TRPV4通道激活增加大鼠血肿瘤屏障通透性

目的研究TRPV4通道激活对脑胶质瘤大鼠血肿瘤屏障(blood tumor barrier, BTB)通透性的作用效果及机制。方法制备脑胶质瘤模型,应用伊文氏蓝(EB)渗透评估TRPV4激动剂GSK1016790A作用后血肿瘤屏障通透性的变化;RT-PCR和Westen blot法检测脑胶质瘤组织中质膜微囊结构蛋白caveolin-1mRNA和蛋白表达的变化。结果 GSK1016790A可引起脑胶质瘤大鼠血肿瘤屏障通透性的改变,灌注15min后BTB通透性开始增加,灌注45min时达高峰,而后有所下降,3h基本恢复灌注前水平。在灌注GSK1016790A 15min后,caveolin-1mRNA和蛋白表达水平开始增加,在45min时表达最多,而后有所下降,在3h基本恢复到灌注前水平。结论 TRPV4通道激活选择性开放血肿瘤屏障可能与跨细胞途径标志蛋白caveolin-1表达水平上调相关,本研究可为人脑胶质瘤的化疗提供新思路。     

颈内动脉移植骨髓间充质干细胞向C6胶质瘤的趋化迁移

目的探讨经颈内动脉移植的骨髓间充质干细胞(BMSC)向C6胶质瘤的趋化迁移能力及小剂量S缓激肽对其影响。方法密度梯度离心法分离BMSCs,体外培养、传代纯化。利用Transwell侵袭小室建立体外趋化迁移模型。立体定向方法建立大鼠C6胶质瘤模型。用BrdU标记BMSCs,经荷瘤侧颈内动脉灌注,观察其向C6胶质瘤组织的趋化能力以及在使用小剂量缓激肽后对其的影响。结果通过密度梯度离心法传至第3代获得了纯化的MSCs。体外模型中BMSCs可通过聚碳酸酯膜向下室内的C6细胞迁移。经颈内动脉灌注后BMSCs可以存活,并表现出了向脑胶质瘤趋化迁移的特性。分布区域主要位于肿瘤内部,在肿瘤与正常脑组织的交界位置亦有分布。使用小剂量缓激肽可以明显增加BMSCs通过血肿瘤屏障的数量。结论BMSCs具有通过血肿瘤屏障向C6胶质瘤趋化迁移的能力,经颈内动脉灌注是其有效的移植途径,小剂量缓激肽选择性开放血肿瘤屏障有助于BMSCs趋化迁移进入C6胶质瘤组织。     

ROCK介导缓激肽开放SD大鼠血肿瘤屏障

目的利用ROCK的特异性抑制剂Y-27632,研究ROCK是否介导缓激肽开放血肿瘤屏障。方法应用ROCK的特异性抑制剂Y-27632预处理大鼠原代脑微血管内皮细胞后,用缓激肽诱导血肿瘤屏障开放,测量跨内皮阻抗值(TEER),辣根过氧化物酶(HRP)渗漏量,分析血肿瘤屏障的通透性的改变;应用Western-blot法检测紧密连接相关蛋白ZO-1的表达;应用免疫荧光方法观察原代大鼠脑微血管内皮细胞紧密连接相关蛋白ZO-1和丝状肌动蛋白结构和分布的改变。结果 Y-27632显著抑制缓激肽诱导TEER值的降低,HRP的升高;Y-27632显著抑制ZO-1的表达;Y-27632抑制ZO-1由内皮细胞的边缘向细胞质转移,抑制丝状肌动蛋白由细胞膜边缘向细胞中央区分布,应力纤维形成明显减少。结论 ROCK介导缓激肽开放血肿瘤屏障。     

低频超声辐照联合小剂量缓激肽选择性开放大鼠血肿瘤屏障及其可能机制

目的研究应用低频超声辐照和小剂量缓激肽颈内动脉灌注非侵袭性、可逆性、靶向最大限度开放血肿瘤屏障的可行性。方法应用伊文氏兰检测血肿瘤屏障通透性,透射电镜观察吞饮小泡数量,Westernblot法检测质膜微囊结构蛋白caveolin-1和caveolin-2的蛋白表达,RT-PCR法检测caveolin-1和caveolin-2的mRNA表达。结果低频超声辐照联合应用小剂量缓激肽后伊文氏兰检测血肿瘤屏障通透性显著增加(P<0.01),透射电镜示吞饮小泡数量显著增加(P<0.01),RT-PCR法、S-P免疫免疫组织化学法、Westernblot法检测质膜微囊结构蛋白caveolin-1和caveolin-2的mRNA和蛋白表达表达显著增加(P<0.01)。结论低频超声辐照联合应用小剂量缓激肽通过增加胞吞饮转运显著增加血肿瘤屏障通透性。     

caveolin-1对低频超声辐照选择性开放血肿瘤屏障中occludin表达及分布的影响

目的探讨低频超声辐照选择性开放血肿瘤屏障过程中,caveolin-1对occludin表达及分布的影响。方法 Western blot检测occludin的表达,测量跨内皮细胞电阻值检测血肿瘤屏障通透性,双重免疫荧光染色法检测caveolin-1及occludin在脑微血管及体外血肿瘤屏障内皮细胞内的定位表达。结果低频超声辐照后occludin表达及跨内皮细胞电阻随时间变化呈先下降后回升趋势,于低频超声辐照后1.5h,二者下降最为显著(P0.01)。以低频超声辐照后1.5h为时间点,抑制caveolin-1的表达,occludin的表达增加(P0.01),跨内皮电阻增高(P0.05)。双重免疫荧光染色显示caveolin-1和occludin在正常和脑胶质瘤大鼠脑微血管共定位,在体外血肿瘤屏障内皮细胞膜上共定位;低频超声辐照后1.5h,caveolin-1在胞质中表达显著增多,并诱导occludin从胞膜转移至胞质。结论低频超声辐照选择性开放血肿瘤屏障的过程中,caveolin-1增加血肿瘤屏障通透性与下调occludin的蛋白表达并诱导其内化相关。     

ATP敏感性钾通道亚基Kir6.1、Kir6.2在MPTP诱导帕金森病小鼠纹状体和黑质内的改变

为探讨ATP敏感性钾通道(KATP)亚基Kir6.1、Kir6.2在帕金森病(PD)病理生理机制中的可能作用。本研究采用蛋白免疫印迹分析(Western blot)对1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)诱导的PD小鼠模型黑质、纹状体Kir6.1、Kir6.2在不同时间点表达变化进行检测,并与酪氨酸羟化酶(TH)的变化进行比较。结果发现:(1)与正常对照组相比,黑质、纹状体TH蛋白的表达在给药后第1d即开始下降,且呈时间依赖性下降(P<0.01);(2)黑质Kir6.1蛋白的表达在给药后第5d才开始下降(P<0.01);而纹状体Kir6.1蛋白的表达在给药后第5d才开始升高(P<0.01);(3)黑质Kir6.2蛋白的表达在给药后第5d才开始明显升高(P<0.01);而纹状体Kir6.2蛋白的表达在给药后第3d轻度升高(P<0.05),第5d又明显降低(P<0.01)。以上结果提示作为KATP通道亚基的Kir6.1、Kir6.2在MPTP的作用下,可能通过参与星形胶质细胞的活化、胆碱能突触传递的抑制以及自身代偿和修复在PD的病理生理过程中发挥了重要的角色。     

缓激肽对脑胶质瘤大鼠紧密连接影响的形态学观察

目的研究缓激肽(BK)对脑胶质瘤大鼠血肿瘤屏障紧密连接的影响。方法采用伊文氏兰(EB)法检测缓激肽作用后血肿瘤屏障(BTB)通透性的变化;应用透射电镜(TEM)观察BK作用后内皮细胞间紧密连接的变化,同时应用硝酸镧[La(NO3)3]和辣根过氧化物酶(HRP)作示踪剂,检测缓激肽作用后,小分子和大分子示踪剂通过紧密连接的情况。结果缓激肽可使血肿瘤屏障对伊文氏兰的通透性增加,在15min时达到高峰,以后逐渐下降。透射电镜显示缓激肽作用15min时,肿瘤组织毛细血管内皮细胞间紧密连接的完整性明显破坏,缝隙指数显著增加,同时可见硝酸镧和辣根过氧化物酶在紧密连接处沉积。结论缓激肽能够通过开放紧密连接选择性增加血肿瘤屏障的通透性。     

大鼠脑微血管内皮细胞的原代培养及缓激肽的作用靶细胞探讨

目的建立稳定的原代大鼠脑微血管内皮细胞培养方法,并在体外探讨不同剂量缓激肽的作用靶细胞,进一步阐释缓激肽开放血脑和血肿瘤屏障的机理。方法运用免疫荧光测定原代培养的脑血管内皮细胞、星形胶质细胞及C6胶质瘤细胞在不同剂量缓激肽作用前后的细胞内钙离子变化,根据给药前后的荧光改变来确定不同剂量缓激肽的作用靶点细胞。结果小剂量缓激肽(终浓度:1μmol/L)可以引起C6胶质瘤细胞内的钙离子水平升高,而只有大剂量(终浓度:10μmol/L~1mmol/L)缓激肽才能触发星形胶质细胞内的钙离子水平升高,脑微血管内皮细胞对大、小剂量缓激肽均无任何反应。结论缓激肽的直接作用靶点是胶质细胞及C6胶质瘤细胞,缓激肽调节脑血管内皮细胞通透性的作用可能需要某些细胞间信使的参与。     

Y-27632抑制缓激肽选择开放血肿瘤屏障的研究

目的研究Rho associated kinase（ROcK）特异性抑制剂Y-27632是否抑制缓激肽开放血肿瘤屏障。方法应用EVOM测定仪测定跨内皮阻抗值，分析血肿瘤屏障的通透性；应用辣根过氧化物酶渗漏实验分析血肿瘤屏障的通透性；应用免疫荧光方法观察原代大鼠脑微血管内皮细胞丝状肌动蛋白结构和分布的改变。结果BK作用15min时，跨内皮阻抗值最低，辣根过氧化物酶流量最高，血肿瘤屏障通透性最高；此时大鼠脑微血管内皮细胞边界的丝状肌动蛋白分布不连续，应力纤维形成增加。ROCK的特异性抑制剂Y-27632显著抑制了由缓激肽引起的血肿瘤屏障通透性升高和应力纤维的增加。结论Y-27632抑制缓激肽引起的血肿瘤屏障通透性升高，可能与丝状肌动蛋白结构和分布的改变和应力纤维的增加相关。     

K_(ATP)通道Kir6.2亚基点突变体Kir6.2AAA在大鼠心肌细胞中的表达

目的制备含K ATP通道亚基点突变的重组腺病毒,并将其在大鼠心肌细胞中表达。方法针对Kir6.2位点的引物,利用Overlap PCR的方法定点突变Kir6.2的GFG氨基酸变成AAA,并将其克隆到pShuttle载体中进行序列分析,经PmeⅠ线性化、连接到腺病毒表达载体pAdEasy-1中,将pAdEasy-1包装进脂质体、转染入大鼠原代心肌细胞,并利用反转录PCR和Western blot法进行检测。结果成功制备了携带大鼠Kir6.2AAA及EGFP基因的重组腺病毒,病毒的滴度为2.64×1011VP/mL。荧光显微镜下可见Kir6.2AAA重组体腺病毒感染后的大鼠心肌细胞表达EGFP而发出绿色荧光,反转录PCR证实重组腺病毒载体Ad-Kir6.2AAA感染的心肌细胞中Kir6.2AAA的表达显著上调,Western blot法证明Kir6.2AAA在大鼠心肌细胞中过表达。结论成功构建了携带EGFP基因的Kir6.2AAA重组腺病毒载体并将其在大鼠心肌细胞中正确表达。     

The properties of the Kir6.1–6.2 tandem channel co-expressed with SUR2A

Functional ATP-sensitive K (KATP) channels have an octameric subunit structure with four pore-forming subunits (Kir6.x) and four sulfonylurea receptors (SURx). In the present study, the properties of the heteromeric KATP channel whose pore subunits are composed of Kir6.1 and Kir6.2 were examined using a heterologous expression system. In COS7 cells co-transfected with Kir6.1, Kir6.2 and SUR2A at a ratio of 1:1:2, KATP channels showed various unitary conductances between those of Kir6.1/SUR2A (33.6+/-4.2 pS) and Kir6.2/ SUR2A (67.1+/-1.6 pS). Kir6.1-6.2 tandem protein, constructed by fusing the C-terminus of Kir6.1 to the N-terminus of Kir6.2 with a ten glutamine linker sequence, also formed a channel with an intermediate conductance (58.9+/-1.5 pS). Kir6.2 and Kir6.1-6.2 showed similar sensitivity to ATP4-: half-maximal inhibition (IC50) was obtained at 14.1+/-12.8 microM and 17.6+/-9.6 microM, respectively. In the presence of Mg2+, Kir6. 1-6.2 was significantly less sensitive than Kir6.2 to MgATP (IC50=95.5+/-49.6 microM versus 18.9+/-5.0 microM). These results suggest that Kir6.1 and Kir6.2 are endowed with the potential to form a heteromeric KATP channel, which has a low sensitivity to MgATP.     

Characterisation of sulphonylurea and ATP-regulated K+ channels in rat pancreatic A-cells

We have monitored whole-cell and single channel ATP-sensitive K+ (KATP) currents in isolated rat glucagon-secreting pancreatic A-cells. Tolbutamide produced a concentration-dependent decrease in the whole-cell KATP conductance (Ki = 6 microM) and initiated action potential firing. The K+ channel opener diazoxide, but not cromakalim or pinacidil, inhibited electrical activity and increased the whole-cell K+ conductance fourfold. ATP applied to the intracellular face of the membrane inhibited KATP channel activity with a Ki of 17 microM, an effect that could be counteracted by Mg-ADP and Mg-GDP. GTP and UTP did not affect KATP channel activity. Phosphatidylinositol 4,5-bisphosphate activated KATP channels inhibited by ATP after a delay of 90 s. In situ hybridisation demonstrated the expression of the mRNA encoding KATP channel subunits Kir6.2 and SUR1 but not Kir6.1 and SUR2. We conclude that rat pancreatic A-cells express KATP channels with the nucleotide-, sulphonylurea- and K+ channel-opener sensitivities expected for a channel formed by Kir6.2 and SUR1 subunits.     

ATP-sensitive potassium channels: structures, functions, and pathophysiology

ATP-sensitive potassium channels (KATP channels) play important roles in various tissues by coupling cell metabolic status to electrical activity. Recently, molecular biological and electrophysiological techniques have revealed the molecular basis of the KATP channels to be a complex of the Kir6.0 subunit, a member of the inwardly rectifying K+ channel subfamily Kir6.0, and the sulfonylurea receptor (SUR) subunit, a member of ATP-binding cassette (ABC) superfamily; the functional diversity of the various KATP channels is being determined by a combination of the Kir6.0 subunit (Kir6.1 or Kir6.2) and the SUR subunit (SUR1 or SUR2) comprising it. Recent studies of the KATP channels have suggested mechanisms of KATP channel regulation and pathophysiology and also a new model in which ABC proteins regulate the functional expression of ion channels.     

Enhanced neuronal damage after ischemic insults in mice lacking Kir6.2-containing ATP-sensitive K+ channels

Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels, incorporating Kir6.x and sulfonylurea receptor subunits, are weak inward rectifiers that are thought to play a role in neuronal protection from ischemic insults. However, the involvement of Kir6.2-containing KATP channel in hippocampus and neocortex has not been tested directly. To delineate the physiological roles of Kir6.2 channels in the CNS, we used knockout (KO) mice that do not express Kir6.2. Immunocytochemical staining demonstrated that Kir6.2 protein was expressed robustly in hippocampal neurons of the wild-type (WT) mice and absent in the KO. To examine neuronal sensitivity to metabolic stress in vitro, and to ischemia in vivo, we 1) exposed hippocampal slices to transient oxygen and glucose deprivation (OGD) and 2) produced focal cerebral ischemia by middle cerebral artery occlusion (MCAO). Both slice and whole animal studies showed that neurons from the KO mice were severely damaged after anoxia or ischemia, whereas few injured neurons were observed in the WT, suggesting that Kir6.2 channels are necessary to protect neurons from ischemic insults. Membrane potential recordings from the WT CA1 pyramidal neurons showed a biphasic response to OGD; a brief hyperpolarization was followed by a small depolarization during OGD, with complete recovery within 30 min after returning to normoxic conditions. By contrast, CA1 pyramidal neurons from the KO mice were irreversibly depolarized by OGD exposure, without any preceding hyperpolarization. These data suggest that expression of Kir6.2 channels prevents prolonged depolarization of neurons resulting from acute hypoxic or ischemic insults, and thus protects these central neurons from the injury.     

mitoK_(ATP)通道经FOXO1-PGC1α通路调节后负荷过载小鼠心肌线粒体的代谢功能

目的:通过ATP依赖的钾离子通道(KATP)亚基-Kir6.2基因敲除小鼠(Kir6.2KO)模型,研究线粒体ATP敏感钾离子通道mitoKATP对心肌线粒体和代谢酶的调控机制。方法:分别将野生型小鼠(WT平行对照组)和Kir6.2KO小鼠(实验组)分为假手术、主动脉横断缩窄(TAC)2周和4周各3个亚组。检测并比较各组心功能、心肌能量代谢酶基因表达水平、信号转导通路中叉头框O1(FOXO1)和转录因子PGC1α水平、线粒体比面积和嵴间距。结果:与平行WT组相比较,TAC前Kir6.2KO小鼠心肌PGC1α表达水平有所降低、FOXO1略提高,能量代谢酶中链乙酰辅酶A脱氢酶(MCAD)、肉碱软脂酰基转移酶1(CPT1)和细胞色素C氧化酶亚单位III(COXIII)明显负表达,线粒体比面积和线粒体嵴间距有所增加(8.45%和3.11%),表现为有氧代谢能力降低,线粒体代偿增生。TAC后2周时,Kir6.2KO组的心肌线粒体比面积没有变化(8.75%vs0.14%),而嵴间距增加幅度低于WT组(18.27%vs11.65%),线粒体失代偿。TAC后4周时,Kir6.2KO组FOXO1和PGC1α的蛋白或mRNA水平均显著降低,下游能量代谢酶mRNA和蛋白显著负调表达,心肌线粒体比面积降低幅度更大(-8.45%vs-23.6%),嵴间距变化与WT组相同(6.60%vs7.17%),心功能障碍更为明显,有氧代谢功能衰竭。结论:阻断mitoKATP降低了心肌线粒体对负荷增加时的增生和正调能量代谢酶的反应能力,这与FOXO1-PGC1α信号通路的弱化有关。说明mitoKATP通过FOXO1-PGC1α信号通路调节负荷过载小鼠心肌线粒体增殖和能量代谢功能。     

Morphological changes in pancreatic islets of KATP channel-deficient mice: the involvement of KATP channels in the survival of insulin cells and the maintenance of islet architecture

The ATP-sensitive potassium channel (KATP channel) is an essential ion channel involved in glucose-induced insulin secretion. The KATP channel is composed of an inwardly rectifying potassium channel, Kir6.2, and the sulfonylurea receptor (SUR 1); in the pancreas it is reported to be shared by all endocrine cell types. A previous study by our research group showed that Kir 6.2-knockout mice lacked KATP channel activities and failed to secrete insulin in response to glucose, but displayed normal blood glucose levels and only mild impairment in glucose tolerance at younger ages. In some aged knockout mice, however, obesity and hyperglycemia were recognizable. The present study aimed to reveal morphological changes in pancreatic islets of Kir 6.2-knockout mice throughout life. At birth, there were no significant differences in the islet cell arrangement between the knockout mice and controls. At 14 postnatal weeks glucagon cells appeared in the central parts of islets, and this image became more pronounced with aging. In animals older than 50 weeks insulin cells decreased in numbers and intensity of insulin immunoreactivity; most islets in 70- and 80-week-old mice were predominantly composed of glucagon cells and peptide YY (PYY)-containing cells. Staining of serial sections and double staining of single sections from these old mice demonstrated the frequent coexpression of glucagon and PYY, which is a phenotype for the earliest progenitor cells of pancreatic endocrine cells. These findings suggest that the KATP channel is important for insulin cell survival and also regulates the differentiation of islet cells.     

Identification of two novel frameshift mutations in the KCNJ11 gene in two Italian patients affected by Congenital Hyperinsulinism of Infancy

Congenital Hyperinsulinism of Infancy (CHI) is a genetically heterogeneous disorder characterized by profound hypoglycemia related to inappropriate insulin secretion. Two histopathologically and genetically distinct groups are recognized among patients with CHI due to ATP-sensitive potassium channel (KATP) defects: a diffuse type (Di-CHI), which involves the whole pancreas, and a focal form (Fo-CHI), which shows adenomatous islet-cell hyperplasia of a particular area within the normal pancreas. The beta-cell KATP channel consists of two essential subunits: Kir6.2 encoded by the KCNJ11 gene which is the pore-forming unit and belongs to the inwardly rectifying potassium channel family, and SUR1 (sulfonylurea receptor 1) encoded by the ABCC8 gene, which belongs to the ATP-binding cassette (ABC) transporter family. The KATP channel is an octameric complex of four Kir6.2 and four SUR1 subunits. More than one hundred mutations have been found in KATP channel genes ABCC8 and KCNJ11, but to date only twenty mutations have been identified in KCNJ11, most of them are missense mutations and only one is a single base deletion. The Fo-CHI has been demonstrated to arise in individuals who have a germline mutation in the paternal allele of ABCC8 or KCNJ11 in addition to a somatic loss of the maternally derived chromosome region 11p15 in adenomatous pancreatic beta-cells, while Di-CHI predominantly arises from the autosomal recessive inheritance of KATP channel gene mutations. Here we describe the molecular findings in nine children who presented, in the neonatal period, with signs and symptoms of hypoglycemia and diagnosed affected by CHI according to international diagnostic criteria. Direct sequencing of the complete coding exon and promoter region of KCNJ11 gene showed, in two Italian patients, two new heterozygous mutations which result in the appearance of premature translation termination codons resulting in the premature end of Kir6.2. Interestingly most of the CHI mutations detected in other population studies are situated in the ABCC8 gene.     

ATP敏感性钾通道突变与新生儿糖尿病iDEND综合征

ATP敏感性钾通道(ATP-sensitive K+chan-nels,KATP)由SUR1和Kir6.2亚基组成,是葡萄糖刺激胰岛β细胞分泌胰岛素的关键部位。新生儿糖尿病iDEND综合征(intermediate developmental delay,epilepsy,and neonatal diabetes syndrome)是由KATP通