人脐带间充质干细胞培养上清对米非司酮处理的人子宫内膜基质细胞存活、凋亡和子宫内膜容受性的影响

doi:10.13481/j.1671-587X.20240110

摘要/Abstract

摘要：

目的探讨人脐带间充质干细胞培养上清（hUCMSCs-Sup）对米非司酮（Ms）处理的人子宫内膜基质细胞（hEndoSCs）增殖、凋亡和子宫内膜容受性的影响，并阐明其可能的作用机制。方法体外培养hEndoSCs，分为对照组和40、60、80及100 μmol·L^－1 Ms组，MTT法检测各组细胞存活率。hEndoSCs分为对照组、40 μmol·L^－1 Ms组和60 μmol·L^－1 Ms组，流式细胞术检测各组细胞凋亡率，Western blotting法检测各组细胞中凋亡相关蛋白B细胞淋巴瘤2（Bcl-2）和Bcl-2相关X蛋白（Bax）蛋白表达水平并计算Bcl-2/Bax比值。hUCMSCs-Sup作用后，hEndoSCs分为对照组、Ms组、Ms+hUCMSCs-Sup组和Ms+hUCMSCs-Sup+3-甲基腺嘌呤（3-MA）组，MTT法检测各组细胞存活率，流式细胞术检测各组细胞凋亡率，Western blotting法检测各组细胞中微管相关蛋白1轻链3B-Ⅱ（LC3B-Ⅱ）和微管相关蛋白1轻链3B-Ⅰ（LC3B-Ⅰ）蛋白表达水平并计算LC3B-Ⅱ/LC3B-Ⅰ比值，实时荧光定量PCR（RT-qPCR）法检测各组细胞中子宫内膜容受性标志分子mRNA表达水平。结果与对照组比较，40、60、80和100 μmol·L^－1 Ms组细胞存活率明显降低（P<0.05），且具有时间和剂量依赖性。与对照组比较，40和60 μmol·L^－1 Ms组细胞凋亡率明显升高（P<0.05），细胞中Bcl-2/Bax比值明显降低（P<0.05）。hUCMSCs-Sup作用后，与对照组比较，Ms组细胞存活率和细胞中LC3B-Ⅱ/LC3B-Ⅰ比值明显降低（P<0.05），细胞凋亡率明显升高（P<0.05），细胞中同源框基因A10（HOXA10）、白血病抑制因子（LIF）和整合素亚基β3（ITGB3）mRNA表达水平明显降低（P<0.05）；与Ms组比较，Ms+hUCMSCs-Sup组细胞存活率和细胞中LC3B-Ⅱ/LC3B-Ⅰ比值明显升高（P<0.05），细胞凋亡率明显降低（P<0.05），细胞中HOXA10、LIF和ITGb3 mRNA表达水平表达明显升高（P<0.05）；与Ms+hUCMSCs-Sup组比较，Ms+hUCMSCs-Sup+3-MA组细胞存活率和细胞中LC3B-Ⅱ/LC3B-Ⅰ比值明显降低（P<0.05）。结论 hUCMSCs-Sup可提高Ms处理后hEndoSCs的存活率，降低其凋亡率，提高子宫内膜容受性，其机制可能与hUCMSCs-Sup激活hEndoSCs自噬有关。

关键词: 脐带间充质干细胞, 人子宫内膜基质细胞, 细胞凋亡, 细胞自噬, 子宫内膜容受性

Abstract:

Objective To discuss the effect of human umbilical cord mesenchymal stem cells culture supernatant （hUCMSCs-Sup） on the proliferation， apoptosis， and endometrium receptivity of the human endometrial stromal cells （hEndoSCs） treated with mifepristone （Ms）， and to clarify the possible mechanism. Methods The hEndoSCs were cultured in vitro and divided into control group and 40， 60， 80， and 100 μmol·L^－1 Ms groups. The survival rates of the cells in various groups were detected by MTT assay. The hEndoSCs were divided into control group， 40 μmol·L^－1 Ms group， and 60 μmol·L^－1 Ms group.The apoptotic rates of the cells in various groups were detected by flow cytometry； the expression levels of apoptosis-related protein B-cell lymphoma-2 （Bcl-2） and Bcl-2-associated X protein （Bax） proteins in the cells in various groups were detected by Western blotting method， and the ratio of Bcl-2/Bax was calculated. After treated with hUCMSCs-Sup， the hEndoSCs were divided into control group， Ms group， Ms+hUCMSCs-Sup group， and Ms+hUCMSCs-Sup+3-methyladenine （3-MA） group.The survival rates of the cells in various groups were detected by MTT assay；the apoptotic rates of the cells in various groups were detected by flow cytometry；the expression levels of microtubule-associated protein 1 light chain 3B-Ⅱ （LC3B-Ⅱ） and microtubule-associated protein 1 light chain 3B-I （LC3B-Ⅰ） proteins in the cells in various groups were detected by Western blotting method， and the ratio of LC3B-Ⅱ/LC3B-Ⅰ was calculated； the expression levels of endometrium receptivity marker molecules mRNA in the cells in various groups were detected by real-time fluorescence quantitative PCR （RT-qPCR） method. Results Compared with control group， the survival rates of the cells in 40， 60， 80， and 100 μmol·L^－1 Ms groups were significantly decreased （P<0.05）in a time-dependent and dose-dependent manner. Compared with control group， the apoptotic rates of the cells in 40 and 60 μmol·L^－1 Ms groups were significantly increased （P<0.05）， and the ratios of Bcl-2/Bax were significantly decreased （P<0.05）. After treated with hUCMSCs-Sup， compared with control group， the survival rate of the cells and ratio of LC3B-Ⅱ/LC3B-Ⅰ in the cells in Ms group were significantly decreased （P<0.05）， the apoptotic rate was significantly increased （P<0.05）， and the expression levels of homeobox A10 （HOXA10）， leukemia inhibitory factor （LIF）， and integrin subunit beta 3 （ITGB3） mRNA in the cells were significantly decreased （P<0.05）；compared with Ms group， the survival rate of the cells and ratio of LC3B-Ⅱ/LC3B-Ⅰ in the cells in Ms+hUCMSCs-Sup group were significantly increased （P<0.05），the apoptotic rate was significantly decreased （P<0.05）， and the expression levels of HOXA10， LIF， and ITGB3 mRNA in the cells were significantly increased （P<0.05）； compared with Ms+hUCMSCs-Sup group， the survival rate of the cells and ratio of LC3B-Ⅱ/LC3B-Ⅰ in the cells in Ms+hUCMSCs-Sup+3-MA group were significantly decreased （P<0.05）. Conclusion hUCMSCs-Sup can increase the survival rate and decrease the apoptotic rate of the hEndoSCs after treated with Ms，and increase the endometrium receptivity，and its mechanism may be associated with the activation of autophagy of the hEndoSCs by hUCMSCs-Sup.

Key words: Umbilical cord mesenchymal stem cell, Human endometrial stromal cell, Apoptosis, Autophagy, Endometrium receptivity

中图分类号:

R711

武梦雪,陈士玲,刘艳,米旭光,林秀英,付建华,方艳秋. 人脐带间充质干细胞培养上清对米非司酮处理的人子宫内膜基质细胞存活、凋亡和子宫内膜容受性的影响[J]. 吉林大学学报(医学版), 2024, 50(1): 79-87.

Mengxue WU,Shiling CHEN,Yan LIU,Xuguang MI,Xiuying LIN,Jianhua FU,Yanqiu FANG. Effect of culture supernatant of human umbilical cord mesenchymal stem cells on survival，apoptosis and endometrium receptivity of human endometrial stromal cells after treated with mifepristone[J]. Journal of Jilin University(Medicine Edition), 2024, 50(1): 79-87.

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Group	Survival rate of cells
Group	(t/h) 0	24	48
Control	100.00±1.36	100.00±1.59	100.00±5.23
Ms(μmol·L^－1)
40	100.00±3.36	94.20±2.18^*△	69.30±1.42^*△#
60	100.00±4.27	69.73±2.09^*△	33.90±1.88^*△#
80	100.00±3.58	55.97±1.13^*△	22.11±2.43^*△#
100	100.00±6.20	54.22±1.02^*△	16.70±1.22^*△#

Group	Apoptotic rate（η/%）	Ratio of Bcl-2/Bax
Control	7.44±0.38	3.02±5.02
Ms(μmol·L^－1)
40	14.61±1.11^*	2.27±0.04^*
60	20.40±1.82^*	1.21±0.14^*

Group	Survival rate	Apoptotic rate
Control	100.00±3.41	6.60±1.25
Ms	68.83±1.47^*	20.43±0.71^*
Ms+hUCMSCs-Sup	85.00±1.41^*△	11.30±0.61^*△

Group	Survival rate (η/%)	Ratio of LC3B-Ⅱ/LC3B-Ⅰ
Control	100.00±1.29	0.39±0.10
Ms	67.12±1.85^*	0.19±0.02^*
Ms+hUCMSCs-Sup	84.67±2.32^△	1.32±0.57^△
Ms+hUCMSCs-Sup+3-MA	35.12±1.67^#	1.03±0.25^#

Group	HOXA10	LIF	ITGB3
Control	1.00±0.33	1.00±0.27	1.00±0.72
Ms	0.58±0.12^*	0.44±0.70^*	0.63±0.02^*
Ms+hUCMSCs-Sup	2.40±0.14^*△	1.93±0.07^*△	2.16±0.16^*△