CAJ | 학술논문

目的观察氟对体外培养的小鼠成骨细胞增殖、分化、骨保护蛋白(OPG)mRNA及核因子κβ受体活化因子配体(RANKL)mRNA表达的影响.方法取出生1～2 d昆明小鼠颅盖骨,分离成骨细胞后进行传代培养.按培养液中加入不同浓度氟化钠(NaF)将成骨细胞分为0(对照)、10-8、10-7、10-6、10-5、10-4、10-3 mol/L组,分别在培养72 h或120 h后检测氟对成骨细胞增殖、分化(碱性磷酸酶,ALP)的影响;提取成骨细胞总mRNA,采用RT-PCR方法分析成骨细胞OPG mRNA、RANKL mRNA表达,并进行半定量分析.组间比较采用单因素方差分析,两两比较采用LSD-t检验.结果培养72 h后,成骨细胞增殖组间比较差异有统计学意义(F=13.806,P<0.05);与对照组(0.434±0.010)比较,10-7～10-4mol/L组成骨细胞增殖明显增加(0.448±0.010、0.453±0.013、0.454±0.016、0.449±0.018,P均<0.05),10-3 mol/L组成骨细胞增殖降低(0.401±0.009,P<0.05).成骨细胞ALP活性组问比较差异有统计学意义(F=9.021,P<0.05);其中10-7～10-5 mol/L组[(2.447±0.756)×106、(2.603±0.183)×106、(2.687±0.886)×106 U/L]ALP活性明显高于对照组[(1.677±0.682)×106U/L,P<0.05或<0.01];10-4mol/L组[(1.479±0.366)×106 U/L]ALP活性明显低于对照组(P<0.05).OPG mRNA表达组间比较差异有统计学意义(F=11.299,P<0.05);与对照组(11000±0.000)比较,10-7～10-4 mol/L组表达增强(1.058±0.027、1.053 ±0.026、1.088±0.055、1.069±0.008,P<0.05或<0.01),10-3mol/L组表达降低(0.941±0.029,P<0.05).成骨细胞RANKL mRNA表达组间比较差异无统计学意义(F=1.311,P>0.05).RANKL mRNA/OPG mRNA比值,在104～10-5mol/L组逐渐降低,10-4、10-3mol/L组升高,但组间比较差异无统计学意义(F=1.376,P>0.05).结论氟对小鼠成骨细胞增殖、分化呈双向调节作用,表现为低剂量刺激而高剂量抑制.低剂量染氟可能通过增加成骨细胞OPG的表达来抑制破骨细胞的分化和成熟,抑制骨吸收;而高剂量可能通过降低OPG的表达来促进破骨细胞的分化、成熟,促进骨吸收. 目的觀察氟對體外培養的小鼠成骨細胞增殖、分化、骨保護蛋白(OPG)mRNA及覈因子κβ受體活化因子配體(RANKL)mRNA錶達的影響.方法取齣生1～2 d昆明小鼠顱蓋骨,分離成骨細胞後進行傳代培養.按培養液中加入不同濃度氟化鈉(NaF)將成骨細胞分為0(對照)、10-8、10-7、10-6、10-5、10-4、10-3 mol/L組,分彆在培養72 h或120 h後檢測氟對成骨細胞增殖、分化(堿性燐痠酶,ALP)的影響;提取成骨細胞總mRNA,採用RT-PCR方法分析成骨細胞OPG mRNA、RANKL mRNA錶達,併進行半定量分析.組間比較採用單因素方差分析,兩兩比較採用LSD-t檢驗.結果培養72 h後,成骨細胞增殖組間比較差異有統計學意義(F=13.806,P<0.05);與對照組(0.434±0.010)比較,10-7～10-4mol/L組成骨細胞增殖明顯增加(0.448±0.010、0.453±0.013、0.454±0.016、0.449±0.018,P均<0.05),10-3 mol/L組成骨細胞增殖降低(0.401±0.009,P<0.05).成骨細胞ALP活性組問比較差異有統計學意義(F=9.021,P<0.05);其中10-7～10-5 mol/L組[(2.447±0.756)×106、(2.603±0.183)×106、(2.687±0.886)×106 U/L]ALP活性明顯高于對照組[(1.677±0.682)×106U/L,P<0.05或<0.01];10-4mol/L組[(1.479±0.366)×106 U/L]ALP活性明顯低于對照組(P<0.05).OPG mRNA錶達組間比較差異有統計學意義(F=11.299,P<0.05);與對照組(11000±0.000)比較,10-7～10-4 mol/L組錶達增彊(1.058±0.027、1.053 ±0.026、1.088±0.055、1.069±0.008,P<0.05或<0.01),10-3mol/L組錶達降低(0.941±0.029,P<0.05).成骨細胞RANKL mRNA錶達組間比較差異無統計學意義(F=1.311,P>0.05).RANKL mRNA/OPG mRNA比值,在104～10-5mol/L組逐漸降低,10-4、10-3mol/L組升高,但組間比較差異無統計學意義(F=1.376,P>0.05).結論氟對小鼠成骨細胞增殖、分化呈雙嚮調節作用,錶現為低劑量刺激而高劑量抑製.低劑量染氟可能通過增加成骨細胞OPG的錶達來抑製破骨細胞的分化和成熟,抑製骨吸收;而高劑量可能通過降低OPG的錶達來促進破骨細胞的分化、成熟,促進骨吸收.
목적 관찰불대체외배양적소서성골세포증식、분화、골보호단백(OPG)mRNA급핵인자κβ수체활화인자배체(RANKL)mRNA표체적영향.방법 취출생1～2 d곤명소서로개골,분리성골세포후진행전대배양.안배양액중가입불동농도불화납(NaF)장성골세포분위0(대조)、10-8、10-7、10-6、10-5、10-4、10-3 mol/L조,분별재배양72 h혹120 h후검측불대성골세포증식、분화(감성린산매,ALP)적영향;제취성골세포총mRNA,채용RT-PCR방법분석성골세포OPG mRNA、RANKL mRNA표체,병진행반정량분석.조간비교채용단인소방차분석,량량비교채용LSD-t검험.결과 배양72 h후,성골세포증식조간비교차이유통계학의의(F=13.806,P<0.05);여대조조(0.434±0.010)비교,10-7～10-4mol/L조성골세포증식명현증가(0.448±0.010、0.453±0.013、0.454±0.016、0.449±0.018,P균<0.05),10-3 mol/L조성골세포증식강저(0.401±0.009,P<0.05).성골세포ALP활성조문비교차이유통계학의의(F=9.021,P<0.05);기중10-7～10-5 mol/L조[(2.447±0.756)×106、(2.603±0.183)×106、(2.687±0.886)×106 U/L]ALP활성명현고우대조조[(1.677±0.682)×106U/L,P<0.05혹<0.01];10-4mol/L조[(1.479±0.366)×106 U/L]ALP활성명현저우대조조(P<0.05).OPG mRNA표체조간비교차이유통계학의의(F=11.299,P<0.05);여대조조(11000±0.000)비교,10-7～10-4 mol/L조표체증강(1.058±0.027、1.053 ±0.026、1.088±0.055、1.069±0.008,P<0.05혹<0.01),10-3mol/L조표체강저(0.941±0.029,P<0.05).성골세포RANKL mRNA표체조간비교차이무통계학의의(F=1.311,P>0.05).RANKL mRNA/OPG mRNA비치,재104～10-5mol/L조축점강저,10-4、10-3mol/L조승고,단조간비교차이무통계학의의(F=1.376,P>0.05).결론 불대소서성골세포증식、분화정쌍향조절작용,표현위저제량자격이고제량억제.저제량염불가능통과증가성골세포OPG적표체래억제파골세포적분화화성숙,억제골흡수;이고제량가능통과강저OPG적표체래촉진파골세포적분화、성숙,촉진골흡수.
Objective To investigate the effect of sodium fluoride(NaF) on proliferation, differentiation and the mRNA expression of osteoprotegerin (OPG) and receptor activator of nuclear factor κβ ligand (RAN KL) of mouse osteoblasts. Methods Osteoblasts were isolated from calvarias of Kunming mice born in 1 - 2 d and cultured. Various concentrations of NaF(0, 10-8, 10-7, 10-6, 10-5, 10-4, 10-3mol/L) were added to the culture medium, the proliferation and activity of alkaline phosphatase(ALP) was determined after 72 h or 120 h. The expression of OPG mRNA and RANKL mRNA was analyzed by semi-quantification RT-PCR. Difference among groups was analyzed by One-Way AN0VA. Difference between two groups was analyzed by LSD-t test. Results There was significant difference in cell proliferation among groups after 72 h(F = 13.806, P < 0.05). Compared with control group(0.434 ± 0.010) , the proliferation was significantly induced in 10-7 - 10-4 mol/L groups treated osteoblasts (0.448 ± 0.010, 0.453 ± 0.013, 0.454 ± 0.016, 0.449 ± 0.018, all P< 0.05), and was significantly suppressed in 10-3 mol/L group(0.401 ± 0.009, P < 0.05). There was statistic difference in the activity of ALP among groups(F = 9.021, P < 0.05). Compared with control group (1.677 ± 0.682), the activity of ALP significantly increased in 10-7 - 10-5 mol/L groups[ (2.447 ± 0.756) × 106, (2603 ± 0.183) × 106, (2.687 ± 0.886) × 106 U/L, P < 0.05 or P < 0.01 ] and significantly decreased in 10-4 mol/L group[ (1.479 ± 0.366) × 106 U/L, P < 0.05 ]. There was significant difference in the expression of OPG mRNA among groups(F = 11.299, P< 0.05). Compared with control group (1.000 ± 0.000), the expression of OPG mRNA was significantly increased in 10-7 - 10-4 mol/L groups( 1.058 ± 0.027, 1.053 ± 0.026, 1.088 ± 0.055, 1.069 ± 0.008, P < 0.05 or P < 0.01) , while significantly decreased in 10-3 mol/L group (0.941 ± 0.029, P< 0.05). There was no difference in RANKL mRNA expression among groups (F= 1.311, P> 0.05). The ratio of RANKL/OPG decreased with increasing doses of fluoride and increased in 10-4, 10-3 mol/L groups, but there was no difference between groups(F = 1.376, P> 0.05). Conclusions A biphasic pattern of proliferation and differentiation has been induced in mouse osteoblasts, which manifests stimulation effect in low doses and suppression in higher doses. Low doses of sodium fluoride suppress differentiation and maturation of osteoblasts by increasing expression of OPG mRNA, while high doses of sodium fluoride enhance differentiation and maturation of osteoblasts by decreasing expression of OPG mRNA.