低氧对大鼠肺动脉平滑肌细胞pHi及细胞增殖、凋亡的影响

目的: 探讨低氧肺血管结构重建时是否伴有PASMCs凋亡.方法: 体外低氧培养大鼠PASMCs,BCECF法测定细胞内pH值、光镜及电镜观察细胞形态、流式细胞仪测细胞周期、原位细胞凋亡(TUNEL法)观察细胞凋亡.结果: PASMCs在低氧早期即出现细胞内碱化,低氧6 h即出现G0/G1期细胞比例减少,G2/M期细胞比例增加,至24 h丝裂活动最强,但细胞凋亡率无显著变化.结论: 低氧PASMCs在细胞内碱化、细胞增殖的过程中不伴有细胞凋亡的改变.     

热带假丝酵母细胞内pH的测定及其与生长代谢活性的关系

应用荧光探针5(6)-双醋酸羧基荧光素 (Carboxyfluorescein diacetate) 测定了产长链二元酸热带假丝酵母 (Candida tropicalis) 细胞内pH (pHi) 值,确定了该探针载入C. tropicalis细胞的适宜条件。用摇瓶培养C. tropicalis细胞,考察了细胞外pH和生长碳源对pH_I的影响,实验结果表明：细胞外pH对pH_I略有影响,而生长碳源对pH_I的影响略为明显。利用5L发酵罐进一步研究了细胞生长代谢活性与pHi的关系,结果表明：细胞比生长速率、CO₂比生产速率和葡萄糖比消耗速率与pHi变化密切相关,pH_I的增加伴随着细胞生长活力的增加,反之亦然。在pH6.0条件下用葡萄糖和醋酸钠共作碳源培养C. tropicalis细胞时,测得的pH_I值维持在5.72～6.15范围内。     

在血清饥饿条件下CHP2调节NHE活性减少细胞死亡

钠氢离子交换蛋白（NHE）是维持细胞内pH值等内环境稳定的重要蛋白;钙调磷酸酶B同源蛋白（CHP）是NHE的一个活性调节亚单位。研究CHP2对NHE1的调节作用时发现,在血清饥饿的条件下,PS120细胞依赖于CHP2的表达来调节外源性NHE1的活性,使细胞维持必要的钠氢交换生理活性和较高水平的细胞内pH值（pHi 7．4）,明显减少细胞因自身的胞浆酸性化而死亡,延长细胞存活时间（70％以上的细胞存活时间超过7天）。实验结果提示,通过研究减少CHP2表达或抑制其活性,可望找到加速细胞死亡的新方法。     

蟾蜍卵母细胞的Na~+/H~+交换

应用pH选择性徽电极和普通檄电极测量了蟾蜍卵母细胞内的pH值(pHi)。当用含NH_4~+而不含HCO_3~-的溶液培灌卵母细胞时,pHi逐渐下降,在停止培灌后,pHi恢复到对照值。这个pHi恢复过程是一个H~+的主动转运,用胆碱离子取代培灌液中的Na~+可以抑制pHi的恢复,这种抑制作用是可逆的。伴随着细胞内pH值的恢复,细胞内Na~+活度(Na_1~+)呈现暂时性增加,而细胞内Cl-活度(Cl_1~+)不变或稍有增加。实验结果提示在蟾蜍卵母细胞膜上存在着Na~+/H~+的交换。     

薄芝糖肽孵育的PBMC抑制胃癌细胞AGS的增殖迁移

探讨薄芝糖肽预孵育的胃癌患者外周血单个核细胞(PBMC)对胃癌细胞AGS形态特征、增殖、迁移的影响,为胃癌治疗新方法的研究提供基础。采用薄芝糖肽注射液预孵育PBMC,去除薄芝糖肽继续培养的PBMC,其培养上清液作为实验因素,未用薄芝糖肽预处理PBMC的上清液作为对照,作用于胃癌细胞AGS及其迁移模型,IPP软件测量AGS的形态学参数、迁移平均宽度,计算迁移率。薄芝糖肽预处理及未预处理的PBMC与AGS混合培养,MTT检测AGS的增殖活性。分析发现,实验组AGS的核浆比、梭形细胞面积、Feret最小值、Feret最大值、Feret平均值均显著低于对照组(p0.01);实验组的划痕宽度显著增加(F=11.277,p0.05);实验组AGS的迁移率显著低于对照组(F=12.96,p0.01)。结果表明,薄芝糖肽可以调节胃癌患者PBMC的功能,使PBMC减小胃癌细胞AGS的核浆比和直径,抑制其增殖和迁移。     

空心二氧化硅纳米微球包裹的photosan对胃腺癌细胞AGS的杀伤作用

目的:探讨空心二氧化硅纳米微球包裹的photosan(简称纳米化-Photosan)介导的光动力疗法(PDT)对人胃腺癌细胞AGS的杀伤作用。方法:采用人胃腺癌细胞AGS作为研究对象,应用MTT法测定AGS胃癌细胞的生存率和流式细胞分析法检测PDT对AGS胃癌细胞凋亡的影响。结果:MTT实验结果显示,纳米化-Photosan和普通Photosan对人胃腺癌细胞均有明显抑制作用,随纳米化-Photosan和普通photosan的浓度增高,胃癌细胞的生存率明显下降(P<0.05),同等浓度的纳米化-Photosan比普通Photosan对细胞的抑制作用更明显;流式法检测细胞凋亡显示光照剂量相同的条件下,同等浓度的纳米化-Photosan更易引起细胞的凋亡。结论:纳米化-Photosan比普通Photosan能更有效的抑制人胃腺癌细胞增殖,更易引起细胞凋亡。     

营养液pH值对非洲菊生长和生理特征的影响

以非洲菊(Gerbera jamesonii Bolus)为材料,采用水培的方法研究了营养液不同pH值(4.0、5.0、6.0、7.0、8.0、9.0)对其生长发育的影响.结果显示:(1)在pH 6.0条件下,非洲菊叶片和根系的鲜重、干重均最大,叶片数多于其他处理,且增加幅度较大;叶面积最大且叶面积增长最快.(2)随着培养天数的增加, pH 4.0～7.0处理非洲菊的叶绿素含量均呈现先上升后下降的趋势,而pH 8.0和pH 9.0处理的叶绿素含量一直呈现下降的趋势;pH 6.0处理的非洲菊在培养30 d时叶绿素含量极显著高于其他处理.(3)在培养45 d时,pH 6.0处理的SOD活性极显著高于其他处理;随着培养时间的延长,根系活力在pH 4.0～7.0处理呈持续上升趋势,在pH 8.0和pH 9.0处理呈先上升后下降的趋势.研究表明,在pH 6.0的微酸性环境下非洲菊的生长状态良好,pH 8.0和9.0的碱性环境对非洲菊的生长具有明显的抑制作用,其叶片发黄;培养介质pH值可能是非洲菊定植后叶片黄化的重要原因之一.     

TMSG-1基因功能的初步研究

TMSG-1是用mRNA差异显示技术克隆的转移相关基因, 它在高转移肿瘤细胞系和有转移的肿瘤组织中表达下降. 以高转移的前列腺癌细胞系PC-3M-1E8为受体细胞, 通过基因转染技术观察了TMSG-1基因表达对细胞V-ATPase活性、细胞内pH值和细胞凋亡情况的影响, 同时利用GFP对TMSG-1细胞内定位进行了分析. 结果表明, V-ATPase在PC-3M-1E8细胞系, 转染空载体和转染反义TMSG-1细胞系的活性无明显差异. 在转染正义TMSG-1的细胞系中, V-ATPase的活性比PC-3M-1E8细胞系, 转染空载体和转染反义TMSG-1细胞系均有明显增高(P < 0.001). 利用pH敏感性荧光探针BCECF测定细胞内的pH值, 结果显示转染正义TMSG-1组的pHi值有明显提高. 细胞凋亡的检测结果表明, 转染正义TMSG-1组细胞凋亡明显增多(P < 0.01), BCL2的表达显著下降. TMSG-1蛋白的细胞内定位分析表明, TMSG-1是一个跨膜蛋白, 定位于内质网、线粒体等细胞质膜系统. 实验结果表明, 前列腺癌细胞系中TMSG-1的上调可以提高细胞V-ATPase的活性, 增加细胞内的pH值, 同时TMSG-1的上调还可抑制BCL2的表达, 促进细胞凋亡的发生.     

新疆家蚕抗菌肽体外抑制人胃癌细胞AGS的增殖

新疆家蚕抗菌肽(Cecropin XJ)具有抑制肿瘤细胞生长的能力。为研究不同浓度Cecropin XJ体外对人胃癌细胞AGS生长的影响,分别采用MTT比色法、软琼脂集落形成实验、流式细胞术、划痕愈合和Transwell实验进行检测。结果表明,20~100μg/mL的Cecropin XJ能够显著抑制AGS细胞的增殖,并具有剂量和时间依赖性。同时,20,50,100μg/mL的Cecropin XJ处理后集落形成率分别降低了(35.81±13.10)%、(48.12±5.68)%和(81.46±6.21)%。AGS细胞经不同浓度Cecropin XJ处理24 h后,凋亡率逐渐上升并且细胞周期阻滞于S期。20μg/mL Cecropin XJ能够显著抑制AGS细胞的迁移和侵袭,100μg/mL Cecropin XJ几乎完全抑制AGS细胞的迁移和侵袭。以上结果说明,Cecropin XJ能够抑制AGS细胞生长、增殖,有可能成为人胃癌治疗的辅助药物。     

pH改变对心肌细胞内Ca~(2 )浓度和细胞长度的影响

目的 :探讨细胞内 pH(pHi)改变对心肌细胞内Ca2 浓度 ([Ca2 ]i)和细胞长度的影响。方法 :心肌细胞内分别灌注 2 0mmol/L丙酸钠和 15mmol/LNH4Cl,建立细胞内酸碱中毒模型。荧光指示剂indo 1和SNARF 1载入大鼠心肌细胞内 ,用荧光显微镜同时测定心肌 [Ca2 ]i、pHi 和细胞长度。结果 :细胞内酸中毒早期 ,收缩期和舒张期[Ca2 ]i 轻度增加 ,细胞缩短 (CS)降低 ,细胞长度增加 ,心肌纤维对Ca2 的敏感性和CS/ [Ca2 ]i 降低 (P <0 .0 1) ;碱中毒时 ,收缩期和舒张期 [Ca2 ]i 均较对照组降低 ,CS增加 ,细胞长度变短 ,心肌纤维对Ca2 的敏感性和CS/[Ca2 ]i 增加 (P <0 .0 1)。结论 :酸中毒早期 [Ca2 ]i 和细胞长度增加 ,碱中毒时 [Ca2 ]i和细胞长度降低。酸、碱中毒对Ca2 敏感性的影响并非线性关系 ,即单位 pHi变化时酸中毒对敏感性的影响较碱中毒小     

Aminoethylated chitooligomers and their apoptotic activity on AGS human cancer cells

In this study, the ability of aminoethylation of chitooligomers (COS) to inhibit the proliferation of AGS human gastric adenocarcinoma cells was evaluated using COS with lower molecular weight (<1 kDa). As water-soluble aminoderivatized COS derivatives, aminoethyl-COS (AE-COS), dimethyl aminoethyl-COS (DMAE-COS) and diethyl aminoethyl-COS (DEAE-COS) were synthesized and confirmed by their IR spectra results in comparison to previous study. Aminoderivatized COS-induced cell death was characterized by cell viability, changes in nuclear morphology and cell morphology. All aminoderivatized COS significantly inhibited cell proliferation of AGS cancer cells in a dose-dependent manner. Moreover, protein and gene expression levels of the regulators involved in apoptosis pathway such as Caspase-9, Bax, p53 and p21 were examined using RT-PCR and Western blot analysis. The exposure of synthesized COS derivatives to AGS cells induced apoptotic activity. Therefore, the present results suggest that all aminoderivatized COS derivatives have a promising potential as valuable as cancer chemopreventive agents.     

Porphyran induces apoptosis related signal pathway in AGS gastric cancer cell lines

Porphyrans, the sulfated polysaccharides, are the main components of Porphyra. The potential apoptotic activities of porphyran were evaluated using AGS human gastric cancer cells. Porphyran did not affect the growth of normal cells, but did induce cancer cell death in a dose-dependent manner. The addition of 0.1% porphyran also reduced DNA synthesis after 24 h of exposure, suggesting that porphyran inhibits cancer cell growth by both decreasing cell proliferation and inducing apoptosis. AGS cells treated with porphyran displayed a marked increase in poly(ADP-ribose) polymerase (PARP) cleavage, as well as caspase-3 activation. The ability of porphyran to promote apoptosis may contribute to its usefulness as an agent capable of significantly inhibiting cell growth in AGS human gastric cancer cells. Insulin-like growth factor-I receptor (IGF-IR) phosphorylation was decreased in porphyran-treated AGS cells compared to control cells, which correlated with Akt activation. Thus, porphyran appears to negatively regulate IGF-IR phosphorylation by causing a decrease in the expression levels in AGS gastric cancer cells, and then inducing caspase-3 activation.     

Anti-proliferative effect of aminoderivatized chitooligosaccharides on AGS human gastric cancer cells

In this study, the ability of aminoethylation of chitooligosaccharide (COS) to inhibit the proliferation of AGS human gastric adenocarcinoma cells was evaluated. Aminoderivatized COSs, aminoethyl-chitooligosaccharide (AE-COS), dimethyl aminoethyl-chitooligosaccharide (DMAE-COS) and diethyl aminoethyl-chitooligosaccharide (DEAE-COS), were synthesized and confirmed by their IR spectra results in comparison to previous study. Aminoderivatized chitooligosaccharides-induced cell death was characterized by cell viability assay, changes in nuclear morphology and changes in cell morphology. According to our results, all aminoderivatized COSs significantly induced cell death in AGS gastric cancer cells. Moreover, protein and gene expression levels of important regulators involved in apoptosis pathway such as Bcl-2, Bax, p53 and p21 were examined using RT-PCR and Western blot analysis. Aminoderivatized COSs showed dose- and time-dependent inhibition of AGS cancer cell proliferation. AE-COS and DEAE-COS showed the higher apoptotic activity than DMAE-COS. The present results suggest all three kinds of water-soluble aminoderivatized COSs have a promising potential as valuable as cancer chemopreventive agents.     

Cellular stress-related protein expression in Helicobacter pylori-infected gastric epithelial AGS cells

Helicobacter pylori infection leads to gastroduodenal inflammation, peptic ulceration, and gastric carcinoma. Moreover, H. pylori may induce disease-specific protein expression in gastric epithelial cells. The present study was aimed at determining differentially expressed proteins in H. pylori-infected gastric epithelial AGS cells. AGS cells were treated with H. pylori at a bacterium/cell ratio of 300:1 for 12 h. Altered protein patterns as separated by two-dimensional electrophoresis using pH gradients of 4-7 were conclusively identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the peptide digests. Four differentially expressed proteins, whose expression levels were increased by more than two-fold in H. pylori-infected cells, were analyzed. These proteins (14-3-3 protein alpha/beta, cullin homolog 3, alpha-enolase, ezrin) are known to be related to cell proliferation, cell adhesion, and carcinogenesis, and may be mediated by cellular stress, such as reactive oxygen species. In conclusion, the identification of these differentially expressed proteins provide valuable information for the understanding of the pathophysiologic mechanisms of H. pylori-induced gastric diseases, and may be useful as prognostic indices of H. pylori-related gastric disorders.     

Ligation or cross-linking of CD40 has different effects on AGS gastric cancer cells

A gastric cancer (GC) cell line, AGS, has high-level expression of CD40, a tumor necrosis factor receptor (TNFR) family member. CD40 is present on the surfaces of a large variety of cells, including B cells, endothelial cells, dendritic cells and some carcinoma cells, and delivers signals regulating diverse cellular responses, such as proliferation, differentiation, growth suppression, and cell death. In this research, we studied the effects of different forms of CD40 stimulation on AGS cells by flow cytometry, Western blotting and siRNA transfection. We found that different forms of CD40 stimulation, either recombinant soluble CD40L (sCD40L, ligation) or agonist anti-CD40 antibody (cross-linking), induced different effects in AGS gastric cancer cells, proliferation or apoptosis. We also showed that VEGF provided a significant contribution to sCD40L-induced proliferation, while agonist anti-CD40 antibody induced GADD45 upregulation and promoted apoptosis.     

Apigetrin induces extrinsic apoptosis,autophagy and G2/M phase cell cycle arrest through PI3K/AKT/mTOR pathway in AGS human gastric cancer cell

Apigetrin is a flavonoid glycoside phytonutrient derived from fruits and vegetables that is well known for a variety of biological activities such as antioxidant and anti-inflammatory activities. In the current study, we determined the effect of apigetrin on AGS gastric cancer cell. Apigetrin reduced cancer cell proliferation and induced G2/M phase cell cycle arrest by regulating cyclin B1, cdc25c and cdk1 protein expression in AGS cell. Apigetrin treatment caused apoptotic cell death in AGS cells, characterized by the accumulation of apoptosis portion, cleavage of caspase-3 and poly ADP-ribose polymerase (PARP). Apigetrin-treated cells increased the expression of extrinsic apoptosis pathway proteins and mRNA. However, intrinsic apoptosis pathway related proteins were not altered. In addition, AGS cells treated with apigetrin increased autophagic cell death, featured by the formation of autophagic vacuole and acidic vesicular organelles. Autophagy marker proteins, such as LC3B-II and beclin-1, were increased, and p62, an autophagy flux marker protein, was also increased by endoplasmic reticulum stress. Also, the phosphorylation of PI3K/AKT/mTOR pathway proteins and its downstream targets in apigetrin-treated AGS cells was identified to be decreased. Taken together, these data suggest that apigetrin-treated AGS cells induced G2/M phase cell cycle arrest, extrinsic apoptosis and autophagic cell death through PI3K/AKT/mTOR pathway, which can lead to the inhibition of gastric cancer development. Thus, our findings strongly indicate that apigetrin is a basic natural derived compound that could be used as a nutrient source with potential anticancer activities against gastric cancer.     

TGF-β1对AGS细胞上皮-间充质转化及体外侵袭的影响

目的观察转化生长因子-β1(TGF-β1)对人胃癌细胞株AGS发生上皮-间充质转化(epithelial-mesenchymal transition,EMT)及体外侵袭的影响。方法将体外培养的AGS用TGF-β1干预后,倒置显微镜下观察细胞形态学的变化,MTT比色法检测TGF-β1对AGS增殖的影响,细胞划痕试验和Transwell侵袭试验检测细胞运动和侵袭力的改变;免疫荧光和Western blot检测snail、E-cadherin(上皮钙粘蛋白)、和N-cadherin(神经钙粘蛋白)表达的变化。结果TGF-β1诱导AGS向间充质细胞形态转化,低浓度促进细胞增殖,而高浓度时细胞增殖率逐步降低,且snail和间充质细胞表型N-cadherin表达上调,而上皮细胞表型E-cadherin表达下调,同时细胞运动和侵袭能力大大增强。结论TGF-β1可诱导AGS发生EMT,从而增加其侵袭、转移的能力。     

Ethanol extract of paeonia suffruticosa Andrews (PSE) induced AGS human gastric cancer cell apoptosis via fas-dependent apoptosis and MDM2-p53 pathways

ABSTRACT: BACKGROUND: The root bark of Paeonia suffruticosa Andrews (PSE), also known as Moutan Cortex, has been widely used in Asia to treat various diseases. The molecular mechanisms by which PSE exerts its anti-oxidant and anti-inflammatory activities are well known, but its anti-cancer activity is not yet well understood. Here, we present evidence demonstrating that PSE can be used as a potent anti-cancer agent to treat gastric cancer. METHODS: The effects of the ethanol extract of PSE on cell proliferation were determined using an MTT (1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan) assay. Cell cytotoxicity induced by the PSE extact is measured using an LDH leakage assay. Flow cytometry was used to analyze the cell cycle and to measure the subG0/G1 apoptotic cell fraction. Apoptosis induced by the PSE extact is also examined using a DNA fragmentation assay. Western blot analysis is used to measure the levels of apoptotic proteins such as Fas receptor, caspase-8, caspase-3, PARP, Bax, Bcl-2, MDM2, and p53. RESULTS: This study demonstrated that treating AGS cells with the PSE extact significantly inhibited cell proliferation and induced cytotoxicity in a dose- and time-dependent manner. The PSE extract also induced apoptosis in AGS cells, as measured by flow cytometry and a DNA fragmentation assay. We found that the PSE extract induced apoptosis via the extrinsic Fas-mediated apoptosis pathway, which was concurrent with the activation of caspases, including caspase-8 and caspase-3, and cleavage of PARP. The MDM2-p53 pathway also played a role in the apoptosis of AGS cells that was induced by the PSE extract. CONCLUSIONS: These results clearly demonstrate that the PSE extact displays growth-suppressive activity and induces apoptosis in AGS cells. Our data suggest that the PSE extact might be a potential anti-cancer agent for gastric cancer.     

Platycodin D induces anoikis and caspase‐mediated apoptosis via p38 MAPK in AGS human gastric cancer cells

Mitogen‐activated protein kinases (MAPKs) cascades play important roles in cell proliferation, death, and differentiation in response to external stimuli. However, the precise role of MAPKs in platycodin D (PD)‐induced cytotoxicity remains unclear. In this study, we investigated the anticancer effect of PD and its underlying mechanism on AGS human gastric cancer cells. PD significantly inhibited cell proliferation and induced anoikis, which is a form of apoptosis in which cells detach from the substrate. It showed phosphatidylserine externalization, DNA fragmentation, increase of sub‐G1 phase, and activation of caspases in a dose‐ and time‐dependent manner. This apoptosis has been associated with the extrinsic pathway via Fas‐L and the intrinsic pathway via mitochondrial Bcl‐2 family members. Moreover, PD led to the phosphorylation of stresses‐activated protein kinases such as JNK and p38, followed by the activation of AP‐1. However, pretreatment with SB203580 (a p38 specific inhibitor) suppressed PD‐induced p38 and AP‐1 activation, and subsequently attenuated the PD‐induced apoptosis in AGS cells. These results suggest that p38 activation is responsible for PD‐induced apoptosis in AGS cells and PD might be useful for the development as the anticancer agent of gastric cancer. J. Cell. Biochem. 114: 456–470, 2013. © 2012 Wiley Periodicals, Inc.