骨髓间充质干细胞对腹膜间皮细胞凋亡的影响

doi:10.11958/20231064

天津医药 ›› 2024, Vol. 52 ›› Issue (2): 113-118.doi: 10.11958/20231064

• 细胞与分子生物学 • 下一篇

骨髓间充质干细胞对腹膜间皮细胞凋亡的影响

王扶凝(), 代会博, 单云, 俞曼殊, 盛梅笑^△()

南京中医药大学附属医院（江苏省中医院）肾内科（邮编210029）

收稿日期:2023-07-17 修回日期:2023-08-29 出版日期:2024-02-15 发布日期:2024-01-26
通讯作者: ^△ E-mail：yfy0075@njucm.edu.cn
作者简介:王扶凝（1997），女，硕士在读，主要从事腹膜透析、间充质干细胞、肾脏病相关方面研究。E-mail：yzwangfuning@126.com
基金资助:
国家自然科学基金资助项目(82074351);江苏省中医院高峰学术人才项目(y2021rc10);江苏省中医药管理局青年人才项目(QN202207);江苏省中医院创新发展基金专项课题(Y2021CX01);江苏省研究生科研创新计划项目(KYCX23_2121);江苏省研究生科研创新计划项目(KYCX22_1939)

Study on the effect and mechanism of bone marrow mesenchymal stem cells on apoptosis of peritoneal mesothelial cells

WANG Funing(), DAI Huibo, SHAN Yun, YU Manshu, SHENG Meixiao^△()

Department of Nephrology, Affilliated Hospital of Nanjing University of Chinese Medicine (Jiangsu Province Hospital of Chinese Medicine), Nanjing 210029, China

Received:2023-07-17 Revised:2023-08-29 Published:2024-02-15 Online:2024-01-26
Contact: ^△ E-mail: yfy0075@njucm.edu.cn

王扶凝, 代会博, 单云, 俞曼殊, 盛梅笑. 骨髓间充质干细胞对腹膜间皮细胞凋亡的影响[J]. 天津医药, 2024, 52(2): 113-118.

WANG Funing, DAI Huibo, SHAN Yun, YU Manshu, SHENG Meixiao. Study on the effect and mechanism of bone marrow mesenchymal stem cells on apoptosis of peritoneal mesothelial cells[J]. Tianjin Medical Journal, 2024, 52(2): 113-118.

摘要/Abstract

摘要：

目的观察大鼠骨髓间充质干细胞（BMSCs）对高糖腹透液（PDF）诱导的大鼠腹膜间皮细胞（PMCs）凋亡的影响及可能作用机制。方法提取并鉴定大鼠原代BMSCs及PMCs，使用高糖腹透液诱导PMCs凋亡，收集BMSCs培养24 h后的细胞上清液作为条件培养基（BMSCs-CM）或通过Transwell小室将PMCs与BMSCs共培养。将PMCs分为空白对照（CON）组、高糖腹透液（PDF）组及间充质干细胞处理（PDF+BMSCs-CM）组，采用CCK-8法测定各组PMCs的增殖活力；JC-1法测定线粒体膜电位的去极化情况；TUNEL染色检测细胞凋亡情况；Western blot检测各组细胞凋亡相关蛋白B细胞淋巴瘤-2（Bcl-2）、Bcl-2相关X蛋白（Bax）、活化的胱天蛋白酶-3（Cleaved Caspase-3）和通路相关蛋白丝氨酸/苏氨酸蛋白激酶（Raf）、丝裂原活化细胞外信号调节激酶（MEK）、细胞外信号调节激酶（ERK）及其磷酸化蛋白的表达水平。结果与CON组比较，PDF组PMCs增殖活力、线粒体膜电位水平降低，而凋亡率、Bax/Bcl-2、Cleaved Caspase-3/Caspase-3、p-Raf/Raf、p-MEK/MEK、p-ERK/ERK比值升高（P<0.05）；与PDF组比较，PDF+BMSCs-CM组PMCs增殖活力、线粒体膜电位升高，而凋亡率、Bax/Bcl-2、Cleaved Caspase-3/Caspase-3、p-Raf/Raf、p-MEK/MEK、p-ERK/ERK比值降低（P<0.05）。结论 BMSCs可减轻高糖腹透液诱导的PMCs凋亡，其机制可能与抑制Raf/MEK/ERK信号通路的活化有关。

关键词: 腹膜透析, 细胞凋亡, 骨髓间充质干细胞, 腹膜间皮细胞

Abstract:

Objective To observe the effect of rat bone marrow mesenchymal stem cells (BMSCs) on the apoptosis of rat peritoneal mesothelium cells (PMCs) induced by high glucose peritoneal dialysis fluid (PDF), and to explore its possible molecular mechanism. Methods The primary BMSCs and PMCs were extracted and identified. Apoptosis of PMCs was induced by high glucose PDF. Cell supernatant from BMSCs after 24 h of culture was collected as the conditioned medium (BMSCs-CM). PMCs were co-cultured with BMSCs by conditioned media or Transwell chambers. PMCs were randomly divided into the control group, the PDF group and the PDF+BMSCs-CM group. The viability of PMCs was measured by CCK-8 in each group. The depolarization of mitochondrial membrane potential was measured by JC-1 method. TUNEL staining was used to detect cell apoptosis. Western blot assay was used to detect the expression levels of apoptosis related proteins B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), Cleaved cysteine aspartase-3 (Cleaved Caspase-3) and pathway related protein serine/threonine protein kinase (Raf), mitogen-activated extracellular signal-regulated kinase (MEK), extracellular-signal regulated protein kinase (ERK) and their phosphorylated proteins in each group. Results Compared with the control group, the proliferative activity and mitochondrial membrane potential of PMCs were decreased in the PDF group, while the apoptosis rate and the ratio of Bax/Bcl-2, Cleaved Caspase-3/Caspase-3, p-Raf/Raf, p-MEK/MEK and p-ERK/ERK were increased (P<0.05). Compared with the PDF group, the proliferative activity and mitochondrial membrane potential of PMCs were increased in the PDF+BMSCs-CM group, while the apoptosis rate and the ratio of Bax/Bcl-2, Cleaved Caspase-3/Caspase-3, p-Raf/Raf, p-MEK/MEK and p-ERK/ERK were decreased (P<0.05). Conclusion BMSCs can reduce the apoptosis of PMCs induced by high glucose PDF, and its mechanism maybe related to inhibiting the activation of Raf/MEK/ERK signaling pathway.

Key words: peritoneal dialysis, apoptosis, bone marrow mesenchymal stem cells, peritoneal mesothelial cells

中图分类号:

R285.5

图/表 8

图1 大鼠原代BMSCs及PMCs鉴定 A：BMSCs形态（×40）；B：成脂分化（油红染色，×200）；C：成骨分化（茜素红染色，×200）；D：PMCs形态（×40）；E：vimentin和Pan-Keratin免疫荧光染色（×200）。

Fig.1 Identification of primary rat BMSCs and PMCs

表1 各组PMCs增殖活力、线粒体膜电位水平及凋亡率比较（n=3，$\bar{x}±s$）

Tab.1 Comparison of relative proliferative activity, mitochondrial membrane potential and apoptosis rate of PMCs between the three groups

组别	增殖活力/%	线粒体膜电位水平	凋亡率/%
CON组	100.00±3.04	3.05±0.22	7.31±1.75
PDF组	62.00±1.36^a	1.03±0.06^a	54.42±0.88^a
PDF+BMSCs-CM组	78.24±2.15^b	1.74±0.16^b	27.36±1.14^b
F	207.551^＊＊	122.875^＊＊	894.079^＊＊

图2 JC-1荧光探针检测各组线粒体膜电位水平（×200）

Fig.2 Results of mitochondrial membrane potential in each group detected by JC-1 fluorescence probe (×200)

图3 TUNEL染色检测各组细胞凋亡情况（×200）

Fig.3 Apoptosis in each group of cells detected by TUNEL staining (×200)

图4 Western blot检测各组凋亡相关蛋白表达

Fig.4 The expression of apoptosis-related proteins detected by Western blot assay

表2 各组细胞凋亡相关蛋白表达水平比较（n=3，$\bar{x}±s$）

Tab.2 Comparison of expression levels of apoptosis-related proteins between the three groups

组别	Bax/Bcl-2	Cleaved Caspase-3/Caspase-3
CON组	0.35±0.01	0.18±0.02
PDF组	0.82±0.08^a	0.30±0.02^a
PDF+BMSCs-CM组	0.44±0.06^b	0.20±0.03^b
F	62.238^＊＊	19.303^＊＊

图5 Western blot检测各组Raf/MEK/ERK信号通路相关蛋白表达

Fig.5 The expression of Raf/MEK/ERK signal pathway related proteins detected by Western blot assay

表3 各组细胞Raf/MEK/ERK信号通路相关蛋白表达水平比较（n=3，$\bar{x}±s$）

Tab.3 Comparison of expression levels of Raf/MEK/ERK signal pathway related proteins between the three groups

组别	p-Raf/Raf	p-MEK/MEK	p-ERK/ERK
CON组	0.18±0.00	0.17±0.01	0.18±0.01
PDF组	0.34±0.03^a	0.32±0.04^a	0.29±0.01^a
PDF+BMSCs-CM组	0.22±0.02^b	0.22±0.02^b	0.21±0.01^b
F	57.076^＊＊	28.990^＊＊	81.448^＊＊

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骨髓间充质干细胞对腹膜间皮细胞凋亡的影响

Study on the effect and mechanism of bone marrow mesenchymal stem cells on apoptosis of peritoneal mesothelial cells

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