低密度培养兔骨髓基质干细胞的生物学特性

Biological character of rabbit bone marrow stromal stem cells by low-density culture

目的探讨低密度体外培养条件下兔骨髓基质干细胞的成骨能力及对Bio-oss胶原复合的影响。方法利用全血贴壁法培养兔骨髓基质干细胞,取第2代细胞进行不同密度的培养,观察细胞克隆的形成及形态变化。细胞消化后进行爬片,测定Stro-1+、CD4+4细胞表面抗原,以流式细胞仪检测正常培养细胞CD3+4、CD4+4表达变化并与非低密度培养组比较。低密度培养细胞成骨诱导后,进行钙化结节诱导,同时进行碱性磷酸酶(ALP)定量测定。把诱导后的细胞与Bio-oss胶原复合,观察与载体材料的生物相容性并进行电镜扫描观察。结果低密度培养的细胞可形成克隆,细胞爬片后CD4+4、Stro-1+免疫荧光测定显示阳性,钙化结节在成骨诱导14 d后形成。低密度培养的细胞CD3+4阳性率为43.83%,而正常培养的细胞CD3+4阳性率为4.20%,二者间有统计学差异(P<0.05)。诱导后细胞ALP定量测定为(1.666±0.015)U/(g.prot),而非诱导细胞为(0.450±0.023)U/(g.prot),二者间有统计学差异(P<0.05)。诱导后的细胞与Bio-oss胶原复合较好,电镜扫描观察细胞表面有大量"伪足"形成。结论低密度培养的骨髓基质干细胞可表达较强干细胞特性和成骨能力,与Bio-oss胶原复合后生长良好。

Objective To observe the osteogenesis of rabbit bone marrow mesenchymal stem cells and its influence on Bio-oss collagen on the condition of low density lipoprotein in vitro. Methods Attachment method of whole blood was used to cuhure bone marrow stromal stem cells of rabbits, taking the 2nd generation cells to be cultured in different density, and formation and morphological change of observation cell clones were observed. Cells were made to climbing sheet after cell dissociation. Cell surface antigen was detected by Stro-1 ± ,CD~. Flow cytometry was used to detected CD3~, CD~ expres- sions in normal cuhured cells, and compared with the non-low-density culture group. After cultivating osteogenic induction in low density, calcified nodules was induced, and alkaline phosphatase （ALP） quantitative was determined. Bio-oss colla- gen integrated cells after induction, observing biocompatibility of carrier materials and electron microscopic scanning was excuted. Results Cells cultured in low density could form clones, cell after climbing CD,~, Stro-1 ± immuno-fluorescence measurement displays positive, calcification nodule formed ation after osteogenic induction for 14 d. The positive rate of CD3] in cells in low density was 43.83 % , whereas 4.2% in cells in normal density in vitro （ P 〈 0.05 ）. ALP was （ 1. 666 ± 0. 015 ） U/（ g · prot） after induction, but （0.450 ± 0.023 ） U/（ g · prot） in non-induced ceils, there was significantly difference between them（P 〈 O. 05 ）. Induced cells and Bio-oss collagen combied very better, cell surface had a large number of ＂pseudo-foot＂ formation in electron microscope. Conclusions Bone marrow stromal stem cells cultured in low density have strong stem cell character and the osteogenic ability, and grow rapidly after combined with Bio-oss collagen.