KLF4在低氧诱导肺动脉平滑肌细胞增殖及表型转化中的作用初步研究  

A preliminary study on the mechanism of KLF4 in the process of hypoxia-induced PASMC proliferationand phenotypic modulation

作　　者：聂欣[1] 施熠炜[2] 张爱珍[1] 杜永成[1] 

机构地区：[1]山西省人民医院呼吸科,太原030012 [2]山西医科大学第一医院呼吸科,太原030001

出　　处：《国际呼吸杂志》2016年第17期1319-1325,共7页International Journal of Respiration

基　　金：基金项目：山西省科技发展计划（社会发展）项目（20120313020-3）;山西省研究生优秀创新项目（20113071）

摘　　要：目的本实验通过观察低氧诱导大鼠原代肺动脉平滑肌细胞（pulmonaryarterysmoothmusclecell，PASMC）增殖与分化标志改变过程中，krfippel样因子4（krfippel-likefactor4，KLF4）表达的变化，并检测KLF4过表达对该过程的影响，探讨KLF4是否在其中担当一定作用。方法采用贴片法培养原代PASMC，低氧（5％O2）培养24h、48h、72h后，以正常氧浓度培养细胞为对照，采用实时荧光聚合酶链反应（real—timePCR）、Westernblot及免疫共沉淀方法，观察低氧24h、48h、72h，PASMC中KLF4乙酰化水平及表达水平的变化。cck-8检测细胞增殖情况，碘化丙啶（PI）染色检测细胞周期，提取细胞总RNA和蛋白，采用real-timePCR、Westernblot方法检测细胞中增殖细胞核抗原（proliferatingcellnuclearantigen，PCNA）的表达水平，综合分析低氧对细胞增殖的影响，同时采用real—timePCR、Westernblot方法检测PASMC分化标志物平滑肌肌动蛋白d（smoothmusclemaction，SMa-actin）的表达水平。通过转染KLF4表达质粒，提高PASMC中KLF4的表达水平，检测细胞周期、增殖活力及PCNA、SMa—actin的变化，观察低氧下KLF4过表达对PASMC增殖、分化的影响。结果低氧24h，PASMC中KLF4表达水平均较常氧培养细胞明显增高，期间SMn—actin表达较常氧培养细胞明显减少，PASMC增殖不明显。随后KLF4表达水平逐渐降低，至72h降低至正常水平，而SMα-actin逐渐增高，同时PASMC增殖活力明显增强，s期细胞逐渐增多。KLF4过表达后，低氧刺激PASMC增殖活力明显降低，SMα-actin表达水平也降低，细胞阻滞于G1期。结论低氧诱导PASMC发生去分化与增殖不同步；KLF4参与调节低氧诱导的PASMC表型转化与增殖过程，该作用涉及KLF4乙酰化水平改变。Objective The initiation step in diseases involving pulmonary vascular remodeling, such aspulmonary hypertension （PH）, was the proliferation of pulmonary arterial smoothmuscle cell （PASMC）. The most common cause of PASMC proliferation ishypoxia. Krtippel-like factor 4 （KLF4） was an extensively expressed zinc fingertranscription factor whichparticipatedin celldifferentiation, celi proliferation,and apoptosis by regulating the expression ofdownstream target genes. In this study, we aimed to clarify whether KLF4inhibits hypoxia-induced PASMC proliferation and phenotypic modulation in the process of pulmonary vascular remodeling. Methods Firstly, rat PASMCs were isolated from rat pulmonary artery and incubated with low-serum SMBM （2 % FBS, 1% SMCGS） under normoxia （21 %,5%CO2,74% N2） and hypoxia （5% O2,5%CO2,90% N2） for 24 h,48 h and 72 h, respectively. Cell proliferation was quantified by cell counting kit-8 （cck-8） assayand flow cytometry. We also examined PCNA,SM a-aetin, klf4 expression by real-time polymerase chain reaction （PCR） and Western blot. Secondly, the alternation of klf4 acetylation levels was detected byco-immunoprecipitation after hypoxicexposure. PASMCs were transfected with KLF4 expression construct by liposome-mediated gene transfer method, and then assessed the effect of KLF overexpression on PASMC proliferation and differentiation marker gene expression. Results The results of cck-8 and the PCNA expression showed that proliferation of PASMCs cultured in 5 % oxygen was inhibited after 24 h but was increased by 72 h ineulture. Cell cycle analysis showed that the S cell population was increased afterhypoxic exposure for 72 h. Real-time PCR and Western blot showed that cultured in 5% oxygen resulted indecreased level of SM a-actin for 24 h, and increased level for 72 h in PASMCs. The results of real-time PCR, co- immunoprecipitation and Western blot assaysshowed that the expression levels of KLF4 were up-regulated drastically in PASMCs after hypoxic exposure for 24 h,

关 键 词：肺动脉 平滑肌 细胞低氧 细胞分化 细胞增殖 KLF4 

分 类 号：R544.1[医药卫生—心血管疾病]