中电导钙激活钾通道(IKCa通道)在HeLa细胞中的表达及其对细胞增殖的影响

目的:本研究旨在探讨中电导钙激活钾通道(IKCa通道)在宫颈癌He La细胞中的表达及其对细胞增殖的作用。方法:以宫颈癌He La细胞株为研究对象,构建包含IKCa通道特异性sh RNA片段的p Genesil-IK质粒;运用荧光定量PCR和Western Blotting比较p Genesil-IK质粒转染干预前后宫颈癌He La细胞中IKCa通道基因和蛋白表达水平的变化;运用WST-1检测技术和流式细胞术观察转染干扰质粒后对He La细胞增殖的影响。结果:1转染p Genesil-IK干扰质粒后He La细胞IKCa通道m RNA及蛋白表达水平较对照组显著降低(P<0.05)。2转染p Genesil-IK1干扰质粒显著抑制He La细胞增殖和细胞周期(P<0.05)。结论 :宫颈癌He La细胞上有较高的IKCa通道表达,抑制宫颈癌He La细胞株IKCa通道表达能有效抑制癌细胞增殖,提示IKCa通道可能是宫颈癌的治疗靶点之一。     

RNA干扰HeLa细胞IKCa1基因对中电导钙激活钾通道电流的影响

目的:本研究旨在探讨RNA干扰He La细胞IKCa1基因对中电导钙激活钾通道电流(IKCa1)的影响。方法:采用携带IKCa1基因的p Genesil-IKCa1质粒及p Genesil-HK对照质粒分别转染He La细胞。实验分为Control组(无质粒转染组),p Genesil-HK组和p Genesil-IKCa1组。在荧光显微镜下观察报告基因GFP在He La细胞中的表达情况并将GFP阳性率作为转染效率的评价指标,采用RT-PCR技术检测IKCa1基因的表达水平,应用膜片钳电生理技术记录IKCa1。结果:1 p GenesilHK组和p Genesil-IKCa1组转染率分别为75.3%和72.4%,两组之间阳性率差异无统计学意义(P>0.05);2 p Genesil-IKca1组IKCa1基因的m RNA表达水平明显低于Control组及p Genesil-HK组(P<0.01);3在He La细胞能记录到IKCal电流,该电流具有钙离子依赖性且能被Clotrimazole完全阻断,与Control组及p Genesil-HK组相比,p Genesil-IKCa1组的IKCa1电流电压曲线下移,钳制电压为+60 m V时电流密度为(10.05±3.37)p A/p F,明显低于Control组(21.78±5.54)p A/p F和p Genesil-HK组(20.54±5.29)p A/p F(P<0.01)。结论:通过RNA干扰能有效抑制宫颈癌He La细胞IKCa1电流,为今后开展以IKCal为靶点的宫颈癌的基因治疗提供新的靶点和电生理实验依据。     

中电导钙激活钾通道的研究进展

中电导钙激活钾通道(KCa3.1),又称IKCa和SK4,广泛分布于机体成纤维细胞、增殖型平滑肌细胞、内皮细胞、T淋巴细胞、浆细胞、巨噬细胞、上皮细胞中,并参与体内血管舒缩、炎症、钙化,组织纤维增生、免疫反应、恶性肿瘤发生和内外分泌腺分泌等病理生理过程.近几年发现通过阻断KCa3.1通道或基因敲除等方法,可明显阻止其参与的病理生理进程,而其特异性阻断剂三芳甲烷-34(TRAM-34)已在动物及人类中应用,表现出了药物的安全性及耐受性,为治疗相关疾病提供了新的方向.本文就近几年KCa3.1通道相关疾病研究进展作一综述.     

中电导钙激活性钾通道在人多发性骨髓瘤细胞增殖中的作用

目的 探讨中电导钙激活性钾通道(intermediate-conductance Ca2+-activated K+channels,IKCa1)在人多发性骨髓瘤(multiple myeloma,MM)细胞增殖中的作用.方法 通过台盼蓝拒染法检测IKCa1阻滞剂CLO对MM细胞株RPMI 8226和U266生存活力的影响,应用流式细胞仪检测CLO作用于RPMI 8226和U266细胞后细胞周期分布及细胞内钙离子浓度([Ca2+]i)变化.结果 CLO以浓度依赖方式显著抑制RPMI 8226和U266细胞生长,10 μmoL/L CLO作用48 h后,RPMI 8226细胞和U266细胞周期明显被阻滞于G0/G1期,S期显著减少.10 μmol/L CLO作用1min后,RPMI 8226和U266细胞内Fluo-3/AM荧光强分别为(448.3 ±32.8)和(675.9 ±45.8),分别与对照组(56.5±7.2)和(31.8±4.5)相比具有显著差异(P＜0.05).结论 钙激活性钾通道阻断剂克霉唑抑制MM细胞增殖,其机制可能与CLO通过调节细胞内钙水平引起细胞周期阻滞于G0/G1期有关.     

IKCa1基因shRNA真核表达载体的构建及其在HeLa细胞中的表达

目的:构建针对钙激活性中电导钾离子通道(intermediate-conductance Ca2+-activated K+channels,IKCa1)的小发夹RNA(small hair RNA,shRNA)真核表达载体,观察其在宫颈癌细胞HeLa中的表达。方法:设计、合成IKCa1基因特异性shRNA序列,插入空载体pGenesil-1.1中,构建重组体,脂质体法转染宫颈癌HeLa细胞,半定量RT-PCR和Western blotting技术检测转染前后HeLa细胞中IKCa1的表达。结果:成功构建IKCa1 shRNA真核表达载体,转染后HeLa细胞中IKCa1的表达受到显著抑制。结论:成功构建IKCa1特异性shRNA表达载体,为进一步研究IKCa1参与宫颈癌发生发展的机制奠定实验基础。     

钙激活性中电导钾离子通道在人宫颈癌组织中的表达及其在细胞凋亡和细胞周期进展中的作用

目的 研究钙激活性中电导钾离子通道(IKCa1)在宫颈癌组织中的表达变化及其在宫颈癌HeLa细胞凋亡和细胞周期进展中的作用.方法 应用RT-PCR技术研究18例正常宫颈组织及15例宫颈癌组织中IKCa1 mRNA的表达;利用IKca1通道的阻断剂--克霉唑处理宫颈癌HeLa细胞,MTT法观察细胞增殖的变化,流式细胞技术检测细胞凋亡和细胞周期变化.结果 宫颈癌组织中IKCa1 mRNA的阳性表达率及表达水平(分别为73.33%、1.14±0.45)明显高于正常宫颈组织(分别为11.11%、0.86±0.23).克霉唑以浓度和时间依赖的方式阻滞HeLa细胞的增殖,随着克霉唑农度的增加,细胞凋亡率增加,G1期细胞比率增加,细胞IKCa1mRNA的表达水平降低.结论 IKCa1的异常表达可能与宫颈癌的发生、发展密切相关,可能通过影响细胞凋亡和细胞周期进展从而参与宫颈癌的发生、发展过程.     

钙激活性中电导钾离子通道在子宫内膜癌细胞周期中的调控作用

目的探讨子宫内膜癌组织中钙激活性中电导钾离子通道(KCa3.1)的表达变化及其在子宫内膜癌细胞周期中的调控作用。方法应用蛋白质印迹法、Real-time PCR技术检测25例正常子宫内膜、26例非典型增生的子宫内膜和25例子宫内膜癌组织中KCa3.1蛋白和mRNA的表达;用克霉唑阻断剂和siRNA干扰两种方法,阻断KCa3.1的作用后,观察子宫内膜癌细胞HEC-1A细胞周期的变化。结果子宫内膜癌组织中KCa3.1 mRNA和蛋白的表达水平明显高于非典型增生的子宫内膜和正常子宫内膜组织(P<0.01);克霉唑呈剂量依赖性方式将细胞阻滞于G0～G1期,使S期细胞减少;转染siRNA细胞KCa3.1蛋白表达量为转染阴性对照siRNA细胞的(40.27±6.09)%,与转染阴性对照siRNA的HEC-1A细胞比较,转染KCa3.1 siRNA可将细胞阻滞于G0～G1期、使S期细胞减少;同时降低cyclin D1蛋白的表达。结论子宫内膜癌组织中KCa3.1的表达增高,KCa3.1可能通过影响cyclin D1蛋白表达参与子宫内膜癌细胞周期调控。     

钙激活钾通道及ATP敏感性钾通道在海马神经元缺氧超极化中的作用

研究缺氧超极化缺氧海马神经元中的电生理机制,以提示缺氧超极化在脑损伤中的作用。方法：应用膜片钳制技术的细胞贴附式膜片记录缺氧海马神经元的单通道电流活动,经p-CLAMP软件进行采样储存数据和数据的分析处理。结果：缺氧引起钙激活钾通道（KCa通道）和ATP敏感性通道（KATP通道）的激活,增加通道的开放概率。结论：缺氧超极化可能是缺血性脑损伤早期的重要代偿机制。     

慢性低氧状态对肾小球足细胞高通量钙激活钾通道表达和功能的影响

目的 探讨慢性低氧对人肾小球足细胞高通量钙激活钾通道（BK 通道）表达和功能的影响,及其在慢性肾脏病进展中的作用。方法 分化完全的条件永生性人类足细胞分别置于常氧（21% O2）和低氧（10%或2% O2）条件下培养6 ～ 48 h,采用实时聚合酶链反应（real-time PCR）和Western blot 检测足细胞BK 通道α、β3 及β4 亚基的表达;用电生理膜片钳技术记录全细胞模式下足细胞BK 通道功能的变化。结果 电生理研究结果显示低氧环境（2% O2 24 h）中,足细胞BK 通道在+80 mV 电压刺激下的电流水平由（14.45±2.06）pS 下降至（4.78±1.12）pS,差异有统计学意义（P <0.05）,且通道激活的时间常数由（4.59±1.67）增加至（25.16±11.04）（P <0.05）;分子生物学研究提示在低氧条件（2% O2 24 h）下足细胞BK 通道的β4 亚基水平升高,且表现为时间依赖性和剂量依赖性。结论 慢性低氧通过上调β4 亚基表达抑制BK 通道活性。     

钙激活钾通道在钙离子致神经细胞损伤中的作用

采用膜片钳单通道记录方法,观察原代培养新生ＳＤ大鼠皮层神经元的钙激活钾通道（ＫＣａ）特征及胞内不同游离钙水平对通道开放动力学的调节。结果显示：培养ＳＤ大鼠皮层神经元的ＫＣａ以大电导活动占优势,胞内游离钙为１０－８ｍｏｌ／Ｌ时,通道几乎不开放,在１０－６ｍｏｌ／Ｌ时达最大激活。由此表明神经元上ＫＣａ通道开放概率明显依赖于胞浆中钙离子和膜电位,ＫＣａ对胞内钙离子浓度变化极为敏感。钾通道的开放剂可能有一定的神经细胞保护作用。     

Effects of isoflurane and ethanol on large conductance Ca^2＋ -activated K^＋ channels

To study the effect of isoflurane and ethanol on large conductance Ca^2 -activated K^ channels (BK channels). Methods: The cRNA of mslol encoding BK channels was injected into Xenopus oocytes. Oocytes were incubated in ND96 (96 mmol/L NaCI, 2.0 mmol/L KCI, 1.8 mmol/L CaCl2, 1.0 mmol/L MgCl2, and 5.0 mmol/L HEPES, pH 7.4) at 4 ℃. Patch clamp recording (outside-out) were performed after 2-3 d. Isoflurane was administrated by the vaporizer driven by air, ethanol was applied by a cloud, manual-controlled administration system. Different test potentials from 0 to 10 mV were given to observe changes of currents. Results: 0.7 mmol/L and 1.2 mmol/L of isoflurane could inhibit BK currents obviously at different command potentials, but 50 mmol/L, 100 mmol/L, or 200 nunol/L of ethanol had no any effect on BK currents. Conclusion: Clinical concentration of isoflurane can distinctly inhibit isolating BK currents.     

Changes of Ca^2＋ activated potassium channels and cellular proliferation in autoge-nous vein grafts

Objective: To investigate changes of Ca2+ activated potassium channels ( KCa) in autogenous vein grafts. Methods: Contraction of venous ring was measured by means of perfusion in vitro. The intimal rabbits proliferation of vascular and proliferation of cultured smooth muscle cells ( vascular smooth muscle cells, VSMCs) were observed by the means of computerised image analysis and MTT method respectively. Furthermore, whole cell mode of patch clamp was used to record KCa of VSMCs isolated from autogenous vein grafts. Results: One week after transplantation there were no significant differences of contraction and intimal relative thickness between autogenous vein grafts and control. Contraction and intimal relative thickness of autogenous vein graft were significantly increased 2 weeks after transplantation (P < 0. 05 , n = 8 vs control) , and they was more enhanced 4 weeks after vein transplantation ( P < 0. 01 , n =8 vs control ). TEA( blocker of Ca2+ activated potassium channels) increased MTT A490 nm     

小电导钙激活钾通道在人心房肌细胞中的表达及功能

目的探讨小电导钙激活钾通道在人心房肌细胞中的表达情况和功能意义。方法收集行心内直视手术的窦性心律先心病患者8例,取右心耳组织,行免疫组化分析3种SK通道亚型(SK1、SK2和SK3)表达情况;使用经典两步酶解法分离心房肌细胞,应用穿孔膜片钳和传统电压钳技术分别记录心房肌细胞动作电位(APs)和全细胞的钙激活钾电流(IK,Ca),比较给予SK通道特异性阻断剂apamin(100 nmol/L)前、后动作电位时程(APD)变化。结果 SK1、SK2和SK3通道亚型在心房肌细胞中均呈阳性表达,膜片钳记录证实IK,Ca的存在,且apamin明显延长复极化90%的动作电位时程(APD90),而对APD50无明显影响。结论 3种SK通道亚型在人心房肌细胞中均有表达;与SK通道在神经元细胞中介导动作电位后超极化过程的功能不同,在心房肌细胞中,SK通道是参与APs复极末期过程的重要离子通道。     

Molecular mechanisms of diabetic coronary dysfunction due to large conductance Ca2+-activated K+ channel impairment

Background  Diabetes mellitus is associated with coronary dysfunction, contributing to a 2- to 4-fold increase in the risk of coronary heart diseases. The mechanisms by which diabetes induces vasculopathy involve endothelial-dependent and -independent vascular dysfunction in both type 1 and type 2 diabetes mellitus. The purpose of this study is to determine the role of vascular large conductance Ca²⁺-activated K⁺ (BK) channel activities in coronary dysfunction in streptozotocin-induced diabetic rats.

Methods  Using videomicroscopy, immunoblotting, fluorescent assay and patch clamp techniques, we investigated the coronary BK channel activities and BK channel-mediated coronary vasoreactivity in streptozotocin-induced diabetic rats.

Results  BK currents (defined as the iberiotoxin-sensitive K⁺ component) contribute (65±4)% of the total K⁺ currents in freshly isolated coronary smooth muscle cells and >50% of the contraction of the inner diameter of coronary arteries from normal rats. However, BK current density is remarkably reduced in coronary smooth muscle cells of streptozotocin-induced diabetic rats, leading to an increase in coronary artery tension. BK channel activity in response to free Ca²⁺ is impaired in diabetic rats. Moreover, cytoplasmic application of DHS-1 (a specific BK channel b₁ subunit activator) robustly enhanced the open probability of BK channels in coronary smooth muscle cells of normal rats. In diabetic rats, the DHS-1 effect was diminished in the presence of 200 nmol/L Ca²⁺ and was significantly attenuated in the presence of high free calcium concentration, i.e., 1 mmol/L Ca²⁺. Immunoblotting experiments confirmed that there was a 2-fold decrease in BK-b₁ protein expression in diabetic vessels, without altering the BK channel α-subunit expression. Although the cytosolic Ca²⁺ concentration of coronary arterial smooth muscle cells was increased from (103±23) nmol/L (n=5) of control rats to (193±22) nmol/L (n=6, P <0.05) of STZ-induced diabetic rats, reduced BK-b₁ expression made these channels less sensitive to intracellular Ca²⁺, which in turn led to enhanced smooth muscle contraction.

Conclusions  Our results indicated that BK channels are the key determinant of coronary arterial tone. Impaired BK channel function in diabetes mellitus is associated with down-regulation of BK-b₁ expression and reduction of the b₁-mediated BK channel activation in diabetic vessels.

     

RNA干扰对HeLa细胞VEGF表达和细胞增殖的影响

目的：探讨RNA干扰对HeLa细胞中血管内皮生长因子(VEGF)表达和细胞增殖的影响,为人宫颈癌的治疗提供理论依据。方法：构建针对VEGF165的RNA干涉质粒,稳定转染HeLa细胞,设HeLa组、对照组和干涉组,用RT-PCR方法检测稳定细胞株中VEGF mRNA的表达,Western blotting和ELISA方法检测VEGF165蛋白的表达,MTT及流式细胞术检测VEGF siRNA作用下的细胞增殖,Hoechst33342染色检测VEGF siRNA作用下细胞凋亡的变化。结果：成功建立了VEGF siRNA的稳定细胞株,与对照组比较,干涉组VEGF的mRNA 和蛋白表达水平均明显降低(P<0.05)。 与对照组比较,稳定转染VEGF siRNA的细胞株在24 、48 和72 h的 细胞生长抑制率分别为12.8%±5.4%、17.4%±3.7%和27.2%±5.7%,细胞增殖明显受到抑制(P＜0.01)。与对照组比较,Hoechst33342染色显示VEGF siRNA对HeLa细胞的细胞凋亡率没有影响。结论：VEGF siRNA可以有效抑制HeLa细胞中VEGF mRNA和蛋白的表达,抑制细胞的增殖,提示VEGF在宫颈癌的发生、发展中具有重要作用,抑制VEGF的表达有望成为一种治疗宫颈癌的途径。     

丁酸钠对宫颈癌HeLa细胞增殖及血管内皮生长因子表达的影响

目的观察丁酸钠对宫颈癌HeLa细胞的数量及血管内皮生长因子（VEGF）mRNA表达水平的影响,探讨丁酸钠对HeLa细胞增殖的作用及相关机制。方法体外分组培养的HeLa细胞分别加入不同浓度的丁酸钠作用72h后,用四甲基偶氮唑盐比色法（MTT）检测细胞生长情况,逆转录聚合酶链反应（RT—PCR）检测各组细胞VEGFmRNA表达情况。结果经不同浓度的丁酸钠作用后,宫颈癌HeLa细胞增殖水平及VEGFmRNA表达水平较对照组均有明显降低,在一定范围内呈现浓度依赖性。结论丁酸钠不但能够抑制宫颈癌HeLa细胞的增殖,还可抑制宫颈癌HeLa细胞VEGF基因的表达水平。丁酸钠抑制VEGF基因表达水平的作用可能是其抑癌机制之一。     

HERG K+ channels expression in gastric cancers and analysis of its regulation in tumor cell proliferation and apoptosis

Objective: To investigate the expression of herg 1 gene in tumor tissues from gastric carcinomas and gastric carcinoma cell lines, and study the relationship between HERG K+ channel expressions and tumor cell proliferation and apoptosis. Methods: RT-PCR and PCR assays were used to detect the expression of herg 1 gene in 64 gastric carcinomas and the gastric cancer cell line SGC-7901. Blocking the HERG K+ channels was used to evaluate their effects on tumor cell proliferation and apoptosis. Results:The statistically significant expression of herg 1 gene was detected in all the gastric cancers and SGC-7901 cells, but not in normal tissues. The HERG K+ channel blocker, E-4031, increased the cell population in G0/G1 (P＜0.05) and the number of apoptotic tumor cells(P＜0.05). Conclusion: HERG K+ channels were expressed in all gastric carcinomas tested and these channels appear to modulate tumor cell proliferation and apoptosis.     

亲环素A在胃癌细胞中的表达及与肿瘤细胞增殖的关系

目的 检测亲环素A(CypA)在胃癌组织和细胞株中的表达情况,采用基因干扰技术抑制胃癌细胞中CypA的表达,探讨CypA对胃癌细胞增殖能力的影响和相关分了机制.方法 实时定量PCR和蛋白质印迹法检测CypA在胃癌组织、癌旁组织和细胞株中的表达;合成针对CypA的靶向siRNA并转染胃癌MKN45细胞株,检测CypA-siRNA对胃癌细胞内源性CypA的抑制作用,同时设置非特异性siRNA对照组和阴性对照组.MTT法检测各组细胞增殖情况,流式细胞术检测各组细胞周期;实时定量PCR和蛋白质印迹法检测各组细胞增殖基因PCNA、P21、P16、Cyclin D1的表达.结果 胃癌组织中CypA的mRNA和蛋白表达高于癌旁组织,胃癌细胞株CypA的mRNA和蛋白表达高于胃上皮细胞株,且在低分化细胞MKN45中表达最高(P＜0.05).CypA-siRNA可有效抑制内源性CypA的表达;CypA-siRNA转染后MKN45细胞增殖能力下降(P＜0.05),G0/G1期细胞比例上升、G2/M期细胞比例下降(P＜0.05),PCNA、Cyclin D1的mRNA和蛋白表达下调,P21mRNA和蛋白表达上调(P＜0.05).结论 胃癌细胞中CypA的表达增强,CypA基因能够通过调节部分增殖基因的表达而促进胃癌细胞增殖.