内质网应激在特发性肺纤维化中的作用

【作者】 王艳勋；

【导师】 徐作军；

【作者基本信息】 北京协和医学院 ， 呼吸内科， 2014， 博士

【摘要】 第一部分内质网应激对C57BL/6小鼠肺纤维化模型的影响研究目的建立博莱霉素小鼠肺纤维化模型,观察衣霉素诱导的内质网应激及Salubrinal阻断内质网应激通路对肺纤维化的干预作用,并探讨可能机制。研究方法1.应用单次气管内给药方法建立小鼠肺纤维化模型。2.观测小鼠呼吸、活动、进食、毛发及体重情况,肺组织病理切片Ashcroft评分及羟脯氨酸测定评估小鼠肺纤维化的严重程度。3.采集小鼠支气管肺泡灌洗液,进行炎症细胞计数；应用ELISA法测定BALF中TGF-p和MMP2蛋白含量；应用TUNEL法检测肺泡上皮细胞细胞凋亡。4.应用免疫组化法及Western blot法检测小鼠肺组织中CHOP、GRP78、α-SMA及E-cadherin蛋白的表达。5.应用Real-Time PCR法检测小鼠肺组织XBP1、Caspase3、α-SMA及TGF-p RNA的表达。研究结果1.与博莱霉素组及对照组小鼠相比,衣霉素干预组小鼠纤维化评分分值明显升高,salubrinal干预组小鼠纤维化评分分值明显减低。2.与博莱霉素组小鼠相比,衣霉素干预组小鼠BALF中TGF-p和MMP2蛋白含量较高、肺泡上皮细胞细胞凋亡较明显,salubrinal干预组小鼠上述指标明显减低。3.与博莱霉素组小鼠相比,衣霉素干预组小鼠肺组织中CHOP、GRP78、α-SMA表达水平较高,E-cadherin蛋白的表达水平较低：Salubrinal干预组CHOP、GRP78、 α-SMA表达水平较低。4.与博莱霉素组小鼠相比,衣霉素干预组小鼠肺组织中Caspase3、α-SMA及TGF-β1蛋白的表达水平较高,Salubrinal干预组小鼠上述指标明显减低。实验结论衣霉素诱导的ERS可加重博莱霉素诱导的C57BL/6小鼠的肺纤维化,机制可能与促进AECs凋亡及诱导EMT相关。Salubrinal阻断内质网应激的PERK通路可减轻博莱霉素及衣霉素联合博莱霉素诱导的C57BL/6小鼠的肺纤维化,在肺纤维化疾病治疗中具有应用前景。第二部分内质网应激在特发性肺纤维化患者中的作用研究目的观察IPF患者肺组织及血浆内质网相关蛋白的表达,探讨内质网应激在IPF患者中的作用及其可能机制,评估相关生物标记物在临床中的应用前景。研究方法1.应用TUNEL免疫荧光染色观察肺泡上皮细胞凋亡情况；2.应用免疫组化方法检测a-SMA、GRP78、CHOP及E-cadherin在肺组织切片中的表达；3.应用ELISA方法检测IPF及正常对照组血浆CK18(M65)及cCK18(M30)的表达。实验结果1. TUNEL免疫荧光染色观察,在100倍荧光显微镜下观察显示IPF患者肺泡上皮区域有散在的凋亡荧光表达。2.IPF患者肺内a-SMA、GRP78、CHOP明显表达,上皮细胞标记物E-cadherin似有表达,但不显著。3.M30/M65比值在吡非尼酮治疗IPF患者治疗12个月有明显下降；M30在IPF的支气管肺泡灌洗液浓度低于其他DPLD。实验结论IPF患者血浆CK18(M65)及cCK18(M30)的检测有一定鉴别意义。IPF患者肺泡上皮细胞存在明显凋亡,可能机制包括内质网应激及凋亡蛋白酶诱导上皮细胞凋亡。第三部分连花清瘟制剂临床用药安全性的系统评价研究目的系统评价连花清瘟制剂临床用药的安全性。研究方法检索中国生物医学文献数据库(CBM)、中国学术期刊全文数据库(CNKI)、索Cochrane图书馆、MEDLINE、EMbase;收集连花清瘟胶囊和颗粒随机对照临床文献,检索时限均从2003年1月至2013年5月。实验结果纳入随机对照临床文献40篇,试验组2592例,对照组2314例。试验组有63例发生不良反应,发生率为2.4%,对照组有100例发生不良反应,不良反应发生率为4.3%。试验组与对照组相比不良反应发生率明显偏低[RR=0.562,95%CI(0.412-0.767)]。纳入的文献中有24篇有不同类别的不良反应报道,统计分析结果提示连花清温制剂不良反应发生率低于其它对照组[RR=0.62,95%CI(0.46,0.82)]。不良反应系统评价亚组分析提示消化系统不良反应发生率低于其它对照组[RR=0.70,95%CI(0.50,0.97)]。实验结论连花清瘟临床常见的不良反应为胃肠道反应和皮疹,在不同疾病用药过程中,不良反应发生率明显低于对照组。更多还原

【Abstract】 PART1:Effects of ERS on Bleomycin-Induced Pulmonary Fibrosis in C57BL/6MiceObjectiveTo determine the effects of tunicamycin induced ERS and Salubrinal blocked ERS pathway on bleomycin-induced pulmonary fibrosis in C57BL/6mice, and discuss the mechanisms.Materials and Methods1. We used a single dosage intratracheal injection in mice.2. The fibrosis degree was determined pathologically by using the Ashcroft scoring method and biochemically by hydroxyproline assay in lung tissue.3. To collect bronchoalveolar lavage fluid, the differential inflammatory cells were counted in BALF and the concentrations of TGF-βand MMP2in BALF were determined by ELISA.4. The expression of CHOP、GRP78、α-SMA and E-cadherin was detected by Immunohistochemistry and Western blot.5. The expression of XBP1、Caspase3、α-SMA and TGF-β mRNA in lung tissue was detected by Real-time PCR.Results1. Compare to the mice administrated by bleomycin and the control group, the Aschcroft score and hydroxyproline content were significantly increased in the mice administered tunicamycin, while salubrinal decreased the effects.2. Compare to the mice administrated by bleomycin, treatment with tunicamycin significantly increased the level of TGF-βand MMP2in BALF, while salubrinal decreased the effects.3. Compared to the bleomycin alone group, treatment with tunicamycin significantly increased the level of CHOP、GRP78、α-SMA and E-cadherin in the lung tissue, while salubrinal decreased the effects.4. Treatment with tunicamycin significantly increased the level of XBP1、Caspase3、α-SMA and TGF-β1in the lungs of mice.ConclusionAdministration of tunicamycin can aggravate bleomycin-induced pulmonary fibrosis in mice, while salubrinal decreased the effects. The mechanisms were possibly by inducing alveolar epithelial cell (AEC) apoptosis and inducing epithelial-mesenchymal transition. PART2:Effects of ERS on IPF PatientsObjectiveTo observe the expression of ERS associated proteins on IPF patients’lung tissue and plasma, discuss the mechanism and to investigate the application of biomarkers like CK18and cCK18in clinical diagnosis and treatment.Materials and Methods1. The apoptosis of AECs was determined pathologically by using TUNEL method;2. The expression of α-SMA、GRP78、CHOP and E-cadherin was detected by immunohistochemical method.3. The expression of CK18(M65) and cCK18(M30) in IPF patients’plasma was detected by ELISA method.Results1. Enhanced green fluorescence proteins was observed at the AECs region of the IPF patients’ lung.2. α-SMA、GRP78、CHOP were obviously expressed in IPF patients’lung tissue, while the expression of E-cadherin was obscure;3. The ratio of M30and M65was significantly decreased in IPF patients’plasma after12months of treatment with pirfinitone, and the expression of M30was lower in IPF patients than in other DPLD patients.ConclusionThe detection of CK18(M65) and cCK18(M30) have important clinical value in diagnosis and differential diagnosis in IPF patients. The AECs region of the IPF patients’ lung had marked apoptosis. PART3:The Safety of Lianhuaqingwen Preparations in Clinical Use:A Systematic ReviewObjectiveTo evaluate the safety of lianhuaqingwen preparations in clinical use.Materials and MethodsWe performed a systematic search of CBM, CNKI, Cochrane Central, MEDLINE and EMbase for trials published between January2003and May2013.ResultsFourty randomized controlled clinical trials including2592cases of the experimental group and2314cases of the control group were evaluated. The experimental group had63cases with adverse reactions(2.4%).While in the control group,100cases had adverse reactions(4.3%). The relative risk of the incidence of adverse reactions in the lianhuaqingwen preparations group was lower than the control group [RR=0.562,95%CI (0.412-0.767)].In the subgroup analysis of24trails, the total adverse effects RR was0.62,95%CI (0.46,0.82),and the adverse reactions of digestive system RR was0.70,95%,CI(0.50,97).The results suggested that the incidence of digestive system adverse reactions was lower in Lianhuaqingwen preparations group.ConclusionThe common adverse reactions of Lianhuaqingwen preparations were gastrointestinal and dermatological reactions.The incidence of those adverse reactions in Lianhuaqingwen preparations group was significantly lower than the control group.更多还原