部PI3K、Akt、mTOR蛋白表达水平。结果与模型组比较,黄连温胆汤高剂量组总胆固醇(TC)、三酰甘油(TG)、低密度脂蛋白胆固醇(LDL-C)均降低(P<0.05)。HE染色显示:模型组主动脉根部斑块明显,呈易损斑块特征,各给药组斑块有不同程度减轻;油红O和Masson染色显示:与模型组比较,雷帕霉素组、黄连温胆汤高剂量组主动脉根部斑块面积减小,胶原纤维含量升高(P<0.05),易损斑块趋于稳定。蛋白免疫组化结果显示:黄连温胆汤中剂量组、黄连温胆汤高剂量组小鼠主动脉PI3K、Akt、mTOR蛋白表达水平降低(P<0.05)。结论黄连温胆汤具有降脂和抗动脉粥样硬化的作用,其机制可能与抑制PI3K/Akt/mTOR信号通路,诱导巨噬细胞自噬有关。     

PI3K/Akt/mTOR信号通路抑制剂在肾癌治疗中的应用进展

磷脂酰肌醇3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白（PI3K/Akt/mTOR）信号通路是细胞内重要的信号转导通路之一，可以调节细胞生长、分化、迁移、存活、血管生成和代谢，并参与多种恶性肿瘤的发生发展。肾癌（RCC）作为泌尿系统常见的恶性肿瘤，有较高的转移率，且对放化疗具有明显的耐受性，随着肿瘤分子生物学的发展，肾癌靶向生物治疗逐渐成为研究的热点。PI3K/Akt/mTOR信号通路抑制剂对RCC的疗效显著，以该信号通路为靶点的RCC药物主要为PI3K抑制剂，Akt抑制剂和mTOR抑制剂，这些药物可通过抑制信号通路的异常激活，阻断肿瘤细胞的增殖和生长，诱导细胞凋亡，并抑制肿瘤的侵袭和血管生成。而PI3K、mTOR双重抑制剂虽然处于临床试验阶段，但是其在RCC治疗的研究中表现出更可靠的前景性。     

5-氮胞苷体外诱导成人脂肪间充质干细胞分化为心肌样细胞的初步研究

目的:探索不同浓度5-氮胞苷(5-aza)在不同诱导时间下对成人脂肪间充质干细胞(ADMSCs)分化为心肌细胞的诱导作用,确定最佳诱导条件。方法:体外分离成人脂肪组织来源的间充质干细胞并进行传代培养,流式细胞仪鉴定细胞CD44、CD34的表达,用含不同浓度5-aza(3、5、10、15、20μmol／L)培养基分别诱导12小时、24小时、48小时、72小时,倒置相差显微镜下逐日观察细胞形态变化;于诱导后14、28天时免疫细胞化学染色鉴定心肌特异性肌钙蛋白I(cTn-I)的表达,分析细胞转化率。结果:分离培养的ADMSCs表达CD44,不表达CD34;5-aza诱导后14天时免疫细胞化学未见有心肌细胞特异性cTn- I表达,诱导28天细胞免疫细胞化学显示cTn-I表达阳性,以10μmol/L 5-aza诱导24小时为最佳体外诱导条件。结论:成人脂肪组织间充质干细胞可在体外5-aza诱导下向心肌细胞分化。     

慢性乙型肝炎患者肝组织PI3K/Akt/mTOR信号通路与HBV病毒载量及肝纤维化程度关系

目的:探讨慢性乙型肝炎(CHB)患者肝组织中磷脂肌醇3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白(PI3K/Akt/mTOR)信号通路与乙型肝炎病毒(HBV DNA)载量及肝纤维化程度的关系。方法:选取2019年4月至2021年4月经肝穿刺的CHB患者74例,另选取同期接受肝穿刺检查的非酒精性脂肪性肝病患者20例作为对照组,采用免疫组化法检测肝组织中PI3K、Akt和mTOR表达水平,分析三者与血清HBV DNA载量和肝纤维化程度的关系。结果:CHB组患者PI3K、Akt、mTOR阳性表达率分别为64.86%、85.14%和75.68%,均显著高于对照组(P<0.05)。根据肝组织活检结果,CHB患者肝纤维化S0～S4级分别为12、31、25、3、3例,各级患者间肝组织PI3K、Akt、mTOR阳性表达率比较,差异有统计学差异(P<0.05),肝纤维化分级升高与PI3K、Akt、mTOR阳性表达率呈正相关。根据HBV DNA载量将CHB组分为4组,HBV DNA<10⁴ IU/ml组、10⁴～10⁵ I...     

PI3K/Akt/mTOR信号通路与低氧性肺动脉高压研究进展

肺动脉高压是一种预后不良的疾病,在我国因各种肺部慢性疾病引起的低氧性肺动脉高压近年来呈上升发病趋势.磷脂酰肌醇3-激酶(phosphatidylionsitol-3-kinases,PI3K)/蛋白质丝氨酸苏氨酸激酶(protein-serine-threonine kinase,Akt)/哺乳动物雷帕霉素蛋白(mammalian target of rapamycin,mTOR)信号通路作为细胞内重要信号转导通路之一,通过影响细胞的增殖、迁移、凋亡以及蛋白合成、细胞周期等活性参与肺动脉血管重塑以及低氧性肺动脉高压的形成.本文综述了PI3K/Akt/mTOR信号通路在低氧性肺动脉高压中的研究现状,以期为低氧性肺动脉高压的发病机制研究及治疗寻找新的思路.     

PI3K/AKT信号通路在RANKL诱导的SGC-7901胃癌细胞迁移中的作用

目的文献报道RANKL/RANK/OPG途径与肿瘤细胞迁移及骨转移密切相关,但RANKL/RANK途径是否参与胃癌细胞迁移,尚无文献报道。本文拟检测RANK在胃癌细胞系SGC-7901细胞中的表达,并进一步探讨磷脂酰肌醇-3-激酶/丝苏氨酸蛋白激酶(PI3K/Akt)信号通路在RANKL诱导的胃癌细胞迁移中的作用。方法 West-ern blot检测SGC-7901细胞表面RANK蛋白的表达;RANKL刺激后磷酸化Akt(P-Akt)及Akt的表达;Transwell法测定RANKL及抑制剂刺激后细胞迁移能力的改变。结果 SGC7901细胞表达RANK蛋白。RANKL(1μg/mL)诱导SGC-7901细胞迁移能力增强,迁移增加率为57.2%±5.9%,RANKL抑制剂rOPG(5μg/mL)显著抑制RANKL诱导的细胞迁移(13.88%±3.57%,P<0.05)。RANKL刺激后30 min～3 h,SGC-7901细胞p-Akt表达升高,应用PI3K的抑制剂LY294002(50 mmol/L)显著抑制RANKL诱导的胃癌细胞SGC-7901的迁移(57.28%±5.91%vs23.18%±2.79%,P<0.05)。结论胃癌细胞系SGC-7901细胞表达受体RANK,PI3K/Akt信号通路参与RANKL诱导的SGC-7901细胞迁移。     

Short-term phytohaemagglutinin-activated mononuclear cells induce endothelial progenitor cells from cord blood CD34+ cells

Endothelial progenitor cells (EPCs) were recently demonstrated to exist in human cord blood. Phytohaemagglutinin (PHA), a potent mitogen for mononuclear cells was used to induce EPCs from unsorted cord blood mononuclear cells (CBMCs). Adherent cells in clusters appeared approximately 24 h after CBMCs were cultured in plain Roswell Park Memorial Institute media containing 10% fetal bovine serum (culture media) and PHA. Adherent cells were further propagated for 1 week in plain culture media. Flow cytometry and Di-I staining analyses showed that CD45-, CD34+, Flk-1+, CD31+ or VE-cadherin+ EPCs were induced and that they were mainly from the CD34+ cell compartment. When enriched CD34+ cells alone were stimulated with culture supernatant of the PHA-activated CBMCs, they neither proliferated readily nor induced EPCs. Because EPCs first appeared within the clustering cells that expressed high levels of fibronectin and vascular endothelial growth factor (VEGF), our data suggest that both cell-cell/cell-matrix interaction and the local VEGF action are important in the induction of EPCs. Thus, we demonstrate for the first time that EPCs are induced from human cord blood stem cell populations that interact with neighbouring PHA-activated CBMCs. This finding may have a significant implication in inflammatory cell-mediated vasculogenesis and angiogenesis in vivo.     

Exercise acutely increases circulating endothelial progenitor cells and monocyte-/macrophage-derived angiogenic cells

OBJECTIVES: We investigated whether a single episode of exercise could acutely increase the numbers of endothelial progenitor cells (EPCs) and cultured/circulating angiogenic cells (CACs) in human subjects. BACKGROUND: Endothelial progenitor cells and CACs can be isolated from peripheral blood and have been shown to participate in vascular repair and angiogenesis. We hypothesized that exercise may acutely increase either circulating EPCs or CACs. METHODS: Volunteer subjects (n = 22) underwent exhaustive dynamic exercise. Blood was drawn before and after exercise, and circulating EPC numbers as well as plasma levels of angiogenic growth factors were assessed. The CACs were obtained by culturing mononuclear cells and the secretion of multiple angiogenic growth factors by CACs was determined. RESULTS: Circulating EPCs (AC133+/VE-Cadherin+ cells) increased nearly four-fold in peripheral blood from 66 +/- 27 cells/ml to 236 +/- 34 cells/ml (p < 0.05). The number of isolated CACs increased 2.5-fold from 8,754 +/- 2,048 cells/ml of peripheral blood to 20,759 +/- 4,676 cells/ml (p < 0.005). Cultured angiogenic cells isolated before and after exercise showed similar secretion patterns of angiogenic growth factors. CONCLUSIONS: Our study demonstrates that exercise can acutely increase EPCs and CACs. Given the ability of these cell populations to promote angiogenesis and vascular regeneration, the exercise-induced cell mobilization may serve as a physiologic repair or compensation mechanism.     

Ex vivo culture of Fancc-/- stem/progenitor cells predisposes cells to undergo apoptosis, and surviving stem/progenitor cells display cytogenetic abnormalities and an increased risk of malignancy

Current strategies for genetic therapy using Moloney retroviruses require ex vivo manipulation of hematopoietic cells to facilitate stable integration of the transgene. While many studies have evaluated the impact of ex vivo culture on normal murine and human stem/progenitor cells, the cellular consequences of ex vivo manipulation of stem cells with intrinsic defects in genome stability are incompletely understood. Here we show that ex vivo culture of Fancc(-/-) bone marrow cells results in a time-dependent increase in apoptosis of primitive Fancc(-/-) progenitor cells in conditions that promote the proliferation of wild-type stem/progenitor cells. Further, recipients reconstituted with the surviving Fancc(-/-) cells have a high incidence of cytogenetic abnormalities and myeloid malignancies that are associated with an acquired resistance to tumor necrosis factor alpha (TNF-alpha). Collectively, these data indicate that the intrinsic defects in the genomic stability of Fancc(-/-) stem/progenitor cells provide a selective pressure for cells that are resistant to apoptosis and have a propensity for the evolution to clonal hematopoiesis and malignancy. These studies could have implications for the design of genetic therapies for treatment of Fanconi anemia and potentially other genetic diseases with intrinsic defects in genome stability.     

Differentiation and expansion of endothelial cells from human bone marrow CD133+ cells

We report a method of purifying, characterizing and expanding endothelial cells (ECs) derived from CD133(+) bone marrow cells, a subset of CD34(+) haematopoietic progenitors. Isolated using immunomagnetic sorting (mean purity 90 +/- 5%), the CD133(+) bone marrow cells were grown on fibronectin-coated flasks in M199 medium supplemented with fetal bovine serum (FBS), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and insulin growth factor (IGF-1). The CD133(+) fraction contained 95 +/- 4% CD34(+) cells, 3 +/- 2% cells expressing VEGF receptor (VEGFR-2/KDR), but did not express von Willebrand factor (VWF), VE-cadherin, P1H12 or TE-7. After 3 weeks of culture, the cells formed a monolayer with a typical EC morphology and expanded 11 +/- 5 times. The cells were further purified using Ulex europaeus agglutinin-1 (UEA-1)-fluorescein isothiocyanate (FITC) and anti-FITC microbeads, and expanded with VEGF for a further 3 weeks. All of the cells were CD45(-) and CD14(-), and expressed several endothelial markers (UEA-1, VWF, P1H12, CD105, E-selectin, VCAM-1 and VE-cadherin) and typical Weibel-Palade bodies. They had a high proliferative potential (up to a 2400-fold increase in cell number after 3 weeks of culture) and the capacity to modulate cell surface antigens upon stimulation with inflammatory cytokines. Purified ECs were also co-cultivated with CD34(+) cells, in parallel with a purified fibroblastic cell monolayer. CD34(+) cells (10 x 10(5)) gave rise to 17,951 +/- 2422 CFU-GM colonies when grown on endothelial cells, and to 12,928 +/- 4415 CFU-GM colonies on fibroblast monolayers. The ECs also supported erythroid blast-forming unit (BFU-E) colonies better. These results suggest that bone marrow CD133(+) progenitor cells can give rise to highly purified ECs, which have a high proliferative capacity, can be activated by inflammatory cytokines and are superior to fibroblasts in supporting haematopoiesis. Our data support the hypothesis that endothelial cell progenitors are present in adult bone marrow and may contribute to neo-angiogenesis.     

Wnt-mediated self-renewal of neural stem/progenitor cells

In this work we have uncovered a role for Wnt signaling as an important regulator of stem cell self-renewal in the developing brain. We identified Wnt-responsive cells in the subventricular zone of the developing E14.5 mouse brain. Responding cell populations were enriched for self-renewing stem cells in primary culture, suggesting that Wnt signaling is a hallmark of self-renewing activity in vivo. We also tested whether Wnt signals directly influence neural stem cells. Using inhibitors of the Wnt pathway, we found that Wnt signaling is required for the efficient cloning and expansion of single-cell derived populations that are able to generate new stem cells as well as neurons, astrocytes, and oligodendrocytes. The addition of exogenous Wnt3a protein enhances clonal outgrowth, demonstrating not only a critical role for the Wnt pathway for the regulation of neurogenesis but also its use for the expansion of neural stem cells in cell culture and in tissue engineering.     

Thrombopoietin stimulation of hematopoietic stem/progenitor cells

The recent cloning of the thrombopoietin gene, and the production of recombinant protein, have allowed studies on both its biological actions and clinical utility. Thrombopoietin not only affects the cells of the megakaryocytic lineage, but has a diverse set of cellular targets. In particular, it stimulates the ex vivo expansion of hematopoietic stem/progenitor cells suggesting that it may play a role in transplantation studies. Pre-clinical but limited clinical studies indicate that under defined conditions, thrombopoietin may accelerate white blood cell count and platelet recoveries following myelosuppression or radiotherapy.