双酚A对子宫内膜间充质干/基质细胞干性的影响及人脐带间充质干细胞源性上清对细胞损伤的改善作用

doi:10.13481/j.1671-587X.20240609

摘要/Abstract

摘要：

目的探讨双酚A（BPA）对子宫内膜间充质干/基质细胞（eMSCs）增殖活性和干性特征的影响，阐明人脐带间充质干细胞源性上清（hUCMSC-Sup）对细胞损伤的改善作用。方法体外培养eMSCs，以0、200、250、300、350、400 μmol·L^－1BPA处理。将eMSCs分为对照组（仅培养液培养）、BPA组（含200 μmol·L^－1 BPA的等体积培养液培养）、BPA+hUCMSC-Sup组（含200 μmol·L^－1 BPA及50%体积比hUCMSC-Sup的等体积培养液培养）和BPA+CHIR-99021组（含200 μmol·L^－1 BPA及10 μmol·L^－1 CHIR-99021的等体积培养液培养），使用干细胞成球培养液培养eMSCs干细胞球，其余细胞均使用DMEM/F12完全培养基培养。噻唑蓝（MTT）法检测各组eMSCs存活率，球体形成实验检测各组eMSCs干细胞球数和直径，CCK-8法检测各组eMSCs干细胞球中细胞增殖活性，流式细胞术检测各组eMSCs中CD73+细胞百分率，实时荧光定量PCR（RT-qPCR）法检测各组eMSCs中性别决定区Y框蛋白2（Sox2）、八聚体结合转录因子4（Oct4）和Nanog mRNA表达水平，Western blotting法检测各组eMSCs中β-连环蛋白（β-catenin）蛋白表达水平。结果 MTT法检测，BPA作用24和48 h，与0 μmol·L^－1 BPA组比较，200、250、300、350和400 μmol·L^－1 BPA组eMSCs存活率均明显降低（P<0.01）。药物作用24 h时，与对照组比较，BPA组eMSCs存活率明显降低（P<0.01）；药物作用48 h时，与对照组比较，BPA组eMSCs存活率明显降低（P<0.01）；与BPA组比较，BPA+hUCMSC-Sup组eMSCs存活率明显升高（P<0.05）。球体形成实验检测，与培养3 d组比较，培养4和5 d组eMSCs干细胞球数和直径均明显增加（P<0.05或P<0.01）；与对照组比较，培养48 h时BPA组eMSCs干细胞球数和直径均明显减少（P<0.05或P<0.01）。CCK-8法检测，处理24和48 h时，与对照组比较，BPA组eMSCs干细胞球中细胞增殖活性均明显降低（P<0.01）；与BPA组比较，BPA+hUCMSC-Sup组eMSCs干细胞球中细胞增殖活性均明显升高（P<0.01）。流式细胞术检测，与对照组比较，BPA组eMSCs中CD73+细胞百分率明显降低（P<0.01）；与BPA组比较，BPA+hUCMSC-Sup组eMSCs中CD73+细胞百分率明显升高（P<0.01）。RT-qPCR法检测，与对照组比较，BPA组eMSCs中Sox2、Oct4和Nanog mRNA表达水平均明显降低（P<0.01）；与BPA组比较，BPA+hUCMSC-Sup组和BPA+CHIR-99021组eMSCs中Sox2、Oct4及Nanog mRNA表达水平均明显升高（P<0.01）。Western blotting法检测，与对照组比较，BPA组eMSCs中β-catenin蛋白表达水平明显降低（P<0.01）；与BPA组比较，BPA+hUCMSC-Sup组和BPA+CHIR-99021组eMSCs中β-catenin蛋白表达水平均明显升高（P<0.01）。结论 BPA能够抑制eMSCs的干性特征，损伤子宫内膜的自我更新及修复作用，其机制可能与下调细胞中Wnt/β-catenin信号通路活性有关。hUCMSC-Sup可以促进受损eMSCs的增殖，并对BPA诱导的eMSCs干性损伤起到改善作用。

关键词: 双酚A, 子宫内膜间充质干/基质细胞, 人脐带间充质干细胞, 干细胞球

Abstract:

Objective To investigate the effects of bisphenol A （BPA） on the proliferation activity and stemness characteristics of endometrial mesenchymal stem/stromal cells （eMSCs）， and to elucidate the improvement effect of human umbilical cord mesenchymal stem cell-derived supernatant （hUCMSC-Sup） on the cell injury. Methods The eMSCs were cultured in vitro and treated with different concentrations of BPA （0， 200， 250， 300， 350， and 400 μmol·L^-1）. The eMSCs were divided into control group（only cultured with culture solution）， BPA group （cultured with isovolumetric culture solution including 200 μmol·L^-1BPA）， BPA+hUCMSC-Sup group （cultured with isovolumetric culture solution including 200 μmol·L^-1BPA and 50% volumetric ratio of hUCMSC-Sup）， and BPA+CHIR-99021 group （cultured with isovolumetric culture solution including 200 μmol·L^-1 BPA and 10 μmol·L^-1CHIR-99021）.The survival rates of eMSCs in various groups were detected by methyl thiazolyl tetrazolium（MTT） assay. The numbers and diameters of the spheroids in various groups were detected by spheroids formation assay， the proliferation activities of the cells in eMSCs stem cell spheroids in various groups were detected by CCK-8 assay； the percentage of CD73+ cells in eMSCs in various groups were detected by flow cytometry； the expression levels of sex determining region Y-box 2 （Sox2）， octamer-binding transcription factor 4（Oct4）， and Nanog mRNA in the eMSCs in various groups were detected by real-time fluorescence quantitative PCR（RT-qPCR） method， the expression levels of β-catenin protein in the eMSCs in various groups were detected by Western blotting method. Results The MTT results showed that after treated with BPA for 24 and 48 h， compared with 0 μmol·L^-1 BPA group， the survival rates of eMSCs in 200， 250， 300， 350，and 400 μmol·L^-1 BPA groups were significantly decreased （P<0.01）. At 24 and 48 h after treatment， compared with control group， the survival rate of the eMSCs in BPA group was significantly decreased （P<0.01）； at 48 h after treatment， compared with BPA group， the survival rate of the eMSCs in BPA+hUCMSC-Sup group was significantly inereased （P<0.05）. The spheroids formation assay results showed that compared with culture 3 d group， the numbers and diameters of stem cell spheroids of the eMSCs in culture 4 d group and culture 5 d group were significantly increased （P<0.05 or P<0.01）； compared with control group， after 48 h of culture， the number and diameter of the cells in eMSCs stem cell spheroids in BPA group were significantly decreased （P<0.05 or P<0.01）. The CCK-8 results showed that after 24 and 48 h of treatment， compared with control group， the proliferation activity of the cells in eMSCs stem cell spheroids in BPA group was significantly decreased （P<0.01）； compared with BPA group， the proliferation activity of the cells in eMSCs stem cell spheroids in BPA+hUCMSC-Sup group was significantly increased（P<0.01）. The flow cytometry results showed that compared with control group， the percentage of the CD73+ cells in the eMSCs in BPA group was significantly decreased （P<0.01）； compared with BPA group， the percentage of the CD73+ cells in eMSCs in BPA+hUCMSC-Sup group was significantly increased （P<0.01）. The RT-qPCR results showed that compared with control group， the expression levels of Sox2， Oct4， and Nanog mRNA in the cells in BPA group were significantly decreased （P<0.01）； compared with BPA group， the expression levels of Sox2， Oct4， and Nanog mRNA in the cells in BPA+hUCMSC-Sup group and BPA+CHIR-99021 group were significantly increased （P<0.01）. The Western blotting results showed that compared with control group， the expression level of β-catenin protein in the eMSCs in BPA group was significantly decreased（P<0.01）； compared with BPA group， the expression levels of β-catenin protein in the eMSCs in BPA+hUCMSC-Sup group and BPA+CHIR-99021 group were signifrcantly inereased （P<0.01）. Conclusion BPA can inhibit the stemness characteristics of the eMSCs， and injury the self-renewal and repair of endometrium； its mechanism may be related to down-regulating the activity of Wnt/β-catenin signal pathway in the cells. hUCMSC-Sup can promote the proliferation of injured eMSCs， and has improvement effect on the stemness injury induced by BPA.

Key words: Bisphenol A, Endometrial mesenchymal stem/stromal cell, Human umbilical cord mesenchymal stem cell, Stem cell spheroid

中图分类号:

Q254

王爱乔,米旭光,林秀英,付建华,刘磊,王琳,张文琦,邓玲,陈士玲,方艳秋. 双酚A对子宫内膜间充质干/基质细胞干性的影响及人脐带间充质干细胞源性上清对细胞损伤的改善作用[J]. 吉林大学学报(医学版), 2024, 50(6): 1557-1564.

Aiqiao WANG,Xuguang MI,Xiuying LIN,Jianhua FU,Lei LIU,Lin WANG,Wenqi ZHANG,Ling DENG,Shiling CHEN,Yanqiu FANG. Effects of bisphenol A on stemness of endometrial mesenchymal stem/stromal cells and improvement effect of human umbilical cord mesenchymal stem cell-derived supernatant on cell injury[J]. Journal of Jilin University(Medicine Edition), 2024, 50(6): 1557-1564.

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Group	Survival rate
Group	(t/h) 24	48
0 μmol·L^－1 BPA	100.00±0.00	100.00±0.00
200 μmol·L^－1 BPA	77.69±3.03^*	75.48±2.93^*
250 μmol·L^－1 BPA	72.82±1.97^*	62.53±2.96^*
300 μmol·L^－1 BPA	63.41±1.58^*	38.59±2.60^*
350 μmol·L^－1 BPA	37.23±3.40^*	29.32±6.56^*
400 μmol·L^－1 BPA	32.41±3.10^*	17.68±3.63^*

Group	Survival rate
Group	(t/h) 24	48
Control	100.00±0.00	100.00±0.00
BPA	72.15±2.59^*	67.39±3.02^*
BPA+hUCMSC-Sup	80.03±4.62	75.21±3.32^△

Group	Spheroid number	Spheroid size (l/μm)
Culture 3 d	18.00±1.00	54.26±2.39
Culture 4 d	29.00±2.00^**	61.25±3.39^*
Culture 5 d	37.33±2.52^**	63.65±2.52^**

Group	Spheroids number		Spheroids size (l/μm)
Group	(t/h) 24	48	24	48
Control	29.00±3.00	38.00±2.65	57.64±1.62	63.97±1.89
BPA	28.67±0.58	31.67±2.08^*	57.18±2.70	52.95±1.21^**

Group	Proliferation activity
Group	(t/h) 0	24	48
Control	1.17±0.04	1.24±0.02	1.29±0.04
BPA	1.18±0.01	1.12±0.03^*	1.09±0.02^*
BPA+hUCMSC-Sup	1.17±0.01	1.22±0.01^△	1.29±0.01^△