摘要
目的观察采用微小RNA-148a(miR-148a)低表达人绒毛膜滋养细胞系HTR-8/SVneo构建的子痫前期(Preeclampsia,PE)模型细胞的细胞活力、焦亡、炎症和氧化应激反应。方法取对数生长期人绒毛膜滋养细胞系HTR-8/SVneo,分为甲、乙、丙、丁组:甲组细胞用抑制miR-148a表达的miR-148a inhibitor转染24 h,加入100 ng/L的LPS培养24 h(建立PE模型);乙组细胞用空白对照NC-inhibitor转染24 h,加入100 ng/L的LPS培养24 h;丙组加入100 ng/L的LPS培养24 h;丁组不做任何处理。培养48 h时,采用qRT-PCR法检测各组细胞miR-148a,采用CCK8法检测各组细胞活力,采用TUNEL法测算各组细胞焦亡率,采用Western Blotting法检测各组细胞焦亡相关蛋白Caspase-1、GSDMD,采用ELISA法检测各组上清液炎症因子[肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、IL-6]及氧化应激指标[谷胱甘肽过氧化物酶(GSH)、超氧化物歧化酶(SOD)、丙二醛(MDA)]。结果与丁组相比,丙组细胞miR-148a相对表达量高,培养24、48、72 h时OD值低,细胞焦亡率高,细胞Caspase-1、GSDMD蛋白相对表达量高(P均<0.05);与乙组相比,甲组细胞miR-148a相对表达量低,培养24、48、72 h时OD值高,细胞焦亡率低,细胞Caspase-1、GSDMD和NLRP3蛋白相对表达量低(P均<0.05)。与丁组相比,丙组细胞上清液TNF-α、IL-1β及IL-6水平高,细胞GSH和SOD表达降低、MDA表达升高(P均<0.05);与乙组相比,甲组细胞上清液TNF-α、IL-1β及IL-6水平低,细胞GSH和SOD表达升高、MDA表达降低(P均<0.05)。结论miR-148a低表达HTR-8/SVneo细胞构建的PE模型细胞活力高,细胞焦亡程度、炎性反应及氧化应激反应低。miR-148a可能是PE的治疗靶点之一。
Objective To observe the cell viability,pyroptosis,inflammation and oxidative stress response of the preeclampsia(Preeclampsia,PE)model cells constructed using the human chorionic trophoblast cell line HTR-8/SVneo with low expression of microRNA-148a(miR-148a).Methods Human chorionic trophoblast cell line HTR-8/SVneo in the logarithmic growth phase were selected and divided into groups A,B,C,and D.Cells in the group A were transfected with miR-148a inhibitor(inhibiting miR-148a expression)for 24 h,followed by the addition of 100 ng/L LPS for 24 h(es⁃tablishing a PE model cell);cells in the group B were transfected with NC inhibitor(blank control)and cultured for 24 h,followed by the addition of 100 ng/L LPS for 24 h;cells in the group C were cultured with 100 ng/L LPS for 24 h;cells in the group D were not treated.At 48 h of culture,qRT-PCR was used to detect miR-148a in cells of each group;CCK-8 was used to detect cell viability in each group;TUNEL method was used to calculate the apoptosis rate in each group;Western blotting was used to detect the apoptosis-related proteins Caspase-1,GSDMD,and NLRP3 in each group;ELISA was used to detect inflammatory factors(tumor necrosis factor-α[TNF-α],interleukin-1β[IL-1β],IL-6)and oxidative stress indicators(glutathione peroxidase[GSH],superoxide dismutase[SOD],and malondialdehyde[MDA])in the su⁃pernatant of each group.Results Compared with the group D,the relative expression level of miR-148a was higher,and the OD values were lower at 24,48,and 72 h of culture,the apoptosis rate was higher,and the relative expression levels of Caspase-1,GSDMD,and NLRP3 proteins in cells were higher in the group C(all P<0.05).Compared with the group B,the relative expression level of miR-148a was lower,and the OD values were higher at 24,48,and 72 h of culture,the apoptosis rate was lower,and the relative expression levels of Caspase-1,GSDMD,and NLRP3 proteins in cells were low⁃er in the group A(all P<0.05).Compared with the group D,the levels of TNF-α,IL-1β,and IL-6 in the cel
作者
郭艳萍
栾媛媛
周巾
蒋天从
GUO Yanping;LUAN Yuanyuan;ZHOU Jin;JIANG Tiancong(Department of Obstetrics and Gynecology,Tangshan Maternal and Child Health Hospital,Tangshan 063000,China)
出处
《山东医药》
CAS
2024年第18期1-5,共5页
Shandong Medical Journal
基金
河北省医学科学研究课题计划(20221770)。
