褪黑激素对增生性瘢痕成纤维细胞增殖的影响及机制研究

目的：探讨褪黑激素对人增生性瘢痕成纤维细胞增殖的影响及作用机制。方法收集人皮肤增生性瘢痕标本,原代培养人皮肤增生性瘢痕成纤维细胞(HSFBs)。将HSFBs细胞根据不同的处理方法分为空白对照组、低浓度褪黑激素组、中浓度褪黑激素组、高浓度褪黑激素组、NVP-BEZ235处理组、NVP-BEZ235+褪黑激素组、褪黑激素+siRNA-PTEN组、褪黑激素+siRNA-scramble组。MTT法检测各组细胞的增殖情况,qRT-PCR和Western blot检测各组细胞中磷酸酶与张力蛋白同源物(PTEN)、CyclinD1和Caspase-3的表达以及PI3k/AKt/mTOR通路相关蛋白p-Akt和p-mTOR的蛋白水平。结果与空白对照组比较,从低到高浓度褪黑激素均能够减少HSFBs细胞同一时间点OD值,上调PTEN和Caspase-3表达量,同时抑制CyclinD1、p-Akt和p-mTOR表达水平,组间差异具有统计学意义(P<0.05)。同时与高浓度褪黑激素组比较,NVP-BEZ235+褪黑激素组中细胞增殖显著被抑制,差异具有统计学意义(P<0.05);沉默PTEN后拮抗褪黑激素对HSFBs细胞增殖和PI3K/Akt/mTOR信号通路有抑制作用,差异具有统计学意义(P<0.05)。结论褪黑激素通过上调PTEN表达,抑制PI3K/Akt/mTOR信号通路活化从而抑制HSFBs细胞增殖。     

姜黄素抑制TGF-β1诱导的人肾小管上皮细胞系表型转化

目的探讨姜黄素抑制TGF-β1诱导的肾纤维化的作用及分子生物学机制。方法应用10 ng/mL TGF-β1单独或联合姜黄素(Cur)(6.25、12.5、25、50和100μmol/L)共同作用人肾小管上皮细胞系(HKCs)72 h,倒置相差显微镜下观察细胞形态,免疫组化检测纤维细胞特异蛋白-1(FSP-1)表达;同时,通过Western blot检测肾小管上皮细胞标志物E-cardhrin和细胞角化蛋白以及基质标志物波形蛋白、α-平滑肌肌动蛋白(α-SMA)和Ⅰ型胶原的表达变化,以及AKT/mTOR信号通路关键蛋白的表达。结果HKCs贴壁增殖,呈铺路石样外观。TGF-β1单独刺激能明显促进细胞由上皮样形态向纤维样细胞形态转化。当TGF-β1与姜黄素联合作用于HKCs时,姜黄素在12.5~50μmol/L浓度时能明显对抗TGF-β1诱导的细胞形态转变而维持HKCs细胞铺路石样外观,同时,TGF-β1能明显抑制HKCs细胞E-cardhrin和角化蛋白基因表达(P<0.05,P<0.01),而促进FSP1阳性细胞显著增加(P<0.05,P<0.01);上调波形蛋白、α-SMA和I型胶原的表达(P<0.05,P<0.01);并且,AKT/mTOR信号通路关键蛋白Akt、mTOR、p70S6K、4E-BP1和eIF4E的磷酸化水平表达也显著增加(P<0.05,P<0.01)。而姜黄素能抑制TGF-β1诱导的HKCs表型转化,同时下调TGF-β1诱导AKT/mTOR信号通路关键蛋白的磷酸化水平(P<0.05)。结论姜黄素通过抑制Akt/mTOR信号通路对抗TGF-β1诱导的上皮细胞-间质细胞转化,可能是姜黄素抗肾纤维化的关键分子机制之一。     

PI3K/AKt通路参与苦参碱抑制raji细胞增殖与凋亡过程的机制研究

[目的]探讨苦参碱体外诱导非霍奇金淋巴瘤raji细胞增殖与凋亡的机制是否与PI3K/AKt信号通路有关.[方法]M TT法检测苦参碱作用后raji细胞的增殖抑制率;流式细胞仪检测苦参碱作用后raii细胞的细胞周期的变化;RT-PCR检测苦参碱作用后PTEN、AKt、caspase3等mRNA水平的变化;Western blot检测PTEN、pBad、Bad、pAKt、AKt、caspase3等蛋白的表达.[结果]苦参碱可抑制raji细胞的增殖,随着药物浓度的提高和作用时问的延长,细胞的增殖抑制率逐渐升高,与对照组比较,差异均有显著(P＜0.001);苦参碱诱导raii细胞周期停滞于G1期(P＜0.05);苦参碱可通过磷酸化AKt来上调PTEN的表达,引起Bad、p21、p27等的变化,与对照组分别比较表达水平差异均有统计学意义(P＜0.05),最终诱导caspase家族级联反应,引发肿瘤细胞的凋亡.[结论]苦参碱可通过PI3K/AKt信号通路抑制非霍奇金淋巴瘤raii细胞的增殖,诱导raji细胞凋亡.     

AKt／mTOR 信号通路中 p70S6K1、4E －BPs 在促进肿瘤发生作用中的研究进展

当受细胞外生长因子等刺激作用后,可激活 PI3K／AKt／mTOR 信号通路,参与控制细胞的生长、增殖、生存、凋亡。AKt／mTOR 信号通路在肿瘤的发生发展过程中起着很重要的促进作用。AKt／mTOR 信号通路在肿瘤细胞中能够通过 p70S6K1和4E －BPs 的作用抑制细胞的凋亡,促进核糖体以及蛋白质的合成、促进肿瘤细胞的侵袭和转移、促进细胞周期进展、促进肿瘤血管的生成,进而促进肿瘤的发生发展。本文就AKt／mTOR 信号通路通过 p70S6K1和4E －BPs 促进肿瘤发生的机制进行综述。     

LPA经PI3K/Akt信号转导通路抑制顺铂诱导卵巢癌细胞凋亡

 目的 研究PI3K/AKT信号转导通路对LPA保护顺铂诱导的卵巢癌SKOV3细胞凋亡作用的影响。 方法 MTT法检测LPA和LY294002对DDP作用后的SKOV3细胞增殖活性的影响;Hoechst33258荧光染色 观察凋亡细胞;FCM分析细胞凋 亡率;凝胶电泳观察凋亡细胞的DNA“梯状”条带;Western blot检测LPA、LY294002对磷酸 化Akt蛋白表达的影响。 结果 LPA+LY294002+DDP组对SKOV3细胞增殖的抑制作用、凋亡小体的产生及细胞凋亡率高于 LPA+DDP组(P<0.05),而与 LY294002+DDP组差异无统计学意义, DNA片段凝胶电泳示LPA作用后不产生明显的凋亡片段, LPA和LY294002同时作用可出现DNA断裂梯形条带。Western blot结果示LPA作用后磷酸化Akt 蛋白表达升高,而LY294002作用后,磷酸化Akt蛋白表达明显下降。 结论 LPA通过激活PI3K/Akt信号转导通路抑制顺铂诱导的卵巢癌细胞的凋亡。     

白细胞介素-6信号通路在银屑病发病机制中的研究进展

银屑病是一种病因不明的自身免疫性炎症性多基因遗传的皮肤疾病,其病理变化的形成需要多种信号通路和细胞因子的共同参与.近年来关于银屑病相关细胞因子信号转导通路及细胞调控信号的研究受到广泛关注,其中IL-6触发的三条途径,包括Ras/Raf/MEK/ERK途径、PI3K/AKt途径和JAK/STAT途径,共同促进银屑病角质形成细胞增殖、炎症反应及血管生成等病理变化;下面针对IL-6信号通路在银屑病发病机制中的研究进展做一综述.     

胡桃醌联合顺铂对宫颈癌HeLa细胞的促凋亡作用及其机制

目的：探讨胡桃醌与顺铂联合应用对宫颈癌HeLa细胞促凋亡作用的机制,阐明其对相关信号转导通路的作用。方法：选取处于对数生长期的HeLa细胞,将其分为对照组、胡桃醌组、顺铂组和胡桃醌＋顺铂组（联合用药组）。采用MTT法观察不同时间点HeLa细胞的增殖抑制率;Hoechst 33258染色法荧光显微镜下观察细胞凋亡形态;Westernblotting法检测各组细胞凋亡相关蛋白Bcl-2、Bax和caspase-3及PI3K/AKt通路中AKt和pAKt表达水平。结果：MTT法,各用药组HeLa细胞增殖于24、48和72h均可被抑制,与对照组比较,胡桃醌组及顺铂组对细胞的抑制较明显,而联合用药组抑制更加显著;与单用药组比较,联合用药组对细胞的抑制更显著。Hoechst 33258染色,胡桃醌组和顺铂组在处理细胞12 h后,出现明显的凋亡细胞形态,联合用药组该现象更明显。Western blotting检测,与对照组比较,12 h后胡桃醌组和顺铂组HeLa细胞中Bcl-2和pAKt蛋白表达水平明显降低,而Bax、Cleave-caspase-3和AKt蛋白表达水平明显升高,联合用药组上述变化更加明显。结论：胡桃醌和顺铂联合应用可通过抑制PI3K/AKt通路,进而促进HeLa细胞凋亡。     

Normal endometrial stromal cells regulate survival and apoptosis signaling through PI3K/AKt/Survivin pathway in endometrial adenocarcinoma cells in vitro

Objective

Stroma–tumor communication plays an important role in the genesis of neoplasia. In the current study, we investigated the effect of normal stromal cells on the survival and apoptosis signaling of endometrial cancer cells and further explored the possible mechanism implied in this communication.

Methods

Using primarily cultured normal endometrial stromal cells and an endometrial adenocarcinoma cell line, Ishikawa cells, we established a 2D-coculture system to observe the stromal cell–tumor cell crosstalk in endometrial carcinomas. Using methyl thiazolyl tetrazolium (MTT) assays, cell counting and colony formation assays, we analyzed the effect of stomal cells on the growth and proliferation of Ishikawa cells under different conditions. Using western blot analysis, we determined the effect of stromal cells on the activity of PI3K/AKt/Survivin signaling in Ishikawa cells under different conditions. Using immunohistochemistry analysis, we determined the expression of Survivin in normal endometria and endometrial adenocarcinomas.

Results

We found that the paracrine factors from normal endometrial stromal cells grown on Matrigel repeatedly and significantly decreased hormone-stimulated activity of PI3K/AKt/Survivin signaling in Ishikawa cells, which were proved to be increased in endometrial adenocarcinoma and essential in hormone-induced cell growth in Ishikawa cells.

Conclusion

Paracrine factors from normal endometrial stromal cells can inhibit hormone-stimulated cell proliferation in Ishikawa cells by regulating cell survival and apoptosis through PI3K/AKt/Survivin signaling.     

Effect of apigenin on apoptosis induced by renal ischemia/reperfusion injury in vivo and in vitro

Objectives: This study aims to investigate the effects and molecular mechanisms of apigenin (ApI) on renal ischemia/reperfusion (I/R) injury in vivo and in vitro.

Methods: In vivo, the left renal artery was clamped for 45?min and the right kidney was removed to study renal I/R injury on Sprague-Dawley (SD) rats. ApI was injected at 60?min before renal ischemia. In vitro, renal tubular epithelial cells (HK-2) were pretreated with or without ApI (20 uM) for 60?min and then treated with hypoxia/reoxygenation (H/R). Renal function, histology, cells apoptosis, and cell viability were tested. Furthermore, the potential molecular mechanisms were assessed.

Results: ApI pretreatment could significantly alleviated the renal function and the pathological damage as well as cells apoptosis after I/R injury. Meanwhile, ApI treatment protects H/R induced HK-2 cell apoptosis in vitro. The results of Western blot showed that ApI significantly increased the expressions of B-cell lymphoma 2 (Bcl-2) and phosphor-AKt (p-AKt), Phosphoinositide 3-kinase (PI3K), while down-regulated the expressions of Caspase3 and Bax induced by H/R injury.

Conclusions: ApI pretreatment can protect renal function against I/R injury and prevent renal tubular cells from apoptosis in vivo and in vitro which might through PI3K/Akt mediated mitochondria-dependent apoptosis signaling pathway.     

PI3K/AKt信号通路与肝细胞癌

PI3K/AKt信号通路与人类肿瘤的发生、发展密切相关。近年来研究表明,肝细胞癌(HCC)中存在PI3K/AKt信号通路异常。此文就PI3K/AKt信号通路的构成、转导途径及其在HCC中的作用作一综述。