Effects of Total Flavonoids ofHippophae RhamnoidesL.on Intracellular Free Calciumin Cultured Vascular Smooth Muscle Cells of Spontaneously Hypertensive Rats and Wistar-Kyoto Rats

To explore the effects of total flavonoids of Hippophae rhamnoides L. （TFH） quercetin （Que） and isorhamnetin （Isor） on the intracellular free calcium （[Ca^2＋]） in vascular smooth muscle cells （VSMC） of spontaneously hypertensive rats （SHR） and Wistar-Kyoto rats （WKY）. Metheds： Fluo 3-acetoxymethylester（Fluo-3/AM） was used to observe the effects of TFH （100mg/L） and its essential monomers, namely Que （10^-4mol/L） and Isor （10^-4mol/L） on changes of [Ca^2＋]1 in cultured SHR and WKY VSMC （abbr. to Ca-SHR ＆ Ca-WKY） following exposure to high K^＋, norepinephrine （NE） and angiotensin Ⅱ （AngⅡ）, and to compare with the effects of verapamil （Ver）. Results： （1） TFH, Que and Isor had inhibitory effects on resting Ca-SHR （P〈0.05）, but had no significant effects on Ca-WKY （P〉0.05）. （2） High K^＋ could increase Ca-SHR more significantly than Ca-WKY （P〈0.05）; TFH, Que and Isor could inhibit the elevation of [Ca^2＋]1 induced by high K^＋ -depolarization, with the effects similar to that of Ver, and the effect on Ca-SHR was more significant than that on Ca-WKY （P〈0.05）. （3） NE and Ang Ⅱ could increase Ca-SHR more significantly than Ca-WKY （P〈0.05）, TFH, Que and Isor had remarkably inhibitory effect on the elevation of Ca-SHR and Ca-WKY induced by NE or Ang Ⅱ. （4） In the absence of extracellular Ca^2＋ , TFH, Que and Isor also had certain inhibitory effect on Ca-SHR and Ca-WKY induced by NE, and the effect on the former was more significant than that on the latter（P〈0.05）. Ceaclusiea： TFH, Que and Isor might decrease the levels of [Ca^2＋], in VSMCs by blocking both voltage-dependent calcium channels （VDC） and receptoroperated calcium channels （ROC） in physiological or pathological state, which may be one of the important mechanisms of their hypotensive and protective effects on target organs in patients with hypertension.

Both Hypoxic Endothelial Cell Conditioned Medium and Hypoxia Elevate Intracellular Free Calcium in Pulmonary Artery Smooth Muscle Cells

By using Ca2+ -sensitive fluorescent probe, Fura-2 , the effects of endothelial cell-conditioned medium and hypoxia on intracellular free calcium ( [Ca2+]i) in cultured pulmonary artery smooth muscle cell (PASMC) were studied. Normoxic porcine pulmonary artery endothelial cell-conditioned medium (NPAECCM) obviously elevated [Ca2+]i in PASMC,whereas the hypoxic porcine pulmonary artery endothelial cell conditioned medium (HPAECCM)significantly elevated [Ca2+]i in PASMC much more than NPAECCM. Both the effects of NPAECCM and HPAECCM were dependent on the cultured endothelial cell extracellular calcium concentrations, ranged from 1.8 mmol/L to 2. 4 mmol/L.Meanwhile, hypoxia directly increased, which was partially inhibited by verapamil,[Ca2+]i in PASMC through Ca2+ influx pathway.The data suggest that the augmented regulation of endothelial cell on PASMC via Ca2+ second messenger system and the hypoxia-induced Ca2+ influx into PASMC,particularly the former, may be components of mechanisms underlying hypoxic pulmonary vasoconstriction and chronic pulmonary hypertension.

Effects of tetrandrine on cytosolic free calcium concentration in corpus cavernosum smooth muscle cells of rabbits

Aim:To study the relaxation mechanisms of tetrandrine(Tet)on the corpus cavernosum smooth muscle.Methods: The corpus cavernosum smooth muscle cells from New Zealand white rabbits were cultured in vitro.[Ca^(2+)]_i was measured by Fluorescence Ion Digital Imaging System,using Fluo-2/AM as a Ca^(2+)-sensitive fluorescent indicator. Results:Tet(1,10 and 100 μmol/L)had no effect on the resting [Ca^(2+)]_i(P>0.05).In the presence of extracellular Ca^(2+)(2.5 mmol/L),Tet(1,10 and 100 μmol/L)inhibited [Ca^(2+)]_i elevation induced by high K^+ and phenylephrine(PE) in a concentration-dependent manner(P<0.05).In calcium free solution containing egtaic acid,Tet(1 and 10 μmol/L) had no inhibitory effects on [Ca^(2+)]_i elevation induced by PE(P>0.05).However,Tet(100μmol/L)inhibited [Ca^(2+)]_i elevation induced by PE(P<0.05).Conclusion:Tet inhibited the Ca^(2+)influx from the extracellular site via voltage- activated Ca^(2+)channel and α_1-adrenoceptor-operated Ca^(2+)channel.At a high concentration,Tet might inhibit the cytosolic calcium pool release in cultured corpus cavernosum smooth muscle cells.This inhibitory action on [Ca^(2+)]_i might be one of the relaxation mechanisms of Tet on the corpus cavernosum smooth muscle.(Asian J Androl 2006 Jul;8:405-409)

ATP Evokes Calcium Transients in Single Pulmonary Artery Endothelial Cell in Primary Cultures

Effects of ATF on cytosclic free calcium ([Ca2+]i) in single porcine pulmonary artery endothelia cell were studied.Using a dual-wavelength excitation on microflurometry.it was found that ATP evoked a rapid transient in[Ca2+]i which was then followed by a maintained elevation of[Ca2+]i.The removal of extracellular Ca2+ abolished the maintained plateform, but exerted no obvious effect on the initial-transient.These results suggest that ATP stimulates both calcium release from intracellular calcium pool(s)and calcium influx across the Plasma membrane from extracellular space.

Effect of Interleukin-1β on the Elevation of Cytoplasmic Free Calcium of the Cultured Hippocampal Neurons Induced by L-Glutamate

To investigate the intracellular mechanism that interleukin 1β (IL 1β) facilitates epileptic seizure and neuronal damage, the effect of IL 1β alone or IL 1β plus glutamate (Glu) on the intracellular free calcium ([Ca 2+ ] i) of single cultured hippocampal neuron was examined by using EPC 9 light electricity measurement system. The results showed that IL 1β of different concentrations (5×10 3 U/L, 10×10 3 U/L, 20×10 3 U/L, 30×10 3 U/L, 50×10 3 U/L, 100×10 3 U/L) failed to affect the neuronal [Ca 2+ ] i, but IL 1β could facilitate the augmentation of neuronal [Ca 2+ ] i induced by Glu in a dose dependent pattern. MK 801 inhibited the effect of Glu on [Ca 2+ ] i, and also inhibited the effect of IL 1β on [Ca 2+ ] i induced by Glu, while verapamil did not influence the effect of Glu or IL 1β. It is concluded that IL 1β, as a neuromodulator, can facilitate the activation of NMDA receptor by Glu, induce the increase of intracellular calcium, which enhances the excitement of neuron.

Effect and Mechanism of Epidermal Growth Factor on Proliferation of GL15 Gliomas Cell Line

The effects of epidermal growth factor （EGF） on proliferation of G15 glioma cells and the possible mechanisms were investigated. GFAP and EGFR expression was detected by immunohistochemical method. After the cells were treated with EGF at different concentrations, cell count method was used to determine the proliferation of glioma cells, cell cycle and apoptosis were analyzed by flow cytometry （FCM）, and laser scan confocal microscope （LSCM） was used to measure the cytoplasmic free calcium. The results showed that GFAP was diffusedly expressed in GLI5 cells and EGFR was over-expressed. EGF at doses of ≤ 1 ng/mL could significantly stimulate cell proliferation, cells in phase G0/G1 decreased, and those in phase S increased. EGF at doses of 10 and 100ng/ml could inhibit the cell proliferation significantly, and the apoptosis ratio in high dose of EGF group was higher than in control group. EGF could significantly induce a quick rise of intracellular free calcium, but the peak value of intracellular free calcium activated by high dose of EGF was higher than by low dose of EGF. It was suggested that EGF had a dual effect on gliomas： low dose of EGF could stimulate the cell proliferation of gliomas, but high dose of EGF could induce the cell apoptosis and inhibit the proliferation of gliomas, which might be contributed to the difference of intracellular free calcium.

小檗碱抑制谷氨酸引起的新生大鼠脑细胞 c-fos 表达及游离钙的升高

目的：研究小檗碱（Ｂｅｒ）对谷氨酸（Ｇｌｕ）引起的新生大鼠脑细胞ｃ－ｆｏｓ表达及游离钙（〔Ｃａ２＋〕ｉ）变化的影响。方法：斑点杂交及Ｆｕｒａ－２技术。结果：Ｇｌｕ能诱导分离的脑细胞ｃ－ｆｏｓ的一过性高表达及〔Ｃａ２＋〕ｉ的显著升高。Ｂｅｒ１０μｍｏｌ·Ｌ－１显著抑制Ｇｌｕ诱导的脑细胞ｃ－ｆｏｓ的高表达，而尼莫地平则没有明显作用。Ｂｅｒ１～１００μｍｏｌ·Ｌ－１能剂量依赖地抑制Ｇｌｕ引起的〔Ｃａ２＋〕ｉ升高。结论：Ｂｅｒ的这种降低脑细胞ｃ－ｆｏｓ基因表达及〔Ｃａ２＋〕ｉ水平的作用可能是其治疗脑缺血性疾病的机制之一。

半枝莲对宫颈癌细胞钙信号系统的影响

为探讨半枝莲对Hela细胞胞内游离钙浓度的影响及其抗癌作用机理。利用荧光染料Fura 2在细胞内能与游离钙结合 ,在一定波长光的激发下可发出荧光的特性 ,根据荧光的强度来检测游离钙的变化 ,结果显示半枝莲对Hela细胞增殖的抑制作用不明显 ,但可通过促进细胞内储藏钙的释放和胞外钙离子的内流 。

灵芝多糖对小鼠T细胞胞浆游离Ca^(2+)浓度和胞内pH的影响

目的通过考察灵芝多糖（ＧＬＰ）对免疫细胞信号转导过程的影响，探讨ＧＬＰ免疫调节作用机制。方法采用激光扫描共聚焦显微镜（ＬＳＣＭ）技术，动态监测ＧＬＰ均一体组分ＧＬＢ７对小鼠Ｔ细胞胞浆游离Ｃａ２＋浓度（［Ｃａ２＋）和胞内 ｐＨ（［ｐＨ］ｉ）的影响。结果 ＧＬＢ７（２０ｍｇ·Ｌ－１）引起小鼠 Ｔ细胞中［Ｃａ２＋］；和［ｐＨ］；明显升高，１ｍｉｎ即分别升高至３３４．７０％±１６，４％（ｎ＝３）、１７１．６％ ± １０．４％（ｎ＝３），之后一直分别维持在该平台期，至 １０ ｍｉｎ仍维持高峰水平。加入ＥＣＴＡ和 ｖｅｒａｐａｍｉｌ处理后， １０ ｍｉｎ ＧＬＢ７ 引起 Ｔ细胞［Ｃａ２＋］；和［ｐＨ］；分别升高为 ２０２．４％± １３．６％（ｎ＝３）。１４０．２％±７．８％（ｎ＝３），与正常对照组比较差异有显著性（Ｐ＜０．０1）。以 ＥＧＴＡ和 ｖｅｒａｐａｍｉｌ处理后，再分别以 ｈｅｐ－ｅｒｉｎ和 ｐrocaine处理细胞 １０ ｍｉｎ，之后以 ＧＬＢ７刺激Ｔ细胞，１０ｍｉｎ［Ｃａ２＋］ｉ值分别为１５１．５％±９．４％（ｎ＝３）、１４３．２％± ８．１ ％（ ｎ＝ ３），与 ＥＧＴＡ和 ｖｅｒａｐａｍｉｌ处理组相比差异有显著性（ Ｐ＜ ０．０１）。

补体膜攻击复合物刺激血管内皮细胞胞浆游离钙离子浓度的变化

目的 在单个细胞水平 ,观测亚溶破补体膜攻击复合物 (MAC)诱导的血管内皮细胞 [Ca2 + ]i的动态变化。方法将原代培养的人脐静脉内皮细胞 ,以Fluo 4 /AM(1～ 6 μmol/L)和 0 .0 2 %PluronicF 12 7进行细胞钙荧光指示剂负载后 ,于 0℃组装亚溶破剂量的MAC。在 37℃条件下 ,以LSCM实时监测MAC沉积引起的胞浆钙荧光强度的变化。结果 MAC沉积后 ,多数细胞的钙荧光强度迅速增强 ,以后随时间的推移 ,升高的钙荧光逐渐下降 ,直至恢复至静息水平。定量分析选定的胞浆区域钙荧光强度的变化发现 ,不同细胞在 [Ca2 + ]i 的变化高度、持续时间和胞内钙振荡的特征等方面存在异质性。结论 MAC沉积到内皮细胞表面后 ,可诱导胞浆游离钙离子的浓度增高 ,且不同细胞 [Ca2 + ]i

蝙蝠葛碱拮抗缓激肽诱导人成神经细胞瘤细胞胞内钙升高的作用

目的 胞内钙离子浓度升高是Alzheimer病 (AD)显著的病理生理学变化之一 ,本研究讨论蝙蝠葛碱对缓激肽诱导人成神经细胞瘤细胞胞内钙升高的影响。方法 采用钙荧光探针Fura 2 /AM定量测试了蝙蝠葛碱 (Dau)对缓激肽诱导的人成神经细胞瘤细胞 (SH SY5Y)胞内钙升高作用的影响。结果 Dau可显著抑制缓激肽所致胞内钙离子浓度 ([Ca2 + ]i)升高 ,并存在剂量 效应关系。结论 Dau有抑制AD样 [Ca2 + ]i升高的作用 ,为AD的发病机制和防治提供了新的线索。

心脉龙注射液对大鼠缺氧-复氧心肌细胞内游离钙离子及脂质过氧化物的影响

目的:观察心脉龙注射液(XML)对大鼠缺氧及缺氧-复氧心肌细胞[Ca2+]及丙二醛(MDA)、超氧化物歧化酶(SOD)的影响,以进一步阐明其作用机制。 方法:采用Fura-2/AM及相应试剂盒测定该药对大鼠缺氧及缺氧-复氧心肌细胞|Ca2+|i,MDA和SOD的影响。结果:7.5×102μg/mL XML能升高缺氧及缺氧-复氧心肌细胞|Ca2+|i(P<0.01,P<0.05),此作用与去乙酰毛花苷相当3.75 × 102,1.5×103μg/mL XML则略降低心肌细胞|Ca2+|i(P>0.05),同时应用维拉帕米均不能抑制去乙酰毛花苷及XML各剂量组对细胞内|Ca2+|i的影响。 3.1 ×102,6.3×102,1.25×103μg/mL XML能升高缺氧及缺氧-复氧心肌细胞悬液的SOD含量(P<0.01,P<0.05),虽能降低MDA,但无统计学意义 结论:对缺氧及缺氧-复氧状态的衰竭心脏,在强心时要严格掌握XML的剂量,其强心作用与心肌细胞|Ca2+|i的升高有关,该药同时还具有一定的抗氧化作用。

大黄素对人增生性瘢痕成纤维细胞的增殖抑制及细胞内游离Ca^(2+)的作用

目的对大黄素的防治瘢痕的生物学作用进行初步探索,为瘢痕的防治开辟新的途径。方法采集我科住院患者的增生性瘢痕标本,进行人增生性瘢痕成纤维细胞培养,分别以不同浓度的大黄素作用于细胞,以MTT法测试药物处理后细胞增殖规律,并以激光共聚焦显微镜观察细胞内[Ca2+]i荧光强度的变化。结果在不同浓度组大黄素的处理下,人增生性瘢痕成纤维细胞的增殖呈现出抑制的趋势(P<0.05);细胞内[Ca2+]i的荧光强度也明显上升,其幅度和药物浓度有一定的关联。结论大黄素对人增生性瘢痕成纤维细胞有显著的抑制作用,其机制可能和细胞内[Ca2+]i水平的改变有关。

苦参碱的正性肌力作用与心肌细胞内游离钙的关系

在离体豚鼠工作心脏上，观察苦参碱对心功能的作用。研究表明，苦参碱０．１、１．０、１０μｍｏｌ·Ｌ－１可浓度依赖地增强心脏的收缩功能，增加冠脉流量，但抑制舒张功能。苦参碱０．０１、０．１、１．０μｍｏｌ·Ｌ－１可浓度依赖性地增加单个乳鼠心肌细胞［Ｃａ２＋］ｉ浓度。提示苦参碱的正性肌力作用与其升高［Ｃａ２＋］ｉ有关。

白术多糖调控钙离子以促进细胞迁移及E-钙黏蛋白表达的研究

目的观察白术多糖对大鼠小肠上皮细胞(IEC-6)钙离子水平、细胞迁移和E-钙黏蛋白表达的影响,探讨益气健脾中药白术促进胃肠黏膜损伤修复的作用机制。方法:流式细胞仪检测白术多糖对细胞游离钙离子水平([Ca^(2+)]cyt)的影响;划痕法复制细胞迁移模型,观察白术多糖对细胞迁移的影响;Western blot法和免疫荧光法检测白术多糖对细胞E-钙黏蛋白表达的影响。结果白术多糖(25,50,100 mg·L^(-1))可增加细胞[Ca^(2+)]cyt、促进细胞迁移、提高E-钙黏蛋白表达,与正常对照组比较,差异有统计学意义(P<0.05,P<0.01);可逆转多胺合成抑制剂α-二氟甲基鸟氨酸(DFMO)所致的[Ca^(2+)]cyt降低、细胞迁移抑制和E-钙黏蛋白表达减少,与DFMO组比较,差异有统计学意义(P<0.05,P<0.01)。结论白术多糖可通过提高细胞钙离子水平以促进细胞迁移和E-钙黏蛋白表达,为探讨益气健脾中药白术修复胃肠黏膜损伤的作用机制提供参考。

阿魏酸钠防护晶状体上皮细胞氧化损伤的信号转导机制

目的:探讨阿魏酸钠对氧化损伤的晶状体上皮细胞内Ca2 +、环磷酸腺苷(cAMP)、环磷酸鸟苷(cGMP)的影响,从细胞信号转导角度揭示天然药物防治白内障的细胞和分子学机制。方法:采用牛晶状体上皮细胞进行晶状体上皮细胞(LEC)原代和传代培养,以含有过氧化氢(H2 O2 )的培养液孵育LEC复制氧化损伤模型,并加入阿魏酸钠单体共同孵育。分别采用四甲基偶氮唑兰(MTT)比色测定法观察在不同时间和浓度条件下LEC活性变化,采用荧光分光光度计测定不同时间细胞内钙离子浓度([Ca2 +]i)以及放射免疫分析法测定不同时间LEC内cAMP、cGMP的含量变化。结果:H2 O2 组可引起LEC吸光度值(A)明显下降(P <0 .0 1) ,阿魏酸钠能明显增强氧化损伤的LEC活性,并呈剂量 效应关系和时间 效应关系。氧化损伤的LEC[Ca2 +]i 升高(P<0 .0 1) ;阿魏酸钠可以降低由H2 O2 引起的细胞[Ca2 +]i 的升高,并呈时间 效应关系。H2 O2 组cAMP浓度升高;cGMP浓度下降(P <0 .0 1) ;阿魏酸钠使cAMP水平下调,cGMP水平上升(P <0 .0 1) ,并呈时间 效应关系。结论:阿魏酸钠可明显抑制LEC氧化损伤及凋亡,其作用机制可能是通过钙信号系统、cAMP信号系统、cGMP信号系统及其相互作用来调节生物学效应。

积雪草总苷对大鼠系膜细胞游离钙的影响

目的:观察积雪草提取物对大鼠肾小球系膜细胞(MC)增殖和钙离子的影响。方法:将血管紧张素Ⅱ(AngⅡ)作用于肾小球系膜细胞,加入不同浓度的积雪草提取物,采用MTT法和Fura-2/AM荧光指示剂负载测定MC增殖和细胞内[Ca2+]I的变化。结果:MC一经AngⅡ刺激后增殖显著,并且Ca2+浓度明显升高,而积雪草提取物对AngⅡ刺激下的MC增殖和Ca2+水平有抑制和降低作用,并呈一定的剂量依赖性。结论:积雪草提取物可能通过降低MC内Ca2+的水平,从而达到抑制MC增殖的目的。

SH－SY5Y细胞胞内钙库特性研究

运用单细胞显微荧光测量技术测量了单个ＳＨ—ＳＹ５Ｙ细胞内游离钙离子浓度的变化。首次报道了ＳＨ—ＳＹ５Ｙ细胞内存在毒蕈碱敏感而非咖啡因敏感的钙库，并研究了它的动力学特征。Ｎ￣￣Ｃ￣ｈｅｔｗＭａｄＵｇｈ＊ＴＯｗｈｏｍＣｂ￣ｐｏｎｄｅｎｅｅｓｈｏｕｌｄｂｅ￣．

钙通道活性与胃泌素促人结肠癌细胞增殖间关系的研究

目的 探讨钙通道活性在胃泌素促人结肠癌细胞增殖中的作用。方法 运用流式细胞仪检测细胞内游离钙离子水平 ,MTT比色法测定细胞增殖情况。结果 2 5× 10 -6M的五肽胃泌素 (PG)可以引起HT2 9细胞内游离钙离子水平明显升高 (P <0 0 1) ,钙通道阻滞剂nifedipine(10 -5M)可以使PG引起的钙离子水平升高幅度明显下降 (P <0 0 1)。同时可以部分抑制PG促增殖作用 (P <0 0 5 )。钙通道激动剂Bayk86 4 4 (10 -6M)可以明显升高细胞内钙离子水平 (P <0 0 1)。并能促进HT2 9细胞增殖 (P <0 0 5 ) ,与PG的促增殖作用相比无显著性差异 (P >0 0 5 )。结论 钙通道作为调节细胞内钙离子变化的主要途径 ,在调控PG促HT2