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1.
破骨细胞是体内唯一的骨吸收细胞,对钙离子跨细胞膜转运及维持正常血钙水平有非常重要的作用。瞬时性受体电位通道香草酸受体6(transient receptor potential vanilloid receptor 6,TRPV6)是TRP家族中的一员,对钙离子有高度选择性,且是钙离子跨细胞转运的限速门控通道。本试验旨在研究TRPV6过表达对破骨细胞钙转运相关基因及细胞凋亡相关基因表达的影响。用TRPV6过表达慢病毒载体(LV-TRPV6)转染培养7 d的破骨细胞构建TRPV6过表达破骨细胞模型,以阐释小鼠破骨细胞中TRPV6与钙离子转运和细胞凋亡的关系。将破骨细胞分为3组:空白对照组、阴性对照组和LV-TRPV6组。结果显示:与空白对照组比较,破骨细胞TRPV6过表达后钙离子转运相关基因(calbindin-D28K,NCX1) mRNA和蛋白表达水平显著增加(P0.05);破骨细胞TRPV6过表达后细胞凋亡相关基因mRNA和蛋白表达水平显著降低(Fas和Fas L除外)(P0.05)。流式细胞仪分析显示,LV-TRPV6组凋亡率显著下降6.33%±0.235%(P0.05)。研究表明,破骨细胞过表达TRPV6基因导致钙转运相关基因表达增加并抑制破骨细胞凋亡。 相似文献
2.
将40羽220日龄ISA蛋鸡分为5组,于产蛋后0、2、4.5、8、16h断头处死,采集蛋壳腺组织,运用Real-time PCR和Western-blot方法检测产蛋循环过程中瞬时性受体单位香草精受体6（Transient receptor potential vanilloid receptor 6,TRPV6）、钙结合蛋白（calbindin D28K,CaBP-D28K）、质膜钙离子ATP酶1b（plasma membrane Ca2＋-ATPase 1b,PMCA 1b）mRNA和蛋白浓度的动态变化。结果显示,卵子未进入蛋壳腺前（产蛋后0~4.5h）,蛋壳腺内TRPV6、CaBP-D28K和PMCA 1bmRNA表达水平较低,随后表达量逐渐升高,在蛋壳钙化过程中达到最大值（产蛋后16h）,与0h相比,TRPV6和CaBP-D28KmRNA表达差异均极显著（P〈0.01）;另外,产蛋循环过程中,TRPV6、CaBP-D28K和PMCA 1b蛋白浓度变化与mRNA变化一致,产蛋后16h到达最大值,其中CaBP-D28K蛋白浓度与0h相比,差异显著（P〈0.05）。结果表明,产蛋循环可调控蛋壳腺内TRPV6、CaBP-D28K和PMCA 1b的表达,并提示钙离子跨细胞转运途径在钙离子进入蛋壳腺形成蛋壳过程中发挥重要作用。 相似文献
3.
本试验旨在研究瞬时性受体电位通道香草酸受体6(transient receptor potential vanilloid receptor 6,TRPV6)基因沉默对蛋鸡十二指肠和空肠跨细胞钙离子转运相关蛋白表达、血浆钙磷浓度、PTH水平及骨密度的影响。将64只220日龄高产蛋鸡随机分为对照组和TRPV6基因沉默组,分别一次性注射生理盐水和pSIRENTRPV6-3质粒,连续观察28d。结果表明,与对照组相比,pSIREN-TRPV6-3质粒注射第7、14和21天,十二指肠和空肠中均检测到ZsGreen蛋白表达;TRPV6、CaBP-D28K mRNA和蛋白表达水平均显著降低(P0.05或P0.01);血浆PTH浓度显著升高(P0.05或P0.01);此外,血浆钙、磷浓度不受影响,股骨和胫骨骨密度有降低趋势,但与对照组相比差异不显著(P0.05)。由此可见,siRNA TRPV6质粒抑制十二指肠和空肠TRPV6和CaBP-D28K的表达,但不影响血液中钙离子浓度和骨密度水平,表明在正常日粮钙水平条件,沉默TRPV6通道蛋白不影响蛋鸡体内正常的钙转运。 相似文献
4.
为制备蛋鸡TRPV6蛋白多克隆抗体,根据其基因序列,设计1对特异性引物,以卵巢组织中提取的总RNA为模板,扩增蛋鸡TRPV6基因1 801～2 176nt的375bp序列,构建原核表达质粒pET-32a（＋）-TRPV6;将重组质粒转化BL21（DE3）,经IPTG诱导表达TRPV6融合蛋白,通过镍离子螯合柱纯化后免疫新西兰大白兔,获得兔抗鸡TRPV6多克隆抗体,分别通过酶联免疫吸附试验（ELISA）法和Western blot检测抗体的效价和抗体特异性。结果表明,试验成功构建原核表达载体pET-32a（＋）-TRPV6,SDS-PAGE蛋白电泳检测发现目的蛋白大小约35 000;Western blot分析显示,表达的TRPV6融合蛋白具有良好的免疫原性,其抗体可与大肠杆菌表达的产物特异性结合;ELISA显示其抗体效价达1∶100 000。获得纯化的融合蛋白和多克隆抗体对研究TRPV6钙离子通道在蛋鸡髓质骨形成的作用机理具有重要作用。 相似文献
5.
为了探讨大鼠前额皮质TRPV4通道蛋白在低氧环境中运动时变化规律,本研究采用H.E.染色和免疫组化方法分析了不同氧浓度环境下递增负荷运动大鼠的前额皮质的病理变化情况以及TRPV4通道蛋白的表达情况。结果显示,模拟4 500 m海拔低氧高度运动至第5级大脑皮质出现静脉淤血、胶质细胞嗜神经现象,与常氧运动第8级情况相似;随着运动负荷的递增,大鼠前额皮质TRPV4的表达量均显著升高;模拟4 500 m海拔低氧组各级运动组TRPV4表达量均显著高于常氧组。结论:与常氧运动相比较,大鼠急性低氧环境递增负荷运动可以导致脑组织病理变化提前发生,并提高脑组织中TRPV4通道蛋白的表达量。这一研究结果将为中枢神经细胞膜钙离子通道在低氧运动中枢疲劳提前这一现象中所起作用的研究提供有价值的资料。 相似文献
6.
TRPV2通道是一种温度敏感型TRP通道,能被高于52℃的伤害性高温所激活,此外还可以响应渗透压、机械和化学等刺激,在维持机体生理功能中具有重要作用。与其他TRP通道相比,TRPV2通道的功能性研究存在重重困难,一方面是由于激活TRPV2通道的高温刺激很难在生理条件下达到,另一方面由于缺乏靶向TRPV2通道的专一性抑制剂或激动剂。我们综述TRPV2通道的结构以及生理学功能方面的研究进展,旨在为全面了解TRPV2作用机制提供参考。 相似文献
7.
哺乳动物钙代谢包括钙平衡与钙稳态。钙平衡是指体内总钙含量保持相对恒定的状态,钙稳态则指细胞内外的钙离子浓度保持稳定。在哺乳动物的泌乳期,大量的钙流失到乳中,导致母体血清钙急剧下降。动物的肠道、肾脏、骨骼作为主要的钙代谢器官,将对此做出适应性改变,以维持哺乳动物在泌乳期的钙代谢稳态。哺乳动物钙代谢紊乱会严重影响母畜及幼崽的营养健康,给畜牧养殖业带来损失,因此,维持钙代谢稳态对泌乳期哺乳动物尤为重要。本文综合近年来国内外哺乳动物钙代谢相关研究,从哺乳动物泌乳期不同器官钙代谢适应性改变、血清钙和血清磷等矿物质以及钙调节因子的变化进行综述,旨在为泌乳期哺乳动物钙代谢调控的相关研究提供思路。 相似文献
8.
试验旨在探究三穗鸭TRPV6基因和SLC4A4基因SNPs突变对蛋壳品质的影响,筛选出与蛋壳品质相关的分子标记。利用PCR扩增和直接测序法相结合的方法检测鸭TRPV6基因和SLC4A4基因的SNPs,并与蛋壳品质进行关联分析。结果显示：在TRPV6基因第2内含子发现的g.81465153 C>G和g.81465176A>G突变分别对蛋壳重和蛋重有显著影响,在第3外显子发现同义突变g.81465450 T>C;3个SNPs联合产生5种单倍型和7种双倍型,位点联合对蛋壳重有显著影响。在SLC4A4基因第24外显子发现同义突变位点g.15125097 C>T,在内含子25发现g.15132523 G>A突变对蛋形指数有极显著影响,在内含子26发现g.15135079G>A突变;3个SNPs联合产生4种单倍型和7种双倍型,位点联合对蛋壳重有极显著影响。2个基因聚合后6个SNPs联合产生8种单倍型和7种双倍型,位点联合对蛋壳重和蛋重均有显著影响。表明TRPV6基因和SLC4A4基因新发现的3个SNPs联合和2个基因的6个SNPs联合均对蛋重和蛋壳重有显著效应,... 相似文献
9.
本研究旨在分析钙离子(Ca2+)跨膜吸收途径相关基因在昆明小鼠胃肠道中的表达模式。选取12只8周龄、平均体重(30.71±2.93)g的雌性昆明小鼠,取胃、十二指肠、空肠、回肠、盲肠和结肠组织样品,利用实时定量PCR法检测维生素D依赖性钙结合蛋白(CaBP-D9k)、瞬时性受体电位通道香草酸受体6(TRPV6)和维生素D受体(VDR)mRNA表达量。结果表明:1)CaBP-D9k、TRPV6和VDR mRNA在胃内属低水平表达,而在盲肠内表达量较高;2)随着小肠的延伸,CaBP-D9k、VDR mRNA表达量逐渐降低,而TRPV6 mRNA则在回肠内高水平表达;3)CaBP-D9k(P<0.05)、VDR(P<0.05)、TRPV6 mRNA的表达量(P>0.05)随着大肠肠段的延伸而不同程度地下降。结果提示,CaBP-D9k、TRPV6和VDR mRNA表达量与胃肠道Ca2+跨膜吸收能力存在关联性。 相似文献
10.
蛋壳的形成是动态时序性过程,在子宫(蛋壳腺)内完成,经过钙化起始、线性沉积和钙化末期3个阶段完成蛋壳的钙化,期间需要持续的提供大量的钙离子。子宫内的钙离子一部分来源于肠道吸收,另一部分来源于髓骨的动员。因此,钙离子代谢是影响蛋壳钙化的重要因素,受多种因素调节。本文从钙离子在肠道的吸收转运、髓骨的动员、子宫的分泌,综述了蛋壳形成的钙代谢机制,并简述其影响因素,以期为蛋壳品质的调控研究提供思路。 相似文献
11.
1. The aim of this study was to investigate the localisation and expression of the epithelial Ca2+ channel TRPV6 (transient receptor potential vanilloid channel type 6) in different intestinal segments and kidney of laying hens during peak lay. 2. Immunohistochemical analysis of the intestine indicated that TRPV6 was localised to the brush-border membranes of the duodenum, jejunum, ileum, caecum, and rectum. Expression was weaker in the rectum, and little or no expression was found in crypt and goblet cells. In addition, TRPV6 mRNA was quantified amongst different intestinal segments, and expression was highest in the duodenum and jejunum. Furthermore, Western blotting indicated that the duodenum expressed the greatest amount of TRPV6 and the rectum the least with the other segments expressing intermediate levels. 3. In the kidney, distinct immunopositive staining for TRPV6 was detected at the apical domain of the distal convoluted tubules (DCT) and medullary connecting tubules (CNT). Interestingly, distribution of TRPV6 extended to the proximal convoluted tubules (PCT). Furthermore, the kidney expressed lower TRPV6 mRNA and protein levels compared with that in the duodenum. 4. In conclusion, the epithelial Ca2+ channel TRPV6 is strongly expressed in the apical cells of the entire intestine and the renal tubules, suggesting that active Ca2+ transcellular transport plays a crucial role in dietary calcium (re)absorption in laying hens. 相似文献
14.
1. The aim of this study was to investigate the localisation of the transient receptor potential vanilloid channel type 6 (TRPV6) in egg shell gland (ESG) and examine the dynamic expression of TRPV6 and Calbindin-d28k (CaBP-D28k), as well as the changes in concentration of total calcium (Ca), total inorganic phosphorus (P), alkaline phosphatase (ALP), parathyroid hormone (PTH) and calcitonin (CT) in plasma during the oviposition cycle. 2. The plasma ALP activity was notably increased at 8 h. In addition, plasma CT was highest at 0 h and significantly lower at 8 h. The change of plasma PTH concentration increased slightly post-oviposition and reached a maximum at 16 h. 3. Immunohistochemical analysis indicated that TRPV6 was strongly localised to the apical luminal epithelium of the mucosa. The mRNA levels of TRPV6 and CaBP-D28k in the ESG remained very low from 0 to 4.5 h, but were significantly increased at 16 h. Furthermore, Western blotting analysis showed that the expression of TRPV6 and CaBP-D28k also reached a maximum at 16 h and was different from the concentration of CaBP-D28k. 4. In conclusion, the epithelial Ca2+ channel TRPV6 is strongly expressed in the epithelial cells of the eggshell gland, and the increase of TRPV6 and CaBP-D28k mRNA and protein expression during eggshell formation suggests that active Ca2+ transcellular transport exerts significant effects in delivering active calcium in the ESG. 相似文献
15.
Sperm cells perform precise chemotactic and thermotactic movement which is crucial for fertilization. However, the key molecules involved in detection of different chemical and physical stimuli which guide the sperm during navigation are not well understood. Ca 2+-signalling mediated by ion channels seem to play important role in motility and other fertility parameters. In this work, we explored the endogenous localization pattern of TRPV channels in the mature spermatozoa of avian species. Using sperm from white pekin duck ( Anas platyrhynchos) as the representative avian model, we demonstrate that duck sperm endogenously express the thermosensitive channels TRPV1, TRPV2, TRPV3, TRPV4, and highly Ca 2+-selective channels TRPV5 and TRPV6 in specific yet differential locations. All of these TRPV channels are enriched in the sperm tail, indicating their relevance in sperm motility. Interestingly, the TRPV3 and TRPV4 channels are present in the mitochondrial region. Calcium selective TRPV5 channel is exclusively present in sperm tail and is most abundant among the TRPV channels. This is the first report describing the endogenous presence of TRPV2 and TRPV3 channels in the sperm of any species. Using confocal imaging and super-resolution imaging, we demonstrate that though the TRPV channels are evolutionarily closely related, they have distinct localization pattern in the duck sperm, which could impact their role in fertilization. 相似文献
16.
Recently, the transient receptor potential vanilloid type 1 (TRPV1) channel was shown to be involved in capacitation, the process that allows mammalian spermatozoa to acquire their fertilizing ability within the female genital tract. Unfortunately, the role of TRPV1 in this process is still unclear. Thus, the aims of the present work were to 1) investigate the function of TRPV1 in the male gamete signaling system and 2) modulate TRPV1 activity by administering a specific activator, capsaicin, or a specific inhibitor, capsazepin, to spermatozoa during in vitro capacitation. Using confocal microscopy, cellular responses were assessed in terms of changes in 1) cell membrane resting potential, 2) intracellular calcium concentrations, and 3) actin polymerization dynamics. As a result, TRPV1 channels were shown to act as specific cationic channels: their activation led to membrane depolarization and, consequently, the opening of voltage-gated calcium channels and an increase in intracellular calcium concentrations. These ionic events promote actin cytoskeletal depolymerization and a loss of acrosome structure integrity. In contrast, TRPV1 inhibition caused a slowing of the capacitation-dependent increase in intracellular calcium concentrations, a reduction in actin polymerization, and acrosome rupture. In conclusion, these results suggest that TRPV1 channels modulate the major pathways involved in capacitation. 相似文献
17.
The activation of α 2 adrenergic receptors contributes to analgesia not only in the central nervous system but also in the peripheral nervous system. We reported that noradrenaline inhibits the activity of transient receptor potential vanilloid 1 (TRPV1) evoked by capsaicin through α 2 receptors in cultured rat dorsal root ganglion (DRG) neurons. However, it is unclear whether activation of TRPV1 expressed in peripheral nerve terminals is inhibited by α 2 receptors and whether this phenomenon contributes to analgesia. Therefore, we examined effects of clonidine, an α 2 receptor agonist, on several types of nociceptive behaviors, which may be caused by TRPV1 activity, and subtypes of α 2 receptors expressed with TRPV1 in primary sensory neurons in rats. Capsaicin injected into hind paws evoked nociceptive behaviors and clonidine preinjected into the same site inhibited capsaicin-evoked responses. This inhibition was not observed when clonidine was injected into the contralateral hind paws. Preinjection of clonidine into the plantar surface of ipsilateral, but not contralateral, hind paws reduced the sensitivity to heat stimuli. Clonidine partially reduced formalin-evoked responses when it was preinjected into ipsilateral hind paws. The expression level of α 2C receptor mRNA quantified by real-time PCR was highest followed by those of α 2A and α 2B receptors in DRGs. α 2A and α 2C receptor-like immunoreactivities were detected with TRPV1-like immunoreactivities in the same neurons. These results suggest that TRPV1 and α 2 receptors are coexpressed in peripheral nerve terminals and that the functional association between these two molecules causes analgesia. 相似文献
18.
In recent years, intestinal transport processes have been studied in detail regarding both, functional and structural aspects. For monosaccharides different systems have been demonstrated for apical uptake: this includes the high-affinity SGLT1 as a distinct d-glucose system and GLUT5 for fructose. Specifically in pigs a low affinity, high-capacity system for d-glucose and d-mannose with no preference for Na + over K + and a very low affinity system are suggested as further uptake systems. As in other species, basolateral extrusion is mediated by GLUT2. The distributions of monosaccharide transport along the gastrointestinal axis as well as the potential role of paracellular monosaccharide absorption have not yet been clarified. Amino acids can principally be absorbed by the paracellular and transcellular pathway whereas transcellular transport can either be mediated by facilitated diffusion or secondary active Na+-coupled transport. This includes different transport systems for neutral, anionic and cationic acids. In addition, the presence of the di-/tripeptides transport system PEPT1 which depends on an inwardly directed H+-gradient has also been confirmed for the pig small intestine, its quantitative proportion is still under debate. Short chain fatty acids (SCFA) are the major end products of microbial carbohydrate fermentation which occurs along the gastrointestinal tract with the highest production rates in the large intestines. At least two uptake mechanisms have to be assumed, i.e., non-ionic diffusion and anionic exchange via SCFA−/HCO3−-exchange. Controversial views still exist to what extent SCFA are metabolized within the epithelial tissue. Segmental differences between small and large intestines have been demonstrated for Na+ absorption. Whereas in the small intestines the major part of Na+ absorption is mediated by coupled nutrient transport systems, aldosterone sensitive Na+ channels and Na+/H+-exchange are the dominant mechanisms in the hindgut. For Cl− paracellular transport and anionic Cl−/HCO3−-exchange are the major absorptive mechanisms. Cl− secretion is mediated by apical channels which may be activated by toxins of different origin. Different types of Cl− channels have been identified, such as Cystic Fibrosis Transmembrane Regulator (CFTR), Ca-activated Cl− channels (CLCA) and Outwardly Rectifying Cl− Channels (ORCC). Whereas CFTR has clearly been shown for jejunal and colonic epithelial and goblet cells controversy still exists on the relevance of CLCA and ORCC in pigs. For Ca2+ there is evidence that both recently published channels TRPV5 and TRPV6 are also expressed in pig intestinal tissues, however, this has not yet been shown on protein level. From several functional approaches it was demonstrated that phosphate uptake can be mediated by both, a Na+-dependent transcellular component and paracellularly. On a molecular basis it is uncertain whether the transport protein of transcellular mechanism belongs to the NaPi-IIb cotransporter family. 相似文献
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