CAJ | 학술논문

目的探讨雌激素和维生素D对成骨细胞MC3T3-E1增殖和分化的协同调节作用及其机制.方法小鼠成骨细胞系MC3T3-E1细胞用无酚红α-MEM完全培养基培养;用MTT法检测细胞增殖率;用实时荧光定量PCR法检测干预前后MC3T3-E1细胞中细胞周期相关基因[细胞周期素E( cyclin E)、增殖细胞核抗原(PCNA)和细胞周期素依赖性激酶抑制物(Cdkn2b)]和成骨细胞分化标志物[Ⅰ型胶原( COL Ⅰ)、碱性磷酸酶(ALP)和骨桥蛋白(OPN)]基因的表达.ALP活性染色用BCIP/NBT显色法.结果单用雌激素17β-雌二醇(E2)可促进MC3T3-E1细胞的增殖,尤其在生理浓度时作用最强,单用维生素D活性产物1,25-二羟维生素D3[1,25-(OH) 2D3]对MC3T3-E1细胞的增殖无影响,1,25 - (OH)2 D3不影响E2对MC3T3-E1细胞的促增殖作用.E2上调MC3T3-E1细胞中cyclin E和PCNA的表达,同时下调Cdkn2b基因的表达,1,25-(OH)2D3单独应用不能影响上述基因表达的变化,也不影响E2对上述基因的调节作用.E2可促进MC3T3-E1细胞中分化标志物(COLⅠ、ALP和OPN)基因的表达,加用1,25-(OH)2D3后可增强此作用.结论雌激素和维生素D作为两种重要的调节骨代谢的激素,在促进成骨细胞增殖方面可能无协同作用,而在促进其分化方面可能有协同作用.
목적 탐토자격소화유생소D대성골세포MC3T3-E1증식화분화적협동조절작용급기궤제.방법 소서성골세포계MC3T3-E1세포용무분홍α-MEM완전배양기배양;용MTT법검측세포증식솔;용실시형광정량PCR법검측간예전후MC3T3-E1세포중세포주기상관기인[세포주기소E( cyclin E)、증식세포핵항원(PCNA)화세포주기소의뢰성격매억제물(Cdkn2b)]화성골세포분화표지물[Ⅰ형효원( COL Ⅰ)、감성린산매(ALP)화골교단백(OPN)]기인적표체.ALP활성염색용BCIP/NBT현색법.결과 단용자격소17β-자이순(E2)가촉진MC3T3-E1세포적증식,우기재생리농도시작용최강,단용유생소D활성산물1,25-이간유생소D3[1,25-(OH) 2D3]대MC3T3-E1세포적증식무영향,1,25 - (OH)2 D3불영향E2대MC3T3-E1세포적촉증식작용.E2상조MC3T3-E1세포중cyclin E화PCNA적표체,동시하조Cdkn2b기인적표체,1,25-(OH)2D3단독응용불능영향상술기인표체적변화,야불영향E2대상술기인적조절작용.E2가촉진MC3T3-E1세포중분화표지물(COLⅠ、ALP화OPN)기인적표체,가용1,25-(OH)2D3후가증강차작용.결론 자격소화유생소D작위량충중요적조절골대사적격소,재촉진성골세포증식방면가능무협동작용,이재촉진기분화방면가능유협동작용.
Objective To assess the coordinated regulation and the molecular mechanisms of 17β-estradiol and 1,25-dihydroxyvitamin D3 [ 1,25-( OH ) 2 D3 ] on the proliferation and differentiation of osteoblastic MC3T3-E1 cells.Methods MC3T3-E1 cells were cultured in phenol-red free α-MEM medium supplemented with 10％ FBS,MTT assay was performed to determine the effects of 17β-estradiol and 1,25-( OH )2 D3 on MC3T3-E1 cells proliferation.After cells were treated with different agents,cell cycle related genes [ cyclin E,proliferation cell nuclear antigen ( PCNA ),and cyclin-dependent kinase inhibitor 2b ( Cdkn2b ) ] and markers of osteoblastic differentiation [ type Ⅰ collagen ( COL Ⅰ ),alkaline phosphatase ( ALP),osteopontin ( OPN ) ] were detected with SYBR green-based quantitative PCR.ALP activity was detected with BCIP/NBT method.Results 17β-estradiol could promote proliferation of MC3T3-E1 cells,which was accordant to its ability to increase cyclin E and PCNA and to inhibit Cdkn2b mRNA expression in MC3T3-E1 cells.However,1,25-( OH)2D3 had no effect on the proliferation of MC3T3-E1 cells and also did not enhance the proliferation of MC3T3-E1 cells stimulated by 17β-estradiol.On the other hand,17β-estradiol promoted the gene expression of differentiation markers Col Ⅰ,ALP,and OPN,and 1,25-(OH) 2 D3 synergistically increased the expression of these genes with 17 β-estradiol.Conclusion As two of the most important hormones which regulate bone metabolism,estrogen and vitamin D may coordinately promote osteoblast differentiation,but may not regulate osteoblasts proliferation synergistically.