LY294002对胆管癌细胞凋亡的影响

目的：观察PI3-Kinase抑制剂LY294002对人胆管癌细胞QBC939的作用。
方法：MTT法检测LY294002对QBC939细胞的生长抑制作用;流式细胞术检测LY294002诱导的细胞凋亡;免疫印迹法分析LY294002对于PI3-K/Akt信号通路中Akt磷酸化及caspase9的表达作用。
结果：LY294002可抑制胆管癌细胞QBC939的增殖,促进其凋亡。Western-blotting显示,LY2940022可抑制Akt磷酸化水平,促进caspase3,9的蛋白表达。
结论：LY294002可通过抑制Akt信号通路的活性,上调促凋亡分子caspase3,9的表达,抑制胆管癌细胞QBC939的增殖,诱导其凋亡。

     

葛根素通过NF-кB信号通路调节破骨细胞的分化

目的探索葛根素抑制破骨细胞分化的实验研究。方法 RAW264.7细胞经sRANKL+M-CSF诱导7 d后向破骨细胞分化;WST-1检测不同浓度的葛根素对破骨细胞增殖活性的影响;TRAP染色检测葛根素对破骨细胞分化的影响;Osteoassay surface Mltiple well plate检测破骨细胞吸收活性;免疫荧光检测葛根素对NF-кB通路核心蛋白p65细胞核内转移的影响;Western blot检测葛根素对p65表达的影响。结果葛根素(10~(-8)mol/L)作用3 h后破骨细胞的增殖活性明显抑制,葛根素抑制破骨细胞的分化和吸收活性,免疫荧光检测显示葛根素抑制p65的细胞核内转位及p65的磷酸化。结论葛根素通过NF-кB信号通路抑制破骨细胞的分化。     

低分子量褐藻糖胶对破骨细胞凋亡的影响

目的探讨低分子量褐藻糖胶(LMWF)对小鼠单核细胞RAW264.7诱导成熟破骨细胞凋亡的影响。方法通过100ng/m L RANKL诱导RAW264.7细胞株分化为破骨细胞,经TRAP特异性染色和骨吸收陷窝对诱导后的细胞进行鉴定。鉴定成功后,用100 ng/m L RANKL诱导RAW264.7细胞株5 d后,使用含有LMWF的培养基继续培养3 d,通过对TRAP阳性细胞计数和分析骨吸收面积来观察低分子量褐藻糖胶对破骨细胞的抑制和骨吸收功能情况;采用流式细胞术检测LMWF对破骨细胞凋亡的影响,capsase-3活性测试试剂盒检测LMWF对capsase-3活性进行测定;RT-PCR检测LMWF对成熟破骨细胞BAX与BCL-2基因表达的影响。结果单纯采用100 ng/m L的RANKL可成功诱导成熟的、有功能的破骨细胞。LMWF可以明显抑制RANKL诱导成熟破骨细胞的形成以及成熟破骨细胞的骨吸收功能;流式细胞术显示LMWF可增加成熟破骨细胞的早期凋亡率;并且能升高capsase-3的活性;PCR显示LMWF可明显下调破骨细胞凋亡相关的BCL-2和上调BAX基因mRNA表达,降低BCL-2/BAX的比值。结论低分子量褐藻糖胶可抑制破骨细胞的活性与骨吸收能力,促进破骨细胞凋亡,其主要机制是通过下调BCL-2和上调BAX mRNA基因表达实现的。     

白细胞介素-21对RAW264.7细胞向破骨细胞分化的影响

目的 了解白细胞介素-21 (IL-21)诱导小鼠巨噬细胞系RAW264.7细胞向破骨细胞分化的作用,并探讨其可能的机制。方法1、以不同浓度IL-21(0,1,10,20,40 ng/ml)处理RAW264. 7细胞,培养5天后进行抗酒石酸酸性磷酸酶(TRAP)染色,Western Blot和免疫组化法检测降钙素受体（CTR)表达,采用Real time PCR法检测CTR和组织蛋白酶（Cathepsin)-K的 mRNA表达水平。2、以信号通路抑制剂AG490、LY294002和PD98059作用30min后再加人IL-21,培养5天后观察破骨细胞形成情况,以Western Blot检测IL-21作用不同时间点时信号通路分子总蛋白和磷酸化蛋白水平,探讨IL-21直接诱导破骨细胞分化的作用机制。结果1、在无RANKL作用的情况下,随着IL-21浓度增加TRAP阳性细胞数逐渐增多,20 ng/ml作用最强,40ng/ml时减弱。IL-21能够诱导破骨标志分子CTR和Cathepsin-K mRNA水平表达上调。Western Blot和免疫细胞化学证实,与阴性组比较,IL-21能促进CTR蛋白水平表达增高。2、PI3K-AKT通路抑制剂（LY294002)可以显著抑制IL-21诱导的 RAW264. 7细胞向破骨细胞分化,IL-21刺激5 ~ 15 min时p-AKT表达增强。结论 IL-21可不依赖RANKL直接诱导小鼠巨噬细胞系RAW264. 7细胞向破骨细胞分化,该作用可能由PI3K-AKT通路介导。     

JNK抑制剂SP600125对骨质疏松症保护作用的实验研究

目的 探讨JNK抑制剂SP600125对骨质疏松症模型成骨细胞和破骨细胞分化及功能的影响。方法 使用CCK8试验检测RAW264.7细胞、BMMs细胞和成骨细胞的增殖活性;使用相关染色试验探究JNK抑制剂SP600125的成骨化作用和对破骨细胞分化及功能的影响;使用RT-PCR分别检测BMMs细胞中破骨细胞及成骨细胞特异性基因mRNA表达水平;使用蛋白印迹试验检测RAW264.7细胞中JNK通路和NF-κB通路的活化水平;使用DCFH-DA法检测RAW264.7细胞中ROS水平。结果 CCK8试验结果显示,当SP600125浓度≤20 μmmol/L,对RAW264.7细胞、BMMs细胞和成骨细胞的增殖活性无显著影响;SP600125不影响成骨细胞钙结节的形成并抑制破骨细胞分化和功能;SP600125抑制的破骨细胞特异性基因的表达但不改变成骨细胞特异性基因的表达;SP600125抑制RANKL诱导的RAW264.7细胞中JNK通路和NF-κB通路的激活及ROS水平的升高。结论 JNK抑制剂SP600125能够抑制破骨细胞的分化及骨吸收功能,但对成骨细胞的分化无显著影响。     

CTGF对破骨前体细胞RAW264.7增殖及碳酸酐酶Ⅱ分化的影响

目的研究结缔组织生长因子(CTGF)对体外培养的破骨细胞前体细胞RAW264.7增殖及对核因子Kappa B配体受体(RANKL)诱导体外培养的破骨细胞前体细胞RAW264.7分化为成熟多核破骨细胞的影响。方法使用200 ng/mLCTGF干预培养的破骨细胞前体细胞RAW264.7,采用3H-TdR掺入法检测RAW264.7细胞增殖率;使用200 ng/mL CTGF与RANKL单独或共同处理RAW264.7细胞,抗酒石酸酸性磷酸酶(TRAP)染色观察TRAP阳性多核细胞,Western blot检测碳酐酶Ⅱ蛋白的表达。结果 CTGF可显著促进RAW264.7细胞增殖;200 ng/mLCTGF与RANKL共同处理RAW264.7细胞可促进RAW264.7细胞分化为成熟多核破骨细胞;200 ng/mL CTGF与RANKL共同处理RAW264.7细胞可促进RAW264.7细胞碳酐酶Ⅱ蛋白的表达。结论 CTGF促进体外培养的破骨细胞前体细胞RAW264.7增殖,促进RANKL诱导的破骨细胞前体细胞RAW264.7分化为成熟多核破骨细胞。     

血小板源生长因子通过PI3K/AKT途径促肝星状细胞增殖与Ⅰ型胶原合成

目的研究磷脂酰肌醇3激酶（P13K）特异性的抑制剂LY294002对血小板源生长因子（PDGF）诱导的肝星状细胞增殖与I型胶原合成的影响，并初步探讨P13K信号通路在PDGF诱导肝星状细胞活化中的机制。方法HSC细胞用LY294002和PDGF—BB共同孵育处理。收集细胞和培养液上清。细胞增殖用噻唑蓝（MTr）法检测，I型胶原蛋白用ELISA法检测，逆转录-聚合酶链反应（RT—PCR）法检测I型胶原mRNA的表达。利用P13K通路下游效应物AKT作为活化指标，ELISA法检测AKT和磷酸化AKT蛋白的表达。结果PDGF—BB能激活P13K通路，并导致AKT磷酸化。LY294002抑制P13K信号通路后，PDGF—BB刺激的HSC—T6增殖与促I型胶原合成作用被抑制。结论PDGF促HSC增殖和I型胶原合成是通过P13K信号通路，其作用可以被P13K抑制剂LY294002抑制。     

高糖通过PI3K-AKT-mTOR信号通路增加足细胞自噬

目的 研究高糖引起足细胞自噬变化及其相关的信号机制.方法 培养的足细胞被分为6组,正常浓度葡萄糖(NG)组、高浓度葡萄糖(HG)组、NG+雷帕霉素(Rap)组、HG+Rap组、NG+LY294002组和HG+LY294002组.观察自噬增强剂Rap和PI3K抑制剂LY294002对高糖条件下培养的足细胞自噬和凋亡的影响.电镜和吖啶橙染色观察细胞内自噬体的形成;Western印迹检测自噬标志蛋白微管相关蛋白1轻链3(LC3)和自噬血管基因Beclin-1的表达;通过阻断自噬的信号通路观察磷脂酰肌醇3激酶-蛋白激酶B-哺乳动物雷帕霉素靶蛋白(PI3K-AKT-mTOR)相关蛋白AKT和mTOR的磷酸化水平的改变.结果 高糖可导致足细胞凋亡增加,促进足细胞内自噬体和自噬相关蛋白表达增加(均P＜ 0.05).与高糖组相比,HG+ Rap组LC3-Ⅱ和Beclin-1的表达增加(均P＜0.05);LY294002部分抑制高糖导致的LC3-Ⅱ和Beclin-1表达增加(均P＜0.05).与高糖组相比,HG+ LY294002组足细胞内AKT磷酸化的水平增加(P＜0.05),mTOR的磷酸化水平降低(P＜0.01);HG+ LY294002组足细胞的AKT和mTOR磷酸化水平较高糖组均降低(均P＜0.05).结论 高糖可促进足细胞的自噬和凋亡,推测高糖诱导的足细胞自噬作用部分通过PI3K-AKT-mTOR信号通路调节实现的.     

Bcl 2和Caspase 3调控他莫昔芬诱导的ER阴性乳腺癌细胞凋亡

目的研究Bcl-2和Caspase-3在他莫昔芬（TAM）诱导ER阴性乳腺癌细胞凋亡中的调控作用。方法在体外培养条件下，用浓度为10μmol／L的TAM作用于雌激素受体（ER）阴性的MDA-MB-231人乳腺癌细胞株12，24，36，48，60h；流式细胞仪检测细胞凋亡率及Bcl-2和Bax的蛋白表达，荧光分光光度仪检测Caspase-3活性，以及加入Caspase-3活性抑制剂Ac—DEVD—CHO后凋亡百分率的变化。结果TAM作用ER阴性乳腺癌细胞后，Bcl-2表达下调，Caspase-3活性增强，细胞凋亡率增加．且有时间依赖性，细胞凋亡在48h达高峰。Bcl-2表达水平与Caspase-3活性变化成负相关（r=-0．921，P〈0．05），但Bax蛋白在药物处理前后无明显变化。加入Ac—DEVD—CHO后，能阻断Caspase-3活化而抑制TAM诱导细胞凋亡。结论TAM通过下调Bcl-2表达而经线粒体途径诱导ER阴性乳腺癌细胞凋亡，Caspase-3的激活在此过程中发挥重要作用。     

姜黄素与LY294002联合应用对前列腺癌PC-3细胞体外生长的影响

【摘要】 目的〓探讨姜黄素与PI3K/Akt抑制剂LY294002联合应用对前列腺癌PC-3细胞体外生长的影响。方法〓根据给药不同将实验分为对照组、姜黄素组、LY294002组和姜黄素组+LY294002联合组,分别加入等量的培养基、25 μmoL/L姜黄素、25 μmoL/L的LY294002和两种混合液。采用MTS法检测各组细胞的增殖情况、流式细胞仪术检测各组细胞凋亡情况、q-PCR测定各组细胞中NF-κB、P53及caspase-9的表达情况。结果〓姜黄素组、LY组和联合组的细胞增值率均较对照组明显降低,且联合组明显低于两个单独用药组(P<0.05)。姜黄素与LY294002联合作用前列腺癌PC-3细胞后,细胞总凋亡率较姜黄素及LY294002单独使用后明显增加(P值均<0.05)。联合用药组的NF-κB表达量明显低于两单独用药组(P<0.05),而P53和caspase-9的表达量均明显高于两单独用药组(P<0.05)。结论〓姜黄素与LY294002联合使用较两种药物单独使用更能有效抑制前列腺癌PC-3细胞的体外生长,作用机制可能是通过抑制NF-κB的表达从而抑制细胞增殖,并增加P53和caspase-9的表达从而促进细胞凋亡来实现的。     

RANKL regulates Fas expression and Fas-mediated apoptosis in osteoclasts.

Osteoclast apoptosis is an influential determinant of osteoclast bone-resorbing activity. RANKL, a critical factor for osteoclastogenesis, is also important in osteoclast survival. However, the mechanisms by which RANKL prevents osteoclast apoptosis remain largely unknown. INTRODUCTION: Fas, a death receptor, mediates apoptosis in multiple types of cells including osteoclasts. Here we report that RANKL acts as a survival factor in osteoclasts by downregulating Fas-mediated apoptosis and Fas expression in mature osteoclasts. MATERIALS AND METHODS: RAW264.7 and mouse bone marrow macrophage/monocyte progenitors and progenitor-derived osteoclasts, in the presence of various concentrations of RANKL, were used in this study. Western blotting, semiquantitative RT-PCR, flow cytometry, nuclear staining, and a fluorescent caspase-3 activity assay were used to assess the effect of RANKL on Fas expression and Fas-mediated apoptosis. The involvement of NF-kappaB in the regulation of Fas by RANKL was analyzed by luciferase assay and EMSA. RESULTS: Mature osteoclasts generated in the presence of a high concentration of RANKL (3.33 nM) failed to respond to Fas-induced apoptosis. The lack of responsiveness in mature osteoclasts is caused by the low level of Fas expression, as detected by both semiquantitative PCR and Western blotting. Fas protein and mRNA expression are inhibited by RANKL in concentration-dependent manners. The downregulation of Fas expression by RANKL is not because of modulation of the stability of Fas protein or mRNA. The regulation of Fas expression by RANKL is biphasic. During the early stage of osteoclastogenesis (1 day) when Fas is expressed at a very low level, RANKL upregulates Fas promoter activity by 2.4 +/- 0.1-fold in a concentration-dependent manner and increases Fas mRNA and protein. This event correlates with regulation of the binding activity of NF-kappaB to the Fas promoter by RANKL, as detected by EMSA. In osteoclast precursors, the induction of Fas promoter activity by RANKL was dramatically reduced when NF-kappaB binding sites on the Fas promoter were mutated. CONCLUSION: RANKL upregulates Fas expression in osteoclast progenitors through NF-kappaB, making osteoclasts targets of Fas-stimulated apoptosis. In differentiated mature osteoclasts, RANKL reduces the levels of Fas expression and Fas-mediated apoptosis, acting as a survival factor.     

Prostate-specific antigen induces apoptosis of osteoclast precursors: potential role in osteoblastic bone metastases of prostate cancer

BACKGROUND: Prostate cancer is frequently associated with bone metastases with marked osteoblastic changes and low osteoclastic activity but its mechanism is not well understood. We previously reported that prostate-specific antigen (PSA) stimulated the proliferation and the activation of osteoblasts. In this study, we investigated the effect of PSA on osteoclastogenesis. METHODS: Two human prostate cancer cell lines and PSA were directly injected into human adult bone (HAB) implanted into NOD/SCID mice, followed by morphological analysis. RAW 264.7 cells, murine osteoclast precursor, were treated with PSA. RESULTS: PSA-producing LNCaP and PSA caused a significant decrease of osteoclast precursors and osteoclasts in HAB accompanied by osteoblast proliferation and new bone formation, while PSA-nonproducing PC3 showed increasing osteoclasts with osteolysis. PSA induced apoptosis of RAW 264.7 cells in vitro. PSA-induced apoptosis was dependent of enzymatic activity of PSA and was specific to immature tartrate-resistant acid phosphatase-negative mononuclear RAW 264.7 cells. CONCLUSIONS: PSA plays a crucial role for osteoblastic bone metastasis by promoting both osteoblasts proliferation and apoptosis of osteoclast precursors.     

The small molecule harmine regulates NFATc1 and Id2 expression in osteoclast progenitor cells

Small molecule compounds that potently affect osteoclastogenesis could be useful as chemical probes for elucidating the mechanisms of various biological phenomena and as effective therapeutic strategies against bone resorption. An osteoclast progenitor cell-based high-throughput screening system was designed to target activation of NFAT, which is a key event for osteoclastogenesis. Orphan ligand library screening using this system identified the β-carboline derivative harmine, which is a highly potent inhibitor of dual-specificity tyrosine-phosphorylation regulated kinase 1A (DYRK1A), to be an NFAT regulator in osteoclasts. RAW264.7 cells highly expressed DYRK1A protein, and in vitro phosphorylation assay demonstrated that harmine directly inhibited the DYRK1A-mediated phosphorylation (in-activation) of NFATc1. Harmine promoted the dephosphorylation (activation) of NFATc1 in RAW264.7 cells within 24h, and it significantly increased the expression of NFATc1 in RAW264.7 cells and mouse primary bone marrow macrophages (BMMs) both in the presence and absence of RANKL stimulation. Although harmine promoted NFATc1 expression and stimulated target genes for osteoclastogenesis, cell-cell fusion and the formation of TRAP-positive multinucleated osteoclasts from RAW264.7 cells and BMMs was significantly inhibited by harmine treatment. Meanwhile, harmine remarkably promoted the expression of inhibitor of DNA binding/differentiation-2 (Id2), which is a negative regulator for osteoclastogenesis, in RAW264.7 cells and BMMs. An Id2-null-mutant showed slightly increased osteoclast formation from BMMs, and the harmine-mediated inhibition of osteoclast formation was abolished in the BMMs of Id2-null-mutant mice. These results suggest that harmine is a potent activator of NFATc1 that interferes with the function of DYRK1A in osteoclast precursors and also up-regulates Id2 protein, which may dominantly inhibit expression pathways associated with cell-cell fusion, thereby leading to the disruption of the fusion events mediating osteoclastogenesis. The small molecule harmine is therefore expected to provide an experimental tool for investigating signaling cascades in osteoclastogenesis, especially those centered on DYRK1A-mediated NFATc1 and Id2 regulation.     

重组人红细胞生成素预处理对肾小管上皮细胞缺血再灌注损伤中PI3K-Akt-GSK-3β通路的调控作用

目的 探讨磷脂酰肌醇-3激酶（PI3K）-蛋白激酶B（Akt）-糖原合成酶激酶3β（GSK-3β）通路对人肾小管上皮细胞（HK-2）缺血再灌注（IR）损伤过程中细胞凋亡的调控及重组人红细胞生成素（rHuEPO）的保护作用。 方法 正常培养的HK-2细胞,分为7组：正常对照组、IR组、LY294002干预组（PI3K-Akt阻断剂,10 μmol/L）、LiCl干预组（GSK-3β阻断剂,20 μmol/L）、rHuEPO干预组（20 U/L）、rHuEPO+LY294002干预组、rHuEPO+LiCl干预组。Western印迹法检测Akt（Ser473）、GSK-3β（Ser9）及半胱氨酸天冬氨酸蛋白酶（caspase-3）活性; MTT法检测细胞活力;Annexin V和PI染色结合流式细胞仪技术检测细胞凋亡。 结果 IR损伤诱导HK-2细胞凋亡率上调（15.20%±1.43％）、Akt活性水平下降、GSK-3β及caspase-3酶活性水平上调,与正常对照组相比,差异有统计学意义（P < 0.05）。与IR组相比,LY294002干预使细胞凋亡率进一步上调（18.20%±2.06％）、Akt活性水平下调、GSK-3β及caspase-3酶活性上调,LiCl干预使细胞凋亡率下调（12.30%±0.85％）、Akt活性水平上调、GSK-3β及caspase-3酶活性下调,差异均有统计学意义（P < 0.05）。rHuEPO干预与IR组相比,细胞凋亡率下降（11.10%±1.62％）、Akt活性水平升高而GSK-3β及caspase-3酶活性下调,差异有统计学意义（P < 0.05）。与rHuEPO干预相比,rHuEPO+LY294002双干预细胞凋亡率升高（13.40%±1.94％）、Akt活性水平下降而GSK-3β及caspase-3酶活性上调,rHuEPO+LiCl双干预细胞凋亡率下调（7.50%±1.31％）、Akt活性水平上升而GSK-3β及caspase-3酶活性下降,差异均有统计学意义（P < 0.05）。 结论 IR损伤可引起肾小管上皮细胞凋亡,Akt活性降低及GSK-3β活性升高,影响caspase-3依赖的外源性凋亡途径可能是其凋亡机制之一。rHuEPO可通过增强Akt活性,降低GSK-3β及caspase-3酶活性,从而减轻细胞凋亡,对HK-2 IR损伤有一定的保护作用。     

BSP and RANKL induce osteoclastogenesis and bone resorption synergistically.

RANKL and BSP are upregulated in several bone resorptive disorders. However, the mechanisms by which these two factors might induce osteoclastogenesis and bone resorption synergistically under pathological conditions remain largely unknown. INTRODUCTION: RANKL and bone sialoprotein II (BSP) have been shown to be upregulated in the serum of individuals with abnormally high osteoclastogenic and bone resorptive activities. Here we provide experimental evidence that RANKL and BSP induce osteoclastogenesis and bone resorption synergistically but mediate opposite effects in osteoclast survival and apoptosis. MATERIALS AND METHODS: RAW264.7 cells and mouse bone marrow-derived monocytes/macrophages were treated with human recombinant BSP in the presence and absence of RANKL. TRACP stainings, bone resorption assays, Western blotting, immunoprecipitation analyses, and semiquantitative RT-PCR were used to evaluate the effects of BSP in osteoclast differentiation and bone resorption. Survival, DNA condensation, and caspase activity assays were used to determine the putative effects of BSP in osteoclast survival and apoptosis. RESULTS AND CONCLUSIONS: RANKL induced osteoclast differentiation and bone resorption at a higher extent in the presence than in the absence of BSP in RAW264.7 cells and bone marrow-derived monocytes/macrophages. c-Src-dependent c-Cbl phosphorylation was 8-fold higher in RAW264.7 cells treated with BSP and RANKL than in those treated with RANKL alone. Furthermore, BSP and RANKL activated the master regulator of osteoclastogenesis nuclear factor of activated T cells (NFAT)-2 and increased the mRNA expression of other differentiation markers such as cathepsin K or TRACP. Inhibition of c-Src activity or chelating intracellular calcium inhibited the synergistic effects in bone resorption and the phosphorylation of the c-Src substrate c-Cbl. Inhibition of calcineurin or intracellular calcium elevation inhibited the synergistic effects in osteoclastogenesis and decreased NFAT-2 nuclear levels. On the other hand, BSP and RANKL mediated opposite effects in osteoclast survival and apoptosis. Thus, BSP increased survival and decreased apoptosis markers in differentiated RANKL-treated RAW267.5 cells and RANKL/macrophage-colony stimulating factor (M-CSF)-treated bone marrow-derived monocytes/macrophages. In addition, RAW267.5 cells treated with BSP and RANKL exhibited decreased activation of the proapoptotic Jun N-terminal kinase pathway and increased activation of anti-apoptotic AKT pathway than cells treated with RANKL or BSP alone. Taken together, our findings suggest that BSP contributes to RANKL-mediated bone resorption by inducing osteoclastogenesis and osteoclast survival and decreasing osteoclast apoptosis.     

nephrin通过PI3K-Akt途径抑制血管紧张素Ⅱ诱导的足细胞凋亡

目的 研究nephrin在血管紧张素Ⅱ（AngⅡ）诱导足细胞凋亡中的作用,以及可能的分子机制。 方法 体外培养永生化小鼠足细胞（MPC）,以不同浓度AngⅡ处理MPC和10-8 mol/L AngⅡ刺激不同时间,用流式细胞仪检测细胞凋亡率;实时定量PCR、免疫荧光和Western印迹法检测nephrin的表达和分布;Western印迹法检测Akt磷酸化水平。脂质体法转染pcDNA3.1-mNPHS1质粒,G418筛选稳定转染细胞系。用Akt抑制剂LY294002或与AngⅡ共孵育刺激MPC和pcDNA3.1-mNPHS1转染细胞,检测Akt磷酸化水平和细胞凋亡率。 结果 （1）AngⅡ以剂量和时间依赖方式诱导MPC凋亡。AngⅡ受体拮抗药氯沙坦与AngⅡ共孵育18 h,显著降低AngⅡ单独刺激的足细胞凋亡率（P < 0.05）。（2）10-8 mol/L AngⅡ刺激12 h后,nephrin mRNA和蛋白较对照组显著降低,24 h nephrin mRNA约为正常对照的50%（P < 0.05）。正常足细胞nephrin主要分布于核膜周围的胞质和细胞膜,随刺激时间延长,胞膜和胞质nephrin表达逐渐降低。（3）10-8 mol/L AngⅡ刺激15 min后,Akt磷酸化显著降低,约为正常对照细胞的50%（P < 0.01）。（4）AngⅡ与LY294002共孵育12 h后,细胞凋亡率显著高于AngⅡ和LY294002单独刺激（均P < 0.05）。（5）pcDNA3.1-mNPHS1稳定转染显著上调足细胞Akt磷酸化水平（P < 0.05）,抑制 AngⅡ诱导的细胞凋亡（P < 0.05）。 结论 AngⅡ通过AngⅡ受体诱导小鼠足细胞凋亡,抑制足细胞nephrin表达。nephrin通过PI3K-Akt信号通路调节足细胞存活状态。