Voltage-gated potassium channel Kv1.3 in rabbit ciliary epithelium regulates the membrane potential via coupling intracellular calcium

Background The cell layer of the ciliary epithelium is responsible for aqueous humor secretion and maintenance. Ion channels play an important role in these processes. The main aim of this study was to determine whether the well-characterized members of the Kvl family （Kv1.3） contribute to the Kv currents in ciliary epithelium. Methods New Zealand White rabbits were maintained in a 12 hours light/dark cycle. Ciliary epithelium samples were isolated from the rabbits. We used Western blotting and immunocytochemistry to identify the expression and location of a voltage-gated potassium channel Kvl.3 in ciliary body epithelium. Membrane potential change after adding of Kvl.3 inhibitor margatoxin （MgTX） was observed with a fluorescence method. Results Western blotting and immunocytochemical studies showed that the Kv1.3 protein expressed in pigment ciliary epithelium and nonpigment ciliary epithelium, however it seemed to express more in the apical membrane of the nonpigmented epithelial cells. One nmol/L margatoxin, a specific inhibitor of Kv1.3 channels caused depolarization of the cultured nonpigmented epithelium （NPE） membrane potential. The cytosolic calcium increased after NPE cell depolarization, this increase of cytosolic calcium was partially blocked by 12.5 μmol/L dantrolene and 10 μmol/L nifedipine. These observations suggest that Kv1.3 channels modulate ciliary epithelium potential and effect calcium dependent mechanisms. Conclusion Kv1.3 channels contribute to K＋ efflux at the membrane of rabbit ciliary epithelium.     

双氯芬酸对人巨噬细胞钾通道Kv1.3、Kir2.1表达的抑制作用及其对膜电位和泡沫细胞形成的影响(英文)

目的分研究双氯芬酸对人巨噬细胞电压依赖性钾通道Kv1.3、内向整流钾通道Kir2.1表达的影响及意义。方法以健康人外周血单核细胞源性巨噬细胞为对象,采用Real-time RT-PCR及Western blot技术研究双氯芬酸对Kv1.3和Kir2.1表达的影响;电压敏感染料膜电位标测技术分析膜电位的变化,并用酶荧光化学法检测摄取氧化修饰低密度脂蛋白(OxLDL)的巨噬细胞内胆固醇酯(CE)的构成比率。结果双氯芬酸(1.5和15μmol/L)抑制巨噬细胞Kv1.3和Kir2.1的表达。同对照组相比,Kv1.3mRNA下降分别超过80%和90%(P<0.05),Kir2.1 mRNA下降分别超过20%和30%(P>0.05);两种钾通道蛋白水平的下降均分别超过10%和60%,且存在明显的剂量依赖性(P<0.05)。同时,双氯芬酸可剂量依赖性减弱巨噬细胞表面的荧光强度,使膜电位分别下降约28%和54%(P<0.05)。巨噬细胞同30 mg/L OxLDL孵育60 h后,细胞体积明显增大,且有许多红色的脂质颗粒沉积于细胞质内,CE/TC的百分比超过50%。1.5和15μmol/L双氯芬酸分别使摄取OxLDL的巨噬细胞内CE的百分比显著减少到(23.624±3.34)%和(13.601±2.916)%,但缺乏明显的量效关系(P>0.05)。结论双氯芬酸显著下调人巨噬细胞Kv1.3和Kir2.1的表达,降低细胞膜电位,并抑制泡沫细胞形成。     

吉非贝齐对人巨噬细胞Kv1.3和Kir2.1表达的差别调节作用及其对膜电位的影响

目的:探讨人巨噬细胞发育过程中Kv1.3、Kir2.1通道的表达和吉非贝齐的调节作用,观察其对膜电位的影响,为动脉粥样硬化（AS）相关性疾病治疗提供电生理学依据。方法：健康人外周血单核细胞源性巨噬细胞随机分为对照5 d组（C 5d）、对照7.5 d组（C 7.5d）和吉非贝齐组（G）,吉非贝齐的终浓度为400 μmol•L^-1;采用Real time RT-PCR及Western blotting技术检测Kv1.3和Kir2.1通道的表达,电压敏感染料膜电位标测技术分析膜电位的变化。结果：与C 5d组比较,C 7.5d组Kv1.3 mRNA/蛋白水平从（1.064±0.275）/（0.227±0.018）升高至（3.067±0.824）/（0.409±0.022）（P<0.05）,但Kir2.1 mRNA/蛋白水平却从（1.024±0.166）/（0.204±0.018）降低至（0.399±0.133）/（0.042±0.008）（P<0.05）;与C 7.5d组比较,吉非贝齐组Kv1.3 mRNA/蛋白水平下降至（1.137±0.067）/（0.143±0.023）（P<0.01）,而Kir2.1 mRNA/蛋白水平却升高至（1.35±0.087）/（0.202±0.033）（P<0.01）;吉非贝齐显著消弱C 5d和C 7.5d组巨噬细胞表面的荧光强度,使其膜电位从（1.000±0.026）分别下降至（0.833±0.046）和（0.481±0.053）（P<0.05）。结论：吉非贝齐差别调节人单核细胞源性巨噬细胞Kv1.3和Kir2.1通道的表达,并降低巨噬细胞膜电位。     

Electrophysiological effects of hydrogen sulfide on human atrial fibers

Background  It has been reported that endogenous or exogenous hydrogen sulfide (H₂S) exerts physiological effects in the vertebrate cardiovascular system. We have also demonstrated that H₂S acts as an important regulator of electrophysiological properties in guinea pig papillary muscles and on pacemaker cells in sinoatrial nodes of rabbits. This study was to observe the electrophysiological effects of H₂S on human atrial fibers.

Methods  Human atrial samples were collected during cardiac surgery. Parameters of action potential in human atrial specialized fibers were recorded using a standard intracellular microelectrode technique.

Results  NaHS (H₂S donor) (50, 100 and 200 μmol/L) decreased the amplitude of action potential (APA), maximal rate of depolarization (V_max), velocity of diastolic (phase 4) depolarization (VDD) and rate of pacemaker firing (RPF), and shortened the duration of 90% repolarization (APD₉₀) in a concentration-dependent manner. ATP-sensitive K⁺ (K_ATP) channel blocker glibenclamide (Gli, 20 μmol/L) partially blocked the effects of NaHS (100 μmol/L) on human atrial fiber cells. The L-type Ca²⁺ channel agonist Bay K8644 (0.5 μmol/L) also partially blocked the effects of NaHS (100 μmol/L). An inhibitor of cystathionine γ-lyase (CSE), DL-propargylglycine (PPG, 200 μmol/L), increased APA, V_max, VDD and RPF, and prolonged APD₉₀.

Conclusions  H₂S exerts a negative chronotropic action and accelerates the repolarization of human atrial specialized fibers, possibly as a result of increases in potassium efflux through the opening of K_ATP channels and a concomitant decrease in calcium influx. Endogenous H₂S may be generated by CSE and act as an important regulator of electrophysiological properties in human atrial fibers.

     

High extracellular potassium ion concentration attenuates the blockade action of ketanserin on Kvl.3 channels expressed in xenopus oocytes

Background Ketanserin (KT), a selective serotonin (5-HT) 2-receptor antagonist, reduces peripheral blood pressure by blocking the activation of peripheral 5-HT receptors. In this study electrophysiological method was used to investigate the effect of KT and potassium ion on Kv1.3 potassium channels and explore the role of blocker KT in the alteration of channel kinetics contributing to the potassium ion imbalances. Methods Kvl.3 channels were expressed in xenopus oocytes, and currents were measured using the two-microelectrode voltage-clamp technique. Results KCI made a left shift of activation and an inactivation curve of Kv1.3 current and accelerated the activation and inactivation time constant. High extracellular [K+] attenuated the blockade effect of KT on Kv1.3 channels. In the presence of KT and KCI the activation and inactivation time constants were not influenced significantly no matter what was administered first. KT did not significantly inhibit Kv1.3 current induced by tetraethylammonium (TEA). Conclusions KT is a weak blocker of Kv1.3 channels at different concentrations of extracellular potassium and binds to the intracellular side of the channel pore. The inhibitor KT of ion channels is not fully effective in clinical use because of high [K+]o and other electrolyte disorders.     

Changes in delayed rectifier K+ channel function and its regulation by protein kinase C pathway in bronchial myocytes from asthmatic rats

Objective To investigate changes in the delayed rectifier K+ channel (Kv) function and the regulation of Kv by the protein kinase C (PKC) pathway in bronchial myocytes from asthmatic rats. Methods The Kv currents and membrane potentials in bronchial myocytes from asthmatic rats and from controls were observed, using whole cell voltage- and current-patch clamp techniques.Results Bronchial myocytes from asthmatic rats showed a significant reduction in Kv-current density (51.6±9.4 pA/pF, n=14, P&lt;0.01) in comparison with those from control rats (72.4±12.3 pA/pF, n=14) at +50 mV. The current-voltage relationship curve exhibited a significant downward shift. Bronchial myocytes from asthmatic rats had no significantly different capacitances (P&gt;0.05), but had more positive membrane potential ( P&lt;0.01) compared with those from controls. 1 μmol/L phorbol 12-myristate 13-acetate, a PKC activator, caused an obvious reduction in Kv-current density (P&lt;0.01) and a significant downward shift in the current-voltage relationship curve, an effect which was partly abolished by 1 μmol/L Ro31-8220 (a PKC inhibitor); 1 μmol/L phorbol 12-myristate 13-acetate caused more positive membrane potential (Em), from -36.8±5.7 mV to -30.4±7.3 mV, in rat bronchial myocytes (P&lt;0.05). This effect was partly abolished by 1 μmol/L Ro31-8220. Conclusions Bronchial myocytes from asthmatic rats have inhibited Kv function, more positive membrane potential, and higher excitability, all of which can also be induced by PKC activation. These characteristics may contribute to the development of airway hyperreactivity in asthma.     

姜黄素抑制硝普钠诱导的大鼠软骨细胞凋亡机制研究

目的 探讨姜黄素抑制硝普钠(SNP)诱导的大鼠软骨细胞凋亡的机制研究。 方法 每组取4只SD大鼠关节软骨，细胞进行原代培养，将软骨细胞分成6组，分别为空白对照组(A组)，SNP损伤组(B组)，SNP+5 μmol/L姜黄素组(C组)，SNP+10 μmol/L姜黄素组(D组)，SNP+20 μmol/L姜黄素组(E组),SNP+30 μmol/L姜黄素组(F组)。用CCK8法检测不同浓度的姜黄素对软骨细胞增殖的影响，同时检测各组中软骨细胞的活性；用RtPCR检测软骨细胞MMP-13、caspase-3、Bax和Bcl-2基因的表达情况；用Hoechst 33342染色观察软骨细胞核凋亡情况；Western Blotting检测线粒体凋亡途径相关蛋白的表达情况。 结果 ①当姜黄素的浓度为20 μmol/L时，姜黄素对软骨细胞的促增殖作用最为明显；②CCK8法检测各组细胞的活性率从大至小依次为A组＞E组＞D组＞C组＞F组＞B组；③RtPCR检测软骨细胞caspase-3、Bax和Bcl-2表达量经姜黄素处理后均有显著变化；④Hoechst 33342染色结果表明姜黄素能改善SNP诱导的软骨细胞核染色质的损伤，减少软骨细胞中的线粒体膜电位；⑤Western boltting检测各组软骨细胞中caspase-3、Bax、蛋白的表达情况，表达量依次为B组＞E组＞A组；Bcl-2蛋白的表达情况，表达量依次为A组＞E组＞B组。 结论 姜黄素通过抑制线粒体凋亡途径，从而减少SNP诱导的大鼠软骨细胞的凋亡。      

Expression of nitric oxide synthase and guanylate cyclase in the human ciliary body and trabecular meshwork

Background  The role played by the nitric oxide (NO) signaling pathway in the aqueous humor dynamics is still unclear. This study was designed to investigate the expression and distribution of NO synthase (NOS) isoforms and guanylate cyclase (GC) in human ciliary body, trabecular meshwork and the Schlemm’s canal.

Methods  Twelve eyes after corneal transplantation were used. Expression of three NOS isoforms (i.e. neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS)) and GC were assessed in 10 eyes by immunohistochemical staining using monoclonal or polyclonal antibody of NOS and GC. Ciliary bodies were dissected free and the total proteins were extracted. Western blotting was performed to confirm the protein expression of 3 NOS isoforms and GC.

Results  Expression of 3 NOS isoforms and GC were observed in the ciliary epithelium, ciliary muscle, trabecular meshwork and the endothelium of the Schlemm’s canal. Immunoreactivity of nNOS was detected mainly along the apical cytoplasmic junction of the non-pigmented epithelium (NPE) and pigmented epithelial (PE) cells. Protein expressions of 3 NOS isoforms and GC were confirmed in isolated human ciliary body by Western blotting.

Conclusions  The expression of NOS isoforms and GC in human ciliary body suggest the possible involvement of NO and cyclic guanosine monophosphate (cyclic GMP, cGMP) signaling pathway in the ciliary body, and may play a role in both processes of aqueous humor formation and drainage.

     

血管紧张素-(1-7)通过抑制ClC-3通道减轻高糖引起的心肌细胞损伤

目的 探讨血管紧张素-(1-7)[Ang-(1-7)]能否通过调控ClC-3通道保护心肌细胞对抗高糖引起的损伤.方法 应用35 mmol/L葡萄糖处理H9c2心肌细胞24 h建立损伤模型.Ang-(1-7)或氯通道抑制剂与心肌细胞共处理24 h观察对高糖诱发的心肌细胞损伤的影响.应用细胞计数试剂盒8检测细胞存活率;Hoechst33258染色荧光显微镜照相术检测凋亡细胞的形态学改变;双氯荧光素染色荧光显微镜照相术测定细胞内活性氧水平;超氧化物歧化酶试剂盒测定活性;罗丹明123染色荧光显微镜照相术检测线粒体膜电位;Western blot法测定心肌细胞ClC-3通道蛋白的表达水平.结果 35 mmol/L葡萄糖处理H9c2心肌细胞24 h明显地增加ClC-3通道蛋白的表达水平(P<0.01);1μmol/LAng-(1-7)与葡萄糖共处理心肌细胞24 h显著地抑制葡萄糖对ClC-3通道蛋白表达的上调作用(P<0.01);1μmol/LAng-(1-7)或100μmol/L氯通道抑制剂氯通道抑制剂与葡萄糖共处理心肌细胞24 h减轻葡萄糖引起的损伤作用,表现为增加细胞存活率和超氧化物歧化酶活性,减小细胞凋亡数量,胞内活性氧水平及线粒体膜电位丢失(P<0.01).结论 ClC-3通道参与葡萄糖引起的心肌细胞损伤;Ang-(1-7)通过抑制ClC-3通道保护心肌细胞对抗葡萄糖引起的损伤.     

阻断Kv1.3和Kir2.1抑制人巨噬细胞源性泡沫细胞分化

目的:研究泡沫细胞分化过程中离子通道Kv1.3和Kir2.1的表达及作用。方法:用30mg/L氧化型低密度脂蛋白(ox-LDL)孵育巨噬细胞60h建立泡沫细胞模型。采用免疫细胞化学、RT-PCR和Western印迹检测人单核细胞源性巨噬细胞和泡沫细胞上Kv1.3和Kir2.1的表达。观察Kv1.3和Kir2.1特异性阻断剂rMargatoxin和BaCl2对巨噬细胞胆固醇代谢的影响。结果:ox-LDL(30mg/L)孵育巨噬细胞60h后,细胞内总胆固醇(TC),游离胆固醇(FC)及胆固醇酯(CE)显著增加,CE/TC从(14.4±6.8)%提高到(57.9±3.5)%(P<0.05);但Kv1.3和Kir2.1的表达水平在巨噬细胞组和泡沫细胞组无明显区别(P>0.05)。Kv1.3和Kir2.1分别被rMargatoxin(0.1,10nmol/L)和BaCl2(75,125μmol/L)阻断后,细胞内TC和CE水平显著降低,CE/TC低于50%(P<0.05)。结论:Kv1.3和Kir2.1对泡沫细胞的分化均起关键作用,特异性阻断后能够抑制人单核细胞源性巨噬细胞向泡沫细胞分化。     

普萘洛尔对兔门静脉收缩反应的影响

本文采用离体兔门静脉环的方法,观察了普萘洛尔(Propranolol,Pro)和维拉帕米(Verapamil,Ver)对门静脉血管的作用。结果表明,Pro对兔门静脉环正常张力无影响。Pro和Ver使兔门静脉环KCl和C_αC_2量效曲线非平行右移,最大反应压低,显示非竞争性拮抗的两种曲线。显著抑制高K~+去极化所致门静脉环的收缩作用。此外Pro100μmol/L和Ver10μmol/L显著抑制兔门静脉条的自律性收缩,而实验证明这种收缩是钙依赖性的。提示较大剂量的Pro可能通过阻断细胞膜上电压依赖通道而发挥钙拮抗作用。     

Kv1.1及Kv1.3通道亚型对小鼠肠系膜微细动脉的调节作用

目的 研究Kv1.1及Kv1.3通道亚型对小鼠肠系膜微细血管的调节作用.方法 以健康6～8周C57BL/6雄性小鼠肠系膜微细动脉作为研究对象,应用丹麦DMT520A离体微血管张力测定系统记录血管张力变化.应用广谱电压依赖性钾通道(Kv)阻断剂4-AP,Kv1.3通道选择性阻断剂PAP-1,Kv1.1通道选择性阻断剂TEA分别作用于静息状态,KCl及NE预收缩动脉,记录血管张力变化情况.应用Kv通道阻断剂4-AP,Kv1.3通道选择性阻断剂PAP-1作用于血管,观察Kv及Kv1.3通道在0 mmol/L Ca2+及2 mmol/L Ca2+ K-H液中对NE收缩曲线的作用.结果 Kv通道阻断剂4-AP可引起静息状态的血管收缩,并进一步收缩KCl预收缩的血管,但浓度依赖性舒张NE预收缩的动脉,并且与对照组相比,4-AP能明显抑制NE在0 mmol/L Ca2+液中所致的血管收缩[(3.45-±0.24)mN vs(0.11±0.02) mN,P＜0.01].Kv1.3通道选择性阻断剂PAP-1未能收缩静息状态的血管,但可使KCl预收缩的血管发生轻微舒张反应,却明显舒张NE预收缩的动脉,而且PAP-1既可抑制NE在0 mmol/L Ca2+ K-H液中所致的血管收缩[对照组vs PAP-1组:(4.28 ±0.53)mN vs(2.75 ±0.49)mN,P＜0.05],又可抑制在2 mmol/L Ca2+液中的收缩张力[对照组vsPAP-1组:(7.08 ±0.58)mN vs(5.90 ±0.80)mN,P＜0.05].Kv1.1通道选择性阻断剂TEA可引起静息状态的血管收缩,但在0 mmol/L Ca2+液中该作用消失,同时对KCl或NE预收缩的动脉均无明显作用.结论 Kv通道可调节小鼠肠系膜动脉的舒缩反应.其中Kv1.1通道主要发挥血管收缩调节作用,可能与促进血管平滑肌细胞外钙内流有关,而Kv1.3通道主要发挥血管舒张调节作用,可能同时抑制平滑肌细胞内钙释放和细胞外钙内流.     

Effects of Mahuang (Herba Ephedra Sinica) and Wuweizi (Fructus Schisandrae Chinensis) medicated serum on chemotactic migration of alveolar macrophages and inters regions macrophages in rats

Objective

To observe the effects of serum containing Mahuang (Herba Ephedra Sinica) or Wuweizi (Fructus Schisandrae Chinensis) on the migration of alveolar macrophages (AM) and interstitial macrophages (IM) from normal rats, and to analyze and compare the mechanisms leading to cell migration differences.

大麻二酚通过促进自噬流减轻棕榈酸诱导的肝细胞损伤

目的 观察大麻二酚(CBD)对棕榈酸(PA)诱导的肝细胞损伤的保护作用,并考察其与自噬流相关的潜在机制.方法 原代培养大鼠肝细胞,分别给予1 μmol/L和5μmol/L的CBD处理24 h,采用蛋白质印迹法检测自噬相关蛋白微管相关蛋白1轻链3(LC3)和p62的表达,考察CBD对细胞自噬的影响.将细胞分为4组并分别给予不同处理:PA组(800μmol/L PA处理细胞)、PA+CBD组(800 μmol/L PA和5μmol/L CBD联合作用)、PA+CBD+CQ组[800 μmol/L PA、5μmol/L CBD和50 nmol/L自噬抑制剂氯喹(CQ)共同作用]和阴性对照组(加入等体积0.03％ DMSO处理细胞),每组作用时间均为24 h;采用蛋白质印迹法检测LC3和p62的蛋白表达,流式细胞术考察细胞凋亡情况,qPCR法检测内质网应激相关因子CCAAT/增强子结合蛋白同源蛋白(CHOP)、葡萄糖调节蛋白78(GRP78)和X盒结合蛋白1(XBP-1) mRNA的表达,Rh123和lucigenin荧光探针分别检测线粒体的膜电位和活性氧簇(ROS)含量.结果 1μmol/L和5μmol/L CBD均不影响肝细胞LC3-Ⅱ/LC3-Ⅰ的比值以及p62蛋白的表达.与阴性对照组相比,PA组肝细胞LC3-Ⅱ/LC3-Ⅰ的比值和p62蛋白表达增加(P＜0.05),细胞凋亡增加(P＜0.05),CHOP、GRP78、XBP-1 mRNA表达增加(P＜0.05),线粒体膜电位降低(P＜0.05),线粒体ROS的生成增加(P＜0.05);与PA组相比,PA+CBD组肝细胞内的自噬流恢复,细胞凋亡减少,内质网应激和线粒体失常减轻(P＜0.05);同时给予CQ处理可以逆转CBD的保护作用(P＜0.05).结论 CBD能够通过促进自噬流减轻PA诱导的肝细胞损伤,改善内质网应激和线粒体功能.     

三硫二苄基抑制头颈部鳞状癌细胞HN30的增殖并诱导其凋亡

目的 探究三硫二苄基（DTS）抑制头颈癌细胞HN30增殖和促进其凋亡的作用机制。方法 通过克隆形成实验检测DTS对不同头颈癌细胞HN30、HN12、SCC25增殖能力的影响,并用MTT实验检测不同浓度DTS对HN30细胞活力的影响;DTS（3、10、30 μmol/L）刺激HN30细胞24 h,经Annexin Ⅴ-FITC/PI双染及JC-1荧光探针染色,采用流式细胞仪检测DTS对HN30 细胞凋亡及线粒体膜电位的影响,并通过免疫印迹实验检测不同浓度 DTS 作用对凋亡相关蛋白 caspase 3、cleavedcaspase-3和Bcl-2表达的影响;通过免疫印迹实验检测HN30细胞在DTS（10 μmol/L）不同作用时间（0、0.5、1、2、4、8、16 h） 下,Akt/p53磷酸化水平的变化。结果 克隆形成实验发现1 μmol/L DTS能够明显抑制头颈癌细胞HN30、HN12及SCC25的增殖。MTT实验发现,与溶剂对照组相比,HN30细胞的细胞活力在DTS作用下呈剂量依赖性降低,在100 μmol/L DTS作用时效果显著（P<0.001）。采用Annexin Ⅴ-FITC/PI双染及JC-1荧光探针染色后,流式细胞术检测发现随着DTS作用浓度增高,HN30细胞的凋亡细胞比例逐渐增高（30 μmol/L DTS作用下,P<0.01）,线粒体膜电位逐渐降低（30 μmol/L DTS作用下, P<0.001）。与溶剂对照组相比,DTS 刺激 24 h 后,HN30 细胞中 cleaved caspase-3 的表达升高（30 μmol/L DTS 作用下,P< 0.01）,Bcl-2的表达降低（30 μmol/L DTS作用下,P<0.001）。与溶剂对照组相比,10 μmol/L DTS刺激HN30细胞16 h后,Akt磷酸化水平被显著抑制（P<0.001）,而p53的磷酸化水平升高（P<0.01）。结论 DTS抑制HN30细胞的增殖,并诱导凋亡,其作用机制可能与Akt/p53信号通路有关。     

Necroptosis与细胞凋亡在激素诱导成骨细胞死亡中的相互作用

目的探讨Necroptosis与凋亡在激素诱导成骨细胞死亡中的相互作用。方法向MC3T3-E1细胞内添加浓度为10-6 mol/L 的地塞米松诱导细胞死亡，随后在本组细胞中分别添加凋亡抑制剂z-VAD-fmk（40 μmol/L）和Necroptosis抑制剂Necrostatin-1 （40 μmol/L），作用2 h 后，AV/PI 双染观察细胞死亡的变化情况，并对细胞进行Hoechst 染色计算凋亡率，用透射电镜观察细 胞超微结构的改变，测定细胞线粒体膜电位和ATP水平，对Necroptosis 和凋亡通路的相关蛋白进行Western blot 检测。结果 10-6 mol/L地塞米松可以诱导MC3T3-E1细胞同时发生凋亡和Necroptosis。AV/PI双染的结果发现，当凋亡受到抑制后，更多的 细胞发生坏死（P<0.01），而透射电镜结果也证实细胞发生了坏死样改变，Hoechst 染色结果发现，凋亡细胞数明显减少（P< 0.01）；当使用Necroptosis特异性抑制剂Necrostatin-1将这种作用阻断后，包括Hoechst染色和AV/PI双染的结果都证实凋亡的 细胞数明显增多（P<0.01），同时，在这一作用过程中伴随着MMP及ATP的明显变化（P<0.01）。结论在激素诱导成骨细胞死亡 过程中，Necroptosis和凋亡在一定条件下可以相互转化，这一转化过程中伴随着线粒体功能的明显改变。     

一氧化氮对豚鼠耳蜗外毛细胞钙通道的作用

目的研究一氧化氮（NO）对豚鼠耳蜗外毛细胞钙通道的影响。方法以硝普钠作为NO的供体,应用全细胞式膜片钳技术记录外毛细胞钙电流。结果 10μmol/L硝普钠使电压依赖性钙通道电流减小,电流电压关系（I-V）曲线上抬;0.05～50μmol/L硝普钠对电流的抑制率与其浓度有关,半数抑制浓度为0.79μmol/L,对电流的最大抑制率为39.48%,其阻断作用是可逆的。结论硝普钠可阻断豚鼠耳蜗外毛细胞电压依赖性钙通道;推测NO在传出神经对外毛细胞功能调节中起重要作用。     

亚硒酸钠对脑胶质瘤细胞生长及线粒体膜电位的影响

目的:研究亚硒酸钠对人脑胶质瘤细胞生长抑制效应及线粒体膜电位的影响。方法:使用不同浓度的亚硒酸钠(0.004、0.020、0.100及0.500μmol/L)对人脑胶质瘤细胞SHG-44进行侵染,通过四甲基偶氮唑盐(MTT)比色法检测亚硒酸钠对SHG-44增殖的影响,提取细胞线粒体后通过激光扫描共聚焦显微镜下观察SHG-44的荧光强度,分析SHG-44的线粒体膜电位。结果:亚硒酸钠对人脑胶质瘤细胞生长具有明显的抑制作用,且随亚硒酸钠浓度的增加抑制作用逐渐加强,0.100及0.500μmol/L染硒组与对照组相比差异均有统计学意义(P<0.05),0.004及0.020μmol/L染硒组与对照组相比差异无统计学意义(P>0.05);0.100、0.500μmol/L染硒组线粒体膜电位显著降低(P<0.05),0.004及0.020μmol/L组与对照组相比差异无统计学意义(P>0.05)。结论:亚硒酸钠能抑制人脑胶质瘤细胞SHG-44生长,并且降低其线粒体膜电位。     

缺氧微环境下钙信号介导鞘氨醇激酶1促进胶质瘤细胞增殖

目的探讨缺氧微环境下鞘氨醇激酶1（SphK1）调控胶质瘤细胞增殖的分子机制。方法缺氧建立缺氧模型;RNA干扰技
术下调SphK1的表达,分别以real-time PCR及蛋白免疫印记方法检测SphK1在mRNA及蛋白水平上的表达;CCK-8检测胶质
瘤细胞增殖;流式细胞术检测细胞周期;Fluo-3/AM孵育,激光共聚焦显微镜检测细胞内Ca2+的动态变化。结果SphK1表达下
调可显著降低缺氧诱导的钙内流,并在缺氧条件下影响胶质瘤细胞增殖;钙通道激活剂OAG可减弱SphK1表达下调引起的细
胞增殖抑制。结论缺氧微环境下SphK1可通过对胞内钙离子的调控影响胶质瘤的增殖。
     

A Preliminary Study of The Short Circuit Current （Isc） Responses of Endometria from Estrum and Bilateral Ovariectomy Female Rats

Objective To investigate the property of the endometrium ion transport in the normal estruation and ovariectomize rats. Methods The basic electrophysiological property of the endometrium was carried out by mean of the short circuit current （Isc） recording with the rats. Results The baseline Lsc and the transepithelial resistance （Rte） were different between the estruation and ovariectomized groups. Apical application of Na＋ channel blocker, amiloride （10 μmol/L）, or CFTR Cl- channel agonist, forskolin （10 μmol/L） did not significantly affect the Lc in the two groups. However, the 1sc in the model group was more sensitive to the CFTR Cl- channel blocker （glibenclamide, 1 mmol/L, apical） decreased about 27.0% （P〈0.05, n=6）, no remarkably changes in the estruation rats. While apical application CA CC CI-channel blocker, DIDS （4, 4 ＇-diisothiocyanostilbene- 2,2 ＇-disulfonic, 100 μmol/L）, the Isc was much more sensitive, percentage almost to 36.52% （P〈0.01） in the model group than that in the control Furthermore, basolateral application of bumetanide （100μmol/L）, a blocker of Na＋-K＋-2Cl- cotransport, didn＇t significantly reduce the Lc in the two groups. Conclusion The baseline Lsc and membrane resistance in the ovariectomize group is much higher than that in the normal estruation group, and the endometrial epithelium CI- secretion may be mediated by predominantly calcium-dependent CI- channels （CACC） and activating adenylate cyclase and apical cAMP-dependent Cl- channels （CFTR） in the model group with minor contributions in the control. Furthermore, the endometrial epithelium responses to different stimulants exists difference in the control and model group a likely mechanism for the ovary hormone.