發(fā)布時間：2018-03-11 11:20

  本文選題：NPC　切入點：NPC　出處：《第三軍醫(yī)大學(xué)學(xué)報》2017年12期 　論文類型：期刊論文

【摘要】：目的采用NPC1、NPC2基因敲低的造血前體細(xì)胞(erythroid myeloid lymphoid,EML)作為模型,探討NPC1、NPC2對EML細(xì)胞增殖、分化的影響。方法通過NPC1 shRNA、NPC2 shRNA重組慢病毒感染EML細(xì)胞獲得NPC1、NPC2基因有效沉默的EML細(xì)胞模型。利用該模型以Filipin染色檢測NPC1、NPC2基因敲低后對EML細(xì)胞內(nèi)膽固醇分布的影響,采用CCK-8和臺盼藍(lán)染色檢測細(xì)胞增殖,采用流式細(xì)胞儀檢測NPC1、NPC2基因沉默后EML細(xì)胞干性標(biāo)志物CD34、sca-1、c-kit及TER-119、CD11b、B220的變化,采用Western blot檢測造血干細(xì)胞因子(stem cell factor,SCF)刺激細(xì)胞后c-kit磷酸化的改變。結(jié)果成功構(gòu)建NPC1、NPC2基因沉默的EML細(xì)胞模型,NPC1、NPC2基因在mRNA和蛋白表達(dá)均顯著降低(P0.01)。NPC1、NPC2敲低的EML細(xì)胞中出現(xiàn)膽固醇蓄積,且在NPC1敲低的EML細(xì)胞模型中膽固醇蓄積更顯著。沉默NPC1、NPC2基因后使得EML細(xì)胞增殖減慢(P0.01),并且能夠降低EML細(xì)胞CD34、sca-1和TER-119、B220的陽性率(P0.01)。經(jīng)SCF刺激后NPC1基因沉默的EML細(xì)胞中c-kit的磷酸化降低顯著(P0.01),但在NPC2基因沉默的EML細(xì)胞中c-kit磷酸化的改變差異無統(tǒng)計學(xué)意義。結(jié)論膽固醇轉(zhuǎn)運相關(guān)基因NPC1通過調(diào)節(jié)細(xì)胞膽固醇流動參與了EML細(xì)胞的增殖和分化調(diào)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.
【作者單位】： 第三軍醫(yī)大學(xué)軍事預(yù)防醫(yī)學(xué)院熱帶醫(yī)學(xué)研究所;第三軍醫(yī)大學(xué)西南醫(yī)院血液科;第三軍醫(yī)大學(xué)西南醫(yī)院整形美容科;
【基金】：國家自然科學(xué)基金面上項目(81170471,31371393) 重慶市自然科學(xué)基金重點項目(CSTC2012jj B0190) 重慶市應(yīng)用開發(fā)計劃項目(CSTC2014yykfa110005)~~
【分類號】：R55

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本文編號：1597938