外周血清素对离体Sprague-Dawley大鼠成骨细胞的影响

Effect of peripheral serotonin on osteoblasts of the isolated SD rats

目的研究不同浓度外周血清素对离体Sprague-Dawley(SD)大鼠成骨细胞(OBs)增殖、分化、矿化的影响。方法 (1)应用贴壁培养法分离培养新生SD大鼠颅骨成骨细胞,观察细胞形态及荧光染色法定性。(2)向P3代细胞培养液中加入不同浓度(10-5 mol/L、10-6 mol/L、10-7 mol/L、10-8 mol/L、10-9 mol/L、0 mol/L)血清素干预。(3)第5天用MTT法反映不同干预浓度对增殖影响。(4)第10天分别用微量酶标法测碱性磷酸酶(ALP)水平和免疫荧光法测Ⅰ型胶原蛋白水平,反映不同水平干预浓度对分化影响。(5)第15天用ELISA法测骨钙素(OCN)水平,反映不同水平干预浓度对矿化影响。收集整理数据,采用SPSS 20.0统计软件进行分析。计量资料以均数±标准差(sx?)表示,不同组间比较采用单因素方差分析,多组间两两之间比较采用LSD-t检验,P<0.05表示差异有统计学意义。结果不同浓度(10-5 mol/L、10-6 mol/L、10-7 mol/L、10-8 mol/L、10-9 mol/L、0 mol/L)血清素干预,MTT法测得平均光密度值分别为:0.400±0.028,0.379±0.023,0.379±0.047,0.401±0.038,0.420±0.038,0.618±0.064,任意组与空白对照组比较,差异有统计学意义(P<0.05);微量酶标法测得ALP平均水平分别为:0.046±0.002,0.017±0.002,0.026±0.002,0.046±0.002,0.051±0.002,0.073±0.003,任意组与空白对照组比较,差异有统计学意义(P<0.05);免疫荧光法测得Ⅰ型胶原蛋白平均水平分别为:45.581±6.378,33.110±1.161,23.327±0.784,15.731±2.243,27.331±2.430,33.592±2.684,除10-6 mol/L组外任意组与空白对照组比较,差异有统计学意义(P<0.05);ELISA法测得OCN平均水平分别为:3.002±1.639,1.711±0.370,2.193±0.551,2.439±1.469,2.703±1.175,8.021±3.036,任意组与空白对照组比较,差异有统计学意义(P<0.05)。结论不同浓度血清素均可抑制离体成骨细胞增殖、分化与矿化,且抑制作用随血清素浓度增高呈先强后弱关系。

Objective To study the effect of the peripheral serotonin at different concentrations on the proliferation, differentiation, and the mineralization of isolated SD rats. Methods(1) Isolated and cultured the neonatal skull osteoblasts of the SD rats by adherent culture, and observed the cell morphology and determined the nature by using fluorescence stain.(2) Added serotonin intervention of different concentrations(10-5 mol/L, 10-6 mol/L, 10-7 mol/L, 10-8 mol/L, 10-9 mol/L, 0 mol/L) to cell culture fluid of the P3 generation.(3) On the fifth day, reflected the effect on the proliferation of different interventions concentration by MTT.(4) On the 10 th day, respectively, measured alkaline phosphatase(ALP) by using micro enzyme labeled methods and detected the type Ⅰ collagen(Col-Ⅰ) by immunofluorescence respectively, which was used to reflect the effect of different intervention concentration on the differentiation.(5)On the 15th day,ELISA were used to measure the osteocalcin(OCN)level,which reflected the effect of different levels of intervention concentrations on mineralization.To collect the data and analyze it by SPSS20.0 statistical software.Statistical data were showed by averages±standard deviation,using single-factor variance to analyze the comparison of among the different groups,P<0.05 indicated that the difference had the statistical significance.Results The average optical density values of the intervention of serotonin of different concentrations(10-5 mol/L,10-6 mol/L,10-7 mol/L,10-8 mol/L,10-9 mol/L,0 mol/L)detected by MTT were 0.400±0.028,0.379±0.023,0.379±0.047,0.401±0.038,0.420±0.038,0.618±0.064,respectively.The difference has statistical significance(P<0.05).The average levels of ALP detected by micro enzyme labeled methods were 0.046±0.002,0.017±0.002,0.026±0.002,0.046±0.002,0.051±0.002,0.073±0.003.The difference between any group and blank control group had statistical significance(P<0.05).The average levels of Col-Ⅰdetected by immunofluorescence method were 45.581±6.378,33.110±1.161,23.327±0.784,15.731±2.243,27.331±2.430 and 33.592±2.684 respectively,the difference had statistical significance(P<0.05)between any group and blank control group(P<0.05)except for 10-6 mol/L.The mean levels of OCN detected by ELISA were 3.002±1.639,1.711±0.370,2.193±0.551,2.439±1.469 and 2.703±0.351respectively.The difference had statistical significance(P<0.05)between any group and the blank control group.Conclusions The serotonin of different concentrations can inhibit the proliferation,differentiation,and mineralization of isolated osteoblast,and the inhibition effect becomes strong at beginning and then weak with the increasing of the concentration of serotonin