Non-muscle myosin II (NMII)[1]

使用牛角膜内皮细胞，研究了靶向 NMII 改善 CEC 迁移的治疗潜力 (BCEC)。通过减少逆行肌动蛋白流动和增强板层足突持久性，肌球蛋白运动的直接抑制剂 blebbistatin 可刺激 CEC 的迁移和定向持久性，并加速体外伤口愈合 [1]。

在兔角膜内皮划痕模型中，blebbistatin（0.05 mL，20 μM；前房注射；每天一次；持续 6 天；新西兰白兔）治疗可改善伤口愈合[1]。

BCEC单层蛋白质渗透性测定[1]
BCEC在对照培养基或含有10μM Y27632或10μMblebbistatin>的培养基中培养，直至细胞在Transwell过滤器（0.4 lm孔径聚碳酸酯过滤器）上融合。在新鲜培养基中洗涤和平衡30分钟后，将浓度为3mg/mL的FITC偶联的4K、40K和500K葡聚糖加入上部隔室。在进一步孵育4小时后，通过荧光法（激发，492nm；发射，520nm）评估来自下部隔室的培养基中的扩散FITC-葡聚糖。

免疫荧光共聚焦显微镜[1]
BCEC在盖玻片上培养，在室温下在指定时间点用4%多聚甲醛（pH 7.4）固定30分钟，用0.5%Triton X-100渗透5分钟，用10%牛血清白蛋白封闭30分钟。然后将细胞与指定的一抗在4°C下孵育过夜。用PBS洗涤两次15分钟后，将样品与Alexa Fluor偶联的二抗在室温下孵育1小时。对于肌动蛋白标记，样品在室温下用Alexa 546鬼笔环肽染色10分钟。几次洗涤后，所有样品都安装在荧光安装溶液中。使用激光扫描共聚焦显微镜获得免疫荧光图像，并使用Zen软件进行分析。对于Y27632或blebbistatin洗脱实验，在开始时将细胞与10μM Y27632和10μMblebbistatin同时接种和孵育。随后，在收获细胞并进行共聚焦显微镜检查之前，如图5f所示，从充满的培养基中取出Y27632或博来司他丁。

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活细胞成像[1]
对于单细胞迁移研究，将BCEC铺在12孔板上，与对照培养基或含有10μM Y27632、10μMblebbistatin和10μM ML-7的培养基一起孵育2天。然后将该板放置在配备蔡司Axiovert 200 M倒置显微镜和电动载物台的加湿、CO2平衡室中。使用10×/0.3 EC Plan Neofluar物镜每30分钟捕获5个场，持续16小时。MetaMorph软件跟踪每个场10个随机选择的细胞（每个条件50个细胞）的核位置。使用Gorelik等人慷慨提供的DiPer程序分析迁移速度、方向性比和均方位移。对于体外伤口愈合研究，将细胞接种在6孔板中，与对照培养基一起孵育直至融合。成像前，用200μl移液管尖端在每个孔中刮擦伤口，并将培养基分别换成含有载体对照、10μM Y27632、10μMblebbistatin或10μM ML-7的无血清培养基。使用10×/0.3 EC Plan Neofluar物镜每30分钟拍摄一次图像，持续16小时。通过ImageJ软件分析伤口区域的变化。

Animal/Disease Models: New Zealand white rabbits (16-20 weeks; 3-3.5 kg)[1]
Doses: 0.05 mL; 20 μM
Route of Administration: Intracameral injection; daily; for 6 days
Experimental Results: Resulted in significant improvement of corneal clarity and corneal edema resolution, implying the restoration of an intact corneal endothelial monolayer.

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Rabbit corneal endothelium wounding model[1]
The rabbit corneal endothelium wounding procedure was performed as described previously with modifications. New Zealand white rabbits aged 16 to 20 weeks and weighing 3 to 3.5 kg were anesthetized with an intramuscular injection of xylazine 5 mg/kg and ketamine 40 mg/kg. Alcaine Ophthalmic Solution (0.5%) was instilled to further minimize ocular pain. The area of endothelial cells to be removed was marked with an 8.0-mm marker. After creating the corneal incision with a 2.75-mm blade, CECs within the 8.0-mm mark were gently removed by using posterior capsule polisher, and the areas of denuded Descemet membrane were confirmed by 0.06% trypan blue staining. Missed areas were debrided again to achieve complete cell removal. After wounding, intracameral injection of 0.05 mL of vehicle control, 20 μM Y27632, or 20 μM blebbistatin was administered immediately after wounding and once daily thereafter in different groups of animals. Topical 0.3% gentamicin sulfate was instilled after the procedure for infection control. During the follow-up evaluation, the corneas were observed and photographed with a surgical microscope. The corneal thickness was measured by ultrasound biomicroscopy. Briefly, after intramuscular and topical anesthesia, the ultrasound gel was applied on the cornea. A 50-MHz transducer-probe, aligned vertically to the corneal surface, was used to acquire the image of the anterior chamber. Only the image of the central cornea was acquired, in which the lens was clearly visible through un-dilated pupil. After acquiring the images, the corneal thickness was measured by drawing a line perpendicular to the corneal apex. The whole procedure was performed by an independent technician without prior knowledge related to the experimental condition, and the corneal thickness derived from the images was further analyzed by the author (W.T.H). The animals were euthanized at the indicated time point, and the corneas were harvested. To quantify the wounding extent, the corneas were stained for denuded Descemet’s membrane with 0.2% trypan blue for 1 min. After capturing the image with operating microscope, the wounding extent was quantified by ImageJ software. For immunofluorescent staining, the corneas were fixed in 4% paraformaldehyde (pH 7.4) for 5 min at room temperature, radially incised to allow flat mounting, and were subjected to immunostaining and observation as described above.

[1]. Targeting non-muscle myosin II promotes corneal endothelial migration through regulating lamellipodial dynamics. J Mol Med (Berl). 2019 Sep;97(9):1345-1357.

[2]. Discovery of the migrasome, an organelle mediating release of cytoplasmic contents during cell migration. Cell Res. 2015 Jan;25(1):24-38.

Blebbistatin is a pyrroloquinoline that is 1,2,3,3a-tetrahydro-H-pyrrolo[2,3-b]quinolin-4-one substituted by a hydroxy group at position 3a, a methyl group at position 6 and a phenyl group at position 1. It acts as an inhibitor of ATPase activity of non-muscle myosin II. It has a role as an inhibitor. It is a pyrroloquinoline, a cyclic ketone, a tertiary alcohol and a tertiary alpha-hydroxy ketone.

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Corneal endothelial cell (CEC) dysfunction causes corneal edema that may lead to blindness. In addition to corneal transplantation, simple descemetorhexis has been proposed to treat centrally located disease with adequate peripheral cell reserve, but promoting the centripetal migration of CECs is pivotal to this strategy. Here, we show that targeting non-muscle myosin II (NMII) activity by Y27632, a ROCK inhibitor, or blebbistatin, a selective NMII inhibitor, promotes directional migration of CECs and accelerates in vitro wound healing. The lamellipodial protrusion persistence is increased, and actin retrograde flow is decreased after NMII inhibition. Counteracting lamellipodial protrusion by actin-related protein 2/3 (ARP2/3) inhibitor abolishes this migration-promoting effect. Although both Y27632 and blebbistatin accelerate wound healing, cell junctional integrity and barrier function are better preserved after blebbistatin treatment, leading to more rapid corneal deturgescence in rabbit corneal endothelial wounding model. Our findings indicate that NMII is a promising therapeutic target in the treatment of CEC dysfunction. KEY MESSAGES: NMII inhibition promotes directional migration and wound healing of CECs in vitro. Lamellipodial protrusion persistence is increased after NMII inhibition. Selective NMII inhibitor preserves junctional integrity better than ROCK inhibitor. Selective NMII inhibitor accelerates corneal deturgescence after wounding in vivo.[1]

C18H16N2O2

292.33

292.121

C, 73.95; H, 5.52; N, 9.58; O, 10.95

674289-55-5

(-)-Blebbistatin;856925-71-8

3476986

Light yellow to yellow solid powder

1.3±0.1 g/cm3

507.3±60.0 °C at 760 mmHg

210-212ºC

260.6±32.9 °C

0.0±1.4 mmHg at 25°C

1.681

1.62

52.9

1

3

1

22

497

0

LZAXPYOBKSJSEX-UHFFFAOYSA-N

InChI=1S/C18H16N2O2/c1-12-7-8-15-14(11-12)16(21)18(22)9-10-20(17(18)19-15)13-5-3-2-4-6-13/h2-8,11,22H,9-10H2,1H3

3a-hydroxy-6-methyl-1-phenyl-2,3-dihydropyrrolo[2,3-b]quinolin-4-one

Blebbistatin; (+/-)-Blebbistatin; 674289-55-5; (+-)-Blebbistatin; CHEBI:75379; UNII-20WC4J7CQ6; 3a-hydroxy-6-methyl-1-phenyl-2,3-dihydropyrrolo[2,3-b]quinolin-4-one; 20WC4J7CQ6;

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)

DMSO : 25 mg/mL (85.52 mM)

配方 1 中的溶解度: 2.5 mg/mL (8.55 mM) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浮液；超声助溶。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80+，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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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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