SPK1基因转染对大鼠骨髓间充质干细胞的影响

目的观察鞘氨醇激酶-1（SPK-1）基因修饰对大鼠骨髓间充质干细胞生物学特性的影响。方法分别用携带SPK-1和绿色荧光蛋白（GFP）的腺病毒载体,感染大鼠骨髓间充质干细胞后用WesternBlot的方法检测SPK1蛋白表达,用[32P]ATP掺入法检测SPK-1酶活性,用MTT法测定细胞增殖,用特殊诱导剂在体外向成骨和成脂细胞诱导后检测碱性磷酸酶的活性及油红O染色,用AnnexinV-PI双标法检测去血清诱导后细胞凋亡比例。结果Ad-SPK感染大鼠骨髓间充质干细胞后可有效表达SPK-1蛋白且有较高的酶活性;基因转染不影响MSC的增殖和向成骨、成脂细胞的分化,但可显著抑制去血清诱导的细胞凋亡。结论腺病毒介导的SPK-1基因转染不影响MSC的增殖、分化等干细胞基本特性,但可显著增强其抗凋亡能力。

Effect of human sphingosine kinase 1 gene transfection on the biological characteristics of rat bone marrow-derived mesenchymal stem cells

Objective To investigate the effect of sphingosine kinase 1（SPK1） gene transfection on the biological characteristics of rat bone marrow-derived mesenchymal stem cells（MSC）.Methods MSC were infected by recombinant adenovirus carrying SPK1.SPK1 expression was identified by Western blot.Cellular SPK-1 activity was detected by gamma-32P adenosine 5＇-triphosphate incorporation assay.Characteristics of gene-transfected MSC including their proliferation, multiple differentiation potentials and their resistance to serum deprivation were investigated by MTT assay, histological staining with oil red O, and flow cytometry, respectively.Results Ad-SPK-infected MSC effectively expressed the SPK-1 protein and exhibited remarkable enzyme activity.In vitro, SPK1 over-expression did not affect MSC in cell proliferation and osteogenic or adipogenic differentiation potentials.Interestingly, SPK1 gene modification reduced MSC apoptosis by 30.71% under the serum starvation condition.Conclusion SPK-1 gene transfection increases the antiapoptotic ability of MSC while maintaining their proliferation and differentiation features.