摘要目的 探讨受体活性修饰蛋白1( receptor activity modifying protein 1,RAMP-1)过表达对降钙素基因相关肽(calcitonin gene-related peptide,CGRP)促进MG-63细胞增殖作用的影响,以期阐明神经多肽系统在口腔颌面部创伤修复中的机制.方法 传代培养MG-63细胞并按完全随机法分为3组:RAMP-1过表达组、空载体组、空白对照组.构建人RAMP-1真核表达载体并稳定转染至MG-63细胞中.实时聚合酶链反应、蛋白质印迹法和免疫荧光法分别检测降钙素受体样受体( calcitonin receptor-like receptor,CRLR)在细胞中的表达及在膜上的分布；在0、24、48、72、96 h时使用细胞计数试剂盒8(cell counting kit-8,CCK-8)分别检测3组细胞增殖情况；分别在0、8、16、24 h时使用流式细胞仪收集并分析细胞周期.对蛋白质印迹法和实时聚合酶链反应结果进行单因素方差分析和多重比较检验,对细胞增殖和细胞S期百分率的结果采用双因素方差分析并对处理因素进行多重比较检验.结果 RAMP-1过表达组MG-63细胞的CRLR蛋白及mRNA表达水平明显高于其他两组(P＜0.05)；RAMP-1过表达组在24、48、72及96 h时的A值分别为0.628±0.175、0.896±0.592、1.055±0.004、1.179±0.618,均显著高于相同时间点其他两组A值(P＜0.05),在72 h时与空白对照组(0.786±0.048)差距最明显.RAMP-1过表达组在0、8、16及24 h时S期百分率分别为(1.25±0.13)％、(68.79±0.56)％、(64.49±1.59)％、( 57.82±0.75)％,均显著高于其他两组(P＜0.01),在8h时与空白对照组[(41.235±1.773)％]差距最显著.结论 RAMP-1过表达能促进MG-63细胞膜上的CRLR分布,增强CGRP对MG-63细胞的促增殖作用.

abstractsObjective To investigate the influnce of receptor activity modifying protein 1 ( RAMP-1 ) overexpression on enhancing effect of calcitonin gene-related peptide on MG-63 cells proliferation. Methods Cultured MG-63 osteoblasts in exponential phase of growth were randomly divided into three groups:RAMP-1 overexpression group,empty vector control group and negative control group. RAMP-1 eukaryotic expression vector was constructed and stalely transfected into MG-63 cells. Realtime-polymerase chain reaction,Western blotting and immunofluroescence were used respectively to detect the expression of calcitonin receptor-like receptor( CRLR ) in the cells and its distribution on cell membrane.The status of proliferation was detected respectively at 0,24,48,72,96 h by cell counting kit-8( CCK-8 ) and cells were collected to analyze their cycle respectively at 0,8,16,24 h by flow cytometry.Results CRLR protein and mRNA expression levels of MG-63 cells in RAMP-1 overexpression group were significantly higher than the other two groups (P＜0.05).The A value of RAMP-1 overexpression group at 24,48,72,96 h were 0.628 ±0.175,0.896 ±0.592,1.055 ±0.004,1.179 ±0.618,respectively,which were significantly higher than that of the other two groups( P ＜0.05).The difference was most pronounced at 72 h.S-phase fraction of RAMP-1 overexpression group was ( 1.25 ± 0.13 ) ％,( 68.79 ± 0.56 ) ％,( 64.49 ± 1.59 ) ％,( 57.82 ± 0.75 ) ％,respectively,which were significantly higher than the other two groups ( P ＜ 0.05 ). The difference was most pronounced at 8 h.Conclusions RAMP-1 overexpression can promote CRLR distribution on MG-63 cell membrane and enhance CGRP's promotion effect on MG-63 cell proliferation.