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

Cholesterol trafficking related genes NPC1 and NPC2 regulate proliferation and differentiation of erythroid myeloid lymphoid cells in vitro

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(P<0.01)。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(P<0.01),并且能够降低EML细胞CD34、sca-1和TER-119、B220的阳性率(P<0.01)。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(P<0.01),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。

Objective To construct lentiviral vectors expressing short hairpin RNAs （shRNAs） targeting mouse NPC1 and NPC2 and investigate their effects on the proliferation and differentiation of erythroid myeloid lymphoid （EML） ceils. Methods The lentiviral vectors expressing NPC1 or NPC2 shRNA were transduced into 293FT cells to produce the lentiviral particles for infecting the EML cells. The mRNA and protein levels of NPC1 and NPC2 in the infected cells were detected with real-time PCR and Western blotting, and cholesterol distribution in the EML cells was analyzed with Filipin staining and laser scanning confocal microscopy. The proliferation of the infected EML cells was tested with cell count kit-8 （CCK-8） and Trypan blue staining. Flow cytometry was used to detect he expression levels of CD34, sca-1, c-kit, TER- 119, CDllb and B220 in the EML cells with NPC1 and NPC2 silencing. The changes in c-kit phosphorylation in the EML cells with NPC1 and NPC2 silencing in response to stem cell factor （SCF） stimulation were detected by Western blotting. Results The lentiviral vectors pLKO. 1/NPC1 shRNA and NPC2 shRNA were successfully constructed. Infection of the EML cells with the lentiviruses resulted in significantly decreased mRNA and protein levels of NPC1 and NPC2 （P 〈0.01 ）. Silencing of NPC1 and NPC2, especially NPC1, caused obvious cholesterol accumulation in the EML cells. NPC1 and NPC2 silencing also resulted in significantly reduced proliferation of the EML cells （P 〈0.01 ） and decreased the number of cells positive for CD34, sca-1 and B220 （ P 〈 0.01 ）. SCF stimulation obviously inhibited the phosphorylation of c-kit in the EML cells with NPC1 silencing but not in cells with NPC2 silencing （ P 〈 0. 01 ）. Conclusion Cholesterol trafficking related gene NPC1 regulates the proliferation and differentiation of EML cells by modulating the cellular distribution of cholesterol.