自噬抑制肿瘤坏死因子α诱导人胎盘胎儿来源间充质干细胞发生凋亡 <sup>*</sup>

doi:10.13523/j.cb.20190909

中国生物工程杂志

2019, Vol. 39

Issue (9): 62-67 DOI: 10.13523/j.cb.20190909

研究报告

自噬抑制肿瘤坏死因子α诱导人胎盘胎儿来源间充质干细胞发生凋亡 ^*

朱永朝^1,^**,陶金^1,^**,任萌萌²,熊燃³,何亚琴¹,周瑜¹,卢震辉¹,杜勇¹,杨芝红^1,^**(

)

1 宁夏医科大学总医院银川 750004
2 银川市妇幼保健院银川 750001
3 深圳精准医疗科技有限公司深圳 518000

Autophagy Protects Against Apoptosis of Human Placental Mesenchymal Stem Cells of Fetal Origin Induced by Tumor Necrosis Fator-α

ZHU Yongzhao^1,^**,TAO Jin^1,^**,REN Meng-meng²,XIONG Ran³,HE Ya-qin¹,ZHOU Yu¹,LU Zhen-hui¹,DU Yong¹,YANG Zhi-hong^1,^**(

)

1 Ningxia Medical University, Yinchuan 750004, China
2 Yinchuan Maternal and Children Health Care Hospital, Yinchuan 750001, China
3 Shenzhen Gentarget Biopharmaceutical Co., Ltd, Shenzhen 518000, China

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摘要：

目的: 探讨TNF-α诱导人胎盘胎儿来源间充质干细胞(hfPMSCs)发生凋亡和自噬的作用,以及自噬对细胞凋亡的调控作用。方法: 利用流式细胞术检测无血清培养hfPMSCs中CD73、CD90、CD105、CD14、CD34、CD45的表达;用终浓度 20 μg /L 的TNF-α处理hfPMSCs 24h,以未处理细胞作为对照组。Annexin V/PI双染色检测TNF-α对hfPMSCs凋亡程度的影响;分别提取各组总蛋白,Western blot 检测自噬标志基因 LC3Ⅰ/Ⅱ的表达;利用mRFP-GFP-LC3 腺病毒感染细胞,观察胞内点状聚集形成的情况;利用Atg5干扰慢病毒(si-Atg5)及阴性对照慢病毒(si-NC)感染hfPMSCs,Annexin V/PI双染色检测TNF-α对慢病毒感染后hfPMSCs凋亡程度的影响。结果： 所培养细胞具有典型的MSCs形态,呈CD73 ⁺CD90 ⁺CD105 ⁺/CD14 ^-CD34 ^-CD45 ^-细胞;Annexin V/PI 染色结果显示,TNF-α作用24 h后,hfPMSCs凋亡数和凋亡率均高于对照组(P<0.05);Western blot检测自噬标志蛋白表达结果表明,TNF-α可增加LC3Ⅱ的表达(P<0.05);荧光共聚焦显微境观察到 TNF-α可显著提高细胞中的点状聚集。利用si-Atg5感染细胞,抑制hfPMSCs自噬的发生,与对照慢病毒si-NC感染细胞比较,可显著促进TNF-α诱导hfPMSCs凋亡的发生(P<0.05)。 结论： TNF-α诱导的自噬抑制人胎盘胎儿来源 MSCs凋亡的发生,具有一定的保护性作用。

关键词： 人胎盘; 间充质干细胞; 肿瘤坏死因子-α; 自噬; 细胞凋亡

Abstract:

Objective: To explore the effects of TNF-α on apoptosis and autophagy of human placenta fetal derived mesenchymal stem cells (hfPMSCs) and the regulation of autophagy on apoptosis.Methods: The expression of CD73, CD90, CD105, CD14, CD34 and CD45 was identified by flow cytometry analysis for fPMSCs cultured in serum-free medium. hfPMSCs were treated with TNF-α at a final concentration of 20 g /L for 24h. Annexin V/PI double staining assay detected cells apoptosis; Each group of total protein was extracted, the expression of autophagy marker protein LC3 Ⅰ/Ⅱ was tested by Western blot; mRFP-GFP-LC3 adenovirus was used to infect fPMSCs, the formation of the puncta light was observed by confocal fluorescence microscopy; hfPMSCs were infected with Atg5 interfering lentivirus (si-Atg5) and negative control lentivirus (si-NC), and Annexin V/PI double staining was used to detect cells apoptosis.Results: The cultured cells possessed typical MSCs morphology and belong to CD73 ⁺CD90 ⁺CD105 ⁺/ CD14 ^-CD34 ^-CD45 ^- cells. Annexin V/PI staining results showed that after TNF-αtreatment for 24 h, the apoptosis number and rate of hfPMSCs were higher than those in the control group (P<0.05). Compared to untreated group, TNF-α increased the deposition number of LC3 Ⅱ (P<0.05),and induced more aggregation of puncta light in cytoplasm.Compared: with the control group, TNF-αinduced apoptosis of hfPMSCs was significantly elevated in autopahgy inhibition group (P<0.05). Conclusion: TNF-αinduced autophagy can inhibit the apoptosis of hfPMSCs, play an important protective function.

Key words: Human placenta Mesenchymal stem cells Tumor necrosis factor-α Autophagy Apoptosis

收稿日期: 2019-08-23 出版日期: 2019-09-20

ZTFLH:

Q291

基金资助: * 国家自然科学基金(81560016)

通讯作者: 朱永朝,陶金,杨芝红 E-mail: zhangpac@163.com

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引用本文:

朱永朝,陶金,任萌萌,熊燃,何亚琴,周瑜,卢震辉,杜勇,杨芝红. 自噬抑制肿瘤坏死因子α诱导人胎盘胎儿来源间充质干细胞发生凋亡 ^*[J]. 中国生物工程杂志, 2019, 39(9): 62-67.

ZHU Yongzhao,TAO Jin,REN Meng-meng,XIONG Ran,HE Ya-qin,ZHOU Yu,LU Zhen-hui,DU Yong,YANG Zhi-hong. Autophagy Protects Against Apoptosis of Human Placental Mesenchymal Stem Cells of Fetal Origin Induced by Tumor Necrosis Fator-α. China Biotechnology, 2019, 39(9): 62-67.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20190909 或 https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I9/62

Fig.1 person placenta fetal source of MSCs morphological observation (phase contrast microscope, ×40)

图2 流式细胞术检测无血清培养人胎盘胎儿来源MSCs表面标志物

图3 TNF-α对人胎盘胎儿来源MSCs凋亡的影响

图4 TNF-α对人胎盘胎儿来源MSCs LC3I/II表达的影响

图5 TNF-α诱导人胎盘胎儿来源MSCs自噬体形成的鉴定

图6 TNF-α诱导的自噬调控人胎盘胎儿来源MSCs凋亡的发生

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