胆固醇转运相关基因NPC1、NPC2调节造血前体细胞的增殖和分化

发布时间：2018-03-11 11:20

本文选题：NPC　切入点：NPC　出处：《第三军医大学学报》2017年12期 　论文类型：期刊论文

【摘要】：目的采用NPC1、NPC2基因敲低的造血前体细胞(erythroid myeloid lymphoid,EML)作为模型,探讨NPC1、NPC2对EML细胞增殖、分化的影响。方法通过NPC1 shRNA、NPC2 shRNA重组慢病毒感染EML细胞获得NPC1、NPC2基因有效沉默的EML细胞模型。利用该模型以Filipin染色检测NPC1、NPC2基因敲低后对EML细胞内胆固醇分布的影响,采用CCK-8和台盼蓝染色检测细胞增殖,采用流式细胞仪检测NPC1、NPC2基因沉默后EML细胞干性标志物CD34、sca-1、c-kit及TER-119、CD11b、B220的变化,采用Western blot检测造血干细胞因子(stem cell factor,SCF)刺激细胞后c-kit磷酸化的改变。结果成功构建NPC1、NPC2基因沉默的EML细胞模型,NPC1、NPC2基因在mRNA和蛋白表达均显著降低(P0.01)。NPC1、NPC2敲低的EML细胞中出现胆固醇蓄积,且在NPC1敲低的EML细胞模型中胆固醇蓄积更显著。沉默NPC1、NPC2基因后使得EML细胞增殖减慢(P0.01),并且能够降低EML细胞CD34、sca-1和TER-119、B220的阳性率(P0.01)。经SCF刺激后NPC1基因沉默的EML细胞中c-kit的磷酸化降低显著(P0.01),但在NPC2基因沉默的EML细胞中c-kit磷酸化的改变差异无统计学意义。结论胆固醇转运相关基因NPC1通过调节细胞胆固醇流动参与了EML细胞的增殖和分化调控。
[Abstract]:Objective to investigate the proliferation of EML cells by using NPC1nPC2 knockout hematopoietic precursor cell line erythroid myeloid lymphoid (EML) as a model, and to investigate the effect of NPC1 / NPC2 gene knockout on the proliferation of EML cells. Methods NPC1 shRNA-NPC2 shRNA recombinant lentivirus was used to infect EML cells to obtain a EML cell model with effective silencing of NPC1nPC2 gene. Filipin staining was used to detect the effect of NPC1nPC2 gene knockout on the distribution of cholesterol in EML cells. CCK-8 and Trypan blue staining were used to detect cell proliferation, and flow cytometry was used to detect the changes of CD34Ca-1C kit and TER-119 CD11bB220 in EML cells after the silencing of NPC1nPC2 gene. Western blot was used to detect the changes of c-kit phosphorylation in cells stimulated by hematopoietic stem cell factor (SCF). Results the EML cell model of NPC1hNPC2 gene silencing was successfully constructed. The expression of NPC1nPC2 gene in mRNA and protein significantly decreased the accumulation of cholesterol in EML cells with low P0.01U. NPC1 and NPC2 knockout. Moreover, cholesterol accumulation was more significant in EML cell model with low NPC1 knockout. Silencing NPC1 + NPC2 gene slowed down the proliferation of EML cells and decreased the positive rates of CD34, sca-1 and TER-119 B220 in EML cells. The c-kit in EML cells silenced by NPC1 gene was stimulated by SCF. However, there was no significant difference in c-kit phosphorylation in EML cells with NPC2 gene silencing. Conclusion the cholesterol transporter related gene NPC1 participates in the regulation of proliferation and differentiation of EML cells by regulating the flow of cholesterol.
【作者单位】：第三军医大学军事预防医学院热带医学研究所;第三军医大学西南医院血液科;第三军医大学西南医院整形美容科;
【基金】：国家自然科学基金面上项目(81170471,31371393) 重庆市自然科学基金重点项目(CSTC2012jj B0190) 重庆市应用开发计划项目(CSTC2014yykfa110005)~~
【分类号】：R55

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