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目的建立人骨髓间充质干细胞(hBM-MSCs)体外培养系统,检测1,4-苯醌(1,4-BQ)对hBM-MSCs中copine 1、copine 3表达的影响。方法采用密度梯度离心和流式细胞技术分离、鉴定hBM-MSCs;10、25、50μmol/L1,4-BQ染毒24 h后,实时定量PCR技术和蛋白免疫印迹检测hBM-MSCs中copine 1、copine 3 mRAN和蛋白质表达情况。结果 hBM-MSCs稳定表达CD29、CD44,几乎不表达CD34、CD45;实时定量PCR的结果证实,在mRNA水平1,4-BQ染毒剂量在10μmol/L即可引起copine 1表达降低,25μmol/L可引起copine 3表达水平降低。蛋白免疫印迹试验结果证实1,4-BQ为25μmol/L和50μmol/L分别引起copine 1和copine 3表达降低。结论 1,4-BQ可在转录水平和蛋白质水平抑制hBM-MSCs中copine 1和copine 3的表达,本研究结果有助于进一步阐明苯及其代谢物1,4-BQ的血液毒性机制。
OBJECTIVE: To establish an in vitro culture system of human bone marrow mesenchymal stem cells (hBM-MSCs) to detect the effects of 1,4-benzoquinone (1,4-BQ) on the expression of copine 1 and copine 3 in hBM-MSCs. Methods The hBM-MSCs were isolated and identified by density gradient centrifugation and flow cytometry. The expressions of copine 1, hBM-MSCs and hBM-MSCs were detected by real-time quantitative PCR and Western blotting after exposed to 10, 25, 50μmol / copine 3 mRAN and protein expression. Results The hBM-MSCs stably expressed CD29 and CD44, but scarcely expressed CD34 and CD45. The results of real-time PCR confirmed that the mRNA expression level of copine 1 was decreased at the dose of 1,4-BQ at mRNA level and decreased at 25μmol / L Can cause a decrease of copine 3 expression level. Results of Western blotting showed that 1,4-BQ at 25 and 50 μmol / L caused a decrease in copine 1 and copine 3, respectively. CONCLUSION: 1,4-BQ can inhibit the expression of copine 1 and copine 3 in hBM-MSCs both at the transcription and protein levels. The results of this study may be helpful to further elucidate the hematological toxicity of benzene and its metabolites 1,4-BQ.