基于mRNA测序筛选先天性膈疝大鼠模型肺PI3K/AKT/eNOS/VEGF的基因表达差异

Screening the differential gene expressions of PI3K/AKT/eNOS/VEGF with mRNA sequencing in lung of congenital diaphragmatic hernia rat model

目的研究除草醚诱导的先天性膈疝大鼠模型的肺组织中磷脂酰肌醇-3-激酶/蛋白激酶B/内皮型一氧化氮合酶/血管内皮生长因子(PI3K/AKT/eNOS/VEGF)基因表达情况。方法制作先天性膈疝大鼠模型,将10只SPF级雌性SD大鼠在怀孕9.5 d时通过随机数字表法分组,分为对照组5只及膈疝组5只,于孕21.5 d时解剖出胎鼠肺组织,通过HE染色检测胎鼠肺组织发育情况;α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)免疫荧光技术检测肺小动脉肌化程度;对照组与膈疝组各取3份胎鼠左肺组织样本行mRNA测序,进行差异表达基因分析,并对差异表达基因行KEGG功能富集分析;采用qRT-PCR技术检测两组胎鼠肺组织PI3K/AKT/eNOS/VEGF mRNA表达水平;采用酶联免疫吸附试验及一氧化氮(NO)试剂盒分别检测eNOS和NO生成量。结果本研究中,对照组获取78只活胎,膈疝组获取64只活胎,膈疝组中37只胎鼠形成膈疝,膈疝率57.8%(37/64);HE染色后膈疝组相较于对照组存在明显肺发育不全及血管重构;免疫荧光显示膈疝组α-SMA表达增高(P<0.01),提示膈疝组胎鼠肺血管中膜厚度增厚且血管肌化水平增加。mRNA测序结果膈疝组与对照组相比,差异基因有4871个,其中下调基因有3741个,上调基因有1130个。这些差异基因主要富集于PI3K/AKT信号通路、MAPK信号通路、Rap1信号通路、cAMP信号通路、催产素信号通路、Apelin信号通路、松弛素(Relaxin)信号通路、环磷酸鸟苷(cGMP-PKG)信号通路等。qRT-PCR显示,膈疝组与对照组相比PI3K/AKT/eNOS/VEGF均下调(P<0.05)。测定eNOS膈疝组浓度(8.720±0.729)ng/ml相比于对照组浓度(11.084±0.605)ng/ml降低;膈疝组NO浓度(2.811±0.213)μmol/gprot相比于对照组浓度(4.598±0.588)μmol/gprot降低,差异均有统计学意义(P<0.01)。结论除草醚诱导的先天性膈疝大鼠模型肺组织中,PI3K/AKT/eNOS/VEGF基因表达下调以及NO生成减少,肺血管重塑及新生血管减少可能与之相关。

Objective To explore the gene expression of phosphatidylinositol-3-kinase/protein kinase B/endothelial nitric oxide synthase/vascular endothelial growth factor(PI3K/AKT/eNOS/VEGF)in lung tissue of herbicide(nitrofen)-induced congenital diaphragmatic hernia(CDH)in rats.Methods Rat model of CDH was established.Ten specific pathogen free(SPF)female Sprague-Dawley(SD)rats were randomized into two groups of blank control(n=5)and CDH(n=5)at 9.5 days of gestation.Lung tissue of fetal rats was dissected at day 21.5.The development of fetal lung tissue was detected after hematoxylin-eosin(HE)staining and muscularization degree of pulmonary arterioles observed after α-smooth muscle actin(α-SMA)immunofluorescence.Three fetal rat left lung samples from control and CDH groups were sequenced by mRNA,differentially expressed genes analyzed by KEGG functional enrichment analysis and the expression levels of PI3K,AKT,eNOS and VEGF mRNA detected by fluorescent quantitative real-time polymerase chain reaction(qRT-PCR);enzyme-linked immunosorbent assay(ELISA)and nitric oxide(NO)kit were utilized for detecting the production of eNOS and NO respectively.Results In this study,78 live fetuses were obtained in control group and 64 live fetuses in CDH group.The incidence of CDH was 57.8%(37/64).As compared with control group,obvious pulmonary hypoplasia and vascular remodeling appeared in CDH group.Immunofluorescence indicated that the expression ofα-SMA increased(P<0.01),suggesting that pulmonary vascular media thickness and level of vascular myogenesis increased in CDH group.mRNA sequencing revealed 4871 differential genes between CDH and control groups,including 3741 down-regulated genes and 1130 up-regulated genes.These differential genes were concentrated in PI3K/AKT signal pathway,MAPK signal pathway,Rap1 signal pathway,cAMP signal pathway,oxytocin signal pathway,Apelin signal pathway,Relaxin signal pathway(Relaxin),cyclic guanosine monophosphate(cGMP-PKG)signal pathway,etc.qRT-PCR indicated that PI3K/AKT/eNOS/VEGF was lower in CDH group than that in blank group(P<0.05).The concentration of eNOS was lower in CDH group than that in blank group[(8.720±0.729)vs(11.084±0.605)ng/ml];NO CDH group was lower than blank group[(2.811±0.213)vs(4.598±0.588)μmol/gprot].Both differences were statistically significant(P<0.01).Conclusions In herbicide-induced CDH rat model,a down-regulation of PI3K/AKT/eNOS/VEGF gene expression and a decline of NO production may be correlated with pulmonary vascular remodeling and neovascularization.