年轻骨髓间充质干细胞可通过细胞融合改善年老骨髓间充质干细胞功能

Aged bone marrow stem cells can be rejuvenated by cell fusion with young bone marrow stem cells

目的:诱导年轻与年老小鼠骨髓间充质干细胞(BMSCs)融合,探讨融合后年轻BMSCs对年老BM-SCs功能的影响。方法:年轻(2-3月龄)及年老(18-24月龄)C57BL/6小鼠BMSCs在红色荧光染料PKH26标记后分别与年轻及年老绿色荧光蛋白转基因C57BL/6小鼠BMSCs通过聚乙烯二醇(PEG1500)诱导,建立细胞融合模型。通过流式细胞仪鉴定细胞融合率和细胞表面特异性抗原的表达。免疫荧光显微镜观察融合细胞形态及细胞核特性。通过细胞计数比较5组细胞(年轻组Y,年老组O,年轻融合组Y-Y,年轻年老融合组Y-O,年老融合组O-O)在2d、4d、6d、8d的细胞增殖能力。诱导细胞向成骨细胞及脂肪细胞分化,比较5组细胞的分化能力。结果:通过PEG诱导,可获得30.45%±4.13%的融合细胞,3组不同年龄融合组的细胞融合率无显著差异。融和细胞表达间充质干细胞表面特异性抗原CD44、Sca-1,而不表达造血干细胞表面特异性抗原CD34、CD117、CD31、CD45。在2d、4d、6d、8d,Y-O组细胞数量增加百分率比O-O组均显著增高。代表成骨细胞分化能力强弱的茜素红染色阳性率和代表脂肪细胞分化能力强弱的油红O染色阳性率,Y-O组比O-O组均显著增高,具体数值分别为:(25.46%±1.52%)vs(13.85%±1.69%),P<0.01;(12.99%±2.61%)vs(6.03%±1.71%),P<0.05。结论:年轻BMSCs可通过细胞融合改善年老BMSCs增殖及分化功能。

AIM：To investigate the function of aged bone marrow mesenchymal stem cells （BMSCs） fused with young BMSCs in mice. METHODS：The cell fusion model,which was made by C57BL/6 mouse BMSCs labeled with PKH26 membrane red fluorescence （young cells,age of 2-3 months,Y） and （old cells,age of 18-24 months,O）,and young and old BMSCs of green fluorescent protein （GFP） transgenic C57BL/6 mouse,was established by the induction of polyethylene glycol 1500 （PEG 1500）. The cell fusion rate and cell surface markers were detected by flow cytometry. The morphology and nuclear characteristics of the fused cells were observed under fluorescence microscopy. In this study,the age dependent changes in BMSCs proliferation and differentiation potential in Y group,O group,and another three fu-sion groups （Y-Y group,Y-O group,O -O group） were examined. The proliferation potentials in 5 groups were compared by counting cell numbers at days 2,4,6,and 8. The osteogenic and adipogenic differentiation potentials of the cells in 5 groups were determined by using standard differentiation procedures. RESULTS：The fusion rate of 30. 45% ± 4. 13% was obtained by PEG 1500 induction. No significant difference of the fusion rates in Y-Y,Y-O and O-O groups was observed. Fused BMSCs coincided with the common BMSCs were reactive to the BMSCs lineage-specific CD44,Sca-1 surface markers and negative for the hematopoietic stem cells （HSCs） lineage-specific surface markers such as CD34,CD117,CD31,and CD45. The percentage of increasing cell numbers in Y-O group was significantly higher than that in O-O group at days 2,4,6,and 8. The positive rate of the area stained with Alizarin red,which represents osteogenic differentiation potential of BMSCs,was significantly higher in Y-O group than that in O -O group [（25. 46% ±1. 52%） vs （13. 85% ±1. 69%）,P 0. 01]. In Y-O group,the higher rate of the positive area stained with oil red O,which represents adipogenic differentiation potential of BMSCs,was observed as compared to that in O-O group [（12. 99% ± 2. 61% ） vs （6. 03% ± 1. 71% ）,P 0. 05]. CONCLUSION：Aged bone marrow stem cells can be rejuvenated by cell fusion with young bone marrow stem cells,particularly the proliferation and differentiation potentials.