酶消化细胞团块法对人胚胎干细胞中OCT4与SOX2蛋白水平的影响

doi:10.3969/j.issn.1674-8115.2021.04.001

摘要/Abstract

摘要：

目的·探究酶消化细胞团块法对人胚胎干细胞（human embryonic stem cell，hESC）的八聚体结合转录因子4（octamer-binding protein 4，OCT4）和性别决定区Y框蛋白2（sex determining region Y-box 2，SOX2）蛋白水平的影响及可能的作用机制。方法·①利用Western blotting分析检测酶消化细胞团块法［分散酶Dispase或胶原酶Ⅳ（collagenase type Ⅳ，COL4）］、酶消化单细胞法（细胞分离溶液Accutase或胰酶）和机械刮取法对hESC中OCT4和SOX2蛋白水平的影响。②使用金属离子螯合剂乙二醇双（2-氨基乙醚）四乙酸（EGTA）破坏hESC-hESC细胞间连接，然后利用Western blotting检测EGTA单独处理或联合COL4处理对hESC中OCT4与SOX2的蛋白水平的影响。③通过基于质谱的定量蛋白质组学技术，检测COL4单独处理或与EGTA联合处理hESC细胞样本中差异表达的磷酸化蛋白和总蛋白，并进行功能富集分析。④利用Western blotting和免疫荧光染色检测蛋白酶体抑制剂bortezomib或溶酶体抑制剂氯喹预处理对COL4引起的hESC中OCT4与SOX2蛋白水平下调的影响。⑤利用Western blotting检测COL4对丝切蛋白（cofilin）的影响，以及Rac1小分子抑制剂EHT1864和EHop-016对SOX2蛋白表达水平的影响。结果·①COL4和Dispase引起OCT4和SOX2的蛋白水平下降；Accutase、胰酶和机械刮取法未观察到此作用。②EGTA处理能够抑制COL4消化hESC引起的OCT4和SOX2蛋白水平下降。③蛋白质谱和功能富集分析发现，COL4消化hESC引起的OCT4和SOX2蛋白水平下降可能与肌动蛋白微丝（actin filament，F-actin）、微管和溶酶体相关蛋白的变化有关。④氯喹能够抑制COL4消化hESC引起的OCT4和SOX2蛋白水平下降，bortezomib则不能。⑤COL4处理或Rac1抑制剂均能下调cofilin和SOX2的蛋白水平。结论·当使用酶消化细胞团块法处理hESC时，OCT4和SOX2蛋白水平下降，Rac1/cofilin/F-actin信号通路和溶酶体可能参与其中。

关键词: 人胚胎干细胞, 细胞外基质, 八聚体结合转录因子4, 性别决定区Y框蛋白2, 溶酶体, 丝切蛋白

Abstract:

Objective· To reveal the effect of enzyme digestion into cell clumps on protein levels of octamer-binding protein 4 (OCT4) and sex determining region Y-box 2 (SOX2) in the human embryonic stem cells (hESCs) and its possible molecular mechanism.

Methods· ① Western blotting was conducted to detect the protein levels of OCT4 and SOX2 in the hESCs treated by enzyme digestion into cell clumps [Dispase or collagenase type Ⅳ (COL4)], enzyme digestion into single cells (Accutase or trypsin), or mechanical scraping dissociation. ② Ethylene glycol tetraacetic acid (EGTA) was used to destruct hESC-hESC interaction, and protein levels of OCT4 and SOX2 in the hESCs treated with EGTA alone or combined with COL4 were detected by Western blotting. ③ Quantitative proteomics analysis based on mass spectrometry was carried out to identify differentially expressed total proteins and phosphorylated proteins in the hESCs treated with COL4 alone or in combination with EGTA. ④ hESCs were pre-treated with proteasome inhibitor (bortezomib) and lysosome inhibitor (chloroquine), respectively, followed by Western blotting and immunofluorescence staining to measure their effects on COL4-induced down regulation of OCT4 and SOX2. ⑤ Western blotting analysis was conducted to test the effect of COL4 treatment on cofilin as well as Rac1 inhibitors (EHT1864 and EHop-016) treatment on the protein level of SOX2 in the hESCs.

Results· ① COL4 or Dispase treatment led to protein levels decrease of OCT4 and SOX2, while Accutase, trypsin and mechanical dissociation did not elicit such effect. ② EGTA rescued OCT4 and SOX2 protein levels decrease indcued by COL4 treatment. ③Quantitative mass spectrometry and functional enrichment analysis suggested that microtubule, actin filament (F-actin) and lysosome-associated proteins might be related to OCT4 and SOX2 protein levels decline in the suspended clumps of hESCs upon COL4 treatment. ④Chloroquine, instead of bortezomib, could block COL4-induced protein levels decrease of OCT4 and SOX2. ⑤The treatment of COL4 or Rac1 inhibitors could downregulate protein levels of cofilin and SOX2.

Conclusion· Upon hESC detachment in cell clumps induced by enzyme treatment, the protein levels of SOX2 and OCT4 decline, which may be related to Rac1/cofilin/F-actin signaling pathway and lysosome.

Key words: human embryonic stem cell (hESC), extracellular matrix (ECM), octamer-binding protein 4 (OCT4), sex determining region Y-box 2 (SOX2), lysosome, cofilin

中图分类号:

R329.2

孙潇智, 李爽, 金颖, 廖兵. 酶消化细胞团块法对人胚胎干细胞中OCT4与SOX2蛋白水平的影响[J]. 上海交通大学学报(医学版), 2021, 41(4): 413-420.

Xiao-zhi SUN, Shuang LI, Ying JIN, Bing LIAO. Effect of enzyme digestion into cell clumps on protein levels of OCT4 and SOX2 in human embryonic stem cells[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(4): 413-420.

图/表 5

图1 Western blotting分析不同细胞传代方法对hESC中OCT4和SOX2蛋白水平的影响

Fig 1 Effects of different cell passage methods on protein levels of OCT4 and SOX2 in hESCs detected by Western blotting

图2 同时或分别破坏hESC-ECM和hESC-hESC的相互作用对OCT4和SOX2蛋白水平的影响Note:A. Protein bands in Western blotting. B. Quantitative analysis of OCT4. C. Quantitative analysis of SOX2. ①P=0.002, ②P=0.013, ③P=0.006, ④P=0.010.

Fig 2 Effect of disruption of hESC-hESC and hESC-ECM interactions simultaneously or respectively on the protein levels of OCT4 and SOX2 in hESCs

图3 蛋白质谱分析鉴定的差异表达磷酸化蛋白和总蛋白的功能富集Note:A. Functional enrichment analysis of differentially expressed phosphorylated proteins between the COL4 group and the Ctrl group. B. Functional enrichment analysis of up-regulated proteins in the COL4 group compared to the Ctrl group. C. Functional enrichment analysis on down-regulated proteins in the COL4+EGTA group compared to the Ctrl group. D. Overlap of differentially expressed proteins identified in B and C. The blue and the red circles represent the differentially expressed proteins identified in B and C, respectively. The identical proteins are listed in the dotted line frame, and the red color denotes lysosome-related proteins. DPP7—dipeptidyl peptidase 7; PKP4—plakophilin 4; GK3P—glycerol kinase 3 pseudogene; SMIM20—small integral membrane protein 20.

Fig 3 Functional enrichment on differentially expressed phosphorylated proteins and total proteins identified by proteomics analysis

图4 氯喹对COL4引起的OCT4和SOX2蛋白水平下降的影响Note:A. Representative Western blotting analysis of OCT4 and SOX2 in the hESCs treated with bortezomib (100 μmol/L) or DMSO (solvent control) for 2 h prior to hESC dissociation as indicated. B. Representative Western blotting analysis of OCT4 and SOX2 in the hESCs treated with or without chloroquine (100 μmol/L) for 2 h prior to hESC dissociation as indicated. C. Immunofluorescence staining in the hESC clumps treated with or without chloroquine for 2 h prior to hESC dissociation as indicated.

Fig 4 Effect of chloroquine on the decrease of OCT4 and SOX2 protein levels induced by COL4

图5 Rac1/cofilin/F-actin信号通路在COL4引起的OCT4和SOX2蛋白水平下降中的作用Note:A. Immunofluorescence staining of F-actin, α-tubulin and SOX2 in the hESCs. Apical—apical side; Middle—lateral side; Basal—basal side. B. Representative Western blotting analysis of integrin-β1, p-cofilin and cofilin in the hESCs collected at the indicated time points after COL4 or COL4+EGTA treatment. C. Representative Western blotting analysis of LIMK2, p-LIMK1/2, cofilin, p-cofilin and SOX2 in the hESCs treated with EHT1864 or EHop016 for 24 h.

Fig 5 Role of Rac1/cofilin/F-actin signaling pathway in the COL4-induced decrease of OCT4 and SOX2 protein levels

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