容积调控氯通道的研究进展

细胞在低渗环境中出现渗透性膨胀 ,随后细胞内的溶质及水分外流 ,使已膨胀的细胞容积向正常容积转化 ,此过程称为调节性细胞容积减小 (RVD) ,它是哺乳动物细胞普遍存在的现象。容积调控氯通道 (VRAC)在这个过程中起重要作用 ,不仅如此 ,最近研究发现VRAC参与了细胞增殖、分化和凋亡过程。本文主要综述了VRAC的生物学特点和生理功能等方面的研究进展     

低渗条件下小肠上皮细胞调节性细胞容积减小的离子通道机制

目的:观察人小肠上皮细胞调节性细胞容积减小(RVD)的过程,探讨参与RVD过程的离子通道机制.方法:将培养的人小肠上皮细胞暴露于低渗溶液, 利用电子细胞体积测量系统测定细胞平均容积变化过程和离子通道的参与过程;采用RT-PCR方法检测人小肠上皮细胞上离子通道的表达.结果:人小肠上皮细胞具有良好的RVD功能; 其RVD过程可被氯通道阻断剂NPPB 和钾通道阻断剂四乙铵所阻断; 进一步的研究发现, 中等电导钙激活性钾通道(IK)的特异性阻断剂Clotrimazole (CLT) (1μmol/L)可以明显抑制细胞的RVD过程,而大电导钙激活性钾通道(BK)和小电导钙激活性钾通道(SK)的特异阻断剂iberiotoxin (100 nmol/L)和apamin (100 nmol/L)对RVD过程无任何抑制作用.RT-PCR的结果也显示, 人小肠上皮细胞只有IK表达, 而无SK和BK的表达.结论:人小肠上皮细胞具有RVD功能,RVD过程的完成有赖于氯通道和钾通道的平行激活, 而其中参与容积调节的钾通道是中等电导钙激活型钾通道IK.     

人乳头瘤病毒与细胞凋亡

近来研究表明病毒感染与细胞凋亡有着密切的关系。在研究中发现,许多病毒参与细胞凋亡的诱导和抑制,在这些过程中相关病毒的基因的表达起着关键作用,而细胞凋亡相关因子直接或间接参与这一过程,对于这些问题有助于阐明病毒感染后病毒和细胞相互作用的分子机理。本文对人乳头瘤病毒的E6、E7、E2、和E5等基因及表达蛋白与细胞凋亡相关因子p53、pRB等相互作用,以阐述HPV的致癌机理。     

动物细胞培养过程中的细胞凋亡

细胞培养过程中的细胞凋 细胞受环境因素的影响而发生的现象。随着对细胞凋亡的分子生物学和细胞生物学了解的深入,显示了有效地控制动物细胞增减保细胞凋亡的巨大潜力。     

细胞凋亡的形态学变化过程及相关的分子调节研究进展

细胞凋亡是细胞自主的有序性死亡,发生凋亡的细胞膜发生皱缩、凹陷,染色质变得致密,最后断裂成小碎片,进一步发展细胞膜将细胞质分割,包绕细胞质和细胞核的断片,形成了凋亡小体。在这个过程中许多分子参与调节。     

细胞凋亡过程中的蛋白水解酶

细胞凋亡过程中的蛋白水解酶李卓玉袁静明（山西大学生物工程实验室,太原０３０００６）关键词细胞凋亡蛋白水解酶细胞凋亡是一种主动清除无用或有害细胞的细胞消亡过程,有一些蛋白水解酶参与其事,下面予以简介：１．ＩＣＥＩＣＥ即白介素－１β－转换酶（ｉｎｔｅｒｌ...     

细胞凋亡中的钙调节

胞质Ca^2 升高是细胞凋亡中Ca^2 调节的经典模式,但近年来有证据表明胞质Ca^2 下降同样能诱导细胞凋亡,目前研究发现,胞内Ca^2 在细胞质,细胞核以及细胞钙库线粒体和内质网的动态平衡破坏和重新分布直接参与细胞凋亡信号的调控,而Bcl-2家族蛋白在细胞凋亡过程的胞内Ca^2 调节及继后的一系列生理效应中发挥特殊作用。细胞凋亡中Ca^2 调节的深入研究不但有助于阐明细胞凋亡的调控机理,同时为相关疾病防治和药物开发提供新的策略。     

动物细胞培养过程中的细胞凋亡

细胞培养过程中的细胞凋亡是细胞受环境因素的影响而发生的现象。随着对细胞凋亡的分子生物学和细胞生物学了解的深入,显示了有效地控制动物细胞培养中细胞凋亡的巨大潜力。包括采用ＤＮＡ重组技术把抗细胞凋亡的基因导入细胞和在培基中加入具有抗细胞凋亡的生存因子或化合物等手段已用于控制细胞培养过程中的细胞凋亡。这些技术将大大延长细胞达到饱和密度后的培养时间,提高细胞培养系统的生产效率。     

支持细胞在生精细胞凋亡过程中的重要作用

精子的生成过程是一个连续的周期性的过程,这包括二倍体的精原细胞进行一系列的有丝分裂,减数分裂最后分化成为成熟的单倍体精子的过程。支持细胞(Sertoli cell)是生精上皮的一种大细胞,它从管周肌样细胞所处的基底膜延伸出去,直到生精小管的管腔内。它的底部与精原细胞相连,胞质的侧面则形成许多突起将互相联系的生精细胞包围起来,成熟的精子就从这儿释放出去。在复杂的动态生精过程中,支持细胞作为精曲小管的重要组成部分指挥着生精细胞的一系列动态过程,包括有丝分裂、减数分裂以及以后的分化。支持细胞通过给生精细胞提供激素、营养以及生理支持来完成它的功能。     

miR-3604在牛子宫内膜上皮细胞容受性、增殖和凋亡中的作用

【目的】研究miR-3604在牛子宫内膜上皮细胞(bovines endometrial epithelial cells,bEECs)容受性中的作用。【方法】利用孕酮(progesterone,P4)和干扰素-tau(interferon tau,IFN-τ)联合诱导bEECs构建容受性细胞模型。在此基础上,对容受态bEECs进行牛miR-3604的过表达和干扰后,分别采用RT-qPCR、流式细胞术、EdU和CCK8技术检测了牛miR-3604在bEECs容受性、细胞增殖和凋亡中的作用。【结果】与对照组相比,容受态bEECs的miR-3604过表达组的细胞容受性标志基因HOXA10、IL-6和VEGF的表达量均显著降低(P < 0.05);细胞凋亡标志基因CASP3和BAX的表达量显著上调(P < 0.05);容受态bEECs的凋亡率极显著降低(P < 0.01),而细胞增殖率显著升高(P < 0.05)。同时,miR-3604干扰实验的结果与过表达实验的结果相反。【结论】miR-3604在容受性bEECs中的表达量升高,从而可抑制bEECs的容受性和细胞凋亡,并促进bEECs的增殖。     

Cell shrinkage and monovalent cation fluxes: role in apoptosis

The loss of cell volume or cell shrinkage has been a morphological hallmark of the programmed cell death process known as apoptosis. This isotonic loss of cell volume has recently been term apoptotic volume decrease or AVD to distinguish it from inherent volume regulatory responses that occurs in cells under anisotonic conditions. Recent studies examining the intracellular signaling pathways that result in this unique cellular characteristic have determined that a fundamental movement of ions, particularly monovalent ions, underlie the AVD process and plays an important role on controlling the cell death process. An efflux of intracellular potassium was shown to be a critical aspect of the AVD process, as preventing this ion loss could protect cells from apoptosis. However, potassium plays a complex role as a loss of intracellular potassium has also been shown to be beneficial to the health of the cell. Additionally, the mechanisms that a cell employs to achieve this loss of intracellular potassium vary depending on the cell type and stimulus used to induce apoptosis, suggesting multiple ways exist to accomplish the same goal of AVD. Additionally, sodium and chloride have been shown to play a vital role during cell death in both the signaling and control of AVD in various apoptotic model systems. This review examines the relationship between this morphological change and intracellular monovalent ions during apoptosis.     

Presence of side-population cells in an immortalized nontumorigenic human liver epithelial cell line

Side-population (SP) cells have been shown to be highly enriched stem cells. We investigated whether an immortalized, nontumorigenic human liver cell line, THLE-5b, contains SP cells. Flow cytometry analysis after Hoechst 33342 staining demonstrated that the THLE-5b line contained a small component of SP cells. These SP cells were essentially eliminated by treatment with verapamil and expressed higher levels of ABCG2 mRNA than non-SP cells. In addition, the level of these SP cells detected by Hoechst 33342 staining was affected by the experimental conditions including the incubation medium. This is the first report of the presence of SP cells in the immortalized, nontumorigenic human liver cell line.     

Effect of H. pylori on the expression of TRAIL, FasL and their receptor subtypes in human gastric epithelial cells and their role in apoptosis

BACKGROUND AND AIMS: In the human stomach expression of TNF-related apoptosis inducing ligand (TRAIL) and its receptors and the modulatory role of Helicobacter pylori are not well described. Therefore, we investigated the effect of H. pylori on the expression of TRAIL, FasL and their receptors (TRAIL-R1-R4, Fas) in gastric epithelial cells and examined their role in apoptosis. MATERIALS AND METHODS: mRNA and protein expression of TRAIL, FasL and their receptors were analyzed in human gastric epithelial cells using RT-PCR, Western blot, and immunohistochemistry. Gastric epithelial cells were incubated with FasL, TRAIL and/or H. pylori, and effects on expression, cell viability and epithelial apoptosis were monitored. Apoptosis was analyzed by histone ELISA, DAPI staining and immunohistochemistry. RESULTS: TRAIL, FasL and their receptor subtypes were expressed in human gastric mucosa, gastric epithelial cell primary cultures and gastric cancer cells. TRAIL, FasL and H. pylori caused a time- and concentration-dependent induction of DNA fragmentation in gastric cancer cells with synergistic effects. In addition, H. pylori caused a selective up-regulation of TRAIL, TRAIL-R1 and Fas mRNA and protein expression in gastric cancer cells. CONCLUSIONS: Next to FasL and Fas, TRAIL and all of its receptor subtypes are expressed in the human stomach and differentially modulated by H. pylori. TRAIL, FasL and H. pylori show complex interaction mediating apoptosis in human gastric epithelial cells. These findings might be important for the understanding of gastric epithelial cell kinetics in patients with H. pylori infection.     

Induction of apoptosis in Caco-2 and HT-29 human intestinal epithelial cells by enterohemolysin produced by classic enteropathogenic Escherichia coli

AIMS: Detect the cytotoxic effects of the Enterohemolysin from enteropathogenic Escherichia coli C3888 (O 26: H-) on Caco 2 and HT-29-human epithelial intestinal cells. METHODS AND RESULTS: The Caco 2 and HT-29 cells, which were treated with Enterohemolysin (EHly) within 10-15 min, became round, lost attachment to substrate, showed extensive surface blebbing, nucleus shrank, and the chromatin became more compact. After 10 min of exposure to the EHly, the cells showed lactate dehydrogenase (LDH) leakage and reduction of mitochondrial activity. The cells showed disorganization of the actin fibers at 15 min. The death of these human epithelial intestinal cells by apoptosis was confirmed by annexin V. CONCLUSIONS: Enterohemolysin induced apoptosis on human epithelial intestinal cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The finding of EHly cytotoxic activity suggests the involvement of this hemolysin in the (Enteropathogenic Escherichia coli) EPEC infection mechanism and may facilitate the understanding of the diarrhea caused by EPEC.     

Parthenolide protects human lens epithelial cells from oxidative stress-induced apoptosis via inhibition of activation of caspase-3 and caspase-9

The apoptosis of lens epithehal cells has been proposed as the common basis of cataract formation, with oxidative stress as the major cause. This study was performed to investigate the protective effect of the herbal constituent parthenolide against oxidative stress-induced apoptosis of human lens epithelial （HLE） cells and the possible molecular mechanisms involved. HLE cells （SRA01-04） were incubated with 50 μM H2O2 in the absence or presence of different doses of parthenolide （10, 20 and 50 μM）. To study apoptosis, the cells were assessed by morphologic examination and Annexin V-propidium iodide double staining flow cytometry; to investigate the underlying molecular mechanisms, the expression of caspase-3 and caspase-9 were assayed by Western blot and quantitative RT-PCR, and the activities of caspase-3 and caspase-9 were measured by a Chemicon caspase colorimetric activity assay kit. Stimulated with H202 for 18 h, a high fraction of riLE cells underwent apoptosis, while in the presence ofparthenolide of different concentrations, dose-dependent blocking of HLE cell apoptosis was observed. The expression of caspase-3 and caspase-9 induced by H202 in HLE cells was significantly reduced by parthenolide both at the protein and mRNA levels, and the activation ofcaspase-3 and caspase-9 was also suppressed by parthenolide in a dose-dependent manner. In conclusion, parthenolide prevents HLE cells from oxidative stress-induced apoptosis through inhibition of the activation ofcaspase-3 and caspase-9, suggesting a potential protective effect against cataract formation.     

Leukotriene-D4 induced cell shrinkage in Ehrlich ascites tumor cells

Summary The nature of the leukotriene-D₄ (LTD₄) induced cell shrinkage in Ehrlich ascites tumor cells has been investigated. LTD₄ treatment of Ehrlich cells induces net loss of cellular KCl and cell shrinkage independent of the initial cell volume. LTD₄ also produces water loss and reduction in cell volume when all extracellular and all intracellular Cl has been replaced by NO₃. On the other hand, LTD₄ fails to produce any significant changes in cell volume in the presence of the K-channel blocker quinine, suggesting that LTD₄ in Ehrlich cells induces Cl-independent K loss through the Ca²⁺-dependent K channels. However, the effect of physiological doses of LTD₄ on cell volume seems not to be as potent in Cl-free, NO₃ cells when compared to Cl-containing cells, indicating that LTD₄ in Ehrlich cells also provokes Cl-dependent K loss. LTD₄ seems not to produce K loss through an electroneutral K⁺/H⁺ exchange system. LTD₄ still produces Cl-independent K loss and cell shrinkage in the presence of the anticalmodulin drug pimozide but not in the presence of the LTD₄ receptor antagonist L-649,923 or the 5-lipoxygenase inhibitor NDGA. Pretreatment of the cells with pertussis toxin, which inactivates inhibitory guanine nucleotide binding proteins (G-proteins), leads to partial inhibition of the LTD₄-induced shrinkage. It is suggested that the LTD₄-induced activation of K and Cl transporting systems in Ehrlich ascites tumor cells is mediated via a G-protein coupled receptor and that LTD₄ might exert its effect through another lipoxygenase product. The Ca²⁺-calmodulin complex is not involved in the LTD₄-induced activation of K and Cl transporting systems.     

Tissue culture of human epithelial cells from benign colonic tumors

Summary Human colonic epithelial cells from three classes of benign tumors have been reproducibly cultured free of fibroblasts for 8 wk using a supplemented Medium 199 (M 199S). The cultured colonic cells were identified as epithelial by the presence of junctional complexes (tight junctions, gap junctions, and desmosomes), a brush border on the apical surface, keratin fibrils, and by both a close-packed columnar or cuboidal morphology and the capability to transport water and ions to form hemicysts. Colony formation was initiated by groups of epithelial cells, not by single cells, and was inhibited by cocultivation with either lethally irradiated 3T3 cells or human diploid fibroblasts. Enhancement of epithelial colony formation was observed following culture on nonadherent, “floating” substrates compared with substrates attached directly to the bottom of the culture dish. Replication of epithelial cells in M 199S from the class of benign colonic tumors least prone to malignancy, the tubular, was significantly enhanced by epidermal growth factor (EGF). In contrast, EGF did not stimulate the growth of cells in M 199S from the other classes of benign tumors, the villotubular and the villous, which exhibit more malignant potential. These data imply that premalignant colonic epithelial cells lose responsiveness to growth modulation by EGF as they progress toward frank carcinoma. This study was supported by NCI Contract N01-CP43366 to M. L. and NCI Grant 1-R26-CA 28822 to E. F.     

Human amniotic epithelial cells induce apoptosis of cancer cells: a new anti-tumor therapeutic strategy

Background aimsAmniotic membrane (AM), the innermost layer of human placenta, is composed of a single layer of epithelial cells, a basement membrane and an avascular stroma. The AM has many functions and properties, among which angiogenic modulatory and immunoregulatory effects are applicable in cancer therapy. Because these functions belong to amniotic epithelial cells, in this study we compared the anti-cancer effect of amniotic epithelial cells and the whole AM.MethodsThe effect of the AM and the amniotic epithelial cells on cancer cell apoptosis was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay, terminal deoxynucleotidyl transferase dUTP nick end labeling assay and immunocytochemistry. The effect of the AM on angiogenesis in conditions both with and without epithelial cells was also evaluated using rat aortic ring assay.ResultsThere was a decrease in cancer cell viability after adding either AM or amniotic epithelial cell supernatant to cancer cells. A significant increase in caspase-3 and caspase-8 expression in cancer cells treated with amniotic epithelial cell supernatant was observed. The recorded media also demonstrated the possible induction of apoptosis in cancer cells treated with the amniotic epithelial cell supernatant. In the aorta ring assay, the AM showed an anti-angiogenic effect in the presence of its epithelial cells; however, this effect was altered to initiate angiogenesis when amniotic epithelial cells were removed from the AM.ConclusionsThese results suggest that amniotic epithelial cells, with their anti-angiogenic effect and induction of apoptosis, are candidates for cancer therapeutic agents in the near future.     

Alkaline stress-induced apoptosis in human pulmonary artery endothelial cells

The effect of alkaline stress, or an increase in extracellular pH (pHext), on cell viability is poorly defined. Human pulmonary artery endothelial cells (HPAEC) were subjected to alkaline stress using different methods of increasing pHext. Viability and mode of cell death following alkaline stress were determined by assessing nuclear morphology, ultrastructural features, and caspase-3 activity. Incubation of monolayers in media set to different pHext values (7.4–8.4) for 24-h induced morphological changes suggesting apoptosis (35–45% apoptotic cells) following severe alkaline stress. The magnitude of apoptosis was related to the severity of alkaline stress. These findings were confirmed with an assessment of ultrastructural changes and caspase-3 activation. While there was no difference in the intracellular calcium level ([Ca²⁺]_i) in monolayers set to pHext 7.4 versus 8.4 following the first hour of alkaline stress, blockade of calcium uptake with the chelator, EGTA, potentiated the magnitude of apoptosis under these conditions. Potentiation of apoptosis was reduced by calcium supplementation of the media. Finally, alkaline stress was associated with an increase in intracellular pH. This is the first report of apoptosis following alkaline stress in endothelial cells in the absence of other cell death stimuli.     

The role of melatonin as an antioxidant in human lens epithelial cells

Abstract

Oxidative stress plays a significant role in pathophysiology of cataracts and also known to affect the phosphatidylinositol-3-kinase/ protein kinase B (PI3K/Akt) signaling pathway. This well-documented pathway is involved in protecting against apoptosis-inducing insults, including oxidative stress. Melatonin (N-acetyl-5-methoxy-tryptamine), the major secretory product of the pineal gland, was identified as a powerful free radical scavenger and a broad-spectrum antioxidant that defends against various oxidative stress-associated diseases. This study was conducted to determine whether melatonin could prevent hydrogen peroxide (H₂O₂)-induced oxidative stress in human lens epithelial cells (HLECs) and to elucidate the molecular pathways involved in this protection. HLECs were subjected to various concentrations of H₂O₂ in the presence or absence of melatonin at different concentrations. Cell viability was monitored by a 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyl-tetrazoliumbromide (MTT) assay, and the apoptosis rate and intracellular reactive oxygen species (ROS) levels were measured by flow cytometry using annexin V-FITC and propidium iodide (PI) staining. The expression levels of HO-1, Nrf-2, CAT, and MDA were measured using Western blot analysis. Akt activation was also evaluated by Western blot analysis. The data from our study showed that cells pretreated with melatonin can reduce H₂O₂-induced intracellular ROS generation and thus protect HLECs from cell apoptosis. Furthermore, we found that melatonin is a potent activator of Akt in HLECs. Our findings suggest that in addition to functioning as a direct free radical scavenger, melatonin can elicit cellular signaling pathways that are protective against oxidative stress-induced cataracts.