p38 MAPK-CDC2途径在重度烧伤血管内皮细胞损伤发生中的作用

We previously reported that p38 MAPK involved in thermal injury induced vascular permeability enhancement, stress fiber formation and ensuing damage of para-cellular tight junction in endothelial cells. When we try to confirm this findings in vivo using electronic microscopy, we found that some of aorta endothelial cells had severe apoptosis. Similar observations were also reported by The Third Military Medical University, that there were endothelial cells damage in major organs and more free endothelial cells in blood circulation in severe burned patients. Our preliminary studies indicated animal vascular damage was very much improved by using adeno-viral approach of dominant negative p38 MAPK to interfere on burns animal model. Using yeast two-hybrid system technique, we found p38 MAPK bound to HPC2 protein. Activation of HPC2 by p38 MAPK inhibited CDC2 protein expression, induced endothelial cells G2/M arrest and ensuing apoptosis. So that we raise a hypothesis that p38 – CDC2 signal pathway involves thermal injury-induced blood vessel endothelial cells damage. In this study, we will further investigate HPC2 phosphorylation site by p38 MAPK, and molecular mechanism on inhibition of CDC2 promoter, including the inhibitory complex formation, such as HPC2 binds to Ring1, Bmi, Rb proteins, and their role on regulation of E2F transcription factor mediated CDC2 expression. Furthermore, we will investigate how the apoptosis induced when CDC2 expression inhibited. Using molecular biology method to interfere this pathways, we will try to develop a new therapeutic method against thermal injury-induced endothelial cell damage, and find new target molecules against sever thermal injury, remote organs and even multiple organs damage.

我们前期报道p38 MAPK参与烧伤引起血管内皮细胞应力纤维形成，造成细胞间紧密联结损伤引起血管通透性增高。同时电子显微镜观察发现，皮肤烧伤能够引起深层主动脉内皮细胞凋亡坏死。国内第三军医大学也报道，重症烧伤病人主要脏器血管内皮细胞均有不同程度损伤和脱落，血液循环中游离内皮细胞明显增加。本课题研究烧伤后血管内皮细胞损伤发生机理。预实验表明，应用失活型p38腺病毒干预，可以大大改善烧伤动物血管损伤；采用酵母双杂交技术发现p38与HPC2蛋白结合，p38激活HPC2可抑制CDC2表达、诱导内皮细胞G2/M期阻滞，导致细胞凋亡。因此我们提出p38-CDC2信号途径参与烧伤血管内皮损伤假说。本项目拟进一步探讨HPC2磷酸化位点、抑制性转录因子复合物包括HPC2与Ring1、Bmi、Rb结合，调控E2F转录因子以及CDC2表达和细胞凋亡；观察阻断这一信号途径防治重症烧伤远端脏器损伤的可能性。

局部烧伤（例如背部烫伤）引起远端，甚至主动脉血管内皮细胞的凋亡坏死。对血管内皮细胞的最基本的功能即维系血液系统的稳态，造成极大损害。我们前期的研究证明p38 MAPK细胞内信号转导系统，对维持血管的稳态具有重要作用。抑制p38 MAPK能够显著降低烧伤引起的血管通透性增高。本项目，我们通过双酵母杂交技术发现p38 MAPK蛋白与HPC2蛋白相结合，而我们的前期研究证明p38蛋白的激活与细胞周期G2期阻滞有关。文献研究发现，HPC2蛋白参与抑制CDC2蛋白表达的调控，CDC2蛋白的表达下降会导致细胞分裂周期停滞在G2/M期，进而引起细胞凋亡。因而我们应用免疫共沉淀等方法，我们证明了双酵母杂交技术的发现，HPC2蛋白是p38 蛋白激酶的底物，并利用磷酸化位点突变技术证明p38磷酸化HPC2的位点与其抑制内皮细胞G2期阻滞的关系；进一步的研究证明，磷酸化的HPC2与Ring1转录因子复合物结合，形成HPC2/Ring1/Bmi/Rb复合体而抑制E2F转录因子与其在CDC2基因启动子上结合位点相结合，从而抑制CDC2蛋白的转录和表达。.烧伤引起的应激反应激活血管内皮细胞p38 MAPK细胞内信号转导系统。体外实验证明烧伤血清促进p38 MAPK在内皮细胞中磷酸化HPC2，通过抑制cdc2蛋白表达从而诱导血管内皮细胞的G2/M期阻滞，进而进一步诱导细胞凋亡坏死。我们应用siRNA技术或突变HPC2的p38蛋白磷酸化位点，可以阻断烧伤血清引起的血管内皮细胞（HUVEC）G2/M期阻滞及细胞凋亡，应用p38 MAPK 阻断剂，可以抑制烧伤血清诱导的血管内皮细胞凋亡坏死，抑制烧伤血清诱导的血管通透性增加。应用腺病毒表达载体阻断p38 MAPK信号转导通路，可以改善烧伤动物的血液循环、改善血管通透性，降低动物的死亡率，提高烧伤动物的存活时间。因而，我们的研究为临床干预烧伤引起的血管通透性增高，提供了新的作用靶点，为基因治疗烧伤提供了实验依据。