SHH信号通路激活对大鼠心脏微血管内皮细胞缺血再灌注损伤血管再生作用研究  

The study of SHH signal pathway activation on rat cardiac microvascular endothelial cells ischemic reperfusion injury angiogenesis

作　　者：国伟[1] 杨军[2] 任法新[2] 梁平平[2] Guo Wei;Yang Jun;Ren Faxin;Liang Pingping(Department of Gerontology,Jining First People's Hospital,Shandong Province,Jining 272011,China;不详)

机构地区：[1]济宁市第一人民医院老年医学科,272011 [2]青岛大学附属烟台毓璜顶医院,264000

出　　处：《疑难病杂志》2021年第1期41-46,共6页Chinese Journal of Difficult and Complicated Cases

基　　金：山东省医药卫生科技发展计划项目(2018WSA08015);济宁市第一人民医院博士科研启动基金。

摘　　要：目的探讨SHH信号通路激活对大鼠心脏微血管内皮细胞(CMECs)缺血再灌注损伤后促血管再生的作用及其潜在机制。方法2016年7月—2017年2月于青岛大学附属烟台毓璜顶医院进行实验。原代培养SD雄性大鼠CMECs,建立氧糖剥夺/复氧(OGD/R)模拟体外缺血再灌注损伤模型,给予外源性重组人SHH蛋白、Cyclopamine(CYC)处理,应用MTT法检测CMECs细胞活力,应用ELISA和RT-PCR方法检测血管新生因子(VEGF、FGF、Ang-1)水平和mRNA表达水平;应用Western-blot方法分别检测SHH信号通路靶分子(SHH、SMO、Patched-1、Gli-1、Gli-2)蛋白表达水平。结果(1)与C组比较,正常氧糖条件下C+SHH组活力增加(P<0.05),但C+CYC组消减(P<0.05);当细胞在OGD/R条件下,OGD组CMECs活力与C组比较减弱(P<0.05),而OGD+SHH组与OGD组比较细胞活力增加(P<0.05);但OGD+CYC显著消减(P<0.05)。(2)与C组比较,正常氧糖条件下C+SHH组血管新生因子VEGF、FGF、Ang-1水平和mRNA表达增加(P<0.01),但C+CYC组上述指标水平和mRNA表达下降(P<0.01);与C组比较,OGD组VEGF、FGF、Ang-1血清水平和mRNA表达显著降低(P<0.01);OGD+SHH组VEGF、FGF、Ang-1血清水平和mRNA表达显著增加(P<0.01),OGD+CYC组上述指标水平和mRNA表达下降(P<0.05)。(3)与OGD组比较,OGD+SHH组在OGD/R条件下,SHH信号通路中靶分子SHH、SMO、Patched-1、Gli-1和Gli-2蛋白表达水平明显升高,差异有统计学意义(P<0.05),但OGD+CYC组SHH信号通路靶分子蛋白表达水平下降(P<0.01)。结论SHH信号通路激活可增强细胞活力,上调血管新生因子VEGF、FGF和Ang-1的表达,在缺血再灌注损伤后血管再生过程中发挥重要作用。Objective To explore the effect of SHH signal pathway activation on the promotion of angiogenesis after ischemia-reperfusion injury of rat cardiac microvascular endothelial cells(CMECs)and its potential mechanism.Methods From July 2016 to February 2017,the experiment was carried out in Yantai Yuhuangding Hospital affiliated to Qingdao University.Primary culture of CMECs in male SD rats was established,oxygen glucose deprivation/reoxygenation(OGD/R)simulated in vitro ischemia-reperfusion injury model was established,exogenous recombinant human SHH protein and Cyclopamine(CYC)were treated,and CMECs were detected by MTT method Cell viability,using ELISA and RT-PCR methods to detect the levels of angiogenesis factors(VEGF,FGF,Ang-1)and mRNA expression levels.Western-blot method was used to detect the protein expression levels of SHH signaling pathway target molecules(SHH,SMO,Patched-1,Gli-1,Gli-2).Results(1)Compared with group C,the viability of C+SHH group increased under normoxic glucose conditions(P<0.05),but the C+CYC group decreased(P<0.05);when cells were under OGD/R conditions,the viability of CMECs in OGD group Compared with group C,it was weakened(P<0.05),while the cell viability of OGD+SHH group was increased compared with OGD group(P<0.05);but OGD+CYC was significantly reduced(P<0.05).(2)Compared with group C,the levels of angiogenesis factors VEGF,FGF,Ang-1 and mRNA expression in C+SHH group increased under normoxic glucose conditions(P<0.01),but the above-mentioned index levels and mRNA expression decreased in C+CYC group(P<0.01);Compared with group C,serum levels of VEGF,FGF,Ang-1 and mRNA expression in OGD group were significantly reduced(P<0.01);serum levels of VEGF,FGF,Ang-1 and mRNA expression in OGD+SHH group increased significantly(P<0.01),the levels of the above indicators and mRNA expression in the OGD+CYC group decreased(P<0.05).(3)Compared with the OGD group,in the OGD+SHH group under OGD/R conditions,the protein expression levels of target molecules SHH,SMO,Patched-1,Gli-1 and Gli-2 in the SH

关 键 词：心脏微血管内皮细胞 缺血再灌注损伤 血管再生 SHH信号通路 细胞活力 大鼠 

分 类 号：R543.7[医药卫生—心血管疾病]