【作者】 曹睿；

【导师】 谢强敏；

【作者基本信息】 浙江大学 ， 药理学， 2011， 硕士

【摘要】 蛋白酪氨酸磷酸酶SHP-2 (protein tyrosine phosphatase SHP-2)是一种由PTPNII基因编码的蛋白酪氨酸磷酸酶。SHP-2其是一种含有2个SH结构域的胞质蛋白酪氨酸磷酸酶,其作为细胞因子、生长因子及其他胞外刺激因素的下游信号分子广泛地表达于机体各种组织和细胞中,参与细胞增殖、分化、死亡等诸多细胞生命活动。近年来的研究发现PTPNII突变将会导致青少年白血病、Noonan综合症,LEOPARD综合症等疾病。香烟烟雾能诱导肺部炎症并导致肺组织的损伤,其是最常见的慢性阻塞性肺部疾病(COPD)的致病因素。而SHP-2与香烟烟雾所引起的肺损伤之间关系的研究无论是整体上还是离体上均未见相关的报道。为研究SHP-2香烟烟雾引起的肺组织损伤之间的关系和作用机制,我们分别建立了香烟烟雾提取物(CSE)气道内滴入引起的急性炎症模型、连续香烟烟雾暴露4天所诱发的COPD模型以及香烟烟雾单次暴露模型进行相关的研究工作,结果发现：(1)通过气道内滴入CSE能使得小鼠产生急性炎症反应,肺泡灌洗液(BALF)中白细胞总数、中性粒细胞数目和巨噬细胞数目均有显著上升。肺组织中匀浆中TNF-a、IL-8、MCP-1的mRNA表达上升。在滴入CSE前0.5h腹腔注射SHP-2抑制剂PHPS1(1mg/kg)能够显著减少BAL液中白细胞和中性粒细胞的数目。通过病理切片观察也发现PHPS1能够显著减少炎症细胞的浸润。1mg/kg PHPS1也可抑制CSE所诱导的肺组织匀浆中MCP-1和IL-8 mRNA表达的上升。(2)使用3R4F标准香烟对C5781/6小鼠进行连续4天香烟烟雾暴露制备COPD模型。在研究中显示：模型组BAL液中白细胞总数上升了约4倍,中性粒细胞和巨噬细胞也都有非常显著的上升。每天在香烟烟雾攻击前0.5h以及香烟烟雾攻击后4h分别给予0.3mg/kg、1mg/kg和3mg/kg的PHPS1以及0.5mg/kg磷酸地塞米松(DXM)进行治疗后,均能显著降低香烟烟雾暴露引起的BALF液中以及病理切片中白细胞总数和中性粒细胞数目的上升(3) COPD模型组的气道阻力与空白组相比有明显的上升,同时模型组引起气道阻力增加200%所需的Mch浓度(RLPC200,其值越小说明实验动物对外来刺激引发肺功能改变越敏感)仅为空白组的37%,而连续每天两次腹腔注射6mg/kgPHPS1的小鼠能显著缓解气道阻力的上升,其RLPC200相对于模型组提高了约1倍。而进一步的免疫组化则表明,PHPS1其能呈剂量依赖性减少香烟烟雾诱导的肺组织支气管周围MMP-9蛋白表达的增加。(4)香烟烟雾单次攻击12h后,小鼠肺组织中KC和IL-8 mRNA表达显著上升,而IL-8的蛋白表达约上升了一倍。在香烟烟雾攻击前30分钟预先腹腔注射6mg/kg PHPS1对KC和IL-8m RNA表达没有显著影响。但腹腔注射3mg/kg和6mg/kgPHPS1后,其能显著抑制香烟烟雾所诱导的肺组织中IL-8蛋白表达的上升。我们的研究首次发现PHPS1可通过抑制肺上皮细胞SHP-2蛋白的活性,显著地减少由香烟烟雾(CS)和香烟烟雾提取物(CSE)所诱导的肺部炎性趋化因子的表达,进而影响炎症细胞的募集和趋化,并缓解肺部的炎症。此外,PHPS1也可呈剂量依赖性缓解COPD模型的气道重塑情况。与模型组相比,每天两次腹腔注射6mg/kg PHPS1可显著降低由乙酰甲胆碱(Mch)诱发的气道阻力的上升,并提高给药组RLPC200数值。通过上述研究,我们首次发现PHPS1可以通过抑制SHP-2的活性,减少香烟烟雾所诱导的炎症介质的释放并减少炎症细胞的浸润,同时减少由香烟烟雾所诱导的小气道重塑并改善小鼠的肺功能。上述发现为进一步明确COPD的病理机制以及为COPD的治疗提供了新的思路和新的方向。更多还原

【Abstract】 The protein tyrosine phosphatase (PTP) SHP-2 is encoded by a gene PTPN11. This PTP contains two tandem Src homology-2 domains and it is widely expressed in most tissues.SHP-2 plays a regulatory role in various cell signaling events that are important for a diversity of cell functions, such as mitogenic activation, metabolic control, transcription regulation, and cell migration. Mutations in this gene are a cause of Noonan syndrome, LEORARD syndrome and acute myeloid leukemia.The primary risk factor for COPD is chronic tobacco smoking. However, the relationship between SHP-2 and diseases caused by cigarette smoke have not been reported in the relevant reports. Thus, our current study was set to study whether a SHP-2 inhibitor-PHPS1 is a novel anti-inflammation drug and whether SHP-2 is a novel therapeutic target for COPD.We used ICR mice intratracheal instillated of CES to observerd the effects of PHPS1 on the CSE-induced inflammation. We also investigate the effects of PHPS1 on 1 day or 4 consecutive days exposed to cigarettes smoke in the C57B1/6 mice. The results include:(1) The CSE instillated from airway can lead to a cute inflammation. The total cells, neutrophil and macrophage numbers were significantly increased in the broncho alveolarlavage fluid (BALF). ICR mice were intratracheal instillated by 8mg/kg CSE 30minutes after an intraperitoneal injection of lmg/kg PHPSl. The total numbers of inflammatory cells and neutrophil were significantly decreased in the lmg/kg PHPS1 group of mice compared to model. IL-8 and MCP-1 mRNA expression were also inhibited by PHPS1 and PHPS1 markedly attenuated CSE-induced eosinophil infiltration in lung tissues.(2) On 4 consecutive days C57B1/6 mice were exposed to CS.30 min before and 4h after CS exposure mice were treated by an intraperitoneal injection of PHPS1. The total numbers of inflammatory cells and neutrophil in BALF were decreased by 0.3,1 and 3 mg/kg PHPS1 in a dose-dependent manner. PHPS1 also markedly attenuated CS-induced eosinophil infiltration in lung tissues and the MMP9 protein were also dose-dependently decreased by PHPS1 in the small airway.(3) On 4 consecutive days C57B1/6 mice were exposed to CS. CS-exposed mice exhibited an obvious AHR with a significant and dose-dependent increase in RL as compared with air-exposed mice. The provocation concentration (PC) of methacholine which cumulatively produced a 200% increase in lung resistance (PC200) in model group was 37% as the value of control group. Intraperitoneal injection of PHPS1 at the doses of 6 mg/kg significantly reduced AHR provoked by MCh. The PC200 of 6mg/kg PHPS1 was twice as much as the value of model group.(4) KC and IL-8 mRNA expression and IL-8 protein expression were increased 12 hours after the CS expose of 10 3R4F reference cigarettes. IL-8 protein level in lung tissues was significantly reduced by 3mg/kg and 6mg/kg PHPS1.In conclusion, our results showed that PHPS1 reduced the expression of inflammatory chemokines induced by cigarette smoke (CS) and cigarette smoke extract (CSE).Meanwhile, PHPS1 can decrese cigarette smoke-induced small airway remodeling. Treatment with BCQB at the doses of 6mg/kg significantly reduced AHR provoked by MCh. It is the first time that we show the regulation of a PTP inhibitor-PHPS1 on cigarette smoke-induced inflammation and airway remodeling. These results imply that SHP-2 may be a novel therapeutic target for COPD and PHPS1 may be a novel anti-inflammation and anti-airway remodeling agent.更多还原