Sonic hedgehog信号通路参与脂多糖诱导肺微血管内皮细胞表达RACK1  

The Sonic hedgehog signaling pathway involved in the expression of RACK1 in the pulmonary microvascular endothelial cells induced by lipopolysaccharide

作　　者：王巾枚 尤青海[1] 牛成成[2] 贾丹[1] 蒋利娟[1] Wang Jinmei;You Qinghai;Niu Chengcheng;Jia Dan;Jiang Lijuan(Department of Respiratory Medicin;Central Terile Supply Cente;the First Affiliated Hospital of Anhui Medical University, Hefei 230022, China)

机构地区：[1]安徽医科大学第一附属医院呼吸内科,合肥230022 [2]安徽医科大学第一附属医院消毒供应中心,合肥230022

出　　处：《中华急诊医学杂志》2018年第7期757-763,共7页Chinese Journal of Emergency Medicine

基　　金：国家自然科学基金(81100053)

摘　　要：目的 探讨脂多糖（lipopolysaccharide, LPS）是否影响大鼠肺微血管内皮细胞（ratpuhnonary microvascular endothelial cells, RPMVEC）表达活化的蛋白激酶C 受体1（protein kinaseC receptor 1, RACK1）及Sonic hedgehog（SHH）信号通路对其表达的影响。方法 取健康雄性、体质量100～120 g、SPF 级SD 大鼠，于安徽医科大学第一附属医院呼吸内科实验室，体外培养RPMVEC，免疫细胞化学法检测RACK1 蛋白在RPMVEC 表达，随机分为：LPS 和SAG 干预实验：（1）LPS 量效组，0.1、1、10 mg/L LPS 与RPMVEC 孵育8 h; LPS 时效组，10 mg/L LPS 与RPMVEC孵育0、2、4、8、12、24 h。（2）SAG 量效组，0.1、1、10 μmol/L RPMVEC 孵育8 h; SAG 时效组，1 μmol/L SAG 与RPMVEC 孵育0、2、4、8、12、24 h。（3） LPS＋SAG 干预组，10 mg/L LPS 预孵育1 h 后加入1 μmol/L SAG 继续孵育8 h; 设空白组、LPS 组和SAG 组为对照。所有干预结束后Western blot 法检测RACK1 蛋白表达及RT-PCR 法检测GLI-1 mRNA 表达，多组变量间比较采用单因素方差分析，组间两两比较采用t 检验，以P〈0.05 为差异有统计学意义。结果 免疫细胞化学染色法显示RPMVEC 表达RACK1。（1） LPS 量效组（0、0.1、1、10 mg/L）：RACK1 蛋白表达量随LPS 浓度增加而升高，组间比较：P〈0.05；GLI-1 mRNA 表达量为（1.109±0.063）、（1.039±0.135）、 （0.813±0.066）、（0.770±0.105 ），1 mg/L 组与10 mg/L 组比较P〉0.05， 其余组间比较P〈0.05 ；LPS 时效组：LPS 作用RPMVEC 诱导RACK1 蛋白自2 h 表达上调（0.370±0.010），12 h 达最高（1.296±0.048），组间比较差异有统计学意义（F=1 272.204，P〈0.05） ；而GLI-1 mRNA 表达自2 h开始下调（0.929±0.007），组间比较差异有统计学意义（F=306.609，P〈0.05）。（2）SAG 量效组（0、0.1、1、10 μmol/L）：RACK1 蛋白表达量未见明显变化，组间比较P〉0.05，GLI-1 mRNA 表达量为（1.109±0.063）、（1.169±0.052）Objective To explore the effect of expression of protein kinase C receptor I（RACK1） induced by lipopolysaccharide （LPS） on Sonic hedgehog（SHH） signaling pathway in rat puhnonary microvascular endothelial cells （RPMVEC）. Methods The healthy male SPF grade SD rat with 100-120 g body weight were gotten from the laboratory animal center of Anhui province. Using immunocytochemistry method, the expression of RACK1 protein in RPMVECs was detected, cultured RPMVECs were randomly divided into different groups as LPS dose-dependent group, SAG（smoothened Agonist, a SHH signaling pathway specific agonist） dose-dependent group, LPS time-dependent group, SAG time-dependent group and LPS＋SAG group. In LPS dose-dependent groups, RPMVECs were cultured with 0.1, 1, 10 mg/L LPS for 8 h. In LPS time-dependent groups, RPMVECs were cultured with 10 mg/L LPS for 0, 2, 4, 8, 12, 24 h. In SAG dose-dependent groups, RPMVECs were cultured with 0.1, 1, 10 μ mol/L for 8 h. In SAG time-dependent groups, RPMVECs were cultured with 1 μmol/L SAG for 0, 2, 4, 8, 12, 24 h. In LPS＋SAG group, RPMVECs were cultured with 1 μ mol/L SAG 8 h after 10 mg/L LPS treatment for 1 h. In addition, blank group, LPS group and SAG group were set for control. Western blot were used to detect the level of RACK1 and RT-PCR were used to detect the expression of GLI-1 mRNA after intervention. Results Immunocytochemistry revealedthat RACK1 were present in RPMVEC. 1. In LPS dose-dependent groups （0, 0.1, 1, 10 mg/L）, the level of RACK1 elevated as LPS dose increased correspondingly with inter-group difference （P〈0.05）; the relative expression levels of GLI-1 mRNA were （1.109 ± 0.063）, （1.039 ± 0.135）, （0.813 ±0.066）, （0.770 ±0.105）, （1 mg/L vs. 10 mg/L, P〉0.05; the rest P〈0.05）. In LPS time-dependent groups, the relative expression level of RACK1 at 2 h （0.370 ± 0.010） was higher than that at 0 h （0.329 ± 0.008）, peaked at 12 h （1.296 ±0.048）, and compared with 0 h, there was significant diff

关 键 词：脂多糖 肺微血管内皮细胞 活化的蛋白激酶C受体1 Sonic HEDGEHOG信号通路 急性呼吸窘迫综合征 

分 类 号：R563.8[医药卫生—呼吸系统]