【作者】 曾静；

【导师】 王晓斌；

【作者基本信息】 泸州医学院 ， 麻醉学， 2013， 硕士

【摘要】 背景与目的：肝肺综合症（hepatopulmonary syndrome，HPS）是慢性肝病导致肺微血管病变引起动脉血氧合障碍的一种综合病征。肺血管重塑是肺微血管病变的主要病理变化，寻找调控HPS肺血管重塑的靶蛋白是目前研究的重点。膜联蛋白A2（annexin A2，ANXA2）是钙依赖的磷脂结合蛋白超家族成员，参与调节多种细胞的分化、增殖、迁移和凋亡，在多个生命过程中发挥重要作用。本研究拟探讨ANXA2在HPS大鼠血清致肺动脉平滑肌细胞（pulmonary arterial smooth muscle cells，PASMCs）增殖中的作用，为阐明HPS肺血管重塑的分子机制及其防治提供依据。方法：原代培养大鼠PASMCs并鉴定后，随机分为2组：对照组（C组）和HPS组，分别用正常大鼠血清培养C组PASMCs，HPS大鼠血清培养HPS组PASMCs，两组PASMCs于血清中孵育24、48、72h。采用氚-胸腺嘧啶核苷掺入法（3H-TdR）、噻唑蓝比色法（MTT）检测大鼠PASMCs增殖变化，分别用RT-PCR和western blot检测大鼠PASMCs中ANXA2mRNA及其蛋白表达含量变化；运用RNA干扰（RNAinterference，RNAi）技术将ANXA2基因沉默，观察对HPS大鼠血清致PASMCs增殖的影响，并用western blot检测ERK1/2、NF-κB磷酸化水平。结果：HPS组PASMCs在血清孵育24、48、72h的3H-TdR以及MTT检测值明显高于C组各时间点，差异具有统计学意义（P<0.05），且随大鼠血清作用时间延长，HPS组PASMCs3H-TdR以及MTT检测值增加更为明显；两组均检测出ANXA2mRNA及其蛋白的表达，但与C组比较，HPS组PASMCs内ANXA2mRNA及其蛋白表达明显增加（P<0.05），且随时间延长，表达量增加更明显。siRNA下调ANXA2表达后，再经HPS大鼠血清孵育，PASMCs MTT以及3H-TdR掺入量明显低于对照组（P<0.05）。与C组比较，HPS大鼠血清可致ERK1/2、NF-κB磷酸化水平明显升高（P<0.05）；而下调ANXA2表达后，HPS大鼠血清致ERK1/2、NF-κB磷酸化明显低于对照组（P<0.05）。结论：HPS大鼠血清可致PASMCs异常增殖；HPS大鼠血清通过ANXA2介导促增殖信号ERK1/2、NF-κB活性改变可能是PASMCs异常增殖的机制之一。更多还原

【Abstract】 Objective: Hepatopulmonary syndrome (HPS) is definedas an arterial oxygenation defect induced by microangiopathy whichassociates with hepatic diseases. Pulmonary vascular remodeling is akey pathophysiological component in microangiopathy, currently, toexplore the crucial target in the pathogenesis has become key point ofstudy. Annexin A2(ANXA2) is member of the annexin superfamily ofcalcium-dependent phospholipid-binding proteins, which involves inmany cellular regulatory processes, such as cell differentiation,proliferation, migration and apoptosis. In this study, we sought toexplore the role of ANXA2in HPS rat serum-induced proliferation ofpulmonary arterial smooth muscle cells （PASMCs） and clarify themolecular mechanism of HPS-associated pulmonary vascularremodeling and provide evidences for prevention and cure. Methods:PASMCs were primary cultured and identified, and then divided to twogroups: group C consisted of PASMCs that were cultured in normal ratserum, group HPS consisted of PASMCs that were incubated in HPS ratserum for various time periods:24,48,72h. The changes of PASMCsproliferation were determined by3H-TdR incorporated way and MTT assay. The levels of ANXA2mRNA and protein expression weredetected by RT-PCR and western blot respectively. Silenced ANXA2expression by RNA interference (RNAi) and further observed the effectof HPS rat serum-induced PASMCs proliferation and examined thealtered phosphorylation level of ERK1/2and NF-κB by western blot.Results: The value of3H-TdR and MTT of PASMCs that incubated inserum for24,48,72h in group HPS were greater than group C at eachtime-point, and the differences were statistically significant (P<0.05).Accompanying the prolongation of incubated time, the increased valueof3H-TdR and MTT were higher than group C. The mRNA and proteinexpression of ANXA2could be detected in both groups, compared togroup C, ANXA2mRNA and protein expression were higher in groupHPS, and the longer PASMCs were incubated in HPS rat serum, thegreater were increasd in expression（P<0.05）. After downregulating theexpression of ANXA2by siRNA and incubating in HPS rat serum, thevalue of3H-TdR and MTT of PASMCs were greatly reduced comparedwith control groups. Compared with group C, the phosphorylation levelof ERK1/2and NF-κB were higher in cultures treated with HPS ratserum, after ANXA2downregulation, HPS rat serum-inducedphosphorylation of ERK1/2and NF-κB were lower than control groups.Conclusion: HPS rat serum induced PASMCs proliferation; the alteredactivation of pro-proliferative signaling such as ERK1/2and NF-κB, in association with HPS rat serum were mediated by ANXA2, suggestingan important mechanism of PASMCs proliferation.更多还原