摘要
目的探讨成纤维细胞(fibroblast,FB)体外成骨表达的诱导因素,为其能否成为骨组织工程的种子细胞提供理论依据。方法分离和纯化新西兰兔肉芽组织FB,按1×105/ml分别接种于含纤维结合蛋白(fibronectin,FN)10、20、40、60、80μg/ml条件培养液中(实验1～5组),对照组为不含FN的无血清培养液。于培养14d后,行细胞组织学观察及钙化结节形成率、趋骨性四环素荧光标记、3H-TdR标记、骨钙素测定及3H-脯氨酸标记,检测FB成骨表型表达的标志。结果组织学观察实验组发现FB由梭形逐渐趋向多突形和圆形,细胞核偏向一侧,细胞重叠成多层结构;其表面有钙盐堆积,逐渐呈云雾状物质,经不断融合和扩大形成不透光的结节。干预培养14d钙化结节形成率:对照组15.35%±3.45%,实验1～5组分别为53.73%±9.49%、75.21%±9.80%、98.34%±15.20%、61.83%±10.04%、45.11%±8.70%,实验组与对照组比较,差异有统计学意义(P<0.05);实验3组与其它各实验组比较,差异有统计学意义(P<0.05)。趋骨性四环素特异性标记为新生骨组织;FB增殖活性,实验3、4、5组7d时,实验2、3、4组14d时与对照组比较,差异有统计学意义(P<0.05)。实验1～5组FB分泌骨钙素,实验2～5组3H-脯氨酸掺入量高于对照组,7、14d时与对照组比较差异有统计学意义(P<0.05)。结论适量的FN可以促进FB增殖、胶原蛋白的合成及骨钙素分泌,FN可以诱导FB成骨表达,适宜浓度为40～60μg/ml。
Objective To explore the regulator factor of osteogenesis induced by the fibroblast in vitro so as to provide enough seeding cells for the bone tissue engineering. Methods The fibroblasts were isolated and purified from granulation of New Zealand rabbits, and they were incubated in the media of fibronectin （FN） 10, 20, 40, 60 and 80 μg/ml, respectively, in the experimental groups 1 5,but there was no FN in the control group. The markers for osteogenic features were investigated by fibroblast morphogenesis, calcium nodules formation ratios, labeling of tetracycline fluorescence, labeling of ^2H TdR, determination of osteocaline, and labeling of ^3H-proline within 2 weeks. Results The morphological changes of the fibroblasts were manifested as transference from a long spindle to a round or multiple form, shifted nucleus increased in number, confluenced and formed muhilayered structure. There was a piling-up of calcium crystals that were gradually merged into foggy substances. The foggy substances increased and formed nodules. The calcium nodules formation ratios were as follows： 15. 35%± 3.45% in the control group, and 53.73%± 9.49%, 75.21%± 9.80%, 98.34%± 15.20%, 61.83%± 10.04%, and 45.11% ±8.70% in the experimental groups 1-5, respectively. There was a significant difference between the control group and the 5 experimental groups at 14 days （P〈0.05）, and a significant difference between the experimental group 3 and the other experimental groups at 14 days （P〈0. 05）. The histochemical study on the nodules with the specific labeling of tetracycline fluorescence indicated that the nodules were composed of new bones. Conclusion Fibronectin can stimulate the fibroblast to proliferate, secrete osteocaline, and synthesize collagen fibrils. Fibronectin, in an optimal dose of 40- 60μg/ml, is capable of inducing the fibroblast to form the bone.
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2006年第2期107-111,共5页
Chinese Journal of Reparative and Reconstructive Surgery
基金
天津市科技发展重点攻关资助项目(033802311)~~
