GFP转基因鼠肌卫星细胞系的构建及基因介导成骨分化的实验研究

Experiment Study on the Osteogenic Differentiation of MSCs Transfected by Ad-BMP2 Gene in Vitro and Vivo

目的评价肌卫星细胞(Muscle satellite cells,MSCs)体内外成骨分化的能力,探讨肌卫星细胞作为骨组织工程新的种子细胞的可能性。方法取新生绿色荧光蛋白转基因小鼠颌面部肌肉,采用酶消化法分离出MSCs,体外原代及传代培养,经腺病毒介导的人骨形成蛋白2(Ad-BMP2)基因转染后,行成骨细胞标志物的活性检测及细胞化学染色,并进行细胞体外矿化及体内成骨的检测。结果转染后细胞碱性磷酸酶(ALP)组织化学染色呈阳性;骨钙素(OC)免疫细胞化学染色呈阳性且OC活性增强(p<0．05);21天后见钙结节形成;转染后的细胞与材料复合物植入裸鼠后肢肌肉内, 4周后见骨组织形成。结论体外培养的肌卫星细胞体内外可以成骨分化,可以成为骨组织工程的新的种子细胞。

Objective To assess the ability of inducing muscle satellite cells into osteoblast-like cells, and to approach the possibility of muscle satellite cells being as a new seed cell for bone tissue engineering. Method The newborn GFP transgenic mouse was acquired to separate muscle satellite cells with enzyme digestion. Cells are cultured and subcultured in vitro. After being transfected by Ad-BMP2 gene, Osteoblastic marker's activity ,cytochemical staining, mineralized capability in vitro and osteoblastic capability in vivo were examined. Results After being transfected, the cells were faint positive with cytochemical staining of ALP and immunocytochemical staining of OC; there was a significant increase in OC activity (p<0.05); Calcium nodules were seen after 21 days in vitro; ectopic osteogenesis were seen after 4 weeks in vivo. Conclusion MSCs have the capacity to be differentiated into osteoblast in vitro and in vivo. MSCs can be regarded as the seed cells for bone tissue engineering.