组织激肽释放酶家族、激肽释放酶—激肽原—激肽系统及其重要应用

目前已明确，人组织激肽释放酶基因家族至少由15个基因组成，都定位于19q13．3—13．4，在基因结构、蛋白水平及三级结构上都有明显同源性。本总结了组织激肽释放酶基因家族、激肽释放酶—激肽原—激肽系统的研究近况，并介绍了其在心血管疾病和肿瘤方面的重要应用。     

组织激肽释放酶家族、激肽释放酶-激肽原-激肽系统及其重要应用

目前已明确,人组织激肽释放酶基因家族至少由15个基因组成,都定位于19q13.3～13.4,在基因结构、蛋白水平及三级结构上都有明显同源性。本文总结了组织激肽释放酶基因家族、激肽释放酶-激肽原-激肽系统的研究近况,并介绍了其在心血管疾病和肿瘤方面的重要应用。     

人组织激肽释放酶

人组织激肽释放酶是一个多基因家族 ,由 15个具有高度同源性的等位基因组成。组织激肽释放酶可在许多组织中表达。在肿瘤细胞中 ,多种组织激肽释放酶受类固醇激素的调节。人类多种疾病中均有组织激肽释放酶表达的改变     

人类组织激肽释放酶家族研究进展

人类组织激肽释放酶家族由 15个成员组成 ,它们位于同一染色体 (19q13.4 )上 ,在核苷酸及蛋白水平上具有很大的同源性 .研究发现它们与多种疾病的发病有关 ,尤其是前列腺癌等恶性肿瘤。     

人组织激肽释放酶单克隆抗体的制备及其ELISA检测试剂盒的建立

目的 制备人组织激肽释放酶(human tissue kallikrein,HK)单克隆抗体(McAb),并研制检测人尿中HK含量的ELISA试剂盒.方法 以一段HK特异多肽和血蓝蛋白(KLH)的耦联物作为抗原免疫BALB/c小鼠,并采用杂交瘤技术制备抗HK的单克隆抗体.然后建立快速检测HK含量的间接竞争ELISA试剂盒.结果 获得了8株可分泌抗HK的杂交瘤细胞株,其产生的抗体效价为1:25 600;经1:1600倍稀释后的竞争抑制率为0.88;经纯化后的纯度为100%,其标准曲线对线良好(r=0.990).结论 成功制备了高效价、高特异性和高纯度的抗HK的单克隆抗体.同时建立了一种可以快速检测人尿中HK含量的ELISA试剂盒.

Abstract：

Objective To prepare monoclonal antibody(McAb) against human tissue kallikrein (HK) and develop an ELISA kit allows for the in vitro quantitative determination of human tissue kallikrein in urine. Methods To generate a monoclonal antibody specific for TK, the synthetic TK peptide consisting of 12 amine acids(12P), was fused to keyhole limpet hemocyanin(KLH) and used for immunization. Using hybridoma screening, monoclonal secreting cell lines were identified and used to generate ascites in BALB/c mouse. Antibody was purified by affinity column chromatography. 12% SDS-PAGE and Western blot were used to visualize the purified antibody. This kit employs indirect competitive ELISA technique and BiotinAvidin System. 12P was fused to bovine serum albumin(BSA) and has been pre-coated onto a microplate at first. Standards and samples were added to the appropriate microplate wells with a biotin-conjugated McAb croplate well. A TMB substrate solution is added to each well. The enzyme-substrate reaction is terminating by the addition of a sulphuric acid solution and the color change is measured spectrotometrically at a wavelength of 450 nm. The concentration of tissue kallikrein in the samples is then determined by comparing the O.D. of the samples to the standard curve. Results 8 hybridoma cell lines secreting mAbs special to HK,SDS-PAGE and Western blot demonstrated successful preparing and purification of McAb( 100% ). The linearity of this ELISA kit is demonstrated(r =0. 990). The range of detection of the assay is 0.008 μg/ml to 0. 5 μg/ml. The assay remained stable, with no change in the values measured, over five cycles of freezing and thawing. Conclusion 8 McAbs against HK have been prepared successfully and possess high titer and specificity. The development of an ELISA kit for detecting HK can meet the needs of detection of HK in urine samples.     

组织激肽释放酶与缺血性脑卒中患者预后的相关性研究

目的:探讨组织激肽释放酶(TK)与缺血性脑卒中(CIS)患者短期预后的关系.方法:采用酶联免疫吸附法检测280例CIS患者(试验组)和50例体检健康者(对照组)血浆TK,并对CIS患者进行神经功能缺损程度评分和牛津残障评分,探讨TK与其相关性.结果:①CIS患者急性期和恢复期血浆TK分别为(0.171±0.031)mg/L和(0.193±0.036)mg/L,均低于对照组(0.251±0.043)mg/L,差异有统计学意义(P<0.05);②CIS患者急性期神经功能缺损评分(22.86±3.21)显著高于恢复期(13.12±1.45),差异有统计学意义(P<0.05);③CIS患者急性期和恢复期血浆TK水平与神经功能缺损评分均显著负相关(r分别为-0.582和-0.473,均P<0.05);④预后良好组急性期和恢复期血浆TK水平分别为(0.176±0.036)、(0.198±0.040)mg/L,均相应高于对照组急性期(0.159±0.023)和恢复期(0.184±0.027)mg/L,差异均有统计学意义(P<0.05).结论:TK水平下降预示CIS患者预后不良.     

检测组织激肽释放酶基因寡核苷酸多态性方法的建立

目的：检测组织激肽释放酶启动子区寡核苷酸多态性，为开展基因诊断高血压研究奠定基础。方法：人外周静脉抗凝血提取基因组DNA，PCR扩增目标启动子片段，ASO(allele-specific oligonucleotide)斑点杂交检测基因型，并与测序法比较了解启动于等位基因多态性。结果：有4例杂交结果阳性，与测序结果吻合。结论：应用ASO斑点杂交技术检测该寡核苷酸多态性与测序法比较具有简便，快速，价廉，特异性高等优点，适于大规模人群筛查，是一种值得推广的研究方法。     

人组织激肽释放酶6的原核表达载体的构建及表达

目的：构建人组织激肽释放酶6原核表达系统。方法：采用RT-PCR技术从人乳腺癌组织中扩增出KLK6基因片段,克隆至原核表达载体pET28b中,经酶切和序列测定后,转化至E.coli BL21,IPTG进行诱导表达,表达产物经SDS-PAGE检测,Ni-NTA亲和层析柱纯化,其纯化产物进行SDS-PAGE和Western-blot检测。结果：pET28b-KLK6原核表达载体构建成功,经IPTG诱导高效表达hK6蛋白,纯化后获得大量高纯度蛋白。结论：成功构建了KLK6的原核表达系统并在E.coli BL21中高效表达,获得高纯度的hK6融合蛋白。     

组织型激肽释放酶基因多态性与长沙地区汉族人群脑出血的关联研究

目的 分析长沙地区汉族人群脑出血与组织型激肽释放酶(kallikrein 1,KLK1)基因多态性的关系.方法 收集273例散发性脑出血患者和140名正常对照者的外周血标本.采用多重单碱基延伸单核苷酸多态分型技术和DNA测序法检测KLK1基因rs5516及rs5517多态性位点在两组人群中的分布.结果 (1)脑出血组及对照组KLK1基因rs5516多态和等位基因频率分布差异无统计学意义(P>0.05);脑出血组组织型KLK1基因rs5517多态A等位基因频率显著高于对照组(P<0.05).(2)对照组rs5517多态AA及GA基因型携带者舒张压水平显著高于GG基因型携带者(P<0.05);而rs5516位点各基因型亚组间血压水平差异无统计学意义(P>0.05).结论 组织型激肽释放酶基因rs5516多态性与脑出血无关,而组织型激肽释放酶基因rs5517多态性与脑出血存在关联,可能通过影响血压水平而参与脑出血的发生发展.     

酸敏感离子通道对海马神经元树突发育的影响

目的 探讨酸敏感离子通道（ASICs）在海马神经元树突发育中的作用。方法 在体外培养第5天的原代海马神经元中转染定位于膜上的绿色荧光蛋白（F-GFP）,随后在神经元培养液中加入ASICs拮抗剂Amiloride和ASIC1a 选择性拮抗剂Psalmotoxin 1（PcTX1）抑制ASICs的功能,观察体外培养8d和14d这两个时间点海马神经元的树突生长、分支复杂程度。结果Amiloride（10-5mol/L）和PcTX1（1∶20 000稀释）处理3d对海马神经元树突分支总长度、树突分支总数均无显著影响,表明在海马神经元发育早期短时间抑制ASICs功能不影响树突发育。Amiloride（10-5mol/L）处理9d可以显著降低树突分支总长度和树突分支总数; PcTX1（1∶20 000稀释）处理9d也可以显著降低树突分支总长度,但对树突分支总数无显著影响。实验结果表明,长时间抑制ASICs功能会影响树突发育。结论 ASICs参与调节树突的发育。     

大鼠破骨细胞的诱导及其酸敏感离子通道的表达

目的:检测酸敏感离子通道(ASICs)在大鼠破骨细胞中的表达情况,探讨其在破骨细胞中的作用.方法:采用1,25-(OH)2D3诱导大鼠骨髓单核细胞分化的破骨细胞.RT-PCR扩增ASICs基因;免疫印迹检测ASICs转录水平;免疫荧光细胞化学方法观察ASICs基因在破骨细胞中的表达和分布.结果:诱导5d后可见抗酒石酸盐酸性磷酸酶(TRAP)阳性多核细胞出现,大鼠破骨细胞在mRNA和蛋白水平均可检测到ASIC1、 ASIC2和ASIC3基因的表达产物.免疫荧光细胞化学显示ASIC1和ASIC2蛋白在细胞膜中有较强的表达,而ASIC3主要定位于细胞质中,少量定位于细胞膜上.结论:大鼠骨髓单核细胞可诱导分化成破骨细胞,能表达ASICs.     

Regulation of acid-sensing ion channel 1a function by tissue kallikrein may be through channel cleavage

Recently, we have demonstrated that serine protease tissue kallikrein (TK) can protect cortical neurons against ischemia-acidosis/reperfusion-induced injury, and that this effect might be mediated by acid-sensing ion channels (ASICs). However, little is known about how TK regulates the function of ASICs. Here we provided evidence that the regulation of ASIC1a function by TK was probably correlated with its cleavage. High concentration of TK (3 μM) partially cleaved the extracellular loop of ASIC1a, followed by a marked decrease of LDH release and an increase of cell survival at pH 6.2. Pretreatment with a protease inhibitor aprotinin inhibited the cleavage of ASIC1a and prevented functional regulation by TK. However, the cleavage of ASIC2a, which was not functionally modified by TK, was not observed. Therefore, we propose that the limited proteolysis of extracellular loop within ASIC1a might be one of the potential regulatory mechanisms of ASIC1a function by TK.     

Characterization of acid-sensing ion channels in medium spiny neurons of mouse striatum

Acid-sensing ion channels (ASICs) regulate synaptic activities and play important roles in neurodegenerative diseases. They are highly expressed in the striatum, where medium spiny neurons (MSNs) are a major population. Given that the properties of ASICs in MSNs are unknown, in this study, we characterized ASICs in MSNs of the mouse striatum. A rapid drop in extracellular pH induced transient inward currents in all MSNs. The pH value for half-maximal activation was 6.25, close to that obtained in homomeric ASIC1a channels. Based on psalmotoxin 1 and zinc sensitivity, ASIC1a (70.5% of neurons) and heteromeric ASIC1a-2 channels (29.5% of neurons) appeared responsible for the acid-induced currents in MSNs. ASIC currents were diminished in MSNs from ASIC1, but not ASIC2, null mice. Furthermore, a drop in pH induced calcium influx by activating homomeric ASIC1a channels. Activation of ASICs increased the membrane excitability of MSNs and lowering extracellular Ca²⁺ potentiated ASIC currents. Our data suggest that the homomeric ASIC1a channel represents a majority of the ASIC isoform in MSNs. The potential function of ASICs in the striatum requires further investigation.     

Two types of acid-sensing ion channel currents in rat hippocampal neurons

Two types of acid-sensing ion channel (ASIC)-like currents in cultured rat hippocampal neurons were recorded and their characteristics were studied by using a whole-cell recording technique. The results revealed that the ASIC-like currents, induced by a quick drop of the extracellular pH, decayed with different time constants (τ) of 229 ± 16 (Type I) and 1209 ± 56 ms (Type II). The ASIC-like currents displayed different sensitivities to extracellular proton (pH_0.5 was 6.17 ± 0.04 for Type I and 5.70 ± 0.07 for Type II) and amiloride, a specific ASIC channel blocker (IC₅₀ was 1.19 ± 0.37 μM for Type I and 0.14 ± 0.02 μM for Type II). Among all the 360 recorded neurons, ASIC-like currents were induced in 314 neurons (87.2%). In the neurons expressing ASICs, Type I currents were evoked from 269 neurons (85.7%) and Type II currents were induced only from 45 neurons (14.3%). As these ASIC-like currents presented various electrophysiological and pharmacological properties, further experiments should be conducted to decipher the complex subunit composition of ASICs in the hippocampus.     

Inhibitory regulation of acid-sensing ion channel 3 by zinc

Acid-sensing ion channel 3 (ASIC3) is a proton-gated, voltage-insensitive Na⁺ channel that is expressed primarily in peripheral sensory neurons and plays an important role in pain perception, particularly as a pH sensor following cardiac ischemia. We previously reported that ASIC3 currents are not affected by zinc at nanomolar concentrations. In this study, we examined the potential role of micromolar zinc in the regulation of ASIC3. In CHO cells expressing ASIC3, we found that ASIC3 currents triggered by dropping the pH from 7.4 to 6.0 were inhibited by pretreatment with zinc in a concentration-dependent manner; the half-maximum inhibitory concentration of zinc was 61 μM. ASIC currents activated by a relatively small drop in pH from 7.4 to 7.2 or 7.0 were also subject to inhibition by zinc. The inhibition was fast and pH independent, and occurred within a relatively narrow range of zinc concentrations between 30 and 300 μM. Further, increasing extracellular Ca²⁺ concentrations from 2 to 10 mM failed to affect inhibition of ASIC3 currents by zinc. Experimentally elevating intracellular zinc levels did not affect the inhibition of ASIC3 currents by equal concentrations of extracellular zinc, and modification of cysteine or histidine residues had no effect on the inhibition of ASIC3 currents by zinc. These collective results suggest that zinc is an important regulator of ASIC3 at physiological concentrations, that zinc inhibits ASIC3 in a pH- and Ca²⁺-independent manner, and that inhibition of ASIC3 currents is dependent upon the interaction of zinc with extracellular domain(s) of ASIC3.     

Modulation of acid-sensing ion channels by Cu(2+) in cultured hypothalamic neurons of the rat

Acid-sensing ion channels (ASICs) are known to distribute throughout the nervous system and serve important roles in various physiological and pathological processes. However, the properties of ASICs in the hypothalamus, an important region of diencephalon, are little known. We herein used whole-cell patch-clamp recordings to characterize proton-induced cation currents in cultured hypothalamic neurons of the rat, and attributed these transient inward currents to ASICs based on their electrophysiological and pharmacological properties. We further examined the effects of Cu(2+), the third most abundant trace element, on ASICs in hypothalamic neurons. Our results showed that this divalent cation reversibly and concentration-dependently inhibited the amplitude of ASIC currents, and slowed down the desensitization of ASIC channels. Our results also displayed that Cu(2+) modulated ASICs independent of change in membrane potential and extracellular protons, suggesting a noncompetitive mechanism. Furthermore, micromolar concentration of Cu(2+) attenuated the acid-induced membrane depolarization. Taken together, our data demonstrate a modulatory effect of Cu(2+) on ASICs in native hypothalamic neurons and suggest a role of this endogenous metal ion in negatively modulating the increased neuronal membrane excitability caused by activation of ASICs.     

酸敏感离子通道基因重组质粒的构建、表达及其活性测定

为了构建含有小鼠酸敏感离子通道(ASIC1a、ASIC2a)基因全长cDNA的重组质粒,并表达有生物学活性的ASIC1a和ASIC2a融合蛋白,本研究通过逆转录聚合酶链反应(RT-PCR)分别获得小鼠ASIC1a和ASIC2a全长cDNA,并将其克隆入真核表达载体pEGFP-N3中,构建含小鼠全长ASIC1a和ASIC2a基因的重组质粒pEGFP-ASIC1a和pEGFP-ASIC2a,经DNA测序鉴定序列正确。脂质体法分别将其转染至中国仓鼠卵巢细胞(CHO),荧光显微镜下观察小鼠ASIC1a和ASIC2a融合蛋白在细胞内的表达分布,Westernblot检测其蛋白表达。酸性处理转染重组质粒细胞,并比较其细胞存活率及乳酸脱氢酶(LDH)的释放来评价融合蛋白的生物学活性。结果显示:本研究成功地分别将小鼠ASIC1a和ASIC2a全长cDNA克隆入pEGFP-N3载体中,并将其转染至CHO细胞,荧光显微镜下观察到CHO细胞膜周围呈强绿色荧光条带,Westernblot显示小鼠ASIC1a和ASIC2a融合蛋白分别在90kD和88kD处有表达。经酸性处理的转染ASIC1a和ASIC2a重组质粒的CHO细胞,分别较其在pH7.4条件下的细胞存活率明显降低,LDH释放量明显增高,且通道阻断剂可抑制该效应。上述结果提示,我们已成功构建出含小鼠ASIC1a和ASIC2a全长cDNA的重组质粒pEGFP-ASIC1a和pEGFP-ASIC2a,并使有生物学活性的ASIC1a和ASIC2a融合蛋白在CHO细胞中表达。     

酸敏感离子通道在中枢化学感受器呼吸调节中的作用

 目的：探讨脑室内注射酸化人工脑脊液（artificial cerebrospinal fluid, aCSF）引起的呼吸效应及酸敏感离子通道（acid sensing ion channels, ASICs）在此过程中的作用。方法：健康成年SD大鼠30只,随机分为aCSF（pH 7.4）对照组、aCSF(pH 6.5)组、ASICs阻断剂阿米洛利（amiloride）对照组、amiloride+aCSF（pH 6.5）组、ASIC1a阻断剂psalmotoxin 1 (PcTx1)对照组及PcTx1+aCSF(pH 6.5)组。通过膈肌肌电记录脑室内注射酸化aCSF后呼吸的变化;通过脑室内先注射阿米洛利和PcTx1再注射酸化aCSF的方法,观察酸敏感离子通道在中枢化学感受器呼吸调节中的作用。结果：脑室内注射酸化aCSF后,呼吸较注射前明显兴奋（P<0.05）;脑室内注射阿米洛利能完全阻断脑室内注射酸化aCSF引起的呼吸兴奋;脑室内注射PcTx1能部分阻断脑室内注射酸化aCSF引起的呼吸兴奋。结论：ASICs是参与中枢化学感受器呼吸调节的关键离子通道,ASIC1a则发挥了部分作用。