Na+/H+交换器1核酶对缺氧大鼠肺动脉平滑肌细胞增殖和凋亡的影响

我们的研究拟观察转染Na /H 交换器 1(NHE 1)核酶基因逆转录病毒载体后 ,缺氧培养的大鼠肺动脉平滑肌细胞(PASMCs)的增殖和凋亡变化。材料与方法　转染表达NHE 1核酶基因的大鼠PASMCs(PRZ组 )为我们前期实验所获得[1] ,同时设立转染pLXSN空载体的大鼠PASMCs(PX组 )和未转染的大鼠PASMCs为对照组。第 4～ 10代细胞用于实验。细胞准备 :用于细胞内pH值 (pHi)测定和原位细胞凋亡检测的细胞接种于 2 4孔培养板内的玻片上 ,其他实验的细胞接种于培养瓶。各组细胞均用含 0 4 %小牛血清的DMEM培养基培养 4 8h ,使细胞处于静止…     

高浓度葡萄糖对人胰岛β细胞凋亡影响的分子机制

目的　初步探讨高浓度葡萄糖对人胰岛 β细胞凋亡的影响及其分子机制。　 方法　分离培养人胰岛细胞 ,并分为对照组、高糖组和高糖 氨基胍组 ,3 7℃ ,5 %CO2 培养 72h ,测定培养液上清液中胰岛素、一氧化氮 (NO)、还原性谷胱甘肽 (GSH)水平。原位末端核苷酸标记法 (TUNEL)和胰岛素免疫组化双染色法及ELISA法检测胰岛 β细胞凋亡 ,RT PCR检测胰岛细胞p5 3、Bcl 2和胰岛素基因启动转录因子 1 (PDX 1 )mRNA表达水平。　结果　高糖组胰岛 β细胞凋亡小体富计因子(1 91± 0 6 9)、β细胞凋亡率 (1 4 8% )、NO〔(1 82 3± 1 5 5 ) μmol/L〕和 p5 3mRNA(0 3 0 6± 0 0 3 9)表达水平均显著高于高糖 氨基胍组〔分别为 1 1 9± 0 3 3、6 8%、(1 5 4 2± 1 9 7) μmol/L、0 1 3 9±0 0 6 9,P <0 0 1〕和对照组〔分别为 1 0 6± 0 2 6、4 2 %、(1 1 7 3± 2 1 7) μmol/L、0 1 2 5± 0 0 1 5 ,P <0 0 5〕 ,而胰岛素释放量、GSH、bcl 2mRNA和PDX 1mRNA表达水平则显著低于高糖 氨基胍组(P <0 0 5 )和对照组 (P <0 0 5 )。　结论　高浓度葡萄糖可通过诱导人胰岛 β细胞凋亡及PDX 1表达降低使胰岛素分泌减少 ,其机制与高糖状态下胰岛 β细胞抗氧化能力降低引起NO介导的p5 3高表达和PDX 1低表     

反义肝素酶基因对胰腺癌细胞体外增殖和侵袭的抑制作用

目的:研究反义肝素酶基因对人胰腺癌SW1990细胞体外增殖和侵袭能力的抑制作用．方法:反义肝素酶基因转染胰腺癌sW1990细胞,并设空载体对照组和空白对照组,以流式细胞仪检测细胞周期;免疫组化、Western blot及RT-PCR检测肝素酶蛋白和mRNA表达;平板克隆形成实验检测细胞增殖活性,Transwell侵袭小室模型检测细胞体外侵袭能力．结果:与空白组和空载组比较,反义组细胞周期中S期比例明显减少(18.8％±2．5％vs 36．3％±2．2％,33．2％±2．1％,P<0．01),G1期细胞比例明显升高(66．0％±2．7％vs30．7％±3．2％,39．8％±4．9％,P<0．01);肝素酶蛋白及mRNA表达分别降低34．3％和37．8％:细胞克隆形成数目减少(12．2±2．8 vs 30．8±4．4,28．3±2．7,P<0．01);Transwell侵袭小室中24 h穿膜细胞数减少(13．0±3．5 vs 34．8±5．8,29．4±5．6．P<0．01)．结论:反义肝素酶基因抑制人胰腺癌SW1990细胞体外增殖及侵袭能力．     

Na+-H+交换体1反义基因磁性纳米微粒体内靶向治疗胃癌的实验研究

目的 探讨Na+-H+交换体1(NHE1)反义基因磁性纳米微粒体内靶向治疗胃癌的可行性.方法 构建NHE1反义基因真核表达载体和NHE1反义基因磁性纳米微粒,以氧化铁磁性纳米颗粒为基因转染载体,将NHE1反义基因导入SGC-7901胃癌细胞中,获得稳定表达NHE1反义基因的7901-AS细胞.研究转染细胞形态学变化及体外生长情况.建立裸鼠胃癌移植瘤模型后,进行体内靶向定位实验,观察抑瘤率.结果 7901-AS细胞在光镜、电镜下的肿瘤细胞形态恶性程度降低.出现显著生长抑制现象,细胞增殖指数降低,凋亡指数明显增高(P相似文献   

单羧酸转运蛋白基因对肿瘤细胞内pH值及生长特性的调节作用

目的 以反义技术抑制肿瘤细胞的单羧酸转运蛋白基因第一亚型(MCT1)表达,观察其对肿瘤细胞内pH值(pHi)调节、乳酸转运及生长性质的影响.方法 (1)应用逆转录-聚合酶链式反应(RT-PCR)从人肺癌细胞系A549中扩增MCT1目的 基因片段,将克隆的基因片段反向插入逆转录病毒载体pLXSN,构建反义表达重组载体pLXSN-MCT1,并对其进行DNA测序验证.(2)通过电穿孔法将pLXSN、重组载体pLXSN-MCT1转染于肺腺癌细胞系A549中,经G418筛选转染细胞阳性克隆.用PCR方法 鉴定pLXSN-MCT1重组载体在基因组的转染整合及表达;以分光光度法测定细胞内pH及乳酸含量变化,并以细胞生长曲线研究细胞的生长情况.结果 (1)对所构建的重组载体进行双酶切电泳分析及DNA序列分析证明反义载体构建成功.(2)与未转染的A549细胞比较,转染MCT1反义表达重组体的细胞pHi降低、乳酸显著升高(P＜0.001);且细胞的生长受到明显抑制.结论 单羧酸转运蛋白MCT1基因在肿瘤细胞pHi调节、乳酸转运及细胞的生长中起着重要的调节作用.     

WSTF过表达的乳腺癌MCF-7细胞增殖、侵袭、迁移能力变化及其机制

目的 观察威廉姆斯综合征转录因子(WSTF)通过调控白细胞介素6(IL-6)/信号传导子及转录激活子3(STAT3)信号通路对乳腺癌MCF-7细胞增殖、侵袭及迁移能力的影响。方法 体外培养乳腺癌MCF-7细胞株,分为WSTF高表达组(转染慢病毒表达载体pLVX-PRDX4)、WSTF低表达组(转染慢病毒干扰载体pLVX-shPRDX4)、空载组(转染空载体pLVX-NC)和对照组(不作特殊处理)。各组转染48 h,采用CCK-8法检测细胞存活率,改良Matrigel Boyden小室实验检测侵袭细胞数,划痕实验检测划痕愈合率,流式细胞术检测细胞凋亡率,qRT-PCR法检测WSTF、IL-6和STAT3 mRNA表达,Western blotting法检测WSTF、IL-6和STAT3蛋白表达。结果 与对照组和空载组比较,WSTF低表达组细胞存活率、划痕愈合率和侵袭细胞数均降低,WSTF高表达组均升高(P均<0.05)。与对照组和空载组比较,WSTF低表达组细胞凋亡率升高,WSTF高表达组降低(P均<0.05)。与对照组和空载组比较,WSTF低表达组WSTF、IL-6和STA...     

TIMP-1抑制大鼠肾小球系膜细胞凋亡与磷脂酰肌醇3激酶/丝/苏氨酸激酶通路的研究

目的探讨磷脂酰肌醇3激酶(phosphoinositide-3-kinase,PI3K)/丝/苏氨酸激酶(serine thre-onine kinase,AKT)通路在基质金属蛋白酶组织抑制剂1(tissue inhibitor of metalloproteinase-1,TIMP-1)抑制高糖诱导大鼠肾小球系膜细胞(rat mesangial cells,RMC)凋亡中的作用和机制。方法(1)脂质体法将正、反义人TIMP-1(hTIMP-1)及空载体转染到大鼠肾小球系膜细胞。(2)Annexin V/PI双染法流式检测系膜细胞凋亡率。(3)Caspase9/Mch6Colorimetric Assay试剂盒检测Caspase9活性。(4)Western blot检测BAD、phospho-BAD、AKT、phospho-AKT蛋白水平。结果与对照组(1·06±0·08)%相比,正义转染组细胞凋亡率降低[(4·51±0·50)%,P<0·05],反义转染组凋亡率显著提高[(24·97±3·64)%,P<0·05],LY294002的加入可部分抵消TIMP-1的抗凋亡作用。TIMP-1的高表达可下调Caspase9活性(0·111±0·064,P<0·05),低表达可上调Caspase9活性(0·461±0·012,P<0·05)。正义TIMP-1能提高细胞phos-pho-BAD、phospho-AKT的蛋白水平,而反义TIMP-1的两种蛋白水平明显减少,且该作用可被LY294002阻断。结论(1)PI3K/AKT通路参与TIMP-1抑制高糖诱导的大鼠肾小球系膜细胞凋亡过程。(2)BAD在其中起重要作用。(3)AKT对Caspase9的抑制亦起主要作用。     

环氧合酶-2反义核酸抑制胃癌细胞的恶性表型

目的　通过环氧合酶 2 (COX 2 )反义核酸基因转染 ,逆转COX 2高表达的人胃癌细胞系中其表达水平 ,观察细胞生物学行为的变化情况 ,以初步探讨COX 2表达在胃癌发生中的某些具体机制。方法　使用脂质体介导的方法 ,用反义重组质粒及空载体分别转染COX 2异常高表达的人胃癌细胞系SGC 790 1(转染细胞分别命名为 790 1 AS及 790 1 P细胞 )。通过免疫细胞化学及RNA斑点杂交试验检测反义核酸转染的细胞中COX 2的蛋白及mRNA表达水平。四唑盐 (MTT)比色试验检测转染细胞的体外增殖速度。应用裸鼠成瘤试验比较转染前后细胞体内成瘤能力的差别。结果　免疫细胞化学及RNA斑点杂交试验证实 :在反义核酸转染的 790 1 AS细胞中COX 2的蛋白及mRNA水平均显著下调。MTT比色试验显示 790 1 AS细胞的增殖速度低于亲本SGC 790 1细胞。裸鼠成瘤试验表明 ,反义核酸转染细胞成瘤潜伏期延长 ,成瘤体积减小。 3组细胞接种裸鼠 30d后 ,瘤体的平均重量( x±s)分别为 (82 6 6 7± 77 6 7)mg(SGC 790 1细胞 ) ,(776 6 7± 30 0 0 6 )mg(790 1 P细胞 )和 (486 6 7±15 2 8)mg (790 1 AS细胞 )。转染反义核酸的细胞成瘤性显著低于未转染细胞及转染载体对照细胞(P <0 0 1)。结论　胃癌细胞中过表达的COX 2与细胞的恶性表型相关。用     

Presenilin-1突变对全反式维甲酸诱导的PC12细胞生长和凋亡的影响

目的　观察Presenilin 1(PS 1)突变型L2 86V对全反式维甲酸 (RA)诱导的PC12细胞生长和凋亡的影响。　方法　运用脂质体介导的基因转染技术建立稳定表达PS 1WT和突变型L2 86V基因的细胞克隆 ,采用MTT法、流式细胞仪检测细胞生长曲线、生长周期及细胞凋亡情况。　结果　 (1)PS 1突变型L2 86V对RA诱导的PC12细胞生长具有抑制作用 ;主要表现为G1期细胞增多、S期细胞减少 ,细胞增殖指数降低 ;(2 )在正常培养条件下 ,对照组细胞凋亡率 (% )为 0 31±0 0 9、野生型组为 0 4 3± 0 11、L2 86V组为 0 6 2± 0 2 7;无血清培养 1、2、3d ,对照组细胞凋亡率 (% )分别为 0 5 5± 0 0 7、1 2 3± 0 4 7、5 5 9± 2 91;野生型组为 1 0 9± 0 73、3 2 1± 1 4 1、7 79± 2 78;L2 86V组为 4 6 5± 1 0 4、10 5 4± 3 18、14 4 7± 3 5 3;PS 1突变型L2 86V对RA诱导的PC12细胞均具有促进细胞凋亡的作用 ,以无血清培养时表现得更为明显。　结论　PS 1突变型L2 86V对RA诱导的PC12细胞生长具有抑制作用 ;在有和无血清培养条件下均具有促进细胞凋亡的作用 ,以无血清培养时表现得更为明显。     

端锚聚合酶1反义寡核苷酸对人肺癌细胞增殖的影响

目的　探讨端锚聚合酶 1(tankyrase 1,TANK1)反义寡核苷酸对人肺癌细胞的影响 ,为肺癌治疗的靶向研究探索新途径。方法　合成TANK1正义寡核苷酸 (TANK1 SODN)、反义寡核苷酸(TANK1 ASODN) ,待人肺癌细胞株CALU在裸鼠蹊部皮下接种成瘤后 ,将成瘤裸鼠随机分成 3组 :对照组 4只 (每天上午于瘤体内多位点注射生理盐水 2 0 0 μl/只 ) ,正义组 (每天同法注射含TANK SODN10 0 μg的生理盐水 2 0 0 μl/只 )和反义组 (每天同法注射含TANK ASODN 10 0 μg的生理盐水 2 0 0 μl/只 )各 5只 ,观察肿瘤的生长情况及组织学改变。用链霉亲和素 生物素 酶复合物 (SABC)法检测肿瘤细胞Ki6 7及端粒酶反转录酶 (hTERT)的阳性表达率。结果　在裸鼠成瘤部位连续注射相应液体 16d后 ,反义组移植瘤体积为 (1 4 6± 0 70 )cm3 ,明显小于对照组的 (3 2 9± 0 4 7)cm3 及正义组的 (3 14±0 70 )cm3 ,差异有显著性 (P均 <0 0 1) ;肿瘤细胞Ki6 7标记指数及hTERT细胞阳性率均明显低于对照组及正义组 (P均 <0 0 1)。结论　人肺癌细胞株端粒酶呈高度表达 ,TANK1 ASODN可降低瘤细胞端粒酶活性 ,抑制肺癌细胞增殖     

Contribution of NHE-1 to cell length shortening of normal and failing rabbit cardiac myocytes

At the same intracellular pH (pHi) Na+/H+ exchange (NHE-1) fluxes of ventricular myocytes of hypertrophied failing hearts (HFH) are increased. We assessed how NHE-1 affected cell length shortening. pHi was measured fluorimetrically in resting and twitching (1-3 Hz) normal and HFH rabbit myocytes. In HEPES-buffered solutions, increased NHE-1 fluxes (P=0.001, n=14) made HFH resting pHi 0.2+/-0.03 units more alkaline than control (n=27). In CO2/HCO3--buffered solutions, HFH resting pHi was not different (7.05+/-0.02, n=30). Twitching myocytes of both groups shortened 15-16% less per 0.1 pH unit acidification. In HEPES-buffered solutions, cariporide depressed cell length shortening of normal myocytes (1-3 Hz) by 16+/-5.4% (n=9, P=0.005). In HFH myocytes cariporide restored the positive force-frequency relationship (n=7, P=0.009), by depressing twitch amplitudes at 1 Hz (16+/-11%, P=0.047) but not at 2 and 3 Hz. The depressions were all caused by pHi acidification. In CO2/HCO3- buffered solutions the cariporide-induced acidification was too small to explain the cell length shortening depression of normal (19+/-5.0%, n=11, P=0.006) and HFH myocytes (14+/-4.7%, n=11, P=0.001). When compared to HEPES-buffered solutions, HFH myocytes in CO2/HCO3--buffered solutions shortened 12+/-6.8% better than expected given the 0.16+/-0.02 units more acidic pHi's at which they twitched. We conclude that in CO2/HCO3--buffered solutions NHE-1 improved cell length shortening of unstretched normal and HFH myocytes via a pHi-independent mechanism. Although NHE-1 was increased in HFH myocytes, the magnitude of the pHi-independent effect of NHE-1 inhibition on cell length shortening was similar in both groups.     

NHE-1 and NBC during pseudo-ischemia/reperfusion in rabbit ventricular myocytes

Despite many studies into the pathophysiology of cardiac ischemia-reperfusion injury, a number of key details are as yet undisclosed. These include the timing and magnitude of the changes in both Na(+)/H(+) exchange (NHE-1) and Na(+) -- HCO(3)(-) -cotransport (NBC) transport rates. We fluorimetrically measured H(i)(+) fluxes (J(NHE-1) and J(NBC)) and Na(i)(+) fluxes in single contracting rabbit ventricular myocytes subjected to metabolic inhibition, pseudo-ischemia (i.e. metabolic inhibition and extracellular acidosis of 6.4), and pseudo-reperfusion. Metabolic inhibition and pseudo-ischemia inhibited NHE-1 by 43 +/- 3.1% and 91 +/- 3.6%, and NBC by 66 +/- 5.4% and 100%, respectively. Inhibition was due to both an acidic shift of the pH(i) at which NHE-1 and NBC become quiescent (set-point pH(i)) and a reduction of the steepness of the pH(i) -- H(i)(+) flux profiles. NHE-1 and NBC did not contribute to Na(i)(+) loading during metabolic inhibition (Na(i)(+) 18 +/- 1.7 mM) or pseudo-ischemia (Na(i)(+) 21 +/- 1.7 mM), because pH(i) acidified less than set-point pH(i)'s. Upon pseudo-reperfusion NBC recovered to 54 +/- 7.3% but NHE-1 to 193 +/- 11% of aerobic control flux, and set-point pH(i)'s returned to near neutral values. Metabolic inhibition and reperfusion caused an acid load of 18 +/- 3.2 mM H(+) 94% of which were extruded by the hyperactive NHE-1. At pseudo-reperfusion Na(i)(+) rose sharply to 31 +/- 5.8 mM within 1.5 min and that coincided with hypercontracture. Cariporide not only prevented the Na(i)(+) transient, but also inhibited pH(i) recovery and prevented hypercontracture. Our results are consistent with the view that NHE-1 is active during metabolic inhibition if, like in whole hearts, pH(i) is driven more acidic than NHE-1 set-point pH(i). Furthermore, either an acidic pH(i) or absence of additional Na(i)(+) loading during reperfusion, or both, limit ischemia-reperfusion injury.     

Inhibitory effect of antisense vascular endothelial growth factor RNA on the profile of hepatocellular carcinoma cell line in vitro and in vivo

AIM:To evaluate the effect of antisense vascularendothelial growth factor(VEGF)RNA(PCMV-FGEV)transfection on the profile of hepatocellular carcinoma(HCC)SMMC-7721 cells in vitro and in vivo.METHODS:SMMC-7721 cells were transfectedwith PCMV-FGEV antisense,PCMV-VEGF sense andempty vector plasmid encapsulated by lipofectamineas antisense group,sense group and control grouprespectively.The positive cell clones were selectedwith G418.The stable transfection and expressionof VEGF in the cells were determined by RT-PCR andimmunohistochemistry.Cell proliferation was observedby MTT assay.FACS analysis was used to determine theeffect of PCMV-FGEV transfection on cell apoptosis.Thegrowth of transfected cells in Wvo was also observed innude mice.RESULTS:VEGF expression was reduced in SMMC-7721transfected with PCMV-FGEV,which was confirmed byRT-PCR and immunohistochemistry.No effect of PCMV-FGEV transfection was found on cell proliferation andcell apoptosis of SMMC-7721 in vitro.The growth of cellstransfected with PCMV-FGEV was slow in nude miceand accompanied with obvious apoptosis.The latenttime of tumors in the antisense group was 25.0±1.8d,which was longer than that in sense and controlgroups(F=19.455,P<0.01).The average tumor weightin antisense group(0.96 g±0.28 g)was the smallestamong the three groups(F=21.501,P<0.01).CONCLUSION:The expression of VEGF can be inhibitedby antisense PCMV-FGEV.Antisense PCMV-FGEV has no effect on cell proliferation and apoptosis of SMMC-7721in vitro but can inhibit tumor growth and induce cellapoptosis in vivo.     

脆性组胺酸三联体基因对人肺腺癌A549细胞恶性表型的影响

目的研究外源性脆性组胺酸三联体(FHIT)基因在体内外对人肺腺癌A549细胞恶性表型的影响。方法用脂质体介导外源FHIT基因转染A549细胞,建立单克隆细胞系FHITA-549和PEGFP-A549。逆转录聚合酶链反应(RTPCR)、免疫组化方法检测外源FHIT基因在A549细胞的表达状况。采用细胞生长曲线、集落形成试验、流式细胞仪及裸鼠移植瘤试验研究外源FHIT基因对A549细胞体外增殖、凋亡、细胞周期和体内成瘤性的影响。结果RTPCR试验证实FHIT-A549中有FHITmRNA表达,免疫组织化学染色显示FHIT-A549细胞FHIT蛋白表达强阳性,而A549细胞和转空载体的PEGFPA549细胞FHIT基因和蛋白表达均阴性。FHIT-A549细胞的集落形成率为2.6%,显著低于A549细胞的50.1%和转染空载体PEGFPA549细胞的53.6%,三者相比差异有统计学意义(P<0.01)。流式细胞仪分析显示FHIT-A549细胞95.8%阻滞在G2期。FHITA549细胞移植瘤的瘤重为(0.04±0.03)g,显著低于A549细胞的(0.24±0.11)g和转染空载体PEGFPA549细胞的(0.25±0.07)g,三者差异有统计学意义(P<0.01)。结论A549细胞内外源FHIT基因的转导并表达能显著抑制其恶性增殖和分裂,诱导其凋亡,调节其细胞周期、抑制成瘤性。     

重构型天冬氨酸特异性半胱氨酸蛋白酶3表达载体诱导人肺腺癌细胞凋亡的意义

目的构建天冬氨酸特异性半胱氨酸蛋白酶3(Caspase3)表达载体,观察Caspase3表达载体对人肺腺癌A549细胞凋亡的影响。方法应用基因重组方法,建立重构型Caspase3基因的真核表达系统pcDNA3.1revCaspase3质粒和野生型pcDNA3.1Caspase3质粒。将实验细胞分为3组转染pcDNA3.1revCaspase3质粒组,转染pcDNA3.1Caspase3质粒组,转染pcDNA3.1质粒空白对照组。前2组用Caspase3抑制剂DEVDfmk干预。应用Caspase3酶活性分析、流式细胞仪及甲基偶氮唑蓝(MTT)比色法检测A549细胞Caspase3酶活性变化及细胞凋亡和增殖情况。结果(1)pcDNA3.1revCaspase3质粒组、pcDNA3.1Caspase3质粒组的A549细胞Caspase3酶活性分别是(11.87±0.92)%、(5.34±0.38)%(t=16.02,P<0.01);用Caspase3抑制剂DEVDfmk干预后,pcDNA3.1revCaspase3质粒组酶活性为(7.04±0.48)%,仍明显高于野生型pcDNA3.1Caspase3质粒组的(4.51±0.20)%(t=11.86,P<0.01)。(2)流式细胞分析结果显示,pcDNA3.1revCaspase3质粒组、pcDNA3.1Caspase3质粒组和pcDNA3.1质粒组的A549细胞凋亡率分别是(20.1±3.5)%、(7.8±2.8)%、(1.4±0.3)%,3组差异有统计学意义(F=44.01,P<0.01)。(3)MTT比色检测显示pcDNA3.1revCaspase3质粒组、pcDNA3.1Caspase3质粒组和pcDNA3.1质粒组的A549细胞生存率分别是(35.7±1.1)%、(72.8±2.9)%、(85.4±4.8)%,转染pcDNA3.1revCaspase3质粒组生存率明显低于其他两组(F=375.07,P<0.01)。结论pcDNA3.1revCaspase3在A549细胞内有较强的自身活化能力,对Caspase3抑制剂抵抗作用较强,可明显诱导A549细胞凋亡并抑制A549细胞生长。     

Increased proximal tubule NHE-3 and H+-ATPase activities in spontaneously hypertensive rats

OBJECTIVE: To investigate renal proximal tubular sodium-hydrogen exchanger 3 (NHE-3) and H+-ATPase activities in young (5-week-old) spontaneously hypertensive rats (SHR) and normotensive Donryu (DRY) rats, in the period during which high blood pressure is developing. METHODS: Five-week-old SHR and DRY rats were weighed and systolic blood pressure recorded. Proximal tubule cells were isolated, loaded with the intracellular pH dye, 2'-7'-bis-carboxyethyl-5(6)-carboxyfluorescein-acetoxymethyl-ester and acidified with a NH4+/NH3 prepulse. Na+-independent intracellular pH recovery rate (H+-ATPase activity) and initial Na+-dependent intracellular pH recovery rate (NHE-3 activity) were assessed. NHE-3 activity was assessed during inhibition of H+-ATPase with Bafilomycin A1 and during inhibition of any possible NHE-1 activity with Hoe 694. RESULTS: Mean body weight and systolic blood pressures of 5-week-old SHR and DRY rats were not significantly different. NHE-3 activity was higher in SHR, 1.08 +/- 0.1 pH units/min compared with DRY rats, 0.73 +/- 0.1 pH units/min (P < 0.05) H+-ATPase activity was also higher in SHR, 0.119 +/- 0.02 pH units/min, compared with DRY rats, 0.051 +/- 0.02 pH units/min (P < 0.05). CONCLUSIONS: Proximal tubule cells of 5-week-old SHR have higher NHE-3 and H+-ATPase activities compared with age-matched DRY rats. Enhanced proximal tubular fluid reabsorption is likely to contribute to development of high blood pressure in young SHR.     

Potassium and activity of the sodium hydrogen exchanger isoform 1 in vascular smooth muscle of hypertensive rats.

Low potassium intake is inversely associated with blood pressure. In vitro, the proliferation of vascular smooth muscle cells (VSMCs) shows an inverse correlation with [K(+)]. In hypertension, many studies have established that the ubiquitous Na(+)/H(+) exchanger isoform 1 (NHE-1) exhibits increased activity and is permissive for cell proliferation. Changes in extracellular [K(+)] lead to altered intracellular Na(+) content, which could affect NHE activity and NHE-1 protein expression. We therefore investigated the effects of altering extracellular [K(+)] on NHE activity and NHE-1 expression in cultured VSMCs of both the spontaneously hypertensive rat (SHR) and its normotensive Wistar-Kyoto counterpart (WKY). Culture of SHR VSMCs for 48 hours in media containing 2, 4, 6, and 8 mmol. L(-1) [K(+)] led to activation of NHE-1 in the low [K(+)] media (NHE-1 activity at [K(+)] 2, 4, 6, and 8 mmol. L(-1) were 34.3 +/- 1.7, 29.5 +/- 1.1, 27.7 +/- 1.4, and 26.1 +/- 2.1 mmol. L(-1) min(-1), P <.006 by analysis of variance [ANOVA]). This was not associated with any significant changes in intracellular pH. By contrast, WKY VSMCs did not exhibit any significant activation of NHE-1 in low [K(+)] media (NHE-1 activity at [K(+)] 2, 4, 6, and 8 mmol. L(-1) were 24.3 +/- 2.9, 22.3 +/- 1.7, 19.0 +/- 1.8, and 18.6 +/- 1.6 mmol. l(-1) min(-1), P = not significant [NS] by ANOVA). Culture of SHR or WKY VSMCs in low [K(+)] media did not alter NHE-1 protein expression, suggesting the enhancement of activity in SHR cells was due to an increased turnover number of NHE-1. This response of NHE-1 in SHR VSMCs to K(+) depletion indicated a direct effect on these cells and could potentially enhance the contractile or proliferative phenotype of these cells in vivo.     

bcl-2对人小细胞肺癌细胞系H446/DDP多药耐药性的影响

目的探讨bc l-2表达上调对人小细胞肺癌(SCLC)多药耐药的影响及相关机制。方法通过定向克隆方法,构建bc l-2正义RNA真核表达载体pLXSN-bc l-2;同时,体外合成bc l-2反义硫代磷酸寡核苷酸(AS-PS-ODNs),然后采用脂质体分别将其转染至人SCLC H446细胞及H446/DDP细胞,用逆转录-聚合酶链反应(RT-PCR)和W estern b lot方法检测转染细胞中bc l-2 mRNA和蛋白的表达水平;流式细胞仪DNA倍性分析观察顺铂(DDP)诱导转染细胞凋亡情况。转染时细胞分3组,pLXSN-bc l-2转染组[或bc l-2 AS-PS-ODNs转染组(AS-PS-ODNs转染组)]、pLXSN转染组[或bc l-2无义硫代磷酸寡核苷酸转染组(NS-PS-ODNs转染组)]和对照组(H446细胞组、H446/DDP细胞组)。结果(1)W estern b lot检测结果表明,对照H446细胞组、pLXSN转染组、pLXSN-bc l-2转染组间Bc l-2蛋白表达水平差异有统计学意义(F=11 221,P<0.05)。转染pLXSN-bc l-2的H446细胞强表达Bc l-2蛋白(灰度值0.854±0.016),与对照H446细胞组(灰度值为0.103±0.005)相比差异有统计学意义(t=2.45,P<0.01);NS-PS-ODNs转染组的H446/DDP细胞,AS-PS-ODNs转染组的H446/DDP细胞,对照H446/DDP细胞组间Bc l-2蛋白表达水平差异有统计学意义(F=11 028,P<0.01)。转染bc l-2AS-PS-ODNs的H446/DDP细胞表达Bc l-2蛋白水平(灰度值为0.695±0.014),与对照H446/DDP细胞组(灰度值为0.942±0.018)相比,显著下降(t=2.26,P<0.01),但仍高于亲代H446细胞Bc l-2表达水平(t=2.31,P<0.01)。(2)流式细胞仪DNA倍性分析结果显示,转染pLXSN-bc l-2的H446细胞,经5μg/m l DDP诱导后凋亡率为(20.9±0.2)%,明显低于对照H446细胞组的(31.1±0.21)%,差异有统计学意义(t=2.45,P<0.01);5μg/m l DDP诱导转染AS-PS-ODNs的H446/DDP细胞的凋亡率为(31.5±0.4)%,对照H446/DDP细胞组的凋亡率为(9.0±0.1)%,转染细胞的凋亡率显著增加(P<0.05)。结论靶向性抑制抗凋亡相关基因bc l-2的表达可能是克服SCLC化疗耐药的重要的基因治疗方法之一。     

Upregulation of myocardial Na+/H+ exchanger induced by chronic treatment with a selective inhibitor

Rats exposed to prolonged administration of the NHE-1 inhibitor cariporide showed enhanced activity of the exchanger in cardiac tissue, as assessed by the rise in the steady-state pHi value in the absence of bicarbonate (7.15+/-0.01 in control vs 7.49+/-0.06 and 7.41+/-0.05 in cariporide-treated for 1 or 2 months, respectively, P<0.05). In the presence of bicarbonate, the change in pHi was blunted due to a compensatory activation of acid loading pHi regulatory mechanisms. The enhancement of NHE activity disappeared after 1 week of the inhibitor withdrawal. The kinetic analysis of H+ fluxes after an acid load revealed an increased net H+ efflux (JH+) at any given pHi value and an alkaline shift of the apparent "set-point" of the exchanger (from 7.11+/-0.02 to 7.38+/-0.04,P <0.05) in treated rats. In the presence of the PKC inhibitor chelerythrine, the "set-point" of the exchanger was normalized in the cariporide-treated rats while JH+ at acidic pHi values persisted elevated. Cardiac NHE-1 mRNA levels and protein expression were increased in cariporide-treated rats. In addition to the increased protein expression after the treatment, the normalization of the augmented "set-point" by chelerythrine suggests an increased turnover rate of the units through a PKC dependent pathway. These data demonstrate that long-term treatment with the NHE-1 inhibitor cariporide enhances the antiporter activity in cardiac tissue through an increase of the number and turnover of functional units. This finding deserves further experimental and clinical evaluations to consider whether it would be advisable a gradual withdrawal of prolonged NHE inhibition to avoid an enhanced response when the exchanger is stimulated.