TRPC6对肝癌细胞增殖的介导

目的 瞬时受体势(TRP)是一种新发现的钙通道,是当前细胞信号转导领域内的研究热点之一.以往有关TRP通道的研究主要集中在神经系统,现已开始向其它研究领域扩展.笔者推测,TRP通道可能与肝癌细胞的增殖有关.该研究将证实此推测.方法 应用TRPC通道抑制剂(SKF-96365)和RNAi技术抑制该通道蛋白在肝癌细胞中的表达和功能,观察TRPC被抑制前后肝癌细胞体外增殖能力的改变.结果 研究发现,应用SKF-96365和siRNA方法阻滞TRPC6的通道功能后,肝癌细胞(HepG2和SMMC-7721)的增殖被明显抑制.结论 该研究证明TRPC6通道介导了肝癌细胞的增殖,为肝癌细胞增殖机制的研究提供了新的思路,探索了肝癌临床治疗新的细胞膜上的靶点,同时也拓展了TRP通道的研究领域.     

RNA干扰PLK1基因表达对肝癌细胞增殖的影响

目的探讨靶向PLK1基因在肝癌基因治疗中的可行性。方法采用PLK1小干扰核糖核酸分子(siRNA)转染人肝癌HepG2细胞,分别以荧光实时定量PCR和Western blot检测PLK1基因和蛋白的表达水平,观察PLK1 siRNA转染对肝癌细胞体外增殖的影响。并于转染不同时间后收集细胞,分别采用琼脂糖凝胶电泳和TUNEL方法检测肝癌细胞的凋亡情况。结果 PLK1基因明显抑制癌细胞体外生长(P0.05)。肝癌HepG2细胞经siRNA转染处理后,PLK1 mRNA和蛋白表达水平明显下降(P0.05)。DNA电泳出现明显的梯度图谱;转染组癌细胞凋亡指数明显增加。结论 PLK1 siRNA转染可明显抑制肝癌细胞增殖,其机制可能与诱导细胞凋亡有关。本结果为肝癌以PLK1基因治疗提供了一条新的思路。     

18β-甘草次酸抑制肝癌细胞生长的实验研究

目的:探讨18β-甘草次酸(GA)对肝癌细胞生长的影响。方法:用不同浓度的GA或阿霉素(ADM)处理肝癌细胞(Hepa1-6)及正常肝细胞(AML-12)不同时间后,分析细胞的抑制情况及半数抑制浓度(IC_(50));根据IC_(50),选择合适浓度的GA处理Hepa1-6细胞一定时间后,检测细胞的凋亡情况。结果:低浓度GA对两种细胞增殖均无明显影响,当GA浓度8μg/m L时,对两种细胞的增殖均有明显的抑制作用,且抑制作用随药物浓度升高、作用时间延长而增加;ADM所有浓度对两种细胞的增殖均有抑制作用,且抑制作用随浓度升高而增加,但随时间延长而减弱。GA对Hepa1-6细胞的IC_(50)低于对AML-12细胞的IC_(50),当药物作用48 h时,GA对两种细胞的IC_(50)的差值最大,而ADM对Hepa1-6细胞的IC_(50)在任何时间都高于对AML-12细胞的IC_(50);选择25μg/m L的GA处理Hepa1-6细胞48 h(该条件下,Hepa1-6细胞的增殖抑制率为50%,而AML-12细胞的增殖抑制率为3.8%),Hepa1-6细胞的明显增加,且主要是早期凋亡(P0.05)。结论:GA能够抑制肝癌的生长,其机制可能与诱导肝癌细胞凋亡有关;在治疗肝癌方面,GA可能较ADM更为安全,毒副作用更小。     

延迟整流性K^＋通道在肿瘤坏死因子α抑制人肝癌细胞增殖中？…

目的 研究延迟整流性Ｋ＾＋通道在肿瘤坏死因子α（ＴＮＦα）抑制人肝癌细胞增殖中的作用。方法 采用膜片钳技术的全细胞记录方式记录人肝癌细胞的跨膜电流，氚标记的胸腺嘧啶核苷掺入技术测定人肝癌细胞的ＤＮＡ合成能力。结果 ＴＮＦα抑制肝癌增殖的同时，救治 有使肝癌细胞膜离子通道的跨膜Ｋ＾＋电流和细胞膜电导降低。Ｋ＾＋通道阻断剂四乙胺不能进一步增加ＴＮＦ抑制ＤＮＡ合成的作用，且无明显量效关系和时间依赖性。结     

平滑肌收缩机制及其与勃起功能障碍的关系

阴茎勃起功能障碍(ED)与平滑肌细胞的收缩与舒张失衡相关。ED发生过程中,除舒张功能减弱外,另一重要原因为平滑肌细胞的收缩功能上调。其中,收缩相关性信号通路、胞膜离子通道、平滑肌细胞表型都参与了平滑肌细胞收缩的调节,其功能变化可引起多种平滑肌细胞相关疾病。收缩相关性信号通路Raf/MEK/ERK1/2与Rho A/Rock通路之间存在交互作用,抑制Rho A蛋白表达或降低Rock2磷酸化水平都可能成为ED的治疗途径。电压依赖性钙通道(VDCCs)、瞬时受体电位(TRP)通道功能失调是高血压、糖尿病ED发病的主要原因之一,多种病理因素上调Ca V1.2、TRPC1及TRPC4表达可使平滑肌收缩功能加强,进而导致ED的发生;阴茎海绵体表型转化也与ED的发生发展有关。本文就近年平滑肌收缩机制与ED关系研究的进展加以综述。     

肝素与无水乙醇抑制肝癌细胞的实验研究

目的通过肝素、无水乙醇对肝癌细胞株生物学行为影响的研究,为临床肝素盐水清洗祛除肝癌术中手术工具肿瘤细胞提供基础实验依据。方法取对数生长期人肝癌细胞株Hepa 1-6,以肝素250U/mL的DMEM、无水乙醇、DMEM、完全培养基刺激细胞3h后,分别测定并比较上述四组细胞增殖、凋亡、趋化运动和侵袭力之间的差异。结果肝素和无水乙醇可以明显抑制Hepa1-6细胞的增殖(与另两组比较,均P0.01);肝素组细胞凋亡数量显著高于另三组(P0.05);经肝素、无水乙醇刺激后,细胞的趋化运动和侵袭能力明显抑制(与另两组比较,均P0.01)。结论肝素、无水乙醇对肝癌细胞株Hepa1-6的体外增殖、趋化运动和侵袭性具有明显的抑制作用;肝素诱导肝癌细胞株Hepa1-6凋亡。     

TRPV4通道蛋白在肿瘤发生发展中的研究进展

TRPV4通道是瞬时受体电位通道家族(TRP)的成员,属非选择性阳离子通道,可被热、机械力、佛波醇酯衍生物等多种理化刺激所激活,参与维持细胞的正常功能。近年来,TRPV4在细胞增殖、分化、凋亡及迁移中的作用研究颇多,其异常表达与肿瘤的发生发展密切相关。笔者就该通道在肿瘤中的研究进展进行综述。     

维生素E琥珀酸酯对人肝癌细胞凋亡和侵袭性的影响研究

目的 研究维生素E琥珀酸酯(VES)诱导体外培养人肝癌细胞凋亡以及对肝癌细胞侵袭性的抑制作用.方法 自2004年至2005年7月,体外培养SMMC7721人肝癌细胞系,在培养液中加入VES共育,用等量的生理盐水作为对照组.培养6、24、48 h后采用流式细胞仪检测肝癌细胞的凋亡,小孔趋化试验检测肝癌细胞的体外侵袭性变化.结果 在加入VES后肝癌细胞的凋亡率与对照组相比明显增加.在48 h加入VES的实验组与对照组相比,肝癌细胞的小孔趋化试验明显受到抑制.结论 VES具有体外诱导人肝癌细胞系凋亡以及抑制肝癌细胞体外侵袭性的作用,是潜在的抗肿瘤因子.     

AG1024对肝癌细胞系增殖与凋亡的影响

目的 探讨胰岛素样生长因子Ⅰ型受体(IGF-IR)酪氨酸激酶阻断剂AG1024(3-溴-5-叔丁基-4-羟基苯叉苹果酸腈)对人肝癌癌细胞的增殖抑制作用和凋亡诱导作用.方法 采用MTY、流式细胞术、细胞侵袭实验、RT-PCR和Western blot等方法检测在不同浓度(0～40 μmol/L)的AG1024作用下,人肝癌细胞系HepG2和SMMC-7721的细胞形态学及分子生物学特性的改变.结果 MTT检测显示,AG1024剂量依赖性地抑制肝癌细胞的增殖.流式细胞术提示,AG1024明显促进肝癌细胞的凋亡.Transwell小室侵袭实验显示,AG1024能明显抑制肝癌细胞系HepG2和SMMC-7721的侵袭能力,与对照组比较差异有统计学意义(P＜0.05).RT-PCR结果显示,肝癌细胞中IGF-IR呈高表达,不同浓度AG1024作用后,剂量依赖性地增加细胞色素C的表达.Western blotting结果显示,AG1024降低胞外途径信号调节激酶(extracellular signal-regulated kinase,ERK)的磷酸化水平,下调procaspase-3的表达,而总ERK保持不变.结论 AG1024阻断胰岛素样生长因子1型受体,从而阻断其下游的信号传导途径,抑制肝癌细胞的增生,诱导凋亡.     

新型增殖性腺病毒的构建及对肝癌细胞的抑制

目的 构建携带p53基因新型增殖性腺病毒CNHK600-p53,研究其对肝癌细胞株抑制效应是否优于Ad-p53.方法 PCR扩增p53基因,利用酶切连接方法 将其插入CNHK600载体,PCR鉴定.经293细胞包装成病毒,抽提病毒DNA,PCR鉴定.氯化铯密度梯度离心法纯化病毒,TCID50 方法 测病毒滴度.病毒增殖实验检测病毒在不同细胞增殖能力.四甲基偶氮唑盐(methyl-thiazolyl tetrazolium assay,MTT)法观察CNHK600-p53、Ad-p53两种病毒分别对肝癌细胞株的抑制率.结果 成功构建新型增殖性腺病毒载体CNHK600-p53;293细胞包装成病毒,PCR方法 鉴定无野生型病毒存在;病毒滴度为1.99×10~（10）pfu/ml;CNHK600-p53在肝癌细胞HepG2、SMMC-7721内复制能力明显高于正常肝细胞HEL-1和L02.对6种肝癌细胞(PLC/PRF5、SMMC7721、HepaG2、BEL-7402、BEL-7404、QGY-7703)而言,随着MOI值的不断增高.其对肝癌细胞的抑制作用也不断增强;在相同MOI情况下,CNHK600-p53组较Ad-p53组的细胞抑制率高(P＜0.05).当细胞抑制率达到80%以上时.两组之间差异无统计学意义(P＞0.05).结论 利用新型增殖性腺病毒CNHK600一p53较 Ad-p53能更有效的抑制肝癌细胞,可能成为肝癌的基因治疗更有效的一种基因治疗手段.     

TRP channels in prostate cancer： the good,the bad and the ugly？

During the last decade, transient receptor potential (TRP) channels emerge as key proteins in central mechanisms of the carcinogenesis such as cell proliferation, apoptosis and migration. Initial studies showed that expression profile of some TRP channels, notably TRP melastatin 8 (TRPM8), TRP vanilloid 6 (TRPV6),TRP canonical (TRPC6) and TRPV2, is changing during the development and the progression of prostate cancer towards the hormone-refractory stages. The link between the change in expression levels and the functional role of these channels in prostate cancer is step by step being elucidated. These recent advances are here described and discussed.     

Podocyte Purinergic P2X4 Channels Are Mechanotransducers That Mediate Cytoskeletal Disorganization

Podocytes are specialized, highly differentiated epithelial cells in the kidney glomerulus that are exposed to glomerular capillary pressure and possible increases in mechanical load. The proteins sensing mechanical forces in podocytes are unconfirmed, but the classic transient receptor potential channel 6 (TRPC6) interacting with the MEC-2 homolog podocin may form a mechanosensitive ion channel complex in podocytes. Here, we observed that podocytes respond to mechanical stimulation with increased intracellular calcium concentrations and increased inward cation currents. However, TRPC6-deficient podocytes responded in a manner similar to that of control podocytes, and mechanically induced currents were unaffected by genetic inactivation of TRPC1/3/6 or administration of the broad-range TRPC blocker SKF-96365. Instead, mechanically induced currents were significantly decreased by the specific P2X purinoceptor 4 (P2X₄) blocker 5-BDBD. Moreover, mechanical P2X₄ channel activation depended on cholesterol and podocin and was inhibited by stabilization of the actin cytoskeleton. Because P2X₄ channels are not intrinsically mechanosensitive, we investigated whether podocytes release ATP upon mechanical stimulation using a fluorometric approach. Indeed, mechanically induced ATP release from podocytes was observed. Furthermore, 5-BDBD attenuated mechanically induced reorganization of the actin cytoskeleton. Altogether, our findings reveal a TRPC channel-independent role of P2X₄ channels as mechanotransducers in podocytes.     

The transient receptor potential superfamily of ion channels

ABSTRACT. The transient receptor potential (TRP) superfamily of proteins is cation-selective ion channels with six predicted transmembrane segments and intracellularly localized amino and carboxyl termini. Members of the TRP superfamily are identified on the basis of amino acid sequence and structural similarity and are classified into TRPC, TRPV, TRPM, TRPP, TRPN, and TRPML subfamilies. TRP channels are widespread and have diverse functions, ranging from thermal, tactile, taste, osmolar, and fluid flow sensing to transepithelial Ca2+ and Mg2+ transport. Mutations of TRP proteins produce many renal diseases, including Mg2+ wasting, hypocalcemia, and polycystic kidney diseases. This review focuses on recent advances in the understanding of their functions.     

On the origin of bladder sensing: Tr(i)ps in urology

The mammalian TRP family consists of 28 channels that can be subdivided into 6 different classes: TRPV (vanilloid), TRPC (canonical), TRPM (Melastatin), TRPP (Polycystin), TRPML (Mucolipin), and TRPA (Ankyrin). TRP channels are activated by a diversity of physical (voltage, heat, cold, mechanical stress) or chemical (pH, osmolality) stimuli and by binding of specific ligands, enabling them to act as multifunctional sensors at the cellular level. Currently, a lot of scientific research is devoted to these channels and their role in sensing mechanisms throughout the body. In urology, there's a growing conviction that disturbances in afferent (sensory) mechanisms are highly important in the pathogenesis of functional problems. Therefore, the TRP family forms an interesting new target to focus on. In this review we attempt to summarize the existing knowledge about TRP channels in the urogenital tract. So far, TRPV1, TRPV2, TRPV4, TRPM8, and TRPA1 have been described in different parts of the urogenital tract. Although only TRPV1 (the vanilloid receptor) has been extensively studied so far, more evidence is slowly accumulating about the role of other TRP channels in the (patho)physiology of the urogenital tract.     

High Glucose�CInduced Oxidative Stress Increases Transient Receptor Potential Channel Expression in Human Monocytes

OBJECTIVE

Transient receptor potential (TRP) channel–induced cation influx activates human monocytes, which play an important role in the pathogenesis of atherosclerosis. In the present study, we investigated the effects of high glucose–induced oxidative stress on TRP channel expression in human monocytes.

RESEARCH DESIGN AND METHODS

Human monocytes were exposed to control conditions (5.6 mmol/l d-glucose), high glucose (30 mmol/l d-glucose or l-glucose), 100 μmol/l peroxynitrite, or high glucose in the presence of the superoxide dismutase mimetic tempol (100 μmol/l). TRP mRNA and TRP protein expression was measured using quantitative real-time RT-PCR and quantitative in-cell Western assay, respectively. Calcium influx and intracellular reactive oxygen species were measured using fluorescent dyes.

RESULTS

Administration of high d-glucose significantly increased reactive oxygen species. High d-glucose or peroxynitrite significantly increased the expression of TRP canonical type 1 (TRPC1), TRPC3, TRPC5, TRPC6, TRP melastatin type 6 (TRPM6), and TRPM7 mRNA and TRPC3 and TRPC6 proteins. High d-glucose plus tempol or high l-glucose did not affect TRP expression. Increased oxidative stress by lipopolysaccharide or tumor necrosis factor-α increased TRP mRNA expression, whereas the reduction of superoxide radicals using diphenylene iodonium significantly reduced TRP mRNA expression. Increased TRPC3 and TRPC6 protein expression was accompanied by increased 1-oleoyl-2-acetyl-sn-glycerol–induced calcium influx, which was blocked by the TRPC inhibitor 2-aminoethoxydiphenylborane. TRPC6 mRNA was significantly higher in monocytes from 18 patients with type 2 diabetes compared with 28 control subjects (P < 0.05).

CONCLUSIONS

High d-glucose–induced oxidative stress increases TRP expression and calcium influx in human monocytes, pointing to a novel pathway for increased activation of monocytes and hence atherosclerosis in patients with diabetes.Cardiovascular complications due to atherosclerotic disease are a frequent cause of morbidity and mortality in patients with diabetes (1). Epidemiologic studies and preliminary intervention studies have shown that hyperglycemia is a direct and independent risk factor for cardiovascular disease (2). Atherogenesis has been considered to be an inflammatory disease with accumulation of monocytes within the artery wall (3). Monocytes are transitional cells, with a short half-life due to rapid differentiation into macrophages, and they are rapidly recruited to sites of inflammation (4,5). Monocyte activation, adhesion to the endothelium, and transmigration into the subendothelial space are key events in early pathogenesis of atherosclerosis. The mechanisms by which high glucose supports monocyte-associated atherosclerosis are only partially known. Mononuclear blood cells from patients with diabetes show increased generation of reactive oxygen species because of chronic high glucose levels (6–8). An increased activation of monocytes from patients with diabetes is associated with elevated protein kinase C activity and increased cytosolic calcium concentrations (9–11). Elevated transmembrane calcium influx may be mediated by increased transient receptor potential canonical (TRPC) channels. Until now, only few studies addressed transient receptor potential (TRP) expression under diabetic high glucose conditions. One study observed TRPC type 1 (TRPC1), TRPC4, and TRPC6 regulation and impaired capacitative calcium entry in vessels of diabetic patients compared with nondiabetic human vessels (12). As TRPC channels have been identified in several cell types including peripheral blood monocytes (13,14), the present study was aimed at elucidating the effects of high glucose and oxidative stress on TRP expression and their functional relevance in mediating calcium influx.     

Canonical transient receptor potential 1 channel is involved in contractile function of glomerular mesangial cells

Contractility of mesangial cells (MC) is tightly controlled by [Ca(2+)](i). Ca(2+) influx across the plasma membrane constitutes a major component of mesangial responses to vasoconstrictors. Canonical transient receptor potential 1 (TRPC1) is a Ca(2+)-permeable cation channel in a variety of cell types. This study was performed to investigate whether TRPC1 takes part in vasoconstrictor-induced mesangial contraction by mediating Ca(2+) entry. It was found that angiotensin II (AngII) evoked remarkable contraction of the cultured MC. Downregulation of TRPC1 using RNA interference significantly attenuated the contractile response. Infusion of AngII or endothelin-1 in rats caused a decrease in GFR. The GFR decline was significantly reduced by infusion of TRPC1 antibody that targets an extracellular domain in the pore region of TRPC1 channel. However, the treatment of TRPC1 antibody did not affect the AngII-induced vasopressing effect. Electrophysiologic experiments revealed that functional or biologic inhibition of TRPC1 significantly depressed AngII-induced channel activation. Fura-2 fluorescence-indicated that Ca(2+) entry in response to AngII stimulation was also dramatically inhibited by TRPC1 antibody and TRPC1-specific RNA interference. These results suggest that TRPC1 plays an important role in controlling contractile function of MC. Mediation of Ca(2+) entry might be the underlying mechanism for the TRPC1-associated MC contraction.     

Transient receptor potential channels in rat renal microcirculation: actions of angiotensin II

BACKGROUND: This study assessed the calcium-activating mechanisms mediating glomerular arteriolar constriction by angiotensin II (Ang II). METHODS: Immunohistochemical and physiological studies were carried out, using antibody against transient receptor potential (TRP)-1 and an isolated perfused kidney model. RESULTS: Immunohistochemical experiments demonstrated that TRP-1 proteins were transcribed on both afferent and efferent arteriolar myocytes. In the first series of physiological experiments, Ang II (0.3 nmol/L) considerably constricted afferent (20.2 +/- 0.9 to 14.9 +/- 0.7 microm) and efferent arterioles (18.4 +/- 0.7 to 14.0 +/- 0.7 microm). The addition of nifedipine (1 micromol/L) restored decrements in afferent (to 20.0 +/- 0.8 microm) but not efferent arteriolar diameters. Further administration of SKF-96365 (100 micromol/L), a TRP channel blocker, reversed efferent arteriolar constriction (to 16.2 +/- 0.8 micromol/L). In the second group, although 2-aminoethoxydiphenyl borate (100 micromol/L), an inhibitor of inositol trisphosphate-induced calcium release (IP3CR), did not alter glomerular arteriolar diameters, it prevented Ang II-induced afferent arteriolar constriction and attenuated efferent arteriolar constriction (18.8 +/- 0.8 to 16.9 +/- microm). Subsequent removal of extracellular calcium abolished residual efferent arteriolar constriction (to 19.1 +/- 0.8 microm). CONCLUSIONS: Our data provide evidence that Ang II elicits IP3CR, possibly inducing a cellular response that activates voltage-dependent calcium channels on afferent arterioles. The present results suggest that Ang II-induced efferent arteriolar constriction involves IP3CR and calcium influx sensitive to SKF-96365.