荧光定量法测量平滑肌细胞内钙离子浓度的探讨

目的 研究平滑肌细胞内Ca²⁺浓度的动力学变化,建立一种定量测量细胞内Ca²⁺浓度的方法。方法 分离肠系膜细动脉血管平滑肌(ASMC)细胞,用荧光探针Indo-1和激光扫描共聚焦显微成像技术测定单个平滑肌细胞Ca²⁺浓度的动力学变化;首先在37 ℃环境下标定Ca²⁺ 探针indo-1的解离常数Kd值,根据荧光-浓度换算公式将测量的荧光强度值换算成Ca²⁺ 浓度值。结果 激光扫描共聚焦显微成像技术测量的细胞荧光图像分析显示,平滑肌细胞[Ca²⁺]_i 在地塞米松的刺激下显著上升,有时会出现自发钙波的现象,并且细胞内出现钙超载的现象(细胞荧光图像呈现白色)。通过测量得到的Kd值结合荧光强度-浓度换算公式可准确地测量细胞内[Ca²⁺]_i。结论 荧光定量法结合共聚焦显微成像技术可以简易、快捷地监测细胞内钙离子的动力学变化,不失为一种定量测量细胞内钙离子的方法。     

滨蒿内酯对豚鼠气管平滑肌细胞细胞内钙离子浓度的影响

目的：探讨滨蒿内酯（Scop)松弛豚鼠气管平滑肌的机制。方法：进行豚鼠气管平滑肌细胞分离。通过负荷钙离子荧光指示剂fluor-3-AM进行[Ca^2 ]i测定。结果：Scop可直接降低豚鼠气管平滑肌[Ca^2 ]ii，并且能抑制磷酸组织胺（His)和咖啡因（caffeine)对气管平滑肌[Ca^2 ]i的升高作用。结论：Scop松弛豚鼠气管平滑肌的主要作用机制是抑制细胞内钙浓度水平。     

组胺刺激的气道平滑肌细胞钙离子浓度和长度变化及机制

目的　观察组胺刺激后大鼠气道平滑肌细胞 (ASMC)内钙离子浓度 ( [Ca2 +]i)和长度的变化 ,并初步探讨其机制。方法　以Fluo 3/AM作为细胞内游离Ca2 +示踪剂 ,通过激光共聚焦显微镜观察不同浓度组胺对大鼠ASMC [Ca2 +]i和长度的影响 ,观察胞外Ca2 +、G蛋白、蛋白激酶C(PKC)、丝裂素活化蛋白 (MAPK)在组胺上述生理效应中的作用。结果　组胺刺激后 ,[Ca2 +]i变化呈现双相反应。组胺刺激后 [Ca2 +]i快速上升至峰值 ,随后稍下降 ,进入一个维持期。 [Ca2 +]i增加的峰值 ( 13.39%± 3.35 %、36 .35 %± 13.35 %、6 4 .6 8%± 11.4 2 % )与组胺浓度 ( 1× 10 -6、1× 10 -5、1× 10 -4mol/L)显著相关 (P <0 .0 5 )。无钙培养基不影响组胺刺激的 [Ca2 +]i上升 ,而在有钙培养基中加入乙二醇二乙醚二胺四乙酸 (EGTA)也不影响组胺刺激诱导的 [Ca2 +]i上升。随组胺浓度 ( 1× 10 -6、1× 10 -5、1× 10 -4mol/L)的上升 ,ASMC最大缩短程度 ( 2 3.14 %、37.6 2 %、4 1.75 % )仅有增加的趋势 (P >0 .0 5 ) ,但在同一组胺浓度下 ,其 [Ca2 +]i变化与细胞缩短程度显著相关 (P <0 .0 1)。百日咳毒素对组胺刺激ASMC [Ca2 +]i和长度变化均有明显的抑制作用。Staurosporine和PD0 980 5 9对组胺刺激的 [Ca2 +]i变化均无明显影响     

荧光比率法测量平滑肌细胞内的钙动力学变化

目的 建立一种荧光比率方法来测量细胞内钙离子的动力学变化，并研究血管平滑肌细胞钙动力学变化在重症休克血管反应性降低中的作用。方法 复制SD大鼠失血性休克模型，分离肠系膜细动脉血管平滑肌(ASMC)。使用荧光探针Fluo-3／AM、FuraRed双标记比率方法结合激光扫描共聚焦显微成像技术测定单个平滑肌细胞钙动力学变化；评估荧光比率法在动态测定胞内钙离子浓度变化中的应用及其观察ATP敏感钾通道(KATP)特异性阻滞剂优降糖对血管反应性和钙动力学影响。结果 休克后2h(失代偿期1，血管反应性明显降低，NE作用带来的平滑肌细胞内钙离子浓度升高的程度明显减弱；加入优降糖可明显提高NE对平滑肌细胞内钙离子的升高作用，改善细动咏对NE的反应性，带来血管反应性部分恢复。在37℃，Flu-o-3／AM、FuraRed浓度为5μmol／L的条件下，肠系膜细动脉平滑肌细胞负载40min左右即可获得良好的标记效果。结论 优降糖特异性的阻滞ATP敏感钾通道的开放，增多细胞外钙内流并提高血管反应性；使用荧光比率法可以使胞内钙离子浓度的测量不受荧光探针浓度、细胞大小、激光漂白等因素的影响，可以准确、实时、动态测量细胞内钙离子浓度变化。     

荧光比率法测量平滑肌细胞内的钙动力学变化

目的建立一种荧光比率方法来测量细胞内钙离子的动力学变化,并研究血管平滑肌细胞钙动力学变化在重症休克血管反应性降低中的作用.方法复制SD大鼠失血性休克模型,分离肠系膜细动脉血管平滑肌(ASMC),使用荧光探针Fluo-3/AM、FuraRed双标记比率方法结合激光扫描共聚焦显微成像技术测定单个平滑肌细胞钙动力学变化;评估荧光比率法在动态测定胞内钙离子浓度变化中的应用及其观察ATP敏感钾通道(KATP)特异性阻滞剂优降糖对血管反应性和钙动力学影响.结果休克后2 h(失代偿期),血管反应性明显降低,NE作用带来的平滑肌细胞内钙离子浓度升高的程度明显减弱;加入优降糖可明显提高NE对平滑肌细胞内钙离子的升高作用,改善细动脉对NE的反应性,带来血管反应性部分恢复.在37℃,Fluo-3/AM、FuraRed浓度为5μnol/L的条件下,肠系膜细动脉平滑肌细胞负载40 min左右即可获得良好的标记效果.结论优降糖特异性的阻滞ATP敏感钾通道的开放,增多细胞外钙内流并提高血管反应性;使用荧光比率法可以使胞内钙离子浓度的测量不受荧光探针浓度、细胞大小、激光漂白等因素的影响,可以准确、实时、动态测量细胞内钙离子浓度变化.     

兔实验性食管炎食管平滑肌细胞内钙离子浓度的变化

目的：观察兔急性实验性反应性食管炎与正常兔食管体部平滑骨细胞内钙离子浓度的变化。方法 ２０只成年新西兰大白兔随机分成两组,一组用０．２ｍｏｌ／Ｌ ＨＣｌ在距食管下括约机（ＬＥＳ）上方６ｃｍ处连续灌注兔食管４ｄ,１ｍＬ／ｍｉｎ,３０ｍｉｎ／ｄ。     

甲基汞导致细胞内钙离子浓度升高机制

汞中毒对人类的危害很大,尤以甲基汞为甚.甲基汞中毒可引起共济失调、视力模糊、听力受损和感觉异常等症状.有研究表明,甲基汞可使细胞膜上钙通道开放,细胞内钙离子浓度升高.本文结合钙离子在细胞内的循环通路和甲基汞的特性对其具体机制进行了探究.     

Ghrelin对大鼠主动脉血管平滑肌细胞内钙离子浓度的影响

目的：研究Ghrelin对大鼠胸主动脉血管平滑肌细胞（VSMCs）内游离钙离子浓度的影响,并揭示Ghrelin血管活性的调节机制。方法：组织贴块法培养大鼠胸主动脉VSMCs,Western blot验证生长激素促泌素受体（GHS-R1a）在VSMCs中的表达。钙荧光染料furo-2AM标记VSMCs,高速钙离子成像分析系统检测Ghrelin对静息状态胞内钙荧光强度（FI）的影响,并观察Ghrelin对KCl和AngII增强胞内钙FI作用的影响,以及腺苷酸环化酶抑制剂Sq22536和GHS-R1a拮抗剂（D-Lys3）-GHRP-6对Ghrelin作用的影响。结果：GHS-R1a在VSMCs内有表达。Ghrelin本身对VSMCs内钙FI无作用,也不能抑制KCl引起的VSMCs内钙FI升高,但可抑制AngII引起的VSMCs内钙FI升高,而这种抑制作用可被Sq22536和GHS-R1a逆转。结论：Ghrelin可抑制由AngII引起的大鼠主动脉VSMCs胞内钙离子的升高,机制可能和激活cAMP/PKA信号通路有关。     

哇巴因对大鼠血管平滑肌细胞内钙离子浓度的影响

目的探讨哇巴因对大鼠胸主动脉血管平滑肌细胞（vascular smooth muscle cells, VSMCs）内Ca2+浓度的影响。方法用组织
块贴壁法进行大鼠胸主动脉VSMCs原代培养,用免疫组织化学方法进行细胞鉴定,采用放射自显影术检测哇巴因对大鼠胸主动
脉VSMCs的结合能力,采用Fluo 3-AM（钙离子荧光探针）法研究哇巴因在短时间内对VSMCs细胞内Ca2+浓度的影响,探讨不同
浓度的钠泵α2亚单位截断性片段的拮抗作用。结果（1）放射自显影术检测结果显示,哇巴因与VSMCs间有一定的结合能力。
在0.1~100 nmol/L浓度范围,随着哇巴因浓度的增加,VSMCs与哇巴因的结合能力增加;（2）Fluo 3-AM检测VSMCs内Ca2+浓度
结果显示：不同浓度的哇巴因在短时间内能够引起VSMCs内Ca2+浓度的变化。在0~3200 nmol/L浓度范围,细胞内Ca2+离子浓
度会随着哇巴因浓度的增加呈现出逐渐升高的趋势;（3）不同浓度的钠泵α2亚单位截断性片段可以拮抗哇巴因引起的VSMCs内
Ca2+浓度的升高作用。结论哇巴因引起的细胞内高钙是高哇巴因高血压发生的细胞学基础,钠泵α2亚单位截断性片段可以拮
抗哇巴因引起的VSMCs内Ca2+浓度的升高作用,为进一步探讨哇巴因的作用机制结高血压的治疗提供了实验依据。
     

低密度脂蛋白对贮脂细胞内钙离子浓度的影响

研究低密度脂蛋白（ＬＤＬ）对大鼠肝脏贮脂细胞（ＦＳＣ）胞内钙离子浓度的影响及钙拮抗剂对其的干预作用。采用Ｆｕｒａ－２／ＡＭ标记法检测不同浓度ＬＤＬ（１０、２５、５０、１００、２００、５００μｇ／ｍｌ）对ＦＳＣ胞内Ｃａ＾２＋浓度峰值的主在细胞外液含钙或不含钙时，１００μｇ／ｍｌＬＤＬ剂量组对ＦＳＣ胞内Ｃａ＾２＋浓度的影响，同时测定钙拮抗剂尼卡地平和维拉帕米对ＬＤＬ引起增钙的拮抗作用，结果：ＬＤＬ能     

胃肠舒对大鼠胃肠平滑肌细胞钙离子浓度的影响

目的通过测定体外培养的SD大鼠胃肠平滑肌细胞内游离Ca^2＋浓度,观察胃肠舒对SD大鼠胃肠平滑肌细胞内游离Ca^2＋浓度的影响,探讨胃肠舒促胃肠动力的作用机制。方法离体培养SD大鼠胃肠平滑肌细胞,应用特异性Ca^2＋荧光探针Fura-2AM负载细胞,激光共聚焦显微镜检测游离Ca^2＋浓度。结果胃肠舒在25、50、100mg/ml时均可升高胃肠平滑肌游离Ca^2＋浓度,且随剂量增加而加强。结论胃肠舒具有升高胃肠平滑肌细胞游离Ca^2＋的作用,Ca^2＋作为细胞内第二信使可能参与了胃肠舒促进胃肠平滑肌细胞的动力机制。     

Batroxobin reduces intracellular calcium concentration and inhibits proliferation of vascular smooth muscle cells

Background Batroxobin (BX), a serine protease used in defibrinogenation and thrombolysis, also has an effect on c-fos gene and growth factor. This study attempted to determine the effects of BX on the proliferation of vascular smooth muscle cells (VSMCs) and calcium metabolism. Methods VSMCs were treated with BX at concentrations of 0.1, 0.3, or 1.0 mmol/L and cell numbers were determined at 0, 24, 48, and 72 hours. Intracellular calcium concentration (［Ca2+］i) was measured using direct fluorescence methods. Results BX was found to suppress proliferation of VSMCs in a dose-dependent fashion with inhibition rates of 18% and 31% by 48 and 72 hours, respectively. In addition, BX decreases basal ［Ca2+］i significantly. The basal level in untreated cells was 162.7±33.8 nmol/L, and decreased to 131.5±27.7 nmol/L, 128.3±28.5 nmol/L, and 125.6±34.3 nmol/L with the three concentrations of BX, respectively. Noradrenaline (NE)-induced ［Ca2+］i stimulation was also attenuated by BX (0.1 mmol/L BX, 20%±8% inhibition; 0.3 mmol/L BX, 54%±11% inhibition; 1.0 mmol/L BX, 62%±15% inhibition). The ability of NE to stimulate ［Ca2+］i was attenuated in cultures in Ca2+-free medium, as was the ability of BX to blunt NE-induced stimulation.Conclusion These findings demonstrate that BX can effectively inhibit proliferation of VSMCs, probably by blocking the release and uptake of Ca2+, thus influencing ［Ca2+］i.     

环匹阿尼酸对大鼠远端肺静脉平滑肌细胞内钙浓度的影响

目的研究环匹阿尼酸(CPA)对大鼠远端肺静脉平滑肌细胞(PVSMC)细胞内钙浓度([Ca2+]i)的影响及可能机制。方法原代培养大鼠远端PVSMC,并通过形态学和免疫荧光染色鉴定,利用荧光显微镜和InCyte细胞内钙浓度检测系统观测高钾(60 mmol/L KCl)溶液及CPA对PVSMC的[Ca2+]i影响。结果原代培养的PVSMC表现出典型血管平滑肌细胞生长特征,并对平滑肌α-actin免疫荧光染色呈阳性反应;高钾溶液使PVSMC的[Ca2+]i显著升高,5μmol/L硝苯地平能完全阻断PVSMC的[Ca2+]i对高钾溶液的反应;10μmol/LCPA能使含5μmol/L硝苯地平的无钙Krebs溶液孵育的PVSMC的[Ca2+]i短暂小幅升高,恢复细胞外Ca2+至2.5 mmol/L后,10μmol/L CPA能使含5μmol/L硝苯地平的Krebs溶液孵育的PVSMC的[Ca2+]i迅速升高。结论 CPA能够使大鼠远端PVSMC的[Ca2+]i升高,其机制可能与Ca2+从肌浆网释放,并诱发细胞外Ca2+通过钙池操纵性钙通道(SOCC)内流有关。     

大鼠气管平滑肌细胞的分离及L-钙通道的记录

目的 建立一种大鼠气管平滑肌细胞急性酶分离法并在分离的细胞上研究其L型钙通道(L-Ca)特性。方法 采用链酶蛋白酶分离大鼠气管平滑肌细胞，用膜片钳单通道记录法记录L-Ca通道电流。结果 该方法分离过程简单，所获细胞数量多，形态正常，在这些细胞上容易记录到正常的L-Ca通道电流。结论 应用本方法易得到单个活性正常的、形态完整的大鼠支气管平滑肌细胞。     

钙超载大肠系膜血管平滑肌细胞钙离子稳态调节的改变

目的 探讨钙超载模型大鼠血管平滑肌细胞（ＶＳＭＣ）胞内是否存在钙离子（Ｃａ＾２＋）稳态调节异常。方法 以Ｆｌｕｏ－３／ＡＭ作为Ｃａ＾２＋批示剂,应用激光扫描共聚焦显微镜技术,比较钙超载模型大鼠肠系膜ＶＳＭＣ（ＣａＯＭＶ）与正常Ｗｉｓｔａｒ大鼠肠系膜ＶＳＭＣ（ＷＭＶ）静息态的胞浆与核内游离Ｃａ＾２＋水平（〖Ｃａ＾２＋〗ｉ,〖Ｃａ＾２＋〗ｎ）,以及在多各药物刺激下Ｃａ＾２＋稳态调节的差异。结果 两种Ｖ     

丙泊酚对肺动脉平滑肌细胞跨膜钙离子移动影响的实验研究

目的 探讨丙泊酚对肺动脉平滑肌细胞跨膜钙离子移动的影响.方法 SD大鼠麻醉后迅速取出心肺,分离肺动脉平滑肌细胞.进行体外培养.应用Fluo-3/AM进行荧光标记.采用激光共聚焦显微镜观察并测定有或无丙泊酚时,氯化钾(KCI)以及去氧肾上腺素(PE)对细胞内钙离子浓度的影响.结果 KCI(60mmol/L)及PE(10 6mol/L)均能诱发体外培养的肺动脉平滑肌细胞内游离钙离子浓度的升高:加入50μmol/L丙泊酚后细胞荧光值由22.0±9.2降至16.4±8.0(P＜0.01).丙泊酚可显著抑制KCI及PE诱发的细胞内钙离子浓度的升高,荧光强度倍率分别由7.11±3.56降至3.43±0.79、1.35±0.14.结论 丙泊酚对肺动脉平滑肌细胞膜上钙离子通道存在一定的抑制作用.     

Mitofusin2 decreases intracellular cholesterol of oxidized LDL-induced foam cells from rat vascular smooth muscle cells

Mitofusin2 (Mfn2) plays a pivotal role in the proliferation and apoptosis of vascular smooth muscle cells (VSMCs). The purpose of this study was to investigate the effects of Mfn2 on the trafficking of intracellular cholesterol in the foam cells derived from rat VSMCs (rVSMCs) and also to investigate the effects of Mfn2 on the expression of adenosine triphosphate-binding cassette subfamily A member 1 (ABCA1), adenosine triphosphate-binding cassette subfamily G member 1 (ABCG1) and peroxisome proliferator-activated receptor gamma (PPARγ). The rVSMCs were co-cultured with oxidized low density lipoprotein (LDL, 80 μg/mL) to produce foam cells and cholesterol accumulation in cells. Before oxidized LDL treatment, different titers (20, 40 and 60 pfu/cell) of recombinant adenovirus containing Mfn2 gene (Adv-Mfn2) were added into the culture medium for 24 h to transfect the Mfn2 gene into the rVSMCs. Then the cells were harvested for analyses. The protein expression of Mfn2 was significantly higher in Adv-Mfn2-transfected group than in untransfected group (P<0.05), and the expression levels significantly increased when the titer of Adv-Mfn2 increased (P<0.05). At 24 or 48 h after oxidized LDL treatment, rVSMCs became irregular and their nuclei became larger, and their plasma abounded with red lipid droplets. However, the number of red lipid droplets was significantly decreased in Adv-Mfn2-transfected group as compared with untransfected group. At 48 h after oxidized LDL treatment, the intracellular cholesterol in rVSMCs was significantly increased (P<0.05), but it was significantly decreased in Adv-Mfn2-transfected group as compared with untransfected group (P<0.05), and it also significantly decreased when the titer of Adv-Mfn2 increased (P<0.05). The mRNA and protein expression levels of ABCA1 and ABCG1 were significantly increased in Adv-Mfn2-transfected group as compared with untransfected group (P<0.05). Though the mRNA and protein expression levels of PPARγ was not significantly increased (P>0.05), the phosporylation levels of PPARγ were significantly decreased in Adv-Mfn2-transfected group as compared with untransfected group (P<0.05). These results suggest that the transfection of Adv-Mfn2 can significantly reduce intracellular cholesterol in oxidized LDL-induced rVSMCs possibly by decreasing PPARγ phosporylation and then increasing protein expression levels of ABCA1 and ABCG1, which may be helpful to suppress the formation of foam cells.     

Effects of Iptakalim on intracellular free calcium concentration of cultured rabbit pulmonary arterial smooth muscle cells

Objective:To explore the effects of Iptakalim on intracellular free calcium concentration and on the proliferation of cultured rabbit pulmonary arterial smooth muscle cells induced by endothelin-1(ET-1) in vitro. Methods:A cell culture model, [3H]-thymidine([3H]-TdR) incorporation test and confocal microscope were used to observe proliferation and intracellular free calcium concentration([Ca2 ]i) of rabbit PASMC induced by ET-1 in vitro. Results:The value of [3H]-TdR incorporation in ET-1 group was increased 1.468 times higher than that in control group. Iptakalim at the concentration of 10-7mol/L,10-6 mol/L,10-5 mol/L lowered [3H]-TdR incorporation by (19.8±4.6)%, (41.2±9.5)%, (54.7±10.1)%, respectively, compared with the value of the cells treated with ET-1(P＜0.01); The intracellular fluorescence intensity of PASMC in ET-1 group was increased from 73.70±10.12 to 143.84±28.23, significantly higher than that in control group(P＜0.01); whereas with Iptakalim,the fluorescence intensity(FI) was only increased from 74.30±10.20 to 86.03±9.82, significantly lower than that in ET-1 group(P＜0.01). Conclusion:Iptakalim inhibited proliferation of PASMC and decreased intracellular free calcium concentration of cultured rabbit PASMC induced by ET-1.