氯化钴模拟法构建绒毛外滋养细胞缺氧模型

Construction of a new model for extravillous trophoblast cell hypoxia using cobalt chloride

目的:探讨适宜的绒毛外滋养细胞(EVT)化学性缺氧模型。方法:选取2022年4月至2023年1月在广西医科大学第一附属医院妇科选择人工流产的正常孕妇(6~9孕周)的绒毛组织,分离和培养原代EVT,利用免疫细胞化学进行纯度鉴定。利用氯化钴(CoCl_(2))构建细胞缺氧模型,CCK-8实验检测不同浓度(0、50、100、150、300μmol/L和600μmol/L)CoCl_(2)在不同时间(6、12、24h和48h)对原代EVT细胞存活率的影响,实时荧光定量聚合酶链反应实验(RT-qPCR)检测缺氧诱导因子-1α(HIF-1α)mRNA表达。结果:成功分离和培养原代EVT。EVT的细胞存活率与CoCl_(2)浓度和缺氧时间有关。相对于对照组(0μmol/L),0~150μmol/L浓度组细胞存活率下降不明显;300μmol/L浓度组作用24h后对细胞生长明显抑制。600μmol/L浓度组的细胞毒性作用出现最早,24h后细胞基本全部死亡。150μmol/L CoCl_(2)缺氧6、12、24、48h各组的HIF-1α mRNA相对表达量分别是对照组(0h)的0.50倍、0.64倍、1.45倍和3.00倍。缺氧时间24h, 50、100、150μmol/L CoCl_(2)各组的HIF-1α mRNA相对表达量分别是对照组(0μmol/L)的1.23倍、1.28倍、1.58倍。结论:CoCl_(2)可用于构建原代EVT的化学性缺氧细胞模型,综合CoCl_(2)的细胞毒性作用及HIF-1α mRNA表达水平,CoCl_(2)的适宜作用浓度是150μmol/L,作用时间是24h。

Objective:To investigate a suitable model of chemical hypoxia of extravillous trophoblast(EVT).Methods:Primary EVTs were isolated and cultured from the villi of normal pregnant women(6~9 weeks)who chose to abortion in the First Affiliated Hospital of Guangxi Medical University from April_(2)022 to January 2023,and the purity was determined by immunocytochemistry.We used cobalt chloride(CoCl_(2))to construct cell hypoxia model,and CCK-8 assay was used to detect the effects of different concentrations(0,50,100,150,300,600μmol/L)of CoCl_(2) on the survival rate of primary EVT cells at different times(6,12,24,48h).Reverse transcription quantitative polymerase chain reaction(RT-qPCR)assay was used to detect the expression of hypoxia-inducible factor-1α(HIF-1α)mRNA.Results:We successfully isolated and cultured primary EVT.The survival rates of EVT were correlated with the concentration and exposure time of CoCl_(2).Compared with control group(0μmol/L),the cell survival rate in 0~150μmol/L concentration groups decreased insignificantly.However,the 300μmol/L concentration group significantly inhibited cell growth after 24h.Cytotoxic effects appeared first in the 600μmol/L group,and almost all cells died after 24h.HIF-1αmRNA relative expression levels in the 150μmol/L CoCl_(2) hypoxia group at 6,12,24h and 48h were 0.50 times,0.64 times,1.45 times,3.00 times of that in the control group(0h),respectively.After 24h hypoxia,the HIF-1αmRNA relative expression levels in 50,100μmol/L and 150μmol/L CoCl_(2) groups were 1.23 times,1.28 times,and 1.58 times of that in control group(0μmol/L),respectively.Conclusion:CoCl_(2) can be used to construct the chemical hypoxia cell model of primary EVT.Based on the cytotoxicity of CoCl_(2) and the expression level of HIF-1αmRNA,the optimal concentration of CoCl_(2) is 150μmol/L,and the optimal time is 24h.