人髓核细胞外泌体诱导人尿源干细胞向髓核样细胞分化的研究

Studies on the differentiation of human urine derived stem cells into nucleus pulposus-like cells induced by human nucleus pulposus cell exosomes

目的探讨人髓核细胞(nucleus pulposus cells, NPCs)外泌体对诱导人尿源干细胞(urine-derived stem cells, USCs)向髓核样细胞分化的作用。方法体外分离培养USCs和NPCs, 提取NPCs外泌体, 以Western-blot检测外泌体标记蛋白;以GFP慢病毒转染标记USCs细胞质、DAPI染料标记USCs细胞核以及PKH26染料标记NPCs外泌体;将USCs与NPCs外泌体共孵育12 h并观察摄取情况, 采用NPCs外泌体和非接触共培养方法诱导USCs分化, 检测各组髓核细胞标志基因mRNA的相对表达量和450 nm波长处的吸光度。结果分离的USCs具备向骨细胞、脂肪细胞、软骨细胞分化的能力, 高表达干细胞标志蛋白CD29(99.57%)、CD44(97.46%)、CD73(97.71%), 低表达干细胞阴性蛋白CD31(0.59%)、CD45(0.19%)。分离的NPCs高表达髓核细胞标志蛋白COL2A1、ACAN、SOX-9。提取的NPCs外泌体高表达标志蛋白CD63、CD81、Tsg101。共孵育12 h后, NPCs外泌体与USCs细胞膜融合并出现在USCs细胞质中。共培养第3、5、7天时, 外泌体组USCs细胞吸光度值(0.44±0.004、0.76±0.004、0.82±0.006)高于共培养组(0.39±0.022、0.63±0.035、0.69±0.012), 差异有统计学意义(P<0.05)。第3、7、14、21天外泌体组USCs髓核标志基因的mRNA相对表达量分别为ACAN(1.80±0.31、3.50±0.21、5.35±0.31、7.46±0.12)、COL2A1(1.43±0.15、4.33±0.23、6.89±0.22、8.11±0.31)、SOX-9(2.21±0.13、3.13±0.11、3.96±0.14、4.52±0.26)、HIF-1α(1.45±0.16、2.14±0.21、4.31±0.41、4.01±0.25)均高于对照组, 差异有统计学意义(P<0.05);第14、21天外泌体组USCs髓核标志基因的mRNA相对表达量为ACAN(5.69±0.21、6.69±0.13)、COL2A1(6.33±0.17、7.89±0.15)、SOX-9(4.19±0.29、4.38±0.12)、HIF-1α(4.49±0.32、4.96±0.26)高于非接触共培养组ACAN(3.69±0.35、5.13±0.23)、COL2A1(3.40±0.16、6.79±0.19)、SOX-9(2.26±0.32、3.69±0.26)、HIF-1α(2.39±0.11、3.96±0.13), 差异均有统计学意义(P<0.05)。结论人髓核细胞外泌体在体外可诱导人尿源干细胞分化为髓核样细胞, 与非接触共培养相比外泌体法具有更高的诱导效率, 并可更好地保持髓核样细胞的增殖活性。

Objective To investigate the effects of exosomes of human nucleus pulposus cells(NPCs)on the differentiation of urine derived stem cells(USCs)into nucleus pulposus-like cells.Methods USCs and NPCs were isolated and cultured in vitro.The exosomes of NPCs were extracted and detected by Western-blot.USCs cytoplasm was transfected with GFP lentivirus,while nucleus was transfected with DAPI dye.The NPCs exosomes were transfected with PKH26 dye.After co-incubation for 12 h,USCs and NPCs exosomes were observed by macroscopy.USCs differentiation was induced by NPCs exosomes and non-contact co-culture methods.The relative expression of marker gene mRNA of nucleus pulposus cells in each group and the absorbance at 450 nm wavelength were detected.Results The isolated USCs had the ability to differentiate into osteocytes,adipocytes and chondrocytes with high expression of marker CD29(99.57%),CD44(97.46%)and CD73(97.71%)and with low expression of negative proteins CD31(0.59%)and CD45(0.19%).The isolated NPCs highly expressed nuclear pulposus cell marker COL2A1,ACAN and SOX-9.The exosomes extracted from NPCs showed high expression of exosome marker CD63,CD81 and Tsg101.After 12 h co-incubation,NPCs exosomes fused with USCs membrane and appeared in the cytoplasm of USCs.At 3,5 and 7 days of co-culture,the absorbance value of USCs cells in exosome group(0.44±0.004,0.76±0.004,0.82±0.006)was higher than that in co-culture group(0.39±0.022,0.63±0.035,0.69±0.012)(P<0.05).The mRNA relative expression of USCs nucleus pulposus marker genes ACAN(1.80±0.31,3.50±0.21,5.35±0.31,7.46±0.12),COL2A1(1.43±0.15,4.33±0.23,6.89±0.22,8.11±0.31),SOX-9(2.21±0.13,3.13±0.11,3.96±0.14,4.52±0.26)and HIF-1α(1.45±0.16,2.14±0.21,4.31±0.41,4.01±0.25)in exosomes group were significantly higher than those in the control group(P<0.05)at the 3rd,7th,14th and 21st days.The mRNA relative expression of USCs nucleus pulposus marker genes ACAN(5.69±0.21,6.69±0.13),COL2A1(6.33±0.17,7.89±0.15),SOX-9(4.19±0.29,4.38±0.12),HIF-1α(4.49±0.32,4.96±0.26)in exosomes group were significantly higher than those ACAN(3.69±0.35,5.13±0.23),COL2A1(3.40±0.16,6.79±0.19),SOX-9(2.26±0.32,3.69±0.26),HIF-1α(2.39±0.11,3.96±0.13)in non-contact co-culture group(P<0.05)at the 14th and 21st days.Conclusion Human nucleus pulposus exosomes could induce differentiation of human USCs into nucleus pulposus-like cells in vitro.Compared with non-contact co-culture,exosomes have higher induction efficiency and can better maintain the proliferation activity of nucleus pulposus-like cells.