LIF和bFGF对脐带间充质干细胞体外自我维持和更新的影响

Effects of Leukemia Inhibitory Factor Combined with Basic Fibroblast Growth Factor on Self-maintenance and Self-renewal of Human Umbilical Cord Mesenchymal Stem Cells In Vitro

目的:研究LIF联合bFGF对人脐带间充质干细胞hUC-MSC增殖、干性维持及衰老的影响。方法:实验分为4组:对照组加入完全培养基(含10%FBS的α-MEM)常规培养;LIF组,加入含10 ng/ml LIF的完全培养液;bFGF组,加入含10 ng/ml bFGF的完全培养液;联合组,加入含10 ng/ml LIF和10 ng/ml bFGF的完全培养液。采用细胞计数试剂盒8(cell counting kit 8,CCK-8)测定各组第4代hUC-MSC生长曲线;在倒置相差显微镜下观察各组hUC-MSC形态变化;β-半乳糖苷酶染色检测各组细胞衰老情况;RT-PCR检测表达情况。结果:4组细胞生长曲线均近似S形,细胞增殖速率表现为联合组>bFGF组>LIF组>对照组。对照组细胞较早出现衰老,增殖减慢;LIF组细胞早期形态维持较好,但增殖相对缓慢,后期大部分细胞衰老;bFGF组部分细胞呈衰老改变,但增殖较快;联合组细胞增殖迅速,能长期维持hUC-MSC形态特征,仅有少量衰老细胞。RT-PC R显示,LIF和bFGF均能上调PCNA、OCT4、NANOG,下调P16、P21、P53的表达,其联合作用效果更明显。结论LIF联合bFGF不仅可以促进hUC-MSC增殖,维持其干性而且能够延缓hUC-MSC的衰老。

Objective:To study the effects of LIF combined with bFGF on the proliferation,sternness and senescence of hUC-MSC.Methods:Experiments were divided into 4 groups:control group,in which the cells were treated with complete medium(α-MEM containing 10%FBS);group LIF,in which the cells were treated with complete medium containing 10 ng/ml LIF;group bFGF,in which the cells were treated with complete medium containing 10 ng/ml bFGF;combination group,in which the cells were treated with complete medium containing 10 ng/ml LIF and 10 ng/ml bFGF.The growth curves of hUC-MSC at passage 4 in different groups were assayed by cell counting kit 8.Cellular morphologic changes were observed under inverted phase contrast microscope;hUC-MSC senescence in different groups was detected by β- galactosidase staining.The expression of PCNA,P16,P21,P53,OCT4 and NANOG genes was detected by RT-PCR.Results:The cell growth curves of each group were similar to the S-shape;the cell proliferation rate from high to low as follows:that in the combination group > group bFGF > group LIF > control group.Senescence and declining of proliferation were observed at hUC-MSC very early in control group;the cells in group LIF maintained good cellular morphology at early stage,but cell proliferation was slow and late senescence was observed;a few cells in group bFGF presented signs of senescence,but with quick proliferation;the cells in combination group grew quickly and maintained cellular morphology of hUC-MSC for long time.The LIF and bFGF up-regulated the expression of PCNA,OCT4 and NANOG,while they down-regulated the expression of P16,P21,P53,and their combinative effects were more significant.Conclusion:LIF combined with bFGF not only can promote the proliferation and maintenance of sternness of hUC-MSC,but also can delay the senescence of hUC-MSC.