1,25-(OH)_2D_3对肺纤维化大鼠肺泡Ⅱ型上皮细胞中Ca^(2+)浓度和PI3K/AKT/mTOR通路的影响  

Effects of 1,25-(OH)_2D_3 on Ca^(2+) concentration and PI3K/AKT/mTOR pathway in type Ⅱalveolar epithelial cells of rat with pulmonary fibrosis

作　　者：董洪亮[1] 刘乃国[1] 苗双[2] 倪娜[1] 郑静[1] 王楠[1] 李新静[3] 

机构地区：[1]滨州医学院附属医院临床医学实验室,滨州理学硕士256603 [2]滨州医学院附属医院肿瘤研究中心,滨州256603 [3]滨州医学院附属医院医务处,滨州256603

出　　处：《医学研究生学报》2016年第10期1040-1045,共6页Journal of Medical Postgraduates

基　　金：山东省自然科学基金(ZR2011HM062);山东省医药卫生科技发展计划项目(2015WS0490);滨州医学院科技计划项目(BY2015KJ39)

摘　　要：目的特发性肺纤维化(idiopathic pulmonary fibrosis,IPF)是一种慢性炎症性疾病,病因不明,缺乏有效的治疗方法。文中旨在探讨肺泡Ⅱ型上皮细胞中Ca^(2+)和PI3K/AKT/mTOR通路在大鼠IPF中的作用,以及1,25-(OH)_2D_3对Ca^(2+)浓度和PI3K/AKT/mTOR通路的影响。方法 150只雄性SD大鼠随机分为:预防组(对照组Ⅰ、模型组Ⅰ、给药组Ⅰ,每组20只)和治疗组(对照组Ⅱ、模型组Ⅱ、给药组Ⅱ,每组30只)。对照组Ⅰ/Ⅱ行气管暴露手术,气管内给以200μL/只的无菌等渗盐水,并分别于第2和14天开始腹腔注射等渗盐水(200μL/只);模型组Ⅰ/Ⅱ气管内灌注博来霉素(5 mg/kg),并分别于手术后第2和14天开始腹腔注射维生素D3的溶剂(0.1%乙醇和99.9%的丙二醇,1μL/g体重);给药组Ⅰ/Ⅱ气管内灌注博来霉素(5 mg/kg),并分别于手术后第2和14天开始腹腔注射1,25-(OH)_2D_3(2μg/kg体重)。大鼠气管内注射博来霉素建立IPF模型,给药组腹腔注射1,25-(OH)_2D_3进行预防和治疗。检测各组大鼠肺组织中羟脯氨酸的含量,分离肺泡Ⅱ型上皮细胞并用Fluo-3AM荧光负载,激光共聚焦显微镜检测细胞内Ca^(2+)浓度。RT-PCR方法检测PI3K、AKT、mTOR mRNA的表达水平。结果模型组Ⅰ/Ⅱ和给药组Ⅰ/Ⅱ中大鼠肺组织中羟脯氨酸的含量、肺泡Ⅱ型上皮细胞内Ca^(2+)浓度以及PI3K、AKT、mTOR mRNA表达,在各时间点与相应对照组Ⅰ/Ⅱ相比均明显升高(P<0.05或P<0.01);给药组Ⅰ/Ⅱ与模型组Ⅰ/Ⅱ相比,上述指标均有显著降低(P<0.05或P<0.01)。模型组Ⅰ/Ⅱ和给药组Ⅰ/Ⅱ中Ca^(2+)浓度与PI3K、AKT、mTOR mRNA表达之间具有显著正相关(r=0.598 8、r=0.623 0、r=0.6603,P<0.01;r=0.7012、r=0.6323、r=0.7403,P<0.01)。结论 PI3K-AKT-mTOR通路在大鼠IPF的发生发展中起重要作用,1,25-(OH)_2D_3可以降低肺泡Ⅱ型上皮细胞内Ca^(2+)的浓度,抑制PI3K-AKT-mTOR通路,进而抑制IPF的发生发展。Objective Idiopathic pulmonary fibrosis （ IPF） is a chronic inflammatory disease with unknown etiology and is lack of effective therapy. The aim of this study is to explore the function of Ca^2＋ and PI3K/AKT/mTOR pathway in the pathogenesis of IPF, and the impact of 1,25-（OH）2D3 on Ca^2＋ and PI3K/AKT/mTOR pathway in type Ⅱ alveolar epithelial cells of rat with IPF. Methods 150 male SD rats were randomly divided into 2 groups： prevention group （ control group Ⅰ,model group Ⅰ,medication group Ⅰ ） and treatment group （ control group Ⅱ , model group Ⅱ , medication group Ⅱ ） . The tracheal exposure surgery was operated in control group Ⅰ/Ⅱ , and then 200 μL sterile physiological saline was administered by intraperitoneal injection of each rats 2 days and 14 days after surgery, separately. Bleomycin（ BLM） （ 5 mg/kg） was injected into the trachea of model group Ⅰ/Ⅱ , and then vitamin D3 solvent（ 0.1 % ethanol and 99.9% glycol propylene, 1 μL /g ） was administered by intraperitoneal injection 2 days and 14 days after surgery, separately. Bleomycin（ BLM） （ 5 mg/kg） was injected into the trachea of medication group Ⅰ/Ⅱ , and then 1,25-（ OH） 2D3（ 2 μg/kg） was administered by intraperitoneal injection 2 days and 14 days after surgery, separately. IPF model was built by injecting Bleomycin into the trachea of rats, 1, 25-（ OH） 2D3（ 2 μg/kg） was used to prevent and treat IPF by intraperitoneal injection in medication group. The hydroxyproline content of lung tissue in each group was measured, type Ⅱ alveolar epithelial cells were separated from lung tissue and labeled with Fluo-3AM, then concentration of Ca^2＋ was detected by Laser scanning confocal microscope. The mRNA levels of PI3K, AKT and mTOR in the type Ⅱ alveolar epithelial cells were tested by RT-PCR. Results Compared with control group Ⅰ / Ⅱ at each time point, hydroxyproline content of lu

关 键 词：1 25-(OH)2D3 SD大鼠 肺纤维化 肺泡II型上皮细胞 CA^2+浓度 PI3K/AKT/mTOR信号通路 

分 类 号：R563[医药卫生—呼吸系统]