CAJ | 학술논문

目的探讨并鉴定骨膜细胞经骨形成蛋白7(BMP7)诱导分化为成骨细胞的基因表达和细胞功能.方法成人胫骨骨膜常规体外细胞培养法,分实验组和对照组,分别加入BMP7加成骨辅助剂和单纯成骨辅助剂进行体外培养.CCK-8法检测细胞增殖活性,第5、10、15和20天分别采用Real Time-PCR检测骨钙素基因表达,ELISA法检测上清液碱性磷酸酶(alkaline phosphatase,ALP)、骨钙素(osteocalcin,OCN)及骨桥蛋白(osteopontin,OPN)的水平,甲苯胺蓝染色检测糖胺聚糖(GAG),Real Time-PCR检测Ⅱ型胶原基因,比色法检测细胞ALP活性,ALP染色法检测ALP,Yon Kossa染色法检测钙结节.结果两组骨钙素基因表达及上清液ALP、骨钙素、骨桥蛋白的含量均增高,实验组与对照组差异有统计学意义(P＜0.05).两组细胞的ALP和钙结节染色阳性率增高,实验组与对照组差异有统计学意义(P＜0.05).甲苯胺蓝染色阳性率及Ⅱ型胶原基因表达表现为先高后低,实验组与对照组差异有统计学意义(P＜0.05).结论骨膜细胞在BMP7诱导下体外大量增殖和分化成骨,表达出明显的成骨特异基因,并具有合成分泌成骨特异蛋白的功能；成骨化过程中,除直接分化成骨外,还存在部分细胞先经软骨形成再钙化成骨的现象；经骨膜细胞-BMP7途径在体外获取大量成骨细胞可用于组织工程的体外构骨及临床应用.
목적 탐토병감정골막세포경골형성단백7(BMP7)유도분화위성골세포적기인표체화세포공능.방법 성인경골골막상규체외세포배양법,분실험조화대조조,분별가입BMP7가성골보조제화단순성골보조제진행체외배양.CCK-8법검측세포증식활성,제5、10、15화20천분별채용Real Time-PCR검측골개소기인표체,ELISA법검측상청액감성린산매(alkaline phosphatase,ALP)、골개소(osteocalcin,OCN)급골교단백(osteopontin,OPN)적수평,갑분알람염색검측당알취당(GAG),Real Time-PCR검측Ⅱ형효원기인,비색법검측세포ALP활성,ALP염색법검측ALP,Yon Kossa염색법검측개결절.결과 량조골개소기인표체급상청액ALP、골개소、골교단백적함량균증고,실험조여대조조차이유통계학의의(P＜0.05).량조세포적ALP화개결절염색양성솔증고,실험조여대조조차이유통계학의의(P＜0.05).갑분알람염색양성솔급Ⅱ형효원기인표체표현위선고후저,실험조여대조조차이유통계학의의(P＜0.05).결론 골막세포재BMP7유도하체외대량증식화분화성골,표체출명현적성골특이기인,병구유합성분비성골특이단백적공능；성골화과정중,제직접분화성골외,환존재부분세포선경연골형성재개화성골적현상；경골막세포-BMP7도경재체외획취대량성골세포가용우조직공정적체외구골급림상응용.
Objective To study and evaluate the gene expression and function of osteoblasts differentiated from human periosteal cells induced by BMP7 in vitro.Methods The periosteal cells isolated from adult tibia periosteum were cultured by the routine method in vitro and divided into experiment group and control group.In the experiment group BMP7 and osteogenic adjuvant were added to the culture media while only osteogenic adjuvant was added in the control group.Cell proliferation was detected by CCK-8.On day 5,day 10,day 15 and day 20,the expression of the osteocalcin gene and type Ⅱ collagen were evaluated by Real TimePCR.Glycosaminoglycan was detected by toluidine blue staining.The levels of ALP,osteocalcin and osteopontin in the supematant were quantified by ELISA.ALP activity,ALP and calcium nodules were detected by colorimetry,ALP staining and Von Kossa staining,respectively.Results The expression of osteocalcin and the level of ALP,osteccalcin and osteopontin in supernatant were both increased with significant difference between the experimental group and control group ( P ＜ 0.05).The positive rate of ALP and calcium nodules staining increased with significant difference between the experimental group and control group ( P ＜ 0.05).The positive rate of toluidine blue staining and the gene expression of collagen Ⅱ in the experimental group both increased temporarily and then decreased,but with significant difference between the experimental group and control group (P ＜ 0.05). Conclusion Human periosteal cells can proliferate and differentiate towards osteoblast when induced by BMP7 in vitro.They express osteogenic specific genes and are capable of synthesizing and secreting osteogenic specific proteins.It appears that apart from a direct osteogenic differentiation pathway,a phenomenon existed that some of the periosteal cells formed chondrocytes first and then calcified into bone.A large number of osteoblasts can be made available in vitro via human periosteal cells combined with BMP7 pathway,which can be used in bone tissue engineering construction and clinical application.