【作者】 陈波；

【导师】 黄巧冰；

【作者基本信息】 第一军医大学 ， 病理生理学， 2004， 硕士

【摘要】 目的： 尽管重症监护和创伤救治方面的研究取得了很大进展，大面积严重烧伤引起的烧伤性休克仍然有着很高的发病率和致死率。血管通透性升高在烧伤引起并发症过程中扮演着重要角色。目前针对这一情况的治疗办法主要依赖于重症护理或液体疗法，缺乏更为切实有效的治疗手段。在血管内皮屏障功能障碍的研究中，肌球蛋白轻链(myosin light chain,MLC)磷酸化引起细胞骨架重组是一个相当重要的环节。肌球蛋白轻链激酶(myosin light chain kinase, MLCK)、Rho激酶(Rho kinase, ROCK)对MLC磷酸化水平的调节作用得到了广泛的重视。。本实验室前期研究还提示烧伤刺激可以引起蛋白激酶C(protein kinase C,PKC)的移位激活，而PKC也是MLCK的上游激活分子之一。本文的研究目的是探讨MLC磷酸化在烧伤血清所致内皮细胞骨架变化中的作用及其PKC信号通路在其中的作用，为烧伤及其并发症的发生机制研究提供更系统的实验数据，为临床治疗提供新的思路。方法： 利用特异性荧光标记探针观察细胞内F-actin分布情况，反映烧伤血清刺激人脐静脉内皮细胞株ECV—304引起的骨架蛋白结构的变化。复制30～40％TBSA的深Ⅱ度以上烧伤SD大鼠模型，烧伤后3h收集血清。分别用不同剂量的烧伤血清刺激人脐静脉内皮细胞株ECV—304，查明烧伤血清引起内皮细胞骨架改变的剂量和时间效应。通过选用利用MLCK的特异性抑制剂ML-7、Rho激酶的特异性抑制剂Y—27632影响MLC磷酸化水平，探讨MLC磷酸化水平的变化在烧伤血清诱导内皮细胞F-actin结构变化中的作用。通过下调细胞内PKC的表达水平，探讨PKC在烧伤引起的肌球蛋白轻链磷酸化在烧伤血清诱导的内皮细胞骨架重组中的作用内皮细胞骨架变化中的作用。使用佛波酷醇(Pho比01一12-mynstate一13一aeetate，pMA)特异性激活细胞内pKC，研究PKC通路是否为烧伤血清所致MLC磷酸化和骨架结果变化的信号途径之一。采用尿素一甘油聚丙烯酞胺凝胶电泳和免疫印迹的方法检测烧伤血清刺激对内皮细胞磷酸化MLC水平的影响，为形态学结果提供直接证明。结果:l、与正常大鼠血清比较，大鼠烧伤血清作用于ECV一304可引起细胞骨架的明显结构变化，在一定浓度范围内(巧%一30%)具有剂量效应。而过高浓度(100%)的正常血清就能够引起内皮细胞收缩，导致单层细胞完整性受到破坏。2、使用巧%的烧伤血清于短时程内(30min一6h)刺激内皮细胞后可导致细胞内骨架结构变化，应力纤维形成。随着刺激时间的延长，细胞内F一actin的改变程度加深，显示一定的时间效应。3、使用ML一7和/或Y一27632预处理细胞30 min后，烧伤血清不能诱导细胞内出现应力纤维。而在烧伤血清刺激细胞30min后再加入ML一7和/或Y一27632后，细胞内的应力纤维结构较烧伤对照组和加入前均明显减少。4、使用PMA预刺激细胞24h，通过负反馈调节反应下调细胞内PKC活性后，烧伤血清刺激内皮细胞30 min不能引起细胞内F一actin分布变化。5、PMA作用内皮细胞巧min以激活PKC，能够诱导细胞内出现应力纤维。在使用ML一7和Y一27632预处理细胞后，PMA刺激不再引起细胞内F一actin结构的改变。ML一7和Y一27632也可以逆转PMA介导的细胞骨架结构变化。6、Westem blot研究结果初步证明，巧%的大鼠烧伤血清刺激30 min能够引起内皮细胞MLC磷酸化水平的升高。结论:1、证明烧伤大鼠诱导内皮细胞内应力纤维形成的过程具有时间和剂量 一3-肌球蛋白轻链磷酸化在烧伤血清诱导的内皮细胞骨架重组中的作用依赖性。明确100%的烧伤血清不适宜用于体外模拟烧伤条件。2、烧伤血清能够诱导内皮细胞内MLC磷酸化水平的升高。在烧伤血清引起内皮细胞内F一ac血重组过程中，MLC的磷酸化是一个重要的步骤，MLCK和孙。激酶可能通过增加MLC磷酸化水平，参与了烧伤血清诱导的内皮细胞内F一actin重组过程。3、使用ML一7和Y一27632降低MLC磷酸化水平，能够逆转烧伤血清在短时间内诱导的内皮细胞内F一actin重组。4、PMA能够在短时间内诱导内皮细胞内应力纤维的形成。PKC可能参与了烧伤血清诱导内皮细胞骨架重组的过程，并位于MLcK和Rho激酶的上游，对其功能进行调节。更多还原

【Abstract】 Objects:Despite the remarkable progress in critical care and wound management, burn shock caused by massive and severe burn injury still has a high rate of morbidity and mortality. Vascular hyperpermeablity plays a central role in the occurrence of syndrome in bum. Fluid resuscitation was the only current accessible clinical method in the therapy of burn shock. Previous studies have approved that MLC phosphorylation is involved in inflammatory mediators-induced alteration of endothelial barrier function, while the level of phosphorylated MLC is regulated by MLC kinase and MLC phosphatase. Activation of Protein kinase C pathway has been approved to contribute to the signal transduction in enhanced microvascular permeability. PKC is also regarded as an important upstream signal for MLC phosphorylation. The objects of this study are to elucidate the property of burn serum induced-endothelial cytoskeleton reorganization and to explore the effect and possible signal pathway of MLC phosphorylation involved in this process. Methods:The serum from Sprague-Dawley rats with II degree burn injury (30-40% TBSA) was used to stimulate ECV-304 cells to observe its time and dose-dependent effects on endothelial cytoskeleton using the specific fluorescent probe of F-actin. ML-7, the specific inhibitor of MLCK, and Y-27632, the specific inhibitor of Rho kinase which could inhibit the activity of MLC phosphatase, were used to reduce the level of phosphorylated MLC and to detect the roles of MLCK and Rho kinase during the process of bum serum-induced endothelial cytoskeltal reorganization. The expression of PKCwas down-regulated by long term (24 h)-PMA stimulation in endothelial cells in order to know whether PKC activation played a role in burn serum induced-cytoskeletal reorganization. PMA treatment for 15min was used to specifically activate PKC so as to clarify the effects and relationship between PKC and MLC phosphorylation in burn serum induced-cytoskeleton morphological changes. Results:1. Compared with normal serum, serum from burn rats could induce remarkable morphological changes of endothelial cytoskeleton in a dose-dependent manner. But even 100% normal serum could cause the contraction of endothelial cells and destruct the integrity of endothelial monolayer.2. 15% burn serum stimulation for 15 min caused the formation of stress fibers in endothelial cells in a time-dependent pattern.3. Reduction of pohsphorylated MLC level in ECs was induced by pretreating the cells with ML-7 or/and Y-27632 for 30min. Burn serum could not induced any changes in the organization of F-actin in these cells. In ECs incubated with ML-7 and/or Y-27632 30 min before the 30 min stimulation of burn serum, few stress fibers could be seen compared with those cells pretreated with vehicle and burn serum or only with burn serum for 30min.4. Down-regulation of PKC activation induced by long-term PMA incubation attenuated the burn serum induced-changes of endothelial cytoskeleton organization.5. Activation of PKC by short-term stimulation with PMA caused the formation of stress fibers in endothelial cells, which resembled the alteration induced by burn serum. In endothelial cells treated with ML-7 or Y-27632,PMA stimulation could not caused the formation of stress fiber. 6. The result of western blot indicated an increased of MLC phosphorylation level in ECs treated with 15% burn serum. Conclusion:1. Burn serum could induce a dose- and time-dependent formation of stress fibers in enthelial cells. 100% serum treatment should not be used in burn model in vitro.2. Burn serum could induced a shift of MLC species from nonphosphorylated form to phosphorylated form, which is very important during the process of endothelial cytoskeletal changes induced by serum from burned rats. The activation of MLCK and Rho kinase may be involved in burn serum-induced cytosketal reorganization in endothelial cells.3. The reduction of MLC phosphorylation level by ML-7 and/or Y-27632 could reverse the cytoskeletal reorga更多还原