Ki: 220/300 nM (ROCK-I/II)[1]

Y-27632具有以剂量依赖方式诱导ADSCs神经元样分化的效力。此外，Y-27632诱导的分化在停药后恢复。用Y-27632处理的ADSCs表达神经元标志物，如NSE、MAP-2和巢蛋白，而未处理的ADSC不表达这些标志物。结论：选择性ROCK抑制剂Y-27632可以增强ADSCs的神经元样分化，表明Y-27632可用于诱导ADSCs向神经元分化，促进ADSCs在组织工程中的临床应用[2]。 Y-27632 在 BrdU 标记出现时的浓度抑制延迟增加了 100 倍，并且延迟持续时间延长。在分别用 10 和 100 μM Y-27632 处理的 Swiss 3T3 细胞中，观察到约 1 和 4 小时的延迟 [1]。 Y-27632 在神经元口腔中促进脂肪组织源性干细胞 (ADSC) 的生长。与 1.0 和 2.5 μM Y-27632 诱导组相比，5.0 μM Y-27632 诱导组具有最多数量的神经样细胞 [2]。在上皮细胞中，肌动蛋白会重塑并上调细胞因子[8]。

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Y-27632[（+）-（R）-反式-4-（1-氨基乙基）-N-（4-吡啶基）环己烷甲酰胺+++二盐酸盐]被广泛用作Rho相关卷曲卷曲形成蛋白丝氨酸/苏氨酸激酶（ROCK）家族蛋白激酶的特异性抑制剂。本研究考察了Y-27632和相关化合物Y-30141[（+）-（R）-反式-4-（1-氨基乙基）-N-（1H-吡咯并[2,3-b]吡啶-4-基）环己酰胺二盐酸盐]的抑制机制和作用特征。Y-27632和Y-30141在体外抑制了ROCK-I和ROCK-II的激酶活性，这种抑制作用被ATP以竞争方式逆转。这表明这些化合物通过与催化位点结合来抑制激酶。通过K（i）值确定的它们对ROCK激酶的亲和力比另外两种Rho效应激酶（柠檬酸激酶和蛋白激酶PKN）的亲和力高至少20至30倍。[（3）H]Y-30141以温度、时间和可饱和的方式被细胞吸收，这种吸收与未标记的Y-27632竞争。没有发现集中的积累，这表明摄取是载体介导的促进扩散。Y-27632在10微M时消除了瑞士3T3细胞中的应力纤维，但在此浓度下，细胞周期和胞质分裂的G（1）-S相变几乎不受影响。Y-30141在抑制激酶活性和应激纤维形成方面的效力是Y-27632的10倍，并且在10微M时导致G（1）-S转换和胞质分裂抑制的显著延迟。[1]

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Y-27632具有以剂量依赖方式诱导ADSCs神经元样分化的效力。此外，Y-27632诱导的分化在停药后恢复。用Y-27632处理的ADSCs表达神经元标志物，如NSE、MAP-2和巢蛋白，而未处理的ADSC不表达这些标志物。 结论：选择性ROCK抑制剂Y-27632可以增强ADSCs的神经元样分化，表明Y-27632可用于诱导ADSCs向神经元分化，促进ADSCs在组织工程中的临床应用 对人类诱导多能干细胞（hIPSC）分化的强有力控制对于实现其针对患者的治疗潜力至关重要。在这里，我们展示了小分子Rho相关蛋白激酶（ROCK）抑制剂Y-27632的一种新应用，以谱系特异性方式显著影响hIPSC的分化。Y-27632在hIPSC上的应用导致肌动蛋白捆绑减少，集落形成以浓度和时间依赖的方式中断。细胞和集落形态的这种变化与细胞-细胞连接蛋白E-钙粘蛋白的表达降低有关，与Y-27632暴露量的增加成正比。有趣的是，多能性标记物如NANOG和OCT4的基因和蛋白质表达在暴露于Y-27632长达36小时后没有下调。同时，上皮间充质（EMT）过渡标志物随着Y-27632的暴露而上调。随着Y-27632暴露时间的延长，细胞中的这些EMT样变化导致随后向中胚层谱系分化的效率显著提高。相比之下，当细胞在长期暴露于Y-27632后经历外胚层分化时，观察到抑制作用。总的来说，这些结果提出了一种新的方法，通过简单应用Y-27632来启动hIPSC以调节其分化潜力。[5]

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c-kit+CSC在CSC培养基中培养3-5天，然后单独用0至10μM Y-27632、0至1.0μM Dox或Y-27632和Dox处理48小时。检测细胞活力、毒性、增殖、形态、迁移、Caspase-3活性、凋亡相关关键蛋白和c-kit+的表达水平。结果显示，单独用Y-27632处理48小时后，c-kit+表达、增殖、Caspase-3活性或凋亡没有发生太大变化；然而，细胞活力显著增加，细胞迁移得到促进。这些效应可能涉及ROCK/Actin通路。相比之下，单独使用Dox治疗48小时会显著增加Caspase-3活性，导致细胞死亡。尽管单独使用Y-27632在基础条件下不影响凋亡相关关键因子（p-Akt、Akt、Bcl-2、Bcl-xl、Bax、切割的Caspase-3和Caspase-3）的表达水平，但它显著抑制了Dox诱导的切割的Caspase3的增加，并减少了Dox处理下的细胞死亡。 结论：我们得出结论，用Y-27632预处理人CSCs可显著降低Dox诱导的细胞死亡，并可能涉及切割的Caspase-3和ROCK/Actin通路。Y-27632的有益作用可应用于基于干细胞的治疗，以提高移植后的细胞存活率，或作为Dox治疗的癌症患者的心脏保护剂。[6]

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RhoA/ROCK信号通路的激活已被证明有助于胚胎和神经干细胞的解离诱导凋亡。我们之前已经证明，在40个Lin（-）Sca-1（+）CD49f（高）（LSC）前列腺基底上皮细胞中，大约有1个具有干细胞自我更新和多谱系分化的能力。我们在这里表明，在体外前列腺集落试验中，用ROCK激酶抑制剂Y-27632处理LSC细胞可将其克隆效率提高8倍。Y-27632治疗允许前列腺集落细胞有效地重新生长，否则不会发生这种情况。Y-27632还提高了前列腺球试验和分离前列腺细胞再生试验中前列腺干细胞的克隆效率。克隆效率的提高是由于抑制了RhoA/ROCK活化介导的前列腺干细胞的解离诱导凋亡。前列腺上皮细胞与细胞外基质的分离增加了PTEN活性并减弱了AKT活性。单独的Y-27632处理足以抑制细胞解离诱导的PTEN活性激活。然而，这并没有提高克隆效率，因为Y-27632处理在类似程度上增加了野生型和Pten-null前列腺细胞的球体形成单位。最后，敲低两种ROCK激酶的表达略微提高了前列腺集落细胞的重铺效率，证实了它们在Y-27632介导的克隆效率提高中起着重要作用。我们的研究表明，根据目前采用的检测方法，具有干/祖细胞活性的前列腺细胞的数量可能被低估，支持解离诱导的凋亡是具有上皮表型的胚胎和体细胞干细胞的共同特征，并强调了环境线索对干细胞维持的重要性。[7]

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在用胶原蛋白（COL）或LPA刺激细胞之前，在0、0.03、0.3、3或10微M的ROCK抑制剂Y27632存在下培养整片禽胚胎角膜上皮。通过Caspase-3活性测定评估细胞凋亡，并用膜联蛋白V结合进行可视化。ROCK抑制剂以剂量依赖的方式阻断了肌动蛋白皮质垫的重建，破坏了基底细胞侧膜，并增加了凋亡标志物膜联蛋白V。此外，使用体外半胱氨酸天冬氨酸蛋白酶-3活性测定来确定，用10微M Y-27632处理的上皮细胞中的半胱氨酸天冬氨酰蛋白酶-3活性高于没有基底膜的上皮细胞或用纤维连接蛋白、COL或LPA刺激的上皮细胞。总之，ECM分子降低了凋亡标志物，抑制了ROCK通路，阻断了ECM刺激的肌动蛋白皮质垫的重建，增加了胚胎角膜上皮细胞的凋亡[8]。

与盐水组相比，Y-27632（5 和 10 mg/kg）显着延长了肌阵挛发作的发作时间。当给予 Y-27632（5 和 10 mg/kg）时，阵挛性惊厥的发作与盐水组相比显着延迟 [3]。与对照组（SS 组）相比，用二甲基亚硝胺（DMN）治疗的大鼠的肝脏重量和体重（DMN-S 组）显着减少。通过口服 Y27632 (30 mg/kg)，大鼠（DMN-Y 组）基本上免受 DMN 引起的体重和肝脏重量损失[4]。

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Y27632治疗显著降低了DMN诱导的肝纤维化的发生率，降低了肝脏中胶原蛋白和羟脯氨酸含量以及α-SMA表达。[3]

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Y27632可防止DMN引起的身体和肝脏重量减轻[3]
口服Y27632对腹腔注射和不注射DMN的大鼠体重和肝脏重量的影响如图1和表1所示。与对照组动物（S-S组）相比，DMN治疗导致大鼠体重和肝脏重量显著降低（DMN-S组）。口服Y27632基本上阻止了DMN诱导的大鼠体重和肝脏减轻（DMN-Y组）。S-S/S-Y组和DMN-Y组之间没有显著差异。第四周结束时，DMN-S组和DMN-Y组的血清ALT水平没有显著差异。

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Y27632降低肝脏胶原蛋白和羟脯氨酸含量[3]
胶原测定证实，Y27632治疗组（DMN-Y组）的肝胶原沉积显著减少（DMN-S为808.5±122.4μg/100mg，而DMN-Y为601.3±67.7μg/100mg，P<0.05）（图3A）。肝纤维化也通过测量肝羟脯氨酸来定量。在这里，发现DMN治疗组（DMN-S）的羟脯氨酸含量显著高于DMN和Y27632治疗组（DMP-Y）（DMN-S为5.95±1.41μmol/100mg，而DMN-Y为2.98±0.91μmol/100mg）（图3B）。此外，DMN未治疗组（S-S、S-Y）和DMN和Y27632治疗组（DMN-Y）的胶原蛋白和羟脯氨酸含量没有显著差异。这些结果表明，Y27632治疗几乎完全预防了DMN诱导的肝纤维化。

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Y27632抑制I型胶原mRNA表达[3]
为了评估Y27632处理对I型胶原mRNA转录的抑制水平，我们通过半定量RT-PCR检测了DMN处理肝脏中α1（I）胶原mRNA的水平。在对照组（S-S组）中，即使只有0.02 fg的竞争对手，在α1（I）胶原蛋白的竞争性RT-PCR中也检测到主要竞争对手特异性带和较弱的α1（Ⅰ）胶原蛋白特异性带（图4A，泳道4）。相比之下，在DMN-S组中，DMN治疗（在没有Y27632的情况下）使α1（I）胶原基因的转录比基础表达（S-S）增强了100多倍，即使在存在2fg竞争对手的情况下，也会产生一条代表α1（Ⅰ）胶原产物的条带（图4B，泳道2）。与DMN治疗相比（在没有Y27632的情况下），Y27632治疗导致了更强烈的竞争带；在0.02fg竞争对手存在的情况下，α1（I）胶原PCR产物的强度与竞争对手相同（图4C，泳道4）。这些结果表明，Y27632治疗可将肝脏中α1（I）胶原mRNA的表达抑制到DMN诱导表达的约10%水平。

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法舒地尔和Y-27632对急性PTZ注射诱导的肌阵挛发作的影响[4]
法舒地尔和Y-27632对肌阵挛发作的影响如图1所示。剂量为25 mg kg−1，法舒地尔显著延长了单剂量PTZ（65 mg）诱导的肌阵挛发作时间 与生理盐水组相比（p<0.05）。然而，在5的剂量下，它没有改变发病时间 mg kg−1。此外，在5和10的剂量下 mg 与生理盐水组相比，Y-27632显著延长了肌阵挛发作时间（P<0.05）。

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法舒地尔和Y-27632对急性PTZ注射诱发的阵挛性惊厥发作的影响[4]
法舒地尔和Y-27632对阵挛性惊厥发作的影响如图1所示。剂量为25 mg kg−1，法舒地尔显著延长了单剂量PTZ（65 mg）诱导的阵挛性惊厥的发作时间 与生理盐水组相比（p<0.05）。然而，在5的剂量下，它没有改变发病时间 mg kg−1。此外，在5和10的剂量下 mg 与生理盐水组相比，Y-27632显著延长了阵挛性惊厥的发作时间（P<0.05）。

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法舒地尔和Y-27632对急性PTZ注射诱导的强直性后肢伸展的影响[4]
PTZ治疗组中，十分之七的小鼠进行了强直性后肢伸展，随后死亡。然而，两种剂量的法舒地尔都能防止强直性后肢伸展和死亡的发生（数据未显示）。与法舒地尔类似，两种剂量的Y-27632也能防止强直性后肢伸展和死亡的发生（数据未显示）。

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法舒地尔和Y-27632对MES系列强直性后肢伸展持续时间和翻正反射恢复潜伏期的影响[4]
两种法舒地尔（25mg 千克-1）和Y-27632（5和10 mg kg−1）与生理盐水治疗组相比，显著缩短了单次应用MES诱导的强直性后肢伸展的持续时间（图2，P<0.05）。此外，与生理盐水组相比，Rho激酶抑制剂法舒地尔和Y-27632也显著降低了翻正反射的恢复潜伏期（图2，P<0.05）。

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急性单次给予法舒地尔或Y-27632对PTZ点燃小鼠肌阵挛发作和阵挛性惊厥的影响[4]
法舒地尔（25mg 千克-1）和Y-27632（5和10 mg kg−1）与生理盐水组相比，改变了肌阵挛抽搐和阵挛性惊厥的发作时间（图3）。

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反复服用法舒地尔或Y-27632对PTZ点燃发展的影响[4]
反复服用法舒地尔或Y-27632对PTZ点燃发展的影响如图4所示。通过组间相互作用注射PTZ对生理盐水有显著影响，25 mg kg−1法舒地尔和5 mg Y-27632组（P<0.05）。如图4所示，法舒地尔不能阻止PTZ点燃的发展，但与生理盐水组相比，它通过降低平均发作阶段，在PTZ点燃发展的第2、3和4天产生了显著影响（P<0.05）。与法舒地尔不同，Y-27632更有效，与生理盐水组相比，从第2天开始，通过降低平均发作阶段，对PTZ点燃的发展有显著影响（P<0.05）。

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法舒地尔和Y-27632对旋转杆性能的影响[4]
法舒地尔（25mg 千克-1）和Y-27632（5和10 mg kg−1）在20℃时，通过旋转杆的性能评估电机协调性发生了变化 下午3点（表1）。

重组ROCK-I、ROCK-II、PKN或柠檬酸激酶通过使用Lipofectamine转染在HeLa细胞中表达为Myc标记蛋白，并通过使用与G蛋白琼脂糖偶联的9E10单克隆抗Myc抗体从细胞裂解物中沉淀出来。在30°C下，在不存在或存在不同浓度Y-27632或Y-30141的情况下，将回收的免疫复合物与不同浓度的[32P]ATP和10 mg组蛋白2作为底物在总体积为30μL的激酶缓冲液中孵育30分钟，该缓冲液含有50 mM HEPES NaOH、pH 7.4、10 mM MgCl2、5 mM MnCl2、0.02%Briji 35和2 mM二硫苏糖醇。在30°C下，在含有50 mM Tris-HCl、pH 7.5、0.5 mM CaCl2、5 mM乙酸镁、25μg/mL磷脂酰丝氨酸、50 ng/mL 12-O-十四烷酰佛波醇-13-乙酸酯和0.001%亮肽的激酶缓冲液中，在不存在或存在不同浓度Y-27632或Y-30141的情况下，将PKCa与5μM[32P]ATP和200μg/mL组蛋白2作为底物孵育10分钟，总体积为30μL。通过加入10μL的43 Laemmli样品缓冲液终止培养。煮沸5分钟后，将混合物在16%凝胶上进行SDS-聚丙烯酰胺凝胶电泳。凝胶用考马斯亮蓝染色，然后干燥。切除与组蛋白2型相对应的条带，并测量放射性[1]。

Y-27632是Rho相关卷曲激酶（ROCK）的特异性抑制剂，已被证明可以促进多种细胞类型的存活和诱导分化。然而，Y-27632对成人脂肪组织来源干细胞（ADSCs）的影响尚不清楚。本研究旨在探讨Y-27632对ADSCs神经元样分化的影响。 方法：从接受整形手术的女性中分离出ADSCs并进行培养。用不同剂量的Y-27632处理ADSCs，并在显微镜下观察形态学变化。通过免疫细胞化学和蛋白质印迹分析检测Y-27632处理的ADSCs中巢蛋白、神经元特异性烯醇化酶（NSE）和微管相关蛋白-2（MAP-2）的表达[2]。 HeLa细胞以每3.5cm培养皿3×104个细胞的密度铺板。在10mM胸苷存在下，将细胞在含有10%FBS的DMEM中培养16小时。用含有10%FBS地DMEM洗涤细胞后，再培养8小时，然后加入40ng/mL的诺考达唑。在诺考达唑治疗11.5小时后，加入不同浓度的Y-27632（0-300μM）、Y-30141或载体，并将细胞再孵育30分钟[1]。

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从接受整形手术的女性中分离出ADSCs并进行培养。用不同剂量的Y-27632处理ADSCs，并在显微镜下观察形态学变化。通过免疫细胞化学和蛋白质印迹分析检测Y-27632处理的ADSCs中巢蛋白、神经元特异性烯醇化酶（NSE）和微管相关蛋白-2（MAP-2）的表达。[2]

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细胞分离和培养[3]
如前所述，分离并培养未接受DMN或Y-27632的雄性Wistar大鼠肝脏的HSC。本研究中描述的实验是在第二和第四系列通道之间的HSC上进行的。在一些实验中，HSC在第一次传代之前使用。

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α-SMA的蛋白质印迹分析[3]
HSC在6cm塑料组织培养板中以1×106个细胞/ml的密度用DMEM（20%FCS）培养。更换培养基后，HSC在有或没有Y-27632的情况下得以维持。全细胞裂解物的制备和蛋白质印迹分析如前所述进行。

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免疫细胞化学[3]
HSC在有或没有Y-27632（30μM）的情况下维持2天。免疫细胞化学如前所述。载玻片用FITC偶联的山羊抗小鼠IgG进行α-SMA染色。

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为了提高初始细胞存活率，将hIPSC在ROCK抑制剂Y-27632存在下以10μM接种12小时，然后将培养基换成常规维持培养基mTeSR™1再接种12小时以获得典型的hIPSC细胞/集落形态。在接下来的预培养期间，细胞暴露于不同浓度和暴露时间的Y-27632中，随后进行基因和蛋白质分析。[5]

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钙黄绿素AM细胞活力测定[6]
在96孔板中培养2天后，用Y-27632（0.0、0.1、1.0和10.0μM）或阿霉素（0.0、0.2、0.4、0.6、0.8和1.0μM）处理细胞48小时，然后用Ham's F12洗涤，并在37°C的黑暗中用2μM钙黄绿素AM孵育30分钟。荧光显微镜和Spectra Max平板阅读器都用于获得染色后的原始荧光图像或相对荧光强度（RFI）。钙黄绿素阳性细胞（绿色）对应于荧光图像上的活细胞。来自平板读数器的RFI用于计算药物治疗组与对照组的比率或倍数变化。

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Caspase-3活性分析[6]
QuantiFluo Caspase-3试剂盒用于测定Caspase-3活性。简而言之，在用Y-27632（0.0、0.1、1.0、10.0μM）、Dox（0.0、0.2、0.4、0.6、0.8、1μM）或Y-27632（0.0、0.1%、1.0、10.0M）加Dox（0.6μM）处理2天后，收集了贴壁和漂浮（死亡）细胞。将细胞颗粒重新悬浮在冰上的200μl裂解缓冲液（50mM HEPES，100mM NaCl，0.5%Triton X-100，pH7.2）中30分钟，然后将细胞悬浮液在-80°C下快速冷冻直至使用。使用Bradford试剂测定蛋白质含量。为了测量Caspase-3活性，将含有20μg总蛋白的细胞提取物等分试样在含有20mM HEPES（pH7.4）、0.1%CHAPS、5mM DTT、2mM EDTA和20μM Caspase底物N-乙酰基-Asp-Glu-Val-Asp-7氨基-4-甲基香豆素（Ac-DEVD-AMC）的反应缓冲液中在室温（RT）下孵育2小时。使用96孔Spectra-Max荧光板阅读器在Ex 360nm和Em 460nm下测量释放的AMC作为RFI。

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膜联蛋白V和丙啶（PI）的凋亡和坏死检测[6]
在12孔板中培养的CSCs用10μMY-27632或Y-27632加0.6μM Dox再处理2天。收集贴壁细胞和漂浮（死亡）细胞，并使用Annexin V试剂盒进行凋亡和坏死分析。简而言之，用冷PBS洗涤细胞两次，在室温下用100ul Annexin V-FITC缓冲液（10mM HEPES、150mM NaCl、5mM KCl、1mM MgCl2、2.5mM CaCl2，补充5μg/ml PI、5μg/ml Hoechst 33342和1:40稀释的Annexin V-FITC，pH7.4）在黑暗中孵育15分钟。用1X结合缓冲液（10M HEPES、150M NaCl、5mM KCl、1mM MgCl2、2.5M CaCl2、pH7.4）洗涤两次后，使用Cytospin 4离心机将细胞在载玻片上进行细胞纺丝。荧光图像用EVOS显微镜 拍摄或用BD FACSAIR流式细胞仪运行。当膜联蛋白V-FITC与碘化丙啶（PI）（一种重要的染料）结合使用时，它能够区分凋亡细胞（膜联蛋白-V-FITC阳性，PI阴性）和坏死/晚期凋亡（膜联素-VITC阳性，P1阳性）细胞。

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使用C-kit抗体进行免疫细胞化学染色[6]
在10μMY-27632存在下培养4天后，用4%PFA固定细胞，然后在室温下用1.5%BSA和0.2%吐温的PBS溶液封闭。将1:100的第一c-kit+抗体在4°c下孵育过夜，然后将第二抗体（1:1000的Alex Fluor 488）和赫斯特在室温下共同孵育2小时。使用EVOS显微镜获得荧光图像。根据用Hoechst染色的细胞核总数计算C-kit+细胞。每张图像大约计数100-200个细胞核，每个实验取6张图像的平均值进行统计分析。

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使用EdU试剂盒进行细胞增殖检测[6]
根据制造商的方案，使用Click-iT EdU Alexa Fluor 488试剂盒测定CSC增殖。简而言之，在12个孔组中用Y-27632治疗2天后，将10μM终浓度的EdU（5-乙炔基-2'-脱氧尿苷）加入对照（0μM Y27632）或治疗（10μM Y-27632）孔中，一式三份。在37°C的培养箱中额外培养16小时后，用温热的PBS洗涤细胞，并在室温下用4%PFA和0.5%Triton X-100固定/渗透20分钟。用250μl Click-iT反应混合物（1X Click-iT反应缓冲液、CuSO4、Alexa Fluor 488叠氮化物和反应缓冲液添加剂）将细胞连续在黑暗中保持30分钟。用3%BSA和PBS洗涤后，用DAPI对细胞核进行复染，并使用EVOS荧光显微镜拍摄荧光图像。计算DAPI染色的细胞核总数中EdU阳性细胞的百分比。每幅图像约计数200个细胞核，每项实验取五幅图像的平均值进行统计分析。

Dissolved in DMSO, and diluted in saline (Rat); 0.9% NaCl (Mice); 30 mg/kg/day (Rat); 0-10 mg/kg (mice); Oral for rats, IP for mice
Male Wistar rats with spontaneous or induced hypertension; Swiss albino mice with Ehrlich ascites carcinoma

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Y-27632 was given orally at 30 mg/kg daily for 4 weeks after the first injection of DMN. The degree of fibrosis was evaluated by image analysis and also by measurements of collagen and hydroxyproline content in the liver. The expression of alpha-smooth muscle actin (alpha-SMA) in the liver and in the primary cultured HSC was also evaluated. Semi-quantitative RT-PCR was performed to evaluate the expression of type I collagen mRNA in the liver.[3]

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Effects of Y-27632 (5-10 mg kg(-1)) and fasudil (5-25 mg kg(-1)) on duration of myoclonic jerks, clonic and tonic convulsions, tonic hindlimb extensions and percentage of tonic convulsion index, as well as recovery latency for righting reflex were investigated in mice stimulated with PTZ (65 mg kg(-1)) or MES (50 Hz, 50 mA and 0.4 s). These inhibitors were also tested on a model of kindling induced by PTZ (35 mg kg(-1), for 11 days). Membrane and cytosolic levels of RhoA protein were measured in brain homogenates from kindled mice.[4]

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Treatment of animals [3]
DMN (1 g/ml) was diluted ten times with saline (final concentration 1%) and 10 mg/kg per day of DMN was injected intraperitoneally (i.p.) on the first 3 days of each week for 4 weeks as described. Y-27632 was dissolved in saline (final concentration 2%) and was given orally once per day at a dose of 30 mg/kg for 4 weeks starting on the day of the first injection of DMN. The dose of 30 mg/kg was selected according to a previous report that this dose corrected hypertension in several rat models without toxicity. Twenty rats were randomized into four experimental groups (n=5 in each group) as follows: (1) S-S (injection of saline i.p. and oral administration of saline); (2) S-Y (injection of saline i.p. and oral administration of Y27632); (3) DMN-S (DMN i.p. and oral administration of saline); (4) DMN-Y (DMN i.p. and oral administration of Y27632). The rats were weighed every week. They were sacrificed at the end of the fourth week and the liver was excised. In addition, a blood sample was taken immediately before the rats were sacrificed.

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Acute PTZ experiments [4]
A group of animals were injected with a single dose of PTZ (65 mg kg−1) to investigate if the two Rho-kinase inhibitors, fasudil and Y-27632, changed the onset of PTZ seizures. Fasudil, Y-27632 or saline was given intraperitoneally 30 min before the PTZ injection. Each mouse was then observed for a 15-min period to measure the onset of the first myoclonic jerk, the onset of the first clonic convulsion and the occurrence of tonic hindlimb extension. Some of the animals died after tonic hindlimb extension, which is an expected outcome of acute PTZ injection. After the observation period, all animals were killed by halothane anaesthesia.

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PTZ kindling experiments [4]
Another group of mice were tested for PTZ kindling. Mice were injected with a sub-convulsive dose of PTZ (35 mg kg−1, i.p.) (on Mondays, Wednesdays and Fridays) of each week for a total of 11 injections. After each PTZ injection, mice were observed for 30 min and the occurrence of convulsive activity was recorded and classified using the scoring system of Fischer and Kittner (1998) as described below. After 30 min, the mice were then injected with either fasudil (25 mg kg−1, i.p.) or Y-27632 (5 mg kg−1, i.p.) and returned to their home cages until the next injection. Control mice for fasudil and Y-27632 received saline. An animal undergoing a stage 5 convulsion was considered to be fully kindled and was not further tested. The seizure stage rating scale was as follows:
Stage 0: no evidence of convulsive activity;
Stage 1: ear and facial twitching, head nodding;
Stage 2: myoclonic jerks;
Stage 3: forelimb clonuses with full rearing;
Stage4: generalized clonic convulsions with loss of righting reflex, rearing, jumping and falling down; and
Stage5: clonic-tonic convulsions with tonic hindlimb extensions.

Another group of kindled mice were injected with a single dose of either fasudil (25 mg kg−1) or Y-27632 (5–10 mg kg−1) to test if acute administration of these Rho-kinase inhibitors could change the seizure susceptibility in kindled mice. At the end of the kindling protocol, all animals were killed by cervical dislocation for the measurement of RhoA translocation in the brain by western blotting.

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MES experiments [4]
To investigate the possible effects of Rho-kinase inhibitors, fasudil and Y-27632, on the tonic hindlimb extensions induced by MES, a group of mice was exposed to an electroshock by ear-clip electrodes. Fasudil, Y-27632 or saline was injected 30 min before electroshock. Immediately after the electroshock, each mouse was put into a Plexiglas chamber to observe the duration of tonic hindlimb extension and the recovery latency for righting reflex for 10 min. After the observation period, all animals were killed by halothane anaesthesia.

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Rota-rod performance test [4]
A separate group of mice received either 25 mg kg−1 of fasudil or 10 mg kg−1 of Y-27632 and was examined in a rota-rod performance test to see if there was any failure of motor coordination induced by Rho-kinase inhibitors.

Y-27632 (5 or 10 mg kg−1) and PTZ (35 or 65 mg kg−1) were dissolved in 0.9% NaCl (saline) and injected intraperitoneally in a volume of 0.1 mL per 10 g body weight. Control animals received saline.

[1]. Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases. Mol Pharmacol. 2000 May;57(5):976-83.

[2]. Rho-associated coiled kinase inhibitor Y-27632 promotes neuronal-like differentiation of adult human adipose tissue-derived stem cells.Chin Med J (Engl). 2012 Sep;125(18):3332-5.

[3]. A selective ROCK inhibitor, Y27632, prevents dimethylnitrosamine-induced hepatic fibrosis in rats. J Hepatol. 2001 Apr;34(4):529-36.

[4]. Antiepileptic effects of two Rho-kinase inhibitors, Y-27632 and fasudil, in mice. Br J Pharmacol. 2008 Sep;155(1):44-51.

[5]. ROCK inhibitor primes human induced pluripotent stem cells to selectively differentiate towardsmesendodermal lineage via epithelial-mesenchymal transition-like modulation. Stem Cell Res. 2016 Sep;17(2):222-227.

[6]. Rho-Associated Kinase Inhibitor (Y-27632) Attenuates Doxorubicin-Induced Apoptosis of Human Cardiac Stem Cells. PLoS One. 2015;10(12):e0144513.

[7]. ROCK inhibitor Y-27632 suppresses dissociation-induced apoptosis of murine prostate stem/progenitor cells and increases their cloning efficiency. PLoS One. 2011;6(3):e18271.

[8]. ROCK inhibitor (Y27632) increases apoptosis and disrupts the actin cortical mat in embryonic avian corneal epithelium. Dev Dyn. 2004;229(3):579-590.

The embryonic chicken corneal epithelium is a unique tissue that has been used as an in vitro epithelial sheet organ culture model for over 30 years (Hay and Revel [1969] Fine structure of the developing Avian cornea. Basel, Switzerland: S. Karger A.G.). This tissue was used to establish that epithelial cells could produce extracellular matrix (ECM) proteins such as collagen and proteoglycans (Dodson and Hay [1971] Exp Cell Res 65:215-220; Meier and Hay [1973] Dev Biol 35:318-331; Linsenmayer et al. [1977] Proc Natl Acad Sci U S A 74:39-43; Hendrix et al. [1982] Invest Ophthalmol Vis Sci 22:359-375). This historic model was also used to establish that ECM proteins could stimulate actin reorganization and increase collagen synthesis (Sugrue and Hay [1981] J Cell Biol 91:45-54; Sugrue and Hay [1982] Dev Biol 92:97-106; Sugrue and Hay [1986] J Cell Biol 102:1907-1916). Our laboratory has used the model to establish the signal transduction pathways involved in ECM-stimulated actin reorganization (Svoboda et al. [1999] Anat Rec 254:348-359; Chu et al. [2000] Invest Ophthalmol Vis Sci 41:3374-3382; Reenstra et al. [2002] Invest Ophthalmol Vis Sci 43:3181-3189). The goal of the current study was to investigate the role of ECM in epithelial cell survival and the role of Rho-associated kinase (p160 ROCK, ROCK-1, ROCK-2, referred to as ROCK), in ECM and lysophosphatidic acid (LPA) -mediated actin reorganization. Whole sheets of avian embryonic corneal epithelium were cultured in the presence of the ROCK inhibitor, Y27632 at 0, 0.03, 0.3, 3, or 10 microM before stimulating the cells with either collagen (COL) or LPA. Apoptosis was assessed by Caspase-3 activity assays and visualized with annexin V binding. The ROCK inhibitor blocked actin cortical mat reformation and disrupted the basal cell lateral membranes in a dose-dependent manner and increased the apoptosis marker annexin V. In addition, an in vitro caspase-3 activity assay was used to determine that caspase-3 activity was higher in epithelia treated with 10 microM Y-27632 than in those isolated without the basal lamina or epithelia stimulated with fibronectin, COL, or LPA. In conclusion, ECM molecules decreased apoptosis markers and inhibiting the ROCK pathway blocked ECM stimulated actin cortical mat reformation and increased apoptosis in embryonic corneal epithelial cells. [1]

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Y-27632 is a monocarboxylic acid amide that is trans-[(1R)-1-aminoethyl]cyclohexanecarboxamide in which one of the nitrogens of the aminocarbony group is substituted by a pyridine nucleus. It has been shown to exhibit inhibitory activity against Rho-associated protein kinase (ROCK) enzyme. It has a role as an EC 2.7.11.1 (non-specific serine/threonine protein kinase) inhibitor. It is a monocarboxylic acid amide, a member of pyridines and a primary amino compound.
Y-27632 is an inhibitor of Rho-associated protein kinase. It inhibits calcium sensitization to affect smooth muscle relaxation.

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Background: p160ROCK is a direct Rho target which mediates Rho-induced assembly of focal adhesions and stress fibers. We previously reported that Rho signaling pathways are involved in the activation of hepatic stellate cells (HSC) in vitro. The aim of the present study was to test the hypothesis that an inhibitor specific for p160ROCK (Y27632) could prevent experimental hepatic fibrosis induced by dimethylnitrosamine (DMN) in rats. Methods: Y27632 was given orally at 30 mg/kg daily for 4 weeks after the first injection of DMN. The degree of fibrosis was evaluated by image analysis and also by measurements of collagen and hydroxyproline content in the liver. The expression of alpha-smooth muscle actin (alpha-SMA) in the liver and in the primary cultured HSC was also evaluated. Semi-quantitative RT-PCR was performed to evaluate the expression of type I collagen mRNA in the liver. Results: Y27632 treatment significantly decreased the occurrence of DMN-induced hepatic fibrosis and reduced the collagen and hydroxyproline content and alpha-SMA expression in the liver. The expression of alpha-SMA in HSC was also suppressed in vitro. Conclusions: These findings indicate that inhibitors of the Rho-ROCK pathway might be useful therapeutically in hepatic fibrosis.[3]

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In this study, we report a previously unexplored field of interest where Y-27632 is identified as a potent small molecule to prime hIPSCs to selectively differentiate towards mesendodermal lineage. By effectively regulating the cell cytoskeleton and cell-cell junction proteins, Y-27632 induces hIPSCs to undergo EMT-like changes which predispose the cells to differentiate towards mesendodermal lineage. Simultaneously, such a disruption of actin and E-cadherin organization results in an inhibition of ectodermal differentiation. These results present a new methodology to enhance the directed differentiation of human PSCs towards mesendodermal target cell types. [5]

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In conclusion, the present study demonstrated that pretreatment of Y-27632 significantly reduces Caspase-3 activity and protects CSCs from Dox-induced apoptosis. The underlying mechanisms are still not completely understood, and probably involve direct and/or indirect interactions between ROCK and Caspase-3, ROCK and LIMK/ADF/Cofilin, or ROCK and p-MCL (Fig 8). The overall consequence or balance among these interactions may result in anti-apoptotic effects on human CSCs. In spite of the fact that many questions remain unanswered, Y-27632 deserves further evaluation in stem cell-based therapy in animal models and the final results may be transferable to human clinical trials.[6]

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Background: Recent clinical trials using c-kit+ human cardiac stem cells (CSCs) demonstrated promising results in increasing cardiac function and improving quality of life. However, CSC efficiency is low, likely due to limited cell survival and engraftment after transplantation. The Rho-associated protein kinase (ROCK) inhibitor, Y-27632, significantly increased cell survival rate, adhesion, and migration in numerous types of cells, including stem cells, suggesting a common feature of the ROCK-mediated apoptotic pathway that may also exist in human CSCs. In this study, we examine the hypothesis that pretreatment of human CSCs with Y-27632 protects cells from Doxorubicin (Dox) induced apoptosis.[6]

C14H25CL2N3O2

338.27

337.132

C, 49.71; H, 7.45; Cl, 20.96; N, 12.42; O, 9.46

331752-47-7

146986-50-7; 129830-38-2 (2HCl); 331752-47-7 (HCl hydrate); 138381-45-0 (racemate HCl); 310898-86-3 (recamate free base)

9797929

Typically exists as solid at room temperature

180-230°C

4.486

77.24

5

4

3

21

268

1

C[C@H](C1CCC(CC1)C(=O)NC2=CC=NC=C2)N.O.Cl.Cl

BLQHLDXMMWHXIT-UYRCGDKNSA-N

InChI=1S/C14H21N3O.2ClH.H2O/c1-10(15)11-2-4-12(5-3-11)14(18)17-13-6-8-16-9-7-13;;;/h6-12H,2-5,15H2,1H3,(H,16,17,18);2*1H;1H2/t10-,11?,12?;;;/m1.../s1

4-[(1R)-1-aminoethyl]-N-pyridin-4-ylcyclohexane-1-carboxamide;hydrate;dihydrochloride

331752-47-7; Y-27632 dihydrochloride monohydrate; Y-27632 Dihydrochloride Hydrate; Y-27632 (hydrochloride hydrate); (R)-(+)-trans-4-(1-Aminoethyl)-N-(4-Pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate; 4-[(1R)-1-aminoethyl]-N-pyridin-4-ylcyclohexane-1-carboxamide;hydrate;dihydrochloride; trans-4-[(R)-1-Aminoethyl]-N-(pyridin-4-yl)cyclohexanecarboxamide dihydrochloride hydrate; Y 27632 Dihydrochloride Hydrate;Y27632 Dihydrochloride Hydrate;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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