丝瓜叶成分对脑缺血大鼠学习记忆障碍及皮层体感诱发电位的影响1

目的:研究丝瓜叶成分L-6a和L-10对脑缺血大鼠学习记忆障碍及皮层体感诱发电位(SEP)的影响。方法:用电针烧灼椎动脉造成永久性闭塞,且夹闭双侧颈总动脉,造成脑缺血大鼠模型,侧脑室埋管及注射(icv)给药。通过穿梭箱主动回避反应(AAR),微机处理,测定学习记忆行为;用SYD_(4228)型生理实验系统测定皮层体感诱发电位。结果:发现L-6a 0.5mg·kg~(-1)显著促进脑缺血大鼠AAR的获得(P<0.05);0.50mg·kg~(-1)及0.25mg·kg~(-1)均显著延缓AAR的消退(P<0.05);脑缺血后SEP波幅逐渐下降,峰潜伏期逐渐延长,但是L-6a给药组较脑缺血不给药组的P_0波幅升高     

高胆红素血症大鼠脑干听觉诱发电位与脑组织NO含量及Na+-K+ATP酶活性的变化

目的： 探讨脑干听觉诱发电位(BAEP)在早期监测高胆红素血症听力和脑损伤中的作用及脑组织一氧化氮(NO)与胆红素诱导的听力和脑损伤的关系。方法: 15 d SD大鼠腹腔注射不同剂量(30 mg/kg、60 mg/kg、90 mg/kg、120 mg/kg和150 mg/kg)胆红素溶液以制备高胆红素血症动物模型，微量胆红素测定仪测定血清胆红素浓度，重氮法测定脑组织胆红素浓度，定磷法测定脑组织中Na+-K+ATP酶活性，硝酸酶还原法测定脑组织NO含量，诱发电位仪检测BAEP。结果: 建模后高剂量组（120 mg/kg和150 mg/kg）部分大鼠出现异常神经行为活动；建模6 h后，除低剂量（30 mg/kg）组外，各实验组大鼠血清和脑组织胆红素浓度及脑组织NO含量显著升高，脑组织Na+-K+ATP酶活性显著降低，BAEP的波峰潜伏期(PL)和波峰间潜伏期(IPL)显著延长；且BAEP的PL与IPL和脑组织NO含量与Na+-K+ATP酶活性的变化均与脑组织胆红素水平显著相关。结论: BAEP的PL和IPL是早期监测高胆红素血症听力和脑损伤的无创性指标，NO的过量产生可能参与了胆红素诱导的听力和脑损伤的发病过程。     

吗啡依赖及戒断对大鼠海马内神经甾体水平的影响

目的:探讨吗啡躯体依赖、精神依赖及戒断对雄性大鼠海马内神经甾体水平的影响。方法:腹腔注射递增剂量吗啡建立大鼠吗啡躯体依赖模型,纳洛酮诱发戒断症状;条件性位置偏爱实验建立吗啡精神依赖模型。高效液相色谱-质谱法测定大鼠海马和血浆中脱氢表雄酮(DHEA)及其硫酸酯(DHEAS)、孕烯醇酮(PREG)及其硫酸酯(PREGS)和别孕烯醇酮(AP)含量。结果:大鼠吗啡躯体依赖形成时海马内DHEA、PREG水平较对照组升高(0·88±0·19/0·67±0·17,t=2·52,P<0·05,10·94±2·02/7·53±2·64,t=3·24,P<0·01),血浆中DHEAS、PREGS水平较对照组升高(115·4±99·7/29·3±8·3,t=3·20,P<0·01;234·5±216·1/38·2±18·8,t=3·39,P<0·01);大鼠吗啡精神依赖形成时海马及血浆中DHEA水平降低(0·90±0·55,15·6±5·0/1·63±0·76,24·5±9·8,t=2·42,2·69,P<0·05),血浆中DHEAS水平显著升高(187·4±44·8/136·7±30·7,t=2·88,P<0·05)。与纳洛酮对照组比较,大鼠吗啡戒断时海马内DHEA、AP、DHEAS、PREGS水平升高(t=2·33~3·96,P<0·05),血浆中PREG、AP、DHEAS和PREGS水平升高(t=3·72~4·82,P<0·01)。结论:吗啡依赖、戒断可影响大鼠海马内某些神经甾体的水平,表明内源性神经甾体可能参与吗啡依赖的形成。     

新城疫病毒对BGC-823胃癌细胞线粒体的影响

目的 探讨新城疫病毒对BGC-823胃癌细胞线粒体结构和三磷酸腺苷(ATP)酶活性等功能的影响.方法 应用电子显微镜观察、线粒体Na+-K+-ATP酶、Ca2+-ATP酶活性变化测定、罗丹明123染色法测定细胞线粒体膜电位及Western Blot测定细胞色素C等方法 进行分析.结果 新城疫病毒感染肿瘤细胞线粒体结构破坏,线粒体Na+-K+ATP酶、Ca2+-ATP酶活性较对照组显著下降(P<0.01).线粒体膜电位及细胞色素C显著下降(P<0.01).结论 对线粒体结构及功能的影响可能是新城疫病毒杀伤BGC-823胃癌细胞的机制之一.     

大鼠中脑导水管周围灰质(PAG)内微量注射八肽胆囊收缩素(CCK-8)对吗啡镇痛的拮抗作用

已知脑室注射CCK-8对吗啡镇痛有明显的对抗作用,但其作用部位迄未阐明。本工作将微量CCK-8注入PAG,观察其对吗啡镇痛的影响。 选用体重200—220g的雄性Wistar大鼠。在PAG埋植不锈钢套管,术后一周进行实验。向PAG内注射药液最均为1μl,在8min内注毕。用辐射热甩尾法测定大鼠痛阈(TFL)。     

PCPA致大鼠失眠后不同时间点神经元线粒体形态和功能的变化

目的探讨大鼠对氯基丙氨酸（PCPA）造模后不同时间点大鼠额叶皮质神经元线粒体形态功能的变化。方法3月龄大鼠进行造模,分别观察PCPA腹腔注射后1、2、3、4d额叶皮质神经元线粒体形态功能的改变;采用超活染色和电镜分别观察线粒体的数目和超微结构变化,同时采用荧光显微镜检测线粒体膜电位改变和WST-1法测定线粒体ATP酶含量。结果3月龄大鼠造模后线粒体数量明显减少,超微结构以第3天变化最为显著;额叶皮层线粒体膜电位和Na＋-K＋-ATP、Ca2＋-Mg2＋-ATP酶活性第2天开始明显下降,第3天下降最明显（P〈0．05）,第4天略有回升。结论以3月龄大鼠进行PCPA制作失眠模型过程中额叶皮层线粒体超微结构和功能变化以连续注射3天最明显。     

高渗葡萄糖处理后脱水红细胞膜Na^＋—K^＋—ATP酶的变化

目的体外观测高渗葡萄糖处理后脱水红细胞膜Na+-K+-ATP酶的变化.方法对全血与50%葡萄糖(50%GS)溶液混合前后红细胞膜Na+-K+-ATP酶的变化,以及脱水红细胞于等渗条件下其Na+-K+-ATP酶的恢复情况进行了测定.结果(1)全血与50%GS溶液混合后其脱水红细胞膜Na+-K+-ATP酶活性比混合前显著降低(0.307μmol/mg.h-1±0.073μmol/rmg.h-1vs.0.396μmol/mg.h-1±0.104μmol/mg.h-1,P＜0.05).(2)等渗条件下脱水红细胞膜Na+-K+-ATP酶的活性由异常逐步恢复至正常(即刻0.349μrrol/mg.h-1±0.0817μmol/mg.h-1;2h0.355μmol/mgh-1±0.0946μmol/m]g.h-1,4h038μmol/mg.h-1±0.0877μmol/mg.h-1,P＜0.01).结论高渗葡萄糖处理后脱水红细胞膜Na+-K+-ATP酶活性显著降低,但其异常变化在等渗条件下是可逆的.     

肾阳虚证患者红细胞LPO、SOD和ATP酶活性的变化

目的 :探讨肾阳虚证患者红细胞LPO、SOD和ATP酶活性的特点及其意义。方法 :观察 19例肾阳虚证患者和 2 1例正常人红细胞LPO、SOD和红细胞膜Na+ K+ ATP酶、Mg2 + ATP酶、Ca2 + ATP酶、Ca2 + Mg2 + ATP酶活性的变化。结果 :与对照组比较 ,肾阳虚证患者红细胞LPO含量升高 (P <0 .0 1) ,红细胞SOD活性降低 (P <0 .0 1) ;红细胞膜Na+ K+ ATP酶活性显著升高 (P <0 .0 1) ,而Mg2 + ATP酶活性变化无显著性差异 ,Ca2 + ATP酶活性升高 (P <0 .0 1) ,Ca2 + Mg2 + ATP酶活性也显著升高 (P <0 .0 1)。结论 :肾阳虚证患者红细胞内脂质过氧化反应增强 ,而抗氧化能力降低 ,红细胞膜Na+ K+ ATP酶、Ca2 + ATP酶和Ca2 + Mg2 + ATP酶活性升高 ;本研究为理解肾阳虚证的病理生理基础提供了初步实验依据     

鞘内注射P2X7受体拮抗剂BBG对大鼠行为学和脊髓PLA2表达的影响

目的:探讨鞘内注射P2X7受体拮抗剂考马斯亮蓝(BBG)在福尔马林模型大鼠中的镇痛作用及对脊髓磷脂酶A2(PLA2)表达的影响。方法:雄性Wistar大鼠鞘内置管成功后鞘内注射P2X7受体拮抗剂考马斯亮蓝(BBG)后所有的大鼠左侧后肢爪掌背侧皮下注射福尔马林(5%,50μl),观察大鼠在第一相(0~10 min)和第二相(10~60 min)的疼痛行为学比,变化(自发缩足次数)。免疫组织化学方法检测脊髓背角PLA2蛋白的表达与变化。结果:各组间大鼠第一相相比自发缩足次数没有明显的差异;第二相与对照组相比鞘内1μg、5μg及腹腔10μg BBG注射组相比,鞘内10μg BBG注射组大鼠的疼痛行为学明显减少(自发缩足次数,P0.05)。与此同时,鞘内10μg BBG注射组大鼠脊髓背角浅层内PLA_2免疫阳性产物的表达与其它组比较明显减少(P0.05)。结论:鞘内注射10μg BBG可减轻福尔马林诱导的炎性痛觉过敏,可能是通过抑制脊髓背角浅层PLA2的表达而发挥作用。     

急性脑梗塞患者血红细胞膜Na~ -K~ ATP酶活力和膜脂质过氧化的研究

本文采用红细胞溶血细胞膜Na －K ATP酶活力孔雀绿比色分析法和膜脂质过氧化测定法检测了22例急性脑梗塞患者红细胞膜Na －K ATP酶活力与膜脂质过氧化值。结果显示，急性脑梗塞患者红细胞膜Ng －K ATP酶活力和腹脂质过氧化值分别为0．11±0.082μM·pi／mg蛋白·小时和0．056±0.021μM硫代巴比土酸反应物／mg蛋白。与对照组相比，急性脑梗塞患者血红细胞膜Na －K 1ATP酶活力明显降低．而膜脂质过氧化值则明显升高，两组p值均＜0．01。提示应注意红细胞膜Na －K ATP酶活力降低和膜脂质过氧化值增高对急性脑梗塞发病过程的影响。     

Lower proportion of CD19+IL-10+ and CD19+CD24+CD27+ but not CD1d+CD5+CD19+CD24+CD27+ IL-10+ B cells in children with autoimmune thyroid diseases

Abstract

Introduction: As it is generally known, regulatory B cells (Bregs) control inflammation and autoimmunity. The significance of Bregs in the population of children with autoimmune thyroid diseases (AITD) still offers plenty of potential to explore. The aim of this study was to estimate the expression of Bregs (phenotype CD19⁺CD24⁺CD27⁺IL-10⁺, CD19⁺IL-10⁺, CD1d⁺CD5⁺CD19⁺IL-10⁺ and CD1d⁺CD5⁺CD19⁺CD24⁺CD27⁺) in a paediatric cohort with AITD and in health controls.

Materials and methods: A total of 100 blood samples were obtained from 53 paediatric patients with Graves’ disease (GD) (N?=?12 newly diagnosed, mean age 12.5?±?3.5 and N?=?17 during methimazole therapy, mean age 12.7?±?4.4), Hashimoto’s thyroiditis (HT) (N?=?10 newly diagnosed, mean age 13.3?±?2.9 and N?=?10 during L-thyroxine therapy, mean age 13.7?±?3.4) and compared with healthy controls (C) (N?=?15, mean age 13.1?±?3.1). The expressions of the immune cell populations were analysed by four-color flow cytometry using a FASC Canto II cytometer (BD Biosciences).

Results: There was a decreasing tendency in the number of lymphocytes B producing IL-10 (B10) cells among all B lymphocytes and more widely, also among all lymphocytes, in each study group, as compared to C. We reported a reduction in IL-10 production in Bregs with the expression of CD19⁺CD24⁺CD27⁺IL-10 and CD1d⁺CD5⁺CD19⁺IL-10⁺ in both untreated and treated AITD.

Conclusions: Our data demonstrate that the reduction in the number of Bregs with CD19⁺CD24⁺CD27⁺IL-10⁺ and CD19⁺IL-10⁺ expression could be responsible for breaking immune tolerance and for AITD development in children.     

Monoclonal antibodies against gastric H+ + K+ ATPase

Monoclonal antibodies were prepared against a purified membrane fraction from hog gastric mucosa containing the H+ + K+ ATPase. On sodium dodecyl sulfate gels the molecular weight of this fraction corresponds to a single band of about 95,000. In contrast, on isoelectric focusing gels three groups of peptides are resolved with isoelectric points of 5.7, 6.2, and 8.5. One of the monoclonal antibodies (HK111) was shown to react selectively with the acidic peptide, whereas another antibody (HK113) reacted with the alkaline peptide, showing that the three peptides were antigenically distinct. Both monoclonal antibodies selectively labeled the parietal cell, and antibody HK111 labeled the tubulovesicles of the resting parietal cell and the microvilli of the secretory canaliculus of the secreting cell. This finding suggests translocation of membrane from the tubulovesicles to the secretory surface on stimulation.     

Ontogeny of Tumor-associated CD4+CD25+Foxp3+ T-regulatory Cells

ABSTRACT

The critical contribution of CD4+CD25+Foxp3+ T-regulatory cells (Treg) to immune suppression in the tumor microenvironment is well-established. Whereas the mechanisms that drive the generation and accumulation of Treg in tumors have been an active area of study, the information on their origin and population dynamics remains limited. In this review, we discuss the ontogeny of tumor-associated Treg in light of the recently identified lineage markers.     

Cytoplasmic Na+-dependent modulation of mitochondrial Ca2+ via electrogenic mitochondrial Na+-Ca2+ exchange

To clarify the role of mitochondrial Na(+)-Ca(2+) exchange (NCX(mito)) in regulating mitochondrial Ca(2+) (Ca(2+)(mito)) concentration at intact and depolarized mitochondrial membrane potential (DeltaPsi(mito)), we measured Ca(2+)(mito) and DeltaPsi(mito) using fluorescence probes Rhod-2 and TMRE, respectively, in the permeabilized rat ventricular cells. Applying 300 nm cytoplasmic Ca(2+) (Ca(2+)(c)) increased Ca(2+)(mito) and this increase was attenuated by cytoplasmic Na(+) (Na(+)(c)) with an IC(50) of 2.4 mm. To the contrary, when DeltaPsi(mito) was depolarized by FCCP, a mitochondrial uncoupler, Na(+)(c) enhanced the Ca(2+)(c)-induced increase in Ca(2+)(mito) with an EC(50) of about 4 mm. This increase was not significantly affected by ruthenium red or cyclosporin A. The inhibition of NCX(mito) by CGP-37157 further increased Ca(2+)(mito) when DeltaPsi(mito) was intact, while it suppressed the Ca(2+)(mito) increase when DeltaPsi(mito) was depolarized, suggesting that DeltaPsi(mito) depolarization changed the exchange mode from forward to reverse. Furthermore, DeltaPsi(mito) depolarization significantly reduced the Ca(2+)(mito) decrease via forward mode, and augmented the Ca(2+)(mito) increase via reverse mode. When the respiratory chain was attenuated, the induction of the reverse mode of NCX(mito) hyperpolarized DeltaPsi(mito), while DeltaPsi(mito) depolarized upon inducing the forward mode of NCX(mito). Both changes in DeltaPsi(mito) were remarkably inhibited by CGP-37157. The above experimental data indicated that NCX(mito) is voltage dependent and electrogenic. This notion was supported theoretically by computer simulation studies with an NCX(mito) model constructed based on present and previous studies, presuming a consecutive and electrogenic Na(+)-Ca(2+) exchange and a depolarization-induced increase in Na(+) flux. It is concluded that Ca(2+)(mito) concentration is dynamically modulated by Na(+)(c) and DeltaPsi(mito) via electrogenic NCX(mito).     

Prognostic Values of CD38+CD101+PD1+CD8+ T Cells in Pancreatic Cancer

Programmed death-1 (PD-1), a key immune checkpoint molecule, has been developed as an oncotherapy target for various carcinomas. However, treatment with anti-PD-1 elicited only a minimal effect in pancreatic ductal adenocarcinoma (PDAC). Subsequent studies revealed the existence of a subset of PD-1⁺ T cells coexpressing CD38 and CD101, representing a fixed dysfunctional subpopulation that are not able to be rescued by anti-PD-1 immunotherapy. However, whether this subpopulation of PD-1 expressing CD8⁺ T cells could be useful in predicting PDAC stage or prognosing survival is unknown. In this study, we used flow cytometry and immunofluorescence assay to analyze the expression of CD38 and CD101 in 183 clinical PDAC samples, including 84 of peripheral blood and 99 of surgical tissues. High coexpression of CD38/CD101 on peripheral PD-1⁺CD8⁺ T cells or tumor-infiltrating lymphocytes (TILs) was found to be most significantly correlated with Tumor/Node/Metastasis (T/N/M) classification and clinical stage, in contrast PD-1⁺CD8⁺ T cells could not correlate with T classification. CD38/CD101 co-repression on TILs also correlated with the poor survival in these PDAC patient samples. Our data suggest that CD38/CD101 might represent a more helpful biomarker than PD-1 alone for diagnosis and prognosis of PDAC.     

Steady state migratory RelB+ langerin+ dermal dendritic cells mediate peripheral induction of antigen-specific CD4+ CD25+ Foxp3+ regulatory T cells

Tolerance to self-antigens expressed in peripheral organs is maintained by CD4(+) CD25(+) Foxp3(+) Treg cells, which are generated as a result of thymic selection or peripheral induction. Here, we demonstrate that steady-state migratory DCs from the skin mediated Treg conversion in draining lymph nodes of mice. These DCs displayed a partially mature MHC II(int) CD86(int) CD40(hi) CCR7(+) phenotype, used endogenous TGF-β for conversion and showed nuclear RelB translocation. Deficiency of the alternative NF-κB signaling pathway (RelB/p52) reduced steady-state migration of DCs. These DCs transported and directly presented soluble OVA provided by s.c. implanted osmotic minipumps, as well as cell-associated epidermal OVA in transgenic K5-mOVA mice to CD4(+) OVA-specific TCR-transgenic OT-II T cells. The langerin(+) dermal DC subset, but not epidermal Langerhans cells, mediated conversion of naive OT-II×RAG-1(-/-) T cells into proliferating CD4(+) CD25(+) Foxp3(+) Tregs. Thus, our data suggest that steady-state migratory RelB(+) TGF-β(+) langerin(+) dermal DCs mediate peripheral Treg conversion in response to epidermal antigen in skin-draining lymph nodes.     

ATP suppresses activity of Ca2+ -activated K+ channels by Ca2+ chelation

Ca²⁺-activated maxi K⁺ channels were studied in inside-out patches from smooth muscle cells isolated from either porcine coronary arteries or guinea-pig urinary bladder. As described by Groschner et al. (Pflügers Arch 417:517, 1990), channel activity (NP _o) was stimulated by 3 M [Ca²⁺]_c (1 mM Ca-EGTA adjusted to a calculated pCa of 5.5) and was suppressed by the addition of 1 mM Na₂ATP. The following results suggest that suppression of NP _o by Na₂ATP is due to Ca²⁺ chelation and hence reduction of [Ca²⁺]_c and reduced Ca²⁺ activation of the channel. The effect was absent when Mg ATP was used instead of Na₂ATP. The effect was diminished by increasing the [EGTA] from 1 to 10 mM. The effect was absent when [Ca²⁺]_c was buffered with 10 mM HDTA (apparent pK _Ca 5.58) instead of EGTA (pK _Ca 6.8). A Ca²⁺-sensitive electrode system indicated that 1 mM Na₂ATP reduced [Ca²⁺]_c in 1 mM Ca-EGTA from 3 M to 1.4 M. Na₂ATP, Na₂GTP, Li₄AMP-PNP and NaADP reduced measured [Ca²⁺]_c in parallel with their suppression of NP _o. After the Na₂ATP-induced reduction of [Ca²⁺]_c was re-adjusted by adding either CaCl₂ or MgCl₂, the effect of Na₂ATP on NP _o disappeared. In vivo, intracellular [Mg²⁺] exceeds free [ATP^4–], hence ATP modulation of maxi K⁺ channels due to Ca²⁺ chelation is without biological relevance.     

Na+ absorption in Aplysia intestine: Na+ fluxes and intracellular Na+ and K+ activities

Microelectrodes were used to measure the potential difference (psi m) across the mucosal membrane of epithelial cells lining the villi of isolated Aplysia californica intestine. In substrate-free NaCl seawater medium psi m was -55.1 +/- 1.2 mV. The cell interior was negative relative to the mucosal bathing solution. Intracellular K+ activity, determined in the absorptive cells with single-barreled liquid ion-exchanger microelectrodes, was 383 +/- 15 mM. Since the calculated K+ equilibrium potential exceeds the membrane potential, K+ is accumulated by the intestinal absorptive cell. Intracellular Na+ activity (aiNa) was also determined in the intestinal cells of Aplysia with single-barreled liquid ion-exchanger microelectrodes and was 17.2 +/- 2.5 mM. aiNa was much less than that predicted by the electrochemical equilibrium value for Na+ across the mucosal membrane. From these data the steady-state transapical Na+ and K+ electrochemical potential differences were calculated. Serosal ouabain abolished net sodium absorption as determined by flux measurements. These results are consistent with the operation of a basolateral Na+ - K+ pump.     

Na+ and K+ concentration of rat parotid saliva

The effects of carbachol and auriculo-temporal stimulation on the Na⁺ and K⁺ concentrations of rat parotid saliva have been compared. The main duct perfused in situ, does not transport Na⁺ or K⁺ and is water impermeable. The Na⁺ concentration of secretion evoked by either stimulus is flow dependent, increasing with increasing flow rate and plateauing at near plasma Na⁺ levels. At low flow rate the carbachol evoked secretion has a higher Na⁺ concentration. This is not due to the release of catecholamines since neither sympathectomy nor adrenoceptor block altered the nature of the secretion. The K⁺ concentration, whilst flow dependent, decreasing with increasing flow rate, was the same for both stimuli.