人脐血清对人脐带血间充质干细胞生物学特征与衰老的影响

Effects of human umbilical cord blood serum on the biological characteristics and senescence of human umbilical cord blood mesenchymal stem cells

摘要:

目的：探讨人脐血清（human umbilical cord blood serum，CBS）对体外培养人脐血间充质干细胞（human umbilical cord blood mesenchymal stem cells，hUCB-MSCs）基本生物学特征的影响。方法：密度梯度离心分离脐带血单个核细胞，以含3%CBS加7%胎牛血清（fetal bovine serum，FBS）（CBS组）或10%FBS（FBS组）的完全培养基培养hUCB-MSCs，流式细胞术检测细胞表面分子与细胞周期，显微镜下测定细胞表面积和表达β-半乳糖苷酶细胞百分率，MTT法检测光密度值、绘制生长曲线。结果：CBS组和FBS组均表达CD44、CD105、CD90、CD73，不表达CD45、CD34、CD11b、CD19和人白细胞DR抗原（human leukocyte antigen DR，HLA-DR），但与FBS组比较，CBS组表达CD90细胞百分率更低，G0/G1期细胞比例较少，细胞增殖指数较高（P<0.05）且细胞表面积也明显更小（P<0.05）。CBS组生长曲线较FBS组左移，峰值更高，细胞倍增时间48.37 h，FBS组则为54.43 h。FBS组β-gal染色阳性细胞百分率明显高于CBS组（P<0.05）。结论：部分CBS合用FBS培养hUCB-MSCs能更好地促进细胞增殖，并更好地保持hUCB-MSCs的基本生物学特征和延缓细胞的衰老。

Abstract:

Objective：To explore effects of human umbilical cord blood serum on the basic biological characteristics and senescence of human umbilical cord blood mesenchymal stem cells（hUCB-MSCs）. Methods：Mononuclear cells were isolated by density gradient centrifugation from human umbilical cord blood. Two groups of hUCB-MSCs were cultured using different complete mediums，one con－sisting of 3% cord blood and 7% fetal bovine serum（CBS group），the other one consisting of 10% fetal bovine serum（FBS group）. Cell surface moleculars and cell cycle were determined by flow cytometry. Then cell surface area and percentage of positive cells expressing β-galactosidase were detected. Absorbance value was measured by MTT methods，and cell growth curve was drawn. Results：hUCB-MSCs expressed CD44，CD105，CD90，CD73 and vimentin in both groups，but not CD45，CD34，CD11b，CD19 and human leukocyte antigen DR（HLA-DR）. Percentage of CD90 expression in the 7th passage hUCB-MSCs was significantly lower in CBS group than in FBS group. Percentage of cells in G0/G1 phase was significantly lower and proliferation index was significantly higher. Cell surface area was significantly smaller in CBS group than in FBS group（P<0.05）. Growth curve of CBS group was shifted to the left with a higher peak. Cell doubling time was 48.37 h in CBS group，but 54.43 h in FBS group. Percentage of β-galactosidase positive cells was higher in FBS than in CBS group. Conclusions：hUCB-MSCs proliferation is promoted by combining partial human umbilical cord blood serum with fetal bovine serum with better maintenance of basic biological characteristic and delayed cell aging.