LacZ转基因小鼠间充质干细胞的分离培养和生物学特性研究

Isolation and characterization of mesenchymal stem cells from LacZ transgenic mouse

目的分离培养LacZ转基因小鼠骨髓间充质干细胞(LacZ-MSC),研究其生物学特性。方法用全骨髓贴壁法分离LacZ-MSC,体外扩增,并观察细胞生长特性,流式细胞仪检测细胞周期,成骨成脂诱导试验分析LacZ-MSC转分化特性,X-gal染色法检测其LacZ基因的表达。结果 LacZ-MSC培养4 d后有散在呈针尖状的贴壁细胞,5～8 d后形成集落或融合呈纤维状;体外扩增每只小鼠原代可获得(1～3)×105个细胞,10代可获得(1～3)×1010个细胞总数,10代后的细胞增殖能力下降;细胞周期分析显示,G0/G1期:占78.44%,G2/M期:占5.20%,S期:占16.36%;成骨成脂诱导试验表明,LacZ-MSC细胞具有向成骨细胞和脂肪细胞分化的潜能;X-gal染色结果表明,LacZ-MSC细胞携带LacZ基因。结论从LacZ转基因小鼠骨髓中成功地分离了携带有LacZ基因的MSC,在体外具有较好的增殖更新能力,3～10代的LacZ-MSC作为组织工程细胞具有广阔的应用前景。

Objective To isolate and culture the bone marrow mesenchymal stem cells from LacZ transgenic mouse（LacZ-MSC） and investigate its biological properties.Methods Adherent method was used to isolate LacZ-MSC from whole bone marrow.After expansion in vitro,the growth characteristics of LacZ-MSC were observed and the cell cycle was analyzed by flow cytometry.The differentiation potential of LacZ-MSC was evaluated by osteogenic and adipogenic induction tests.The LacZ gene expression of LacZ-MSC was detected by X-gal staining.Results After LacZ-MSC were cultured for 4 days,scattered needle-like adherent cells were observed,and colonies or fibrous fusion were formed between 5 and 8 days after culturing.During expansion in vitro,1 to 3×10^5 cells can be obtained after primary culture and the cell counts increased 105-fold at the tenth passage.Then the proliferation ability of cells was gradually decreased.Flow cytometry analysis showed that the percentages of cells in G0/G1,G2/M and S phases were 78.44%,5.20% and 16.36%,respectively.LacZ-MSC cells had the potentiality to differentiate into bone cells and fat cells.X-gal staining showed that the cells expressed LacZ gene.Conclusion LacZ-MSC was successfully isolated from the bone marrow of LacZ transgenic mouse,and the isolated cells could proliferate in vitro,indicating that broad application of LacZ-MSC between 3 and 10 passages may be prospected in tissue engineering.