青龙藤提取物对小白鼠胰岛β细胞ATP敏感钾（KATP）通道的影响

青龙藤 (Ｂｉｏｎｄｉａｈｅｎｒｉｙｉ,ＢＨ)为萝科青龙藤属植物 ,具有活血舒筋 ,理气祛风的功能 :主治跌打损伤、下肢冷痛、风湿手足麻木、牙痛。在其它实验中我们发现青龙藤提取物可以刺激胰岛 β细胞单位放电增加 ,促进胰岛素分泌 ,使糖尿病模型大鼠血浆中的胰岛素水平由 (18.8± 2 .3)ｍｕ/Ｌ升高到 (4 3 .2± 3.5 )ｍｕ/Ｌ。已知当血糖浓度升高 ,胰岛 β细胞膜上ＫＡＴＰ通道关闭 ,引起胰岛素分泌。为探讨作用机制 ,用记录小白鼠胰岛 β细胞膜上的ＫＡＴＰ通道电流为指标 ,观察对ＫＡＴＰ通道的影响。1　材料和方法(1)细胞的分离　体…     

木黄酮对小鼠胰岛β细胞电压依赖性钙通道表达和电流的影响

目的:研究长期抑制酪氨酸激酶活性对胰岛β细胞中电压依赖性钙通道的影响,探讨酪氨酸激酶在胰岛β细胞中的作用.方法:原代培养小鼠胰岛和胰岛β细胞,经0.1 mmol/L酪氨酸激酶抑制剂木黄酮处理12 h后,运用全细胞电流记录的方法观察电压依赖性钙电流以及动作电位的改变,RT-PCR方法观察电压依赖性钙通道α1亚单位的表达改变.结果:木黄酮处理12 h后,小鼠胰岛β细胞的电压依赖性钙电流明显减小(13.83±1.515pA/pFvs 7.012±1.502 pA/pF,P＜0.01,n=6),动作电位幅度明显减弱(38.50±7.46 mV vs 15.95±4.39 mV,P＜0.01,n=6).木黄酮处理12 h后,小鼠胰岛中电压依赖性钙通道的α1亚单位的表达明显减少,降低为对照组的0.792±0.078(P＜0.01,n=5).结论:木黄酮处理可以抑制小鼠胰岛β细胞中电压依赖性钙通道的表达和电流,提示长期抑制酪氨酸激酶活性在胰岛β细胞功能损害中具有重要作用.     

胰岛α细胞对β细胞功能的影响

众多研究表明,胰岛移植的短期效果较显著,但长期效果仍不尽人意。在胰岛移植的早期,绝大多数病人可以获得明显的治疗效果,而后大多数的病人发生进行性的胰岛功能丧失,产生这种现象的原因至今仍不甚明了。如何解决移植胰岛的长期存活问题对于胰岛移植的临床应用至关重要。近年来人们对胰岛移植失败的原因进行了不断探索,发现胰岛移植后期的失败不仅与免疫反应有关,而且与移植物的细胞组成有密切关系,即胰岛细胞间的相互作用对移植物的功能维持可能有重要影响。因此,本文就胰岛α细胞及其分泌的胰高血糖素对β细胞功能的影响作一综述。     

胰岛β细胞的电生理学特性

神经、肌肉以及某些感觉细胞具有电压依从性的跨膜内向电流,产生动作电位(AP)。1968年Dean等首先观察到胰岛β细胞具有电兴奋性,即具有象神经、肌肉细胞等那样产生AP的能力,才揭开了内分泌细胞电生理研究的序幕。十几年来的研究表明,胰岛素(In)的释放与胰岛β细胞的Ca~(2 )依从性AP有关,后者在刺激—分泌偶联(stimulus—secretion coupling)中起着关键性作用。     

乙酰胆碱对小鼠胰岛B细胞电活动作用的分析

用细胞内电位记录和细胞外微电泳技术,研究乙酰胆碱对小鼠胰岛Ｂ细胞电活动的作用,微电泳ＡＣｈ使Ｂ细胞胞膜去极化５－１０ｍＶ和锋电位电位发放数增加１１－１７／３０ｓ。这种效应具有葡萄糖依赖性,并被阿托品完全阻断,而哌仓西平可阻抑ＡＣｈ效应的７０％。ＡＣｈ的膜去极化作用不依赖于细胞外Ｃａ＾２＋,而可被河豚毒阻断;ＡＣｈ增加锋电位数的效应依赖于细胞外Ｃａ＾２＋,但不被异捕定阻断。结果表明：ＡＣｈ增强Ｂ细胞     

IL-1β对体外培养NIT-1β细胞胰岛素分泌的影响

利用小鼠胰岛β细胞系NIT 1细胞,对不同时间、不同浓度IL 1β作用下细胞的生长和胰岛素的分泌进行观察,以研究IL 1β对胰岛β细胞胰岛素分泌的影响,探讨细胞因子对胰岛β细胞功能的抑制作用.结果显示在IL 1β作用下,各组NIT 1细胞分泌胰岛素呈不同程度降低,表明IL 1β对NIT 1β细胞系胰岛素的分泌具有显著抑制作用.     

软脂酸对胰岛β细胞凋亡及Akt途径的影响

目的：探讨软脂酸（PA）对胰岛β细胞（MIN6细胞）凋亡及Akt信号途径的影响。方法：细胞采用小鼠胰岛素瘤细胞株MIN6,不同浓度PA（0-1．6mmol／L）干预24、48h,MTT、法测定各组细胞存活率。干预48h后Hoechst-PI染色法和Annexin—V／PI双标流式测定法测定各组细胞凋亡率,Western-blot法测定p-Akt、Akt、Bax、Bcl-2。结果：随着PA浓度的增高①MIN6细胞存活率逐渐减小、凋亡率逐渐增大;②MIN6细胞中p-Akt和Bcl-2的表达逐渐减少,而Akt、Bax无明显改变。结论：长时间PA作用引起MIN6细胞凋亡,并呈现一定的量效关系;这一效应可能是通过Akt／Bcl-2产生作用的。     

Reg基因家族蛋白对胰岛β细胞生长的影响

Reg基因家族蛋白,属于C型凝集素超家族,具有相同的钙依赖性碳水化合物识别域,在损伤、感染、糖尿病及肿瘤中发生作用.近年来,已有18个Reg基因家族成员被克隆和鉴定.本文综述Reg基因家族蛋白的分类和基因表达调节,以及Reg基因家族蛋白对胰岛β细胞增殖与在自身免疫中的作用.Reg I和人胰岛再生相关蛋白(INGAP)在体外或体内参与胰腺的再生.在1型糖尿病发生中,Reg I和Reg II可做为自身抗原.尽管Reg基因蛋白的功能尚不清楚,但为1型糖尿病的治疗带来新的希望.     

RNAi介导的Nrf2表达下调对亚砷酸钠诱发的小鼠胰岛β细胞毒性损伤的影响

目的探讨用RNA干扰技术下调核转录因子Nrf2的表达,对亚砷酸钠诱发的小鼠胰岛口细胞毒性损伤的影响。方法将针对Nrf2和非针对阴性对照的shRNA慢病毒颗粒稳定转染至小鼠胰岛β细胞MIN6,利用Real—time PCR和Western Blot检测细胞的Nrf2的表达,筛选Nrf2基因沉默（Nrf2-KD）和阴性对照（Scr）的细胞;应用4μmol／L亚砷酸钠（NaAsO2）作用于Nrf2-KD和Scr细胞24h,通过光学显微镜观察细胞的形态和贴壁数量的改变;四甲基偶氮唑盐（MTT）法检测细胞生长情况;利用Western Blot检测细胞内cleaved—caspase-3蛋白的表达。结果成功建立稳定转染的Nrf2基因沉默的小鼠胰岛8细胞系MIN6,与Scr细胞相比,Nrf2-KD细胞的Nrf2基因和蛋白表达水平显著降低。4μmol／L亚砷酸钠暴露24h后,与Scr细胞相比,Nrf2-KD细胞贴壁数量、细胞体积、细胞生长活性都显著下降,而细胞内的cleaved—caspase-3蛋白表达明显升高。结论Nrf2基因沉默的小鼠胰岛口细胞对亚砷酸钠暴露诱发的细胞毒性损伤更敏感。     

氟对小鼠胰岛β细胞增殖和胰岛素分泌的影响

目的:观察不同剂量氟化钠(NaF)对体外培养的小鼠胰岛β细胞增殖活力和胰岛素分泌的影响。方法:选用小鼠胰岛β细胞株Beta-TC-6作为实验对象,分别以0、0.1、0.5、1.0、2.0、4.0、8.0、16.0 mg/LNa F干预24 h、48 h、72 h、96 h观察对β细胞形态学的影响,采用四唑蓝[3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide,MTT]比色法,检测不同剂量NaF对β细胞增殖活力的影响;用酶联免疫吸附(enzyme linked immunosorbent assay,ELISA)法测定不同剂量NaF对β细胞胰岛素分泌的影响。结果:0.5 mg/L、1.0 mg/L的NaF作用72 h时,可使胰岛β细胞增殖活力和胰岛素分泌较对照组明显增强(P0.05);≥8.0 mg/L时随着NaF剂量的增加和作用时间的延长,胰岛β细胞的增殖活力和胰岛素分泌明显减弱(P0.05)且随着NaF剂量的增加和时间的延长,细胞生长缓慢,数量减少,不易贴壁或融合成片,多边形细胞减少,可见较多椭圆或圆形细胞。结论:NaF对胰岛β细胞的增殖和胰岛素分泌呈剂量效应关系,随着剂量的增大和时间的延长对细胞增殖活力和胰岛素分泌能力的抑制逐渐增强。     

Dual effects of glucose on the cytosolic Ca2+ activity of mouse pancreatic beta-cells

The cytosolic Ca2+ activity of mouse pancreatic beta-cells was studied with the intracellular fluorescent indicator quin2 . When the extracellular Ca2+ concentration was 1.20 mM, the basal cytosolic Ca2+ activity was 162 +/- 9 nM. Stimulation with 20 mM glucose increased this Ca2+ activity by 40%. In the presence of only 0.20 mM Ca2+ or after the addition of the voltage-dependent Ca2+ -channel blocker D-600, glucose had an opposite and more prompt effect in reducing cytosolic Ca2+ by about 15%. It is concluded that an early result of glucose exposure is a lowering of the cytosolic Ca2+ activity and that this effect tends to be masked by a subsequent increase of the Ca2+ activity due to influx of Ca2+ through the voltage-dependent Ca2+ channels.     

The adipoinsular axis: effects of leptin on pancreatic beta-cells

The prevalence of obesity and related diabetes mellitus is increasing worldwide. Here we review evidence for the existence of an adipoinsular axis, a dual hormonal feedback loop involving the hormones insulin and leptin produced by pancreatic beta-cells and adipose tissue, respectively. Insulin is adipogenic, increases body fat mass, and stimulates the production and secretion of leptin, the satiety hormone that acts centrally to reduce food intake and increase energy expenditure. Leptin in turn suppresses insulin secretion by both central actions and direct actions on beta-cells. Because plasma levels of leptin are directly proportional to body fat mass, an increase of adiposity increases plasma leptin, thereby curtailing insulin production and further increasing fat mass. We propose that the adipoinsular axis is designed to maintain nutrient balance and that dysregulation of this axis may contribute to obesity and the development of hyperinsulinemia associated with diabetes.     

Effects of Cl- deficiency on the membrane potential in mouse pancreatic beta-cells

The membrane potential of mouse pancreatic beta-cells was measured with microelectrodes. In the resting cell (3 mM D-glucose), the membrane potential was -63 +/- 3 mV (mean +/- S.E. for four experiments). In the presence of 3 mM D-glucose, total Cl- substitution by isethionate induced a depolarization by 3-4 mV, and readmission of Cl- induced a hyperpolarization by 3-5 mV. At 10 mM glucose, reduction of Cl- to 12 mM by substituting isethionate for Cl- reversibly shifted the repolarization potential by 6-9 mV in the positive direction and stimulated the burst activity during the initial 2-3 min by increasing the fraction of plateau phase. This was followed by a gradual inhibition of electrical activity, including decrease in fraction of plateau phase and slow wave amplitude. Total substitution of Cl- by isethionate or methyl sulphate reversibly shifted the repolarization potential by 3-4 mV in the positive direction and rapidly inhibited the electrical burst pattern without any initial stimulation. Glucose-induced (10 mM) insulin release (15 min) and 45Ca2+ uptake (3 min) were strongly inhibited by reducing the Cl- concentration to 10 mM (isethionate as substitute) and were further inhibited by further reduction of the Cl- concentration. It is suggested that beta-cells are equipped with on electrogenic Cl- flux, which can affect the burst pattern of electrical activity. The inhibitory effects of Cl- substitution may be explained by an influence of Cl- on the voltage-controlled Ca2+ channels.     

Pre- and postsynaptic effects of nicotine on the mouse phrenic nerve-diaphragm preparation

Nicotine at less than or equal to 33 microM enhanced the single twitch response to indirect stimulation but potentiated the blocking effect of tubocurarine. Failure of tetanic contraction (tetanic fade) occurred on stimulation at 100 Hz. At 76 microM, nicotine induced a first phase rapid (10 min) inhibition of twitch response followed later (60-90 min) by a second phase complete block. Neostigmine partially restored the response at either phase of block whereas diaminopyridine completely antagonized the blockade. The end-plate was depolarized maximally by only 10-15 mV within 30 min with 43 microM nicotine. The depolarization was maintained but was antagonized by tubocurarine. The twitch response induced by direct stimulation was unchanged indicating no depolarization block ensued. The amplitudes of both EPP (0.7 Hz) and MEPP were markedly depressed in parallel indicating a curare-like postsynaptic inhibition without an effect on the release of transmitter. It is concluded that nicotine blocks the neuromuscular transmission by a dual mechanism by its partial agonist action. At higher frequencies of transmission, nicotine (greater than or equal to 22 microM) also produced a remarkable run-down of EPP just like other receptor antagonists suggesting that the nerve terminal acetylcholine receptors are not particularly sensitive to nicotine as those on the autonomic ganglia.     

Analysis of the effects of firing rate and synchronization on spike-triggered averaging of multidirectional motor unit torque

Spike-triggered averaging (STA) of muscle force transients has often been used to estimate motor unit contractile properties, using the discharge of a motor unit within the muscle as the triggering events. For motor units that exert torque about multiple degrees-of-freedom, STA has also been used to estimate motor unit pulling direction. It is well known that motor unit firing rate and weak synchronization of motor unit discharges with other motor units in the muscle can distort STA estimates of contractile properties, but the distortion of STA estimates of motor unit pulling direction has not been thoroughly evaluated. Here, we derive exact equations that predict that STA decouples firing rate and synchronization distortion when used to estimate motor unit pulling direction. We derive a framework for analyzing synchronization, consider whether the distortion due to synchronization can be removed from STA estimates of pulling direction, and show that there are distributions of motor unit pulling directions for which STA is insensitive to synchronization. We conclude that STA may give insight into how motoneuronal synchronization is organized with respect to motor unit pulling direction. Action Editor: David Terman