幼龄及成龄鼠骨髓基质干细胞生物学特性的比较

Comparison of Biological Characteristics of Bone Marrow Stromal Cells in Fetal and Adult SD Rats

目的比较幼龄及成龄大鼠骨髓基质干细胞体外培养过程中生物学特性的差异。方法取3周及3月龄SD大鼠股骨骨髓原代培养基质干细胞。第2代细胞添加诱导成骨培养液,分别通过相差显微镜观察细胞生长,MTT法检测细胞增殖,钙钴法、茜素红染色了解体外矿化,测定细胞总蛋白含量,RT-PCR测Ⅰ型胶原mRNA表达,透射电镜了解细胞及胞外基质分泌来比较两者差异。结果幼鼠细胞增殖更快,倍增时间4.8 d;培养第7天出现较多集落;钙钴法、茜素红染色深染细胞数目多,结节典型;细胞总蛋白含量高。成龄鼠细胞增殖相对慢,倍增时间5.4 d;未见细胞集落,钙化呈片状;细胞总蛋白含量较低。两者琼脂糖电泳均可显示Ⅰ型胶原mRNA条带;超微结构内质网丰富,胞外基质为前胶原样结构。结论不同鼠龄的骨髓基质干细胞经体外诱导均可分化为成骨细胞,考虑到细胞增殖及成骨潜能,幼鼠基质干细胞是研究细胞/生物材料相互作用更理想的工具。

Objective To compare the biological properties in vitro of bone marrow stromal cells （BMSC） derived from fetal and adult rats. Methods The second passage cells primarily cuhured from 3 weeks and 3 months old rat femoral marrow were induced by osteogenesis medium and compared with respects to cellular growth observed by phase-contrast microscopy. The proliferation capacity was determined by MTT method. Mineralization in vitro was assessed by Von Kossa and Alizarin red staining. Total protein level, expression of mRNA of collagen I was detected by RT-PCR and ultrastructure was examined by transmission electron microscopy. Results Cells from fetal rats grew faster than those from adults, their doubling times were 4.8 d and 5.4 d, respectively. After seven days many colonies were formed among fetal cells, which could not be observed among adult cells. Strong positive cells stained by von Kossa and Alizarin red and total protein level of the former were more frequently observed than those of the latter. Expressions of mRNA of Collagen I were demonstrated by agarose electrophoresis by Day7 and many intra-cellu- lar reticula and precollagen extracellular matrices were showed by electron microscopic examination. Conclusion Both cells can be transformed into osteoblasts in vitro, but considering the proliferation capacity and osteogenic potency, BMSCs derived from fetal rat are most suitable for studies on cell/biomaterial interaction.