Novel insights into D-Pinitol based therapies:a link between tau hyperphosphorylation and insulin resistance

Alzheimer’s disease is a neurodegenerative disorder characterized by the amyloid accumulation in the brains of patients with Alzheimer’s disease.The pathogenesis of Alzheimer’s disease is mainly mediated by the phosphorylation and aggregation of tau protein.Among the multiple causes of tau hyperphosphorylation,brain insulin resistance has generated much attention,and inositols as insulin sensitizers,are currently considered candidates for drug development.The present narrative review revises the interactions between these three elements:Alzheimer’s disease-tau-inositols,which can eventually identify targets for new disease modifiers capable of bringing hope to the millions of people affected by this devastating disease.

TRIB3靶向AKT磷酸化调控高糖条件下小鼠RAW264.7巨噬细胞极化的机制研究

目的探讨TRIB3介导高糖条件下巨噬细胞促炎性M1型极化的下游机制。方法以小鼠巨噬细胞RAW264.7为研究对象:1)细胞分为对照(CON)组和高糖(HG)组,Western blot检测TRIB3、p-AKT和AKT蛋白;在各组内分为DMSO组和SC79组/MK2206组,使用AKT激动剂SC79或抑制剂MK2206处理细胞,Western blot检测p-AKT和AKT蛋白。2)细胞随机分为Control vector-DMSO组、TRIB3 overexpress-DMSO组、Control vector-SC79组、TRIB3 overexpress-SC79组、Control vector-MK2206组和TRIB3 overexpress-MK2206组,CCK8检测细胞活性,相差显微镜观察细胞形态并采集图像,Western blot检测TRIB3、pAKT、AKT、iNOS和Arg-1蛋白,ELISA检测细胞培养液中IL-1β和IL-10分泌。结果1)与CON组相比,HG组TRIB3显著增加、p-AKT/AKT显著下降。HG-SC79组p-AKT/AKT显著高于HG-DMSO组且与CON-SC79组无显著差异;HG-MK2206组pAKT/AKT显著低于HG-DMSO组。2)与对应的Control vector组相比,TRIB3 overexpress组TRIB3均显著增加、p-AKT/AKT均显著下降;与对应的DMSO组相比,SC79组p-AKT/AKT均显著增加、MK2206组p-AKT/AKT均显著下降。与Control vector-DMSO组相比,TRIB3 overexpress-DMSO组出现较多长梭形和不规则形细胞,iNOS和IL-1β显著增加,IL-10显著减少。与TRIB3overexpress-DMSO组相比,TRIB3 overexpress-SC79组长梭形和不规则形细胞明显减少,iNSO和IL-1β显著下降,IL-10显著增加;TRIB3 overexpress-MK2206组长梭形和不规则形细胞进一步增加,Arg-1和IL-10显著下降,IL-1β显著增加。结论高糖环境下巨噬细胞中激活的TRIB3蛋白通过靶向负调控AKT磷酸化水平发挥诱导巨噬细胞M1型极化、抑制M2型极化的促炎作用。

益肺宣肺降浊方通过EGFR调节PI3K/Akt-MAPK/Erk通路改善血管性痴呆大鼠的记忆

目的 探讨益肺宣肺降浊方通过表皮生长因子受体(Objective Epidermal growth factor receptor, EGFR)调节PI3K/Akt-MAPK/Erk通路改善对血管性痴呆(VaD)的作用及机制。方法 建立大脑中动脉栓塞法(Middle Cerebral Artery Occlusion, MCAO)大鼠血管性痴呆模型;给予益肺宣肺降浊方联合PTEN、EGFR和MAPK抑制剂,进行行为学实验,并运用Western Blot、免疫荧光、电镜透射等技术检测益肺宣肺降浊方对VaD可能通过PI3K/Akt和MAPK/Erk信号通路作用机制进行探索。结果 益肺宣肺降浊方呈剂量依赖性改善VaD大鼠的记忆能力及脑海马组织的病理、降低神经元凋亡。上调EGFR激活PI3K/Akt信号通路与MAPK/Erk信号通活性。结论 益肺宣肺降浊方对VaD大鼠的作用可能通过VEGF受体EGFR调节PI3K/Akt信号通路与MAPK/Erk信号通活性,以及PI3K/Akt与raf1-MAPK/Erk的串扰作用,增强Erk1/2磷酸化,进而促进神经细胞存活、神经元形态,最终达到修复记忆能力,治疗血管性痴呆的作用。

亚甲蓝通过增强ERK1/2和AKT磷酸化降低IL⁃1β水平改善糖尿病大鼠视网膜病变

目的探究亚甲蓝对糖尿病大鼠视网膜病变的神经保护作用。方法将30只SD大鼠随机分为空白组、对照组和实验组。对照组和实验组采用链脲佐菌素(STZ)腹腔注射构建糖尿病大鼠模型。造模成功6周后,在实验组动物玻璃体内按照[0.2 mg/(kg·d)]剂量注射亚甲蓝,对照组玻璃体内注射等量二甲基亚砜(DMSO),均连续处理7 d。采用ELISA检测大鼠的视网膜超氧化物歧化酶(SOD),8⁃异前列腺素F2α(iPF2α)和白细胞介素1β(IL⁃1β)的水平,Western blot法检测视网膜磷酸化的细胞外信号调节激酶1/2(p⁃ERK1/2)和磷酸化的蛋白激酶B(p⁃AKT)表达,过碘酸希夫(PAS)染色检测视网膜形态变化。结果与空白组大鼠相比,对照组和实验组大鼠中的视网膜SOD活性明显降低,iPF2α和IL⁃1β水平增加,p⁃ERK1/2水平增加、p⁃AKT磷酸化水平降低;与对照组相比,实验组大鼠SOD活性增加,iPF2α和IL⁃1β水平降低,p⁃ERK1/2、p⁃AKT水平明显升高,视网膜层的整体厚度和视网膜神经节细胞的数量显著减少。结论亚甲蓝通过降低视网膜氧化应激和增强ERK1/2和AKT磷酸化改善糖尿病大鼠视网膜病变。

甘草查尔酮A通过Akt/ERK信号通路抑制骨肉瘤增殖

目的 探究甘草查尔酮A(licochalcone A,LCA)对骨肉瘤细胞增殖和凋亡的影响及相关分子机制。方法 体外培养骨肉瘤HOS和U2OS细胞,通过MTT实验检测不同浓度LCA处理不同时间对HOS和U2OS细胞增殖的影响;加入终浓度分别为对照组(含0.1%DMSO的培养液)、5、10及20μmol·L^(-1)的LCA处理细胞48 h,流式细胞术检测LCA对HOS和U2OS细胞凋亡的影响,通过Western blot检测LCA对细胞凋亡相关蛋白cleaved PARP1、Bcl-2、Bax以及Akt和ERK蛋白表达水平的影响;通过裸鼠荷瘤实验从体内水平探究LCA的抗肿瘤作用。结果 MTT实验结果表明,LCA可抑制骨肉瘤HOS和U2OS细胞的增殖,并且呈时间和剂量依赖性;流式细胞术的结果表明,LCA可引起细胞凋亡。Western blot结果显示,ERK和Akt的磷酸化被抑制,cleaved PARP1和Bax的表达量升高,Bcl-2的表达量降低。裸鼠荷瘤实验表明,注射LCA后肿瘤体积明显减小(P<0.05),肿瘤的质量明显减小(P<0.01)。结论 LCA可以抑制骨肉瘤细胞的增殖并诱导凋亡,其作用机制可能与抑制Akt/ERK信号通路有关。

Reversal of Multidrug Resistance and Inhibition of Phosphorylation of AKT in Human Ovarian Cancer Cell Line by Wild-type PTEN Gene

The reversing effect of wild-type PTEN gene on resistance of C 13K cells to cisplatin and its inhibitory effect on the phosphorylation of protein kinase B （AKT） were studied. The expression of PTEN mRNA and protein in OV2008 cells and C13K cells were semi-quantitatively detected by using RT-PCR and Western blotting. Recombinant eukaryotic expression plasmid containing human wild-type PTEN gene was transfected into C13K cells by lipofectamine2000. The expression of PTEN mRNA was monitored by RT-PCR and the expression of PTEN, Akt, p-Akt protein were ana- lyzed by Western blotting in PTEN-transfected and non-transfected C13K cells. Proliferation and chemosensitivity of cells to DDP were measured by MTT, and cell apoptosis was detected by flow cytometry after treatment with cisplatin. The expression of PTEN mRNA and protein in OV2008 cells were significantly higher than those in C13K cells. After transfection with PTEN gene for 48 h, the expression of PTEN mRNA and protein in C 13K cells were 2.04 ± 0.10, 0.94± 0.04 respectively and the expression of p-Akt protein （ 0.94± 0.07） was lower than those in control groups （1.68 ±0.14, 1.66± 0.10） （P〈 0.05）. The IC50 of DDP to C 13 K cells transfected with PTEN （7.2± 0.3 la mol/L） was obviously lower than those of empty-vector transfected cells and non-transfected cells （12.7±0.4 lamol/1, 13.0±0.3 lamol/L） （P〈0.05）. The apopototis ratio of wild-type PTEN-transfected, empty vector transfected and non-transfected C13K cells were （41.65___0.87）%, （18.61 ±0.70）% and （15.28±0.80）% respectively, and the difference was statistically significant （P〈0.05）. PTEN gene plays an important role in ovarian cancer multidrug resistance. Transfection of PTEN could increase the expression of PTEN and restore drug sensitivity to cisplatin in human ovarian cancer cell line C 13K with multidrug-resistance by decreasing the expression of p-Akt.

Correlation between Loss of PTEN Expression and PKB/AKT Phosphorylation in Hepatocellular Carcinoma

The clinical significance of phosphatase and tensin homolog deleted on chromosome ten (PTEN) protein expression and the correlation between the expression of PTEN and phosphorylation of protein kinase B (PKB/AKT) in human hepatocellular carcinoma (HCC) were investigated. The expression of PTEN and phospho-AKT was detected by SP immunohistochemical technique and Western blotting in 35 cases of HCC, 15 cases of liver cirrhosis and 8 cases of normal tissues. The correlation between the expression of PTEN and PKB/AKT in HCC was analyzed. The results showed that the positive expression of PTEN in HCC (62.9 %, 0.085±0.021) was significantly lower than that in liver cirrhosis and normal tissues (P<0.01). The expression level of PTEN was related to the differentiation degree of HCC and the status of metastasis (P<0.05). Western blotting revealed a significant inverse correlation between PTEN and phospho-AKT (r=-0.818, P<0.01). These results demonstrated that down-regulation or loss of PTEN, which may not be able to effectively inhibit the hyper-phosphorylation of PKB/AKT, might play an important role in tumorigenesis and progression of HCC.

ENO1 expression and Erk phosphorylation in PDAC and their effects on tumor cell apoptosis in a hypoxic microenvironment

Objective: Hypoxia is an important feature of pancreatic ductal adenocarcinoma(PDAC). Previously, we found that hypoxia promotes ENO1 expression and PDAC invasion. However, the underlying molecular mechanism was remains unclear.Methods: The relationship between ENO1 expression and clinicopathological characteristics was analyzed in 84 patients with PADC. The effects of CoCl2-induced hypoxia and ENO1 downregulation on the apoptosis, invasion, and proliferation of PDAC cells were evaluated in vitro and in vivo. Hypoxia-and ENO1-induced gene expression was analyzed by transcriptomic sequencing.Results: The prognosis of PDAC with high ENO1 expression was poor(P < 0.05). High ENO1 expression was closely associated with histological differentiation and tumor invasion in 84 PDAC cases(P < 0.05). Hypoxia increased ENO1 expression in PDAC and promoted its migration and invasion. Apoptotic cells and the apoptosis marker caspase-3 in the CoCl_(2)-treated ENO1-sh group were significantly elevated(P < 0.05). Transcriptomic sequencing indicated that CoCl_(2)-induced PDAC cells initiated MAPK signaling. Under hypoxic conditions, PDAC cells upregulated ENO1 expression, thereby accelerating ERK phosphorylation and inhibiting apoptosis(P < 0.05). Consistent results were also observed in a PDAC-bearing mouse hindlimb ischemia model.Conclusions: Hypoxia-induced ENO1 expression promotes ERK phosphorylation and inhibits apoptosis, thus leading to PDAC survival and invasion. These results suggest that ENO1 is a potential therapeutic target for PDAC.

人参皂苷Rh2通过激活Akt和Erk通路促进神经分化

目的研究人参皂苷Rh2的促神经分化作用,初步明确相关分子机制。方法将细胞分为对照组、实验组,使用MTT检测Rh2对细胞毒性的影响,筛选最适药物浓度,用倒置相差显微镜观察拍照,使用Image-J软件对照片进行细胞分化率和神经突起长度统计分析,用Western Blotting检测其促神经分化所激活的信号通路,之后用相关通路抑制剂加以干预,检测神经突起长度、分化率和相关通路的蛋白表达变化。结果人参皂苷Rh2浓度在2~6μM不影响细胞存活,能够促进神经分化,随剂量依赖性增加,其中6μM的效果最显著,其通过激活PI3K/Akt、MEK/Erk发挥作用,其中p-Akt和p-Erk在30 min表达最明显,Rh2可以降低由于PI3K/Akt、MEK/Erk抑制剂所致p-Akt、p-Erk抑制的效果。结论人参皂苷Rh2促进神经突起生长、增加和延长,具有较好促进神经分化的效果,具有开发为治疗神经退行性疾病的潜力。

uPAR expression under hypoxic conditions depends on iNOS modulated ERK phosphorylation in the MDA-MB-231 breast carcinoma cell line

尿激 plasminogen 使活跃之物受体(uPAR ) 戏在癌症侵略和转移和 uPAR 表示的一个主要角色在各种各样的癌症类型与差的预后被相关。而且， uPAR 的表示在 hypoxic 条件下面被增加。氮的氧化物(没有) 并且它可诱导的氮的氧化物 synthase (i NOS ) 生产的代谢物是 hypoxic 应力的重要产品，并且可以不激活或调制细胞外的信号调整 kinase (英皇家空军之阶级最低之兵) 。这里，我们在 MDA-MB-231 人的乳癌房间线在 hypoxic 条件，和 i NOS 并且不的调节效果下面在 A， uPAR，和激活的英皇家空军之阶级最低之兵层次上面评估了。房间在 hypoxic 或 normoxic 孵卵器被孵化并且与 PD98059 对待(一个 MEK 1/2 禁止者，它废除英皇家空军之阶级最低之兵 phosphorylation ) 并且 aminoguanidine (选择 i NOS 禁止者) 。uPAR 表示，英皇家空军之阶级最低之兵 phosphorylation，并且在 A 上面，活动被发现在 hypoxic 条件下面被增加。而且当房间在 hypoxic 条件下面与 PD98059 被对待时， uPAR 低调整，而 aminoguanidine 显著地以一种剂量依赖者方式增加了英皇家空军之阶级最低之兵 phosphorylation。而且， aminoguanidine 增加了 uPAR 表示并且由 PD98059 阻止了 uPAR 表示的抑制。这些结果证明 uPAR 被组织缺氧导致，那增加的 uPAR 表情被英皇家空军之阶级最低之兵 phosphorylation 调停，它被 iNOS/NO 接着在 MDA-MB-231 房间调制。我们结束那 iNOS/NO 在下面经由英皇家空军之阶级最低之兵小径调整在 hypoxic 条件下面调整 uPAR 的表示。

ERK phosphorylation functions in invadopodia formation in tongue cancer cells in a novel silicate fibre-based 3D cell culture system

To screen for additional treatment targets against tongue cancer, we evaluated the contributions of extracellular signal-related kinase(ERK), AKT and ezrin in cancer development. Immunohistochemical staining showed that ERK and ezrin expressions were significantly higher in invasive squamous cell carcinoma than in carcinoma in situ. To investigate the roles of ERK and ezrin in cancer development, we used the non-woven silica fibre sheet Cellbedwith a structure resembling the loose connective tissue morphology in a novel 3 D culture system. We confirmed that the 3 D system using CellbedTMaccurately mimicked cancer cell morphology in vivo. Furthermore, cell projections were much more apparent in 3 D-cultured tongue cancer cell lines than in 2 D cultures. Typically, under conventional 2 D culture conditions, F-actin and cortactin are colocalized in the form of puncta within cells.However, in the 3 D-cultured cells, colocalization was mainly observed at the cell margins, including the projections. Projections containing F-actin and cortactin colocalization were predicted to be invadopodia. Although suppressing ezrin expression with small interfering RNA transfection caused no marked changes in morphology, cell projection formation was decreased, and the tumour thickness in vertical sections after 3 D culture was markedly decreased after suppressing ERK activity because both the invasion ability and proliferation were inhibited. An association between cortactin activation as well as ERK activity and invadopodia formation was detected. Our novel 3 D culture systems using Cellbed? are simple and useful for in vitro studies before conducting animal experiments. ERK contributes to tongue cancer development by increasing both cancer cell proliferation and migration via cortactin activation.

GubenyiliuⅡ inhibits breast tumor growth and metastasis associated with decreased heparanase expression and Akt phosphorylation

OBJECTIVE GubenyiliuⅡ(GYⅡ),a traditional Chinese medicine(TCM)formula used in our hospital,has shown beneficial effects in cancer patients.In this study,we investigated the molecular mechanisms underlying the beneficial effects of GYⅡon murine breast cancer models.METHODS Inhibition of tumor growth and metastasis was evaluated by assessment of tumor weight and analysis of bioluminescent signal after a homograft inoculation.Viability of cultured breast cancer cells was determined using MTT assay andreal-time cell analysis(RTCA).Cell migratory ability was evaluated by Transwell?assay and wound healing assay.Subsequently,the potential anti-tumor and anti-metastatic mechanism was investigated by Western blotting and Immunohistochemistry.RESULTS GYⅡshowed significant inhibitory effects on tumor growth and metastasis in the murine breast cancer model.And GYⅡsuppressed theproliferation of 4T1 and MCF-7 cells in a dose-dependent manner.A better inhibitory effect on 4T1 cells proliferation and migration was found in sub-fractions(SF)of GYⅡ.Moreover,heparanase expression and degree of angiogenesis were reduced in tumor tissues.Western blotting analysis showed decreased expression of heparanase and growth factors in the cells treated with GYⅡand its sub-fractions(SF2 and SF3),there by a reduction in phosphorylation of ERK and AKT.CONCLUSION GYⅡexerts anti-tumor growth and anti-metastatic effects on murine breast cancer model.Sub-fractions 2 and 3 exhibits higher potency of the anti-tumor activity that is,at least partly,associated with decreased heparanase and growth factor sexpression,which subsequently sup-pressed activation of ERK and AKT pathways.

USP9X对Akt磷酸化水平及血小板活化的影响

目的 探讨血小板中去泛素化酶USP9X的表达及其对血小板功能的影响。方法 通过聚合酶链式反应和免疫印记方法检测人和小鼠血小板中USP9X的表达情况;分离制备年轻小鼠和老年小鼠的血小板,检测USP9X的表达变化;采用USP9X特异性抑制剂检测USP9X对血小板聚集释放及铺展功能的影响;收集激活不同时间点的血小板蛋白裂解液,并进行免疫印记检测磷酸化Akt的变化。结果 人血小板与小鼠血小板在mRNA水平和蛋白水平均表达USP9X。相对于年轻小鼠,老年小鼠血小板中USP9X的表达明显升高(0.87±0.099 vs 1.05±0.980,P<0.05)。使用USP9X的特异性抑制剂提前30 min孵育血小板,之后检测血小板的聚集、释放及铺展功能,结果显示,相比于对照组,USP9X抑制剂处理组的血小板的聚集和释放水平明显下降(P<0.05);相比于对照组,抑制剂处理组45 min的铺展面积显著降低。最后,免疫印记结果显示,USP9X抑制剂处理组的血小板的Akt磷酸化水平明显下降。结论 人和小鼠的血小板表达USP9X,USP9X可促进血小板的聚集释放及铺展,并可影响Akt的磷酸化水平,USP9X的抑制剂或可成为潜在的临床血栓干预靶点。

杜仲叶通过激活ERK及AKT磷酸化促进大鼠成骨细胞增殖的研究

目的研究杜仲叶提取物对大鼠成骨细胞的增殖作用及其分子机制。方法选取新生SD大鼠的乳鼠颅骨通过消化法分离出乳鼠成骨细胞,并用碱性磷酸酶染色进行细胞鉴定;先用杜仲叶提取物分别以0 mg/ml,5 mg/ml,20 mg/ml,40 mg/ml,60 mg/ml五种不同浓度梯度对大鼠成骨细胞干预,2d后用MTT方法检测细胞增殖情况;用无血清无酚红的培养基饥饿大鼠成骨细胞2 h后,分别以0mg/ml,5 mg/ml,20 mg/ml,40 mg/ml,60 mg/ml五种不同浓度杜仲叶提取物干预大鼠成骨细胞,2 h后Western blot检测ERK和AKT的活化情况。结果碱性磷酸酶染色后的成骨细胞呈紫红色(特异性染色),MTT法结果显示杜仲叶提取物促进大鼠成骨细胞的增殖,并具有浓度依赖性;Western blot结果显示杜仲叶提取物可使ERK及AKT磷酸化水平提高,并具有浓度依赖性。结论杜仲叶提取物通过ERK通路及AKT通路促进大鼠成骨细胞的增殖。

利拉鲁肽通过PI3K/Akt和MAPK/ERK通路促进心肌微血管内皮细胞的增殖和迁移

目的研究胰高血糖素样肽-1类似物利拉鲁肽对原代大鼠心肌微血管内皮细胞（CMECs）增殖和迁移的影响及其机制。方法使用双酶消化法体外分离培养SD大鼠心肌微血管内皮细胞,差速贴壁法进行纯化,CD31和Ⅷ因子抗体进行免疫细胞化学染色鉴定。采用不同浓度利拉鲁肽（0~1000 nmol/L）干预1代细胞,MTT绘制细胞生长曲线,Western blot检测利拉鲁肽对PI3K/Akt和MAPK/ERK通路的激活作用,Brd U荧光标记法和划痕实验观察药物作用下细胞增殖和迁移能力,并使用相应的通路阻断剂LY294002和PD98059来进一步证实。结果体外分离原代CMECs,CD31及Ⅷ因子免疫荧光染色示双阳性率大于95%,MTT示细胞于种植48 h后进入对数生长期,利拉鲁肽以浓度依赖方式促进CMECs的体外增殖,100 nmol/L为其最适生长浓度（P〈0.05）。予以100 nmol/L利拉鲁肽预干预细胞24 h后,Western blot显示胞内Akt和ERK磷酸化水平与正常细胞组相比有明显升高（P〈0.05）,加入相应通路阻断剂LY294002和PD98059,其磷酸化水平相较于利拉鲁肽组明显降低（P〈0.05）。Brd U和划痕实验均说明利拉鲁肽能促进CMECs的增殖和迁移能力（P〈0.05）,使用LY294002和PD98059预处理后,增殖迁移能力均显著低于利拉鲁肽组（P〈0.05）。结论利拉鲁肽是通过激活胞内PI3K/Akt和MAPK/ERK通路从而促进原代大鼠心肌微血管内皮细胞的增殖和迁移。

Ghrelin通过AKT/ERK信号通路对LPS诱导的肺泡上皮细胞凋亡的影响

目的:研究Ghrelin对内毒素(Lipopolysaccharide,LPS)所致的肺泡Ⅱ型上皮细胞(A549)凋亡的影响及其机制。方法:CCK-8(Cell Counting Kit-8)法检测LPS刺激对A549的细胞毒性;原位末端标记法(TUNEL)检测细胞凋亡率;流式细胞术检测细胞内一氧化氮(NO)的产生;Western blot检测诱导型一氧化氮合成酶(iN OS)、AKT、ERK、p-AKT、p-ERK信号通路蛋白以及cleaved caspase-3、Bax、Bcl-2凋亡相关蛋白的表达。结果:CCK-8检测结果显示LPS可显著抑制A549细胞的增殖,降低细胞活力;TUNEL检测发现Ghrelin可显著抑制LPS导致的A549细胞的凋亡(P<0.05);LPS可以促进iN OS的表达,增加细胞内NO的产生,并同时抑制AKT、ERK通路的活性,上调下游促凋亡蛋白Bax以及终末凋亡蛋白cleaved caspase-3的表达,下调抗凋亡蛋白Bcl-2的表达,而应用Ghrelin预处理后可以逆转LPS对AKT、ERK通路活性的抑制,继而下调Bax以及cleaved caspase-3的表达,上调Bcl-2的表达,差异均具有统计学意义(P<0.05),但Ghrelin对细胞内NO的产生无明显影响。结论:Ghrelin可以通过上调AKT及ERK通路的活性抑制LPS诱导的肺泡上皮细胞凋亡,但不能降低iN OS诱导产生NO的水平。

自分泌IL-6经Ras/MEK/ERK、PI3K/Akt通路促进卵巢癌细胞黏附和侵袭功能的研究

目的探讨内源性IL-6表达改变对卵巢癌细胞黏附和侵袭功能的影响及相关信号转导通路。方法利用以往构建的内源性过表达IL-6的人卵巢癌A2780细胞系和内源性抑制IL-6表达的人卵巢癌SKOV-3细胞,分别采用细胞体外黏附实验、Transwell小室体外侵袭实验、免疫印迹技术等观察IL-6对卵巢癌细胞黏附、侵袭能力的影响,并对其可能的信号通路进行研究。结果与对照组相比,内源性过表达IL-6可促进卵巢癌细胞的体外黏附和侵袭功能;抑制IL-6表达则可抑制卵巢癌细胞的上述功能。过表达或抑制表达IL-6可增加或减少卵巢癌细胞磷酸化ERK、Akt的表达水平,应用ERK或Akt特异性信号阻断剂可显著抑制高表达IL-6的卵巢癌细胞黏附和侵袭作用,提示可能与其活化Ras/MEK/ERK、PI3K/Akt通路有关。结论卵巢癌细胞产生的IL-6可经Ras/MEK/ERK和PI3K/Akt通路增强自身的黏附和侵袭能力,调节IL-6表达及相关信号转导通路可能是未来临床控制卵巢癌进展的一种良好策略。

七叶皂苷钠通过抑制Akt和ERK信号通路诱导HeLa细胞凋亡及对死亡受体表达的影响

目的:探讨七叶皂苷钠诱导宫颈癌HeLa细胞凋亡的作用及其分子机制。方法:采用MTT法检测七叶皂苷钠对宫颈癌HeLa细胞的生长和增殖抑制作用;倒置显微镜观察细胞形态改变;利用annexin V-FITC/PI流式细胞术检测细胞凋亡率;采用DAPI单染法荧光显微镜下观察细胞核变化情况;利用Western blotting检测凋亡相关蛋白[聚(ADP-核糖)聚合酶(PARP)、cleaved caspase-8、cleaved caspase-9、pro-caspase-3]和细胞存活相关信号通路(Akt、ERK)以及TRAIL受体(DR4、DR5)的变化情况。结果:七叶皂苷钠以剂量依赖的方式显著抑制宫颈癌HeLa细胞的生长和增殖;七叶皂苷钠作用于HeLa细胞后,可见典型的凋亡细胞形态学特征,细胞凋亡率显著增加;随着七叶皂苷钠浓度升高,cleaved PARP、cleaved caspase-8和cleaved caspase-9明显增多,pro-caspase-3显著减少,p-Akt和p-ERK激活减少,细胞内DR4和DR5总蛋白水平上调。结论:七叶皂苷钠通过抑制细胞存活相关信号通路,上调死亡受体水平,抑制细胞增殖,促进细胞凋亡。

内质网应激下肝癌细胞Akt和ERK通路间的cross-talk研究

目的研究内质网应激介导的肝癌细胞PI3K/AKt和M EK/ER K途径间的信号交流。方法采用PI3K抑制剂LY 294002/Akt激活型突变载体m yr-Akt和M EK抑制剂U 0126分别阻断/激活内质网应激介导的Akt和ER K活化,并利用W estern blot分析内质网应激条件下PI3K/Akt和M EK/ER K途径间的信号交流。结果阻断PI3K/Akt明显促进内质网应激介导的M EK/ER K活化,而过度激活PI3K/Akt则抑制内质网应激介导的M EK/ER K活化。阻断M EK/ER K对内质网应激介导的PI3K/Akt活化无影响。结论 PI3K/Akt和M EK/ER K信号途径间在内质网应激肝癌细胞中存在信号交流。

SDF-1通过PI3K/Akt和MAPK/Erk信号通路对人微血管内皮细胞功能产生影响

目的 :观察基质细胞衍生因子-1(stromal cell derived factor-1,SDF-1)对人微血管内皮细胞(human microvascular endothelial cell line-1,HMEC-1)功能的影响,并对相关机制进行初步探讨,从而明确SDF-1在糖尿病血管病变中的作用。方法:体外培养HMEC-1,分别在培养基中加入不同浓度的SDF-1(0、25、50、100μg/L),Western blot检测HMEC-1中PI3K/Akt和MAPK/Erk信号通路的活化情况,MTT和流式细胞分析检测HMEC-1增殖和凋亡能力的变化情况,划痕愈合实验检测HMEC-1迁移能力的变化情况。采用PI3K/Akt和MAPK/Erk信号通路抑制剂LY294002和U0126阻断HMEC-1中PI3K/Akt和MAPK/Erk信号通路后,再以SDF-1处理HMEC-1,MTT和流式细胞分析检测HMEC-1增殖和凋亡能力的变化情况;划痕愈合实验检测HMEC-1迁移能力的变化情况。结果:Western blot结果显示,25μg/L的SDF-1处理即可显著活化HMEC-1中的PI3K/Akt和MAPK/Erk信号通路,且随着SDF-1处理浓度的升高,PI3K/Akt和MAPK/Erk信号通路活化水平逐渐升高。同0μg/L的SDF-1处理组相比,25、50、100μg/L的SDF-1均可显著加强HMEC-1的增殖和迁移能力(P<0.01),并抑制HMEC-1的凋亡水平(P<0.01),且这种作用具有剂量依赖性。当采用LY294002和U0126阻断HMEC-1中的PI3K/Akt和MAPK/Erk信号通路后,SDF-1促HMEC-1增殖和迁移能力及抑制HMEC-1凋亡的能力均显著降低(P<0.01)。结论:SDF-1可通过活化PI3K/Akt和MAPK/Erk信号通路,进而促进HMEC-1的增殖和迁移,并抑制HMEC-1的凋亡水平,从而在糖尿病血管病变中发挥一定的作用。