风湿性瓣膜病心房颤动患者右心房超极化激活电流密度及其通道表达的变化

目的比较风湿性心脏瓣膜病心房颤动患者和窦性心律患者右心房单个心肌细胞超极化激活电流（If）密度及超极化激活的环核苷酸门控（HCN）通道表达的变化。方法应用全细胞膜片钳技术记录25例房颤患者和16例窦性心律患者右心耳单个心房肌细胞If的变化，并用Westernblot法检测两组患者右心耳组织HCN通道的表达水平。结果相同激活电位时，房颤组患者右心耳单个心房肌细胞If电流密度明显大于窦性心律组，且随电位增加激活更快，尤其在-70mV时，房颤组和窦性心律组If电流密度分别为（一43．74±2．87）和（-26．81±1．41）pA／pF，此时两组比较差异有统计学意义（P〈0．01）。房颤组HCN2、HCN4通道表达水平分别为0．44±0．20和0．53±0．20，与窦性心律组的0．23±0．13和0．36±0．18比较均明显增高（P〈0．01）。结论心房组织If电流密度增大及HCN通道表达上调参与了心房电重构，在房颤发生和维持中发挥作用。

HERG通道在爪蟾卵母细胞的持续表达与电流变化

目的:探索一个HERG通道在爪蟾卵母细胞持续稳定表达HERG通道的方法,研究表达培养不同时期卵母细胞膜静息电位和通道电流特性的变化。方法:用含HERG片段的pSP64载体质粒体外转录制备的mRNA注射表达于卵母细胞,用双电极电压钳技术记录通道电流。结果:①本方法可成功在卵母细胞持续表达与HERG通道电生理特性一致的功能性通道,并可在10～15d内稳定用于电生理记录。②在培养的第3、6和9d细胞膜静息电位负值逐渐升高,在第12d又开始降低。异源表达的HERG通道内向整流的最大电位在第3、6和9d逐渐向正向转移,在第12d又回复,激活曲线的半最大激活电压(V1/2)在培养的第3、6和9d逐渐向负向转移,在第12d又逐渐回复,其改变和膜静息电位的变化趋势一致。结论:本研究提供了一种HERG基因在爪蟾卵母细胞长期持续表达的方法,并为HERG通道的分子位点和药物作用研究在不同实验条件电生理实验数据的差异提供依据。

虎鼬肾组织结构及水通道蛋白1,2的表达

虎鼬（Vormela peregusna）属哺乳纲（Mammalia）食肉目（Carnivora）鼬科（Mustelidae）虎鼬属（Vormela）,是虎鼬属唯一一种小型哺乳动物,主要分布在东欧到中国西部的广大干燥地区,主要以啮齿动物为食（雷刚等,2009）。目前国内外有关虎鼬的研究比较少,包括核型分析（许可芬和高行宜,1986）、分子遗传学研究（Rozhnov et al.,2006）、

高温和HgCl2复合处理对水稻花期生理特性和水通道蛋白基因表达的影响

以转OsPIN1a的基因水稻‘中花11’为材料,设置3个处理:适温处理(CK),昼夜温度为30℃/28℃;高温处理(TR1),昼夜温度为38℃/33℃;复合处理(TR2),昼夜温度为38℃/33℃,配以0.1 mmol/L HgCl2处理,研究高温胁迫和水通道蛋白抑制剂(HgCl2)对水稻花期生理特性、水通道蛋白基因表达、稻穗和剑叶相对含水量及淀粉粒发育的影响。结果表明:与适温处理相比,高温与HgCl2胁迫均能降低稻穗和剑叶相对含水量,降低结实率和千粒质量,降低柱头上花粉粒数量及柱头和花粉粒的线粒体活性,抑制OsTIPs和OsPIPs家族多个基因的表达,使稻穗水分供给减少,导致胚乳淀粉粒发育不良,形成不规则圆球形,以高温与HgCl2复合处理时影响最大。水通道蛋白基因家族表达受抑制后,加重了高温胁迫对水稻植株花期生长发育的伤害,致使花粉粒和柱头活性下降,导致结实率降低和籽粒充实不良。

水通道蛋白基因在胎肺发育过程中的表达

【目的】测定水通道蛋白基因(Aquaporins,AQPs)是否在中晚期妊娠羊胎肺中表达及其表达模式。【方法】应用荧光定量 PCR 技术对妊娠80d,100d,120d,140d 的羊胎外周肺组织各4例及4例成年羊外周肺组织行 AQP1,3,4和5基因的 mRNA 表达的定量检测;应用 Western 免疫蛋白杂交对妊娠100d,130d的羊胎的外周肺组织行 AQP1,3,4和5基因的蛋白表达的检测。【结果】AQP1,3,4,5基因 mRNA 及蛋白质均在妊娠中晚期羊胎肺中表达;AQP5mRNA 在胎肺的表达显著高于成羊肺。【结论】AQPs 基因在胎肺中表达及 AQP5在胎肺中高于成年肺的特定表达模式提示 AQPs 在胎肺液体产生及吸收中发挥一定作用。

STZ诱导的糖尿病对大鼠冠状动脉Kv1.2和Kv1.5通道蛋白表达的影响

目的:研究链脲佐菌素(STZ)诱导的糖尿病对大鼠冠状动脉平滑肌细胞钾离子通道Kv1.2和Kv1.5通道蛋白表达的影响。方法:实验采用STZ诱导的大鼠Ⅱ型糖尿病,并剥离冠状动脉,应用western blot方法测定糖尿病大鼠与正常大鼠冠状动脉平滑肌细胞电压依赖性钾离子通道Kv1.2和Kv1.5蛋白的含量差异。结果:成功的建造糖尿病大鼠模型,成模率100%,血糖全部为High值。Western blot分析显示,冠状动脉平滑肌细胞Kv1.2与Kv1.5的含量均明显低于对照组。结论:糖尿病大鼠冠状动脉平滑肌细胞钾离子通道蛋白含量显著降低。

不同钾肥对烤烟叶片钾吸收规律的影响

为探究在水肥一体化技术下追施不同钾肥对烤烟叶片钾吸收规律的影响,为烤烟质量调控提供参考。以云烟99为研究对象,设置不同钾肥为试验因素,以当地常用钾肥(硫酸钾)作为对照(CK),设置磷酸二氢钾(T1)、焦磷酸钾(T2)、黄腐酸钾(T3)3个钾肥处理,研究不同处理对烤烟生育期叶片钾含量、叶片钾积累量与积累速率、钾通道基因表达量及烤后烟常规化学成分的影响。结果表明,与移栽后30 d相比,在烤烟移栽后90 d, CK、T1、T2、T3处理叶片钾含量分别降低了60.19%、52.75%、56.31%、48.54%,降低幅度表现为CK>T2>T1>T3。在烤烟移栽后45~60、75~90 d, T3处理烤烟叶片钾的积累量、积累速率高于其他处理。在烤烟移栽后50 d, T1处理NtKC1基因相对表达量为CK处理的5.15倍。在烤烟移栽后65 d, T1、T3处理的NtHAK基因相对表达量分别为CK处理的4.27、3.77倍,T3处理NtNHX2基因的相对表达量为CK处理的3.46倍。在烤烟移栽后90 d, T3处理NtKC1基因的相对表达量为CK处理的7.98倍。在T3处理下,烤后烟上、中部叶钾含量分别较CK处理提高了10.00%、33.33%,烤后烟中部叶钾氯比较CK处理提高了107.41%。在水肥一体化技术下追施黄腐酸钾可以显著降低烤烟生育后期叶片钾的流失量,提高叶片钾的积累量及积累速率,诱导NtHAK、NtNHX2基因表达,并提高烤后烟中上部叶钾含量、中部叶钾氯比。

Construction of Maize Universal Expression Vector PGM-35Sbar and Maize Transformation

[Objective] The paper was to construct maize universal expression vector, in order to provide basis for using transgenic methods to improve abiotic stress tolerance of maize. [Method] Based on the transformation of existing pGreen0229 plant expression vector, phosphinothricin-resistant selectable marker-bar gene driving by CaMv35S promoter was constructed, which could be used to connect target gene of maize expression vector PGM-35Sbar, and transform Ji444 maize inbred lines by pollen tube pathway. [Result] The universal expression vector for PGM-35Sbar maize had been successfully constructed. When the maize plants were transformed, 14 resistant plants were obtained, and 12 plants were identified to be positive plants by PCR detection. [Conclusion] The study provided basis for rapid construction of maize expression vector containing specific target gene.

钙信号抑制剂加剧低钾胁迫对烟草幼苗光合特性及钾吸收的影响

【目的】研究低钾胁迫抑制钙信号传导对植株幼苗叶片光合特性及钾吸收的影响,为缓解作物的低钾胁迫提供理论依据。【方法】以烟草品种K326为试验材料进行了室内水培试验。烟草幼苗先在营养液正常钾(K^(+)5 mmol/L)和缺钾(K^(+)0.15 mmol/L)营养液中生长8天,然后在营养液中分别施加钙通道有机阻断剂Vp、钙通道无机阻断剂LaCl_(3)和钙离子螯合剂(EGTA)钙信号抑制剂,继续生长4天后取样,测定植株干鲜重、电导率、活性氧含量、叶片抗氧化酶活性、叶片光合特性等指标和植株地上、地下部钾素浓度及钾离子吸收相关基因表达量。【结果】(1)不添加钙通道抑制剂或螯合剂条件下,低钾胁迫的烟株地上部和根系干重较常钾水平显著降低了59.29%、39.82%,鲜重显著降低了51.58%、48.19%,除水汽压饱和亏外的其余5项光合特性指标显著降低,植株地上部和根系钾含量分别显著降低了39.31%和77.05%,而叶中可溶性蛋白、相对电导率、过氧化氢含量则显著增加,NKT2、NtKC1、NtHAK1、NtHAK5基因的相对表达量分别增加了2.09、3.32、3.23、12.34倍。(2)常钾水平下,3个钙抑制剂处理未明显影响地上部和根系干鲜重,而低钾水平下,钙抑制处理的地上部鲜重和干重显著高于无钙抑制剂对照。两个钾水平下,与各自对照相比,3个钙抑制剂处理的叶片相对电导率、活性氧积累量均显著增加,抗氧化酶SOD和POD活性、叶绿素含量显著减少,光合特性指标(除水汽压饱和亏外)明显降低。(3)两个钾水平下,3个钙信号抑制剂处理的烟株地上部和根系钾含量均显著低于其对照,低钾胁迫下平均降低幅度分别达到25.54%和53.36%。低钾胁迫下,钾离子通道基因NKT2、NtKC1和转运蛋白基因NtHAK1、NtHAK5在根系中的相对表达量分别平均降低了60.60%、50.62%、38.61%、69.79%,钙通道相关基因NtCNGC1和NtCNGC11在根中的相对表达量分别平均降低了95.36%和87.04%。【结论】低钾胁迫明显影响烟株幼苗生长、光合作用及对钾素的吸收和积累。低钾水平下,外施钙通道抑制剂或螯合剂可进一步降低烟草叶片抗氧化酶活性,减弱烟株叶片光合作用,并抑制根中钾吸收相关基因的表达和烟株对钾素的吸收。

Aberrant expression of ether à go-go potassium channel in colorectal cancer patients and cell lines

AIM: To study the expression of ether à go-go (Eag1) potassium channel in colorectal cancer and the relation- ship between their expression and clinico-pathological features. METHODS: The expression levels of Eag1 protein were determined in 76 cancer tissues with paired non- cancerous matched tissues as well as 9 colorectal adenoma tissues by immunohistochemistry. Eag1 mRNA expression was detected in 13 colorectal cancer tissues with paired non-cancerous matched tissues and 4 colorectal adenoma tissues as well as two colorectal cancer cell lines (LoVo and HT-29) by reverse transcription PCR. RESULTS: The frequency of positive expression of Eag1 protein was 76.3% (58/76) and Eag1 mRNA was 76.9% (10/13) in colorectal cancer tissue. Expression level of Eag1 protein was dependent on the tumor size, lymphatic node metastasis, other organ metastases and Dukes’ stage (P < 0.05), while not dependent on age, sex, site and degree of differentiation. Eag1 protein and mRNA were negative in normal colorectal tissue, and absolutely negative in colorectal adenomas except that one case was positively stained for Eag1 protein. CONCLUSION: Eag1 protein and mRNA are aberrantly expressed in colorectal cancer and occasionally expressed in colorectal adenoma. The high frequency of expression of Eag1 in tumors and the restriction of normal expression to the brain suggest the potential of this protein for diagnostic, prognostic and therapeutic purposes.

Aquaporin-8 expression is reduced in ileum and induced in colon of patients with ulcerative colitis

AIM： To study susceptibility genes which may play a potential role in the pathogenesis and etiology of inflammatory bowel disease （IBD）. METHODS： To identify potential susceptibility genes we performed global gene expression profiling in patients with IBD and control specimens. For determination of an intrinsic gene expression profile in ulcerative colitis （UC） and Crohn＇s disease （CD） compared to normal subjects, mucosal biopsies of non-inflamed regions of the colon and the terminal ileum were subjected to DNA microarray analysis. Real-time RT-PCR and immunohistochemistry were used for verification of selected regulated candidate genes and a genetic analysis was performed. RESULTS： We could show that aquaporin-8 （AQP8） mRNA and protein levels were significantly increased in the colon of UC patients compared to controls. Genetic analysis of the six exons and the promoter region of AQPS, however, revealed no mutations or polymorphisms in IBD patients. CONCLUSION： Our results suggest that upregulation of AQP8 in the colon of UC patients represents a secondary phenomenon which may, due to altered water exchange of the distal intestinal mucosa, disturb the physiologic colonic mucus barrier and thus lead to chronic inflao mmation and ulceration.

Function of chloride intracellular channel 1 in gastric cancer cells

AIM:To investigate the effect of chloride intracellular channel 1(CLIC1) on the cell proliferation,apoptosis,migration and invasion of gastric cancer cells.METHODS:CLIC1 expression was evaluated in human gastric cancer cell lines SGC-7901 and MGC-803 by real time polymerase chain reaction(RT-PCR).Four segments of small interference RNA(siRNA) targeting CLIC1 mRNA and a no-sense control segment were designed by bioinformatics technology.CLIC1 siRNA was selected using Lipofectamine 2000 and transfected transiently into human gastric cancer SGC-7901 and MGC-803 cells.The transfected efficiency was observed under fluorescence microscope.After transfection,mRNA expression of CLIC1 was detected with RT-PCR and Western blotting was used to detect the protein expression.Proliferation was examined by methyl thiazolyl tetrazolium and apoptosis was detected with flow cytometry.Polycarbonate membrane transwell chamber and Matrigel were used for the detection of the changes of invasion and migration of the two cell lines.RESULTS:In gastric cancer cell lines SGC-7901 and MGC-803,CLIC1 was obviously expressed and CLIC1 siRNA could effectively suppress the expression of CLIC1 protein and mRNA.Proliferation of cells transfected with CLIC1 siRNA3 was enhanced notably,and the highest proliferation rate was 23.3%(P = 0.002) in SGC-7901 and 35.55%(P = 0.001) in MGC-803 cells at 48 h.The G2/M phase proportion increased,while G0/G1 and S phase proportions decreased.The apoptotic rate of the CLIC1 siRNA3 group obviously decreased in both SGC-7901 cells(62.24%,P = 0.000) and MGC-803 cells(52.67%,P = 0.004).Down-regulation of CLIC1 led to the inhibition of invasion and migration by 54.31%(P = 0.000) and 33.62%(P = 0.001) in SGC-7901 and 40.74%(P = 0.000) and 29.26%(P = 0.002) in MGC-803.However,there was no significant difference between the mock group cells and the negative control group cells.

Expression of hNav1.8 sodium channel protein in affected nerves of patients with trigeminal neuralgia

Objective: To explore the pathogenesis of trigeminal neuralgia (TN) and to provide a new target for the drug treatment of TN by studying the expression of tetrodotoxin-resistant hNav1. 8 sodium channel protein in affected nerves of patients with TN. Methods: Twelve affected inferior alveolar nerves were obtained from patients with idiopathic TN, to whom the drug therapy was not effective. As negative control, one nonnal inferior alveolar nerve was obtained from patients who accepted the combined radical neck dissection with glossectomy and mandibulectomy. One muscle sample was obtained as normal control. One dorsal root ganglion from rat was as positive control. These tissues and prepared hNav1. 8 antibody were conducted immunohistochemistry response. Results: hNav1.8 channel protein was expresses in all the 12 specimens of the affected nerves of patients with TN, but not in the muscle sample and the normal inferior alveolar nerve. Conclusion: The abnormal expression of hNav1. 8 channel protein in the affected nerves of patients with TN may play an important role in the pathogenesis of TN.

Analytic expression of the chirped sampled function used to produce the equal chirp in the specific reflection channel in uniform period fiber Bragg gratings

The general analytic expression of the chirped sampled function is derived based on coupled mode theory. This function can be used to describe how to use uniform period fiber Bragg grating to produce the equal chirp at will in the specific reflection channel. As an example,the exact sampled function expression that produces a linear chirped at the +4 channel is given. The simulation results by using the transfer-matrix show that the theory is correct.

Tumor necrosis factor alpha increases intestinal permeability in mice with fulminant hepatic failure

AIM:To determine the effect of tumor necrosis factor alpha(TNF-α) on intestinal permeability(IP) in mice with fulminant hepatic failure(FHF),and the expression of tight junction proteins.METHODS:We selected D-lactate as an index of IP,induced FHF using D-galactosamine/lipopolysaccharide and D-galactosamine/TNF-α,assessed the results using an enzymatic-spectrophotometric method,transmission electron microscopy,immunohistochemistry,Western blotting and real-time quantitative polymerase chain reaction.The effect of the administration of antiTNF-α immunoglobulin G(IgG) antibody,before the administration of D-galactosamine/lipopolysaccharide,on TNF-α was also assessed.RESULTS:IP was significantly increased in the mouse model of FHF 6 h after injection(13.57 ± 1.70 mg/L,13.02 ± 1.97 mg/L vs 3.76 ± 0.67 mg/L,P = 0.001).Electron microscopic analysis revealed tight junction(TJ) disruptions,epithelial cell swelling,and atrophy of intestinal villi.Expression of occludin and claudin-1 mRNA was significantly decreased in both FHF models(occludin:0.57 ± 0.159 fold vs baseline,P = 0.000;claudin-1:0.3067 ± 0.1291 fold vs baseline,P = 0.003),as were the distribution density of proteins in the intestinal mucosa and the levels of occludin and claudin-1 protein(occludin:0.61 ± 0.0473 fold vs baseline,P = 0.000;claudin-1:0.6633 ± 0.0328 fold vs baseline,P = 0.000).Prophylactic treatment with antiTNF-α IgG antibody prevented changes in IP(4.50 ± 0.97 mg/L vs 3.76 ± 0.67 mg/L,P = 0.791),intestinal tissue ultrastructure,and the mRNA levels of occludin and claudin-1 expression(occludin:0.8865 ± 0.0274 fold vs baseline,P = 0.505;claudin-1:0.85 ± 0.1437 fold vs baseline,P = 0.1),and in the protein levels(occludin:0.9467 ± 0.0285 fold vs baseline,P > 0.05;claudin-1:0.9533 ± 0.0186 fold vs baseline,P = 0.148).CONCLUSION:Increased in IP stemmed from the downregulation of the TJ proteins occludin and claudin-1,and destruction of the TJ in the colon,which were induced by TNF-α in FHF mice.

2型糖尿病大鼠心房钙通道及钙电流的变化

目的研究2型糖尿病大鼠心房组织钙离子通道的表达及钙电流的改变情况。方法 30只雄性Wistar成年大鼠随机分为对照组和糖尿病组。其中糖尿病组高脂高糖饮食4周后给予小剂量链脲佐菌素35mg/kg腹腔注射,建立2型糖尿病模型。8周后取两组大鼠的血清测定血糖、胰岛素、总胆固醇、甘油三酯、低密度脂蛋白以及高密度脂蛋白,判定两组的代谢情况。将固定后的大鼠心房组织切成冰冻切片,通过免疫荧光双标染色法测定两组的Cav1.2,Cav3.1和Cav3.2的表达情况。采用Langendorff装置进行急性分离成年大鼠心房细胞,通过全细胞膜片钳技术分别测定两组L型和T型钙电流的峰值电流、I-V曲线、稳态激活和失活电流曲线等数据。结果与对照组相比,糖尿病组有严重的代谢紊乱,Cav1.2的蛋白表达降低,Cav3.1的蛋白表达升高,Cav3.2的蛋白表达未见明显改变。另外,糖尿病组L型钙电流的峰值电流显著降低(P<0.05)、电流-电压(I-V)曲线下移、稳态激活和失活电流曲线未见明显差异(P>0.05),T型钙电流的峰值电流显著升高(P<0.05)、电流-电压(I-V)曲线上移、稳态激活电流曲线未见明显差异(P>0.05)。结论 2型糖尿病大鼠心房肌细胞有着明显的钙通道重构,导致L型钙电流密度下降,T型钙电流密度上升。

Effect of electronic stimulation at Neiguan(PC 6) acupoint on gene expression of adenosine triphosphate-sensitive potassium channel and protein kinases in rats with myocardial ischemia

OBJECTIVE:To investigate the effects of electronic stimulation at acupoints Neiguan(PC 6) and Lieque(LU 7) on the gene expression of the adenosine triphosphate(ATP)-Sensitive potassium channel(KATP:Kir6.1,Kir6.2,SUR2 A,and SUR2B) and protein kinases(PKA,PKG,and PKCβ2) in myocardial cells of rats with myocardial ischemia(Ml) induced by isoproterenol(ISO).METHODS:Rats were randomly divided into a control,model,Neiguan(PC 6),Lieque(LU 7),and non-acupoint groups.The Ml model was established by injecting rats with ISO.Electro-acupuncture treatment was given to the acupuncture groups,once a day for 7 days.Gene expression was analyzed with real-time PCR.RESULTS:The gene expression of KATP and protein kinases in the model group was higher than those in the control group(P < 0.05).After acupuncture treatment,the KATP and protein kinase expression levels were significantly lower in the Neiguan(PC 6)and Lieque(LU 7) groups compared with the model group[P < 0.05).The Neiguan(PC 6) group lowered these levels significantly more than that of the Lieque(LU 7) group(P < 0.05).No significant differences were observed between the model and non-acupoint groups(P > 0.05).CONCLUSION:Our findings suggest that electronic needling of Neiguan(PC 6) can both reduce the gene expression of KATP and protein kinases in rats with ISO-induced Ml.

Astragaloside IV Regulates Expression of ATP-sensitive Potassium Channel Subunits after Ischemia-reperfusion in Rat Ventricular Cardiomyocytes

Objective: Astragaloside IV (AsIV) is the major effective component extracted from the Chinese herb Astragalus membranaceus, which has been widely used to treat cardiovascular disease. Recent studies have shown that AsIV can potentially protect the heart from myocardial ischemic injury, but the mechanisms of action are unknown. ATP-sensitive potassium (KATP) channels are activated during ischemia and exert a compensatory protective effect on cardiomyocytes. We therefore examined the effects of AsIV on KATP channel currents and channel expression in isolated rat ventricular cardiomyocytes after ischemia-reperfusion injury. Methods: Forty Wistar rats were divided into five groups: control group, ischemia-reperfusion (IP) group, IP + glibenclamide group, IP + pinacidil group and IP + AsIV group. The ischemia-reperfusion injury model was established in enzymatically isolated ventricular cardiomyocytes by perfusion with calcium-free Tyrode solution for 10 min, arrest for 30 min, and reperfusion for 45 min. The different drugs were applied for 10-15 min, and the KATP channel current (IKATP) was recorded with voltage-clamp mode by whole-cell patch-clamp technique. Protein and mRNA expression of the KATP channel subunits Kir6.1, Kir6.2, SUR2A and SUR2B was quantified using western blotting and real-time PCR. Results: The KATP current in IP group was significantly greater than that in control group (211.45±33.67 vs 83.51±23.67 pA; P<0.01). Glibenclamide (10 μmol/L) blocked KATP currents, whereas both AsIV (1 mg/L) and the known channel opener pinacidil (50 μmol/L) significantly increased IKATP (P<0.05). Consistent with this, AsIV significantly up-regulated protein and mRNA expression of Kir6.1, Kir6.2, SUR2A, SUR2B (P<0.01 vs IP group). Conclusion: The protective effects of AsIV in ischemia-reperfusion injury may be related to the up-regulation of several KATP channel subunits and facilitation of KATP currents.