丝裂原活化蛋白激酶对大鼠成骨细胞TRPV6表达的影响

Effects of MAPK on Expression of TRPV6 in Osteoblast of Rat

目的探讨丝裂原活化蛋白激酶对大鼠成骨细胞瞬时性受体电位香草精受体6(transient receptor poten-tial vanilloid receptor 6,TRPV6)表达的影响。方法采用Wistar大鼠乳鼠,连续酶消化法提取成骨细胞并培养。根据组织形态学、改良Kaplow氏成骨细胞碱性磷酸酶染色、矿化结节茜素红染等方法进行鉴定。实验分组:a)正常对照组;b)PD组(PD98059 5μmol/L;PD98059 10μmol/L;PD98059 20μmol/L);c)SB组(SB203580 5μmol/L;SB20358010μmol/L;SB203580 20μmol/L);d)PD+SB组(PD98059 5μmol/L+SB203580 5μmol/L;PD98059 10μmol/L+SB203580 10μmol/L;PD98059 20μmol/L+SB203580 20μmol/L)。细胞计数法、MTT法检测细胞活性,RT-PCR检测成骨细胞TRPV6 mRNA。所得数据用x-±s表示,采用t检验进行统计学分析。结果随着PD98059和/或SB203580浓度的增加,细胞增殖活性有下降的趋势,二者联合作用时细胞活性低于二者单独作用。细胞计数法检测显示,PD98059与SB203580联合作用时,三种组合中细胞数均显著低于空白对照组,P<0.01;同时也明显低于二者单独作用组细胞数量。碱性磷酸酶染色显示,联合用药组较单独用药细胞数明显减少,细胞体积变小,突起狭长,ALP染色阳性颗粒减少,随药物作用浓度增加上述变化更加显著。茜素红染色显示,钙结节与对照组比较数量少、体积小;10、20μmol/L联合作用组,细胞数减少更加明显,无钙结节形成。SB203580与PD98059二者联合应用显著抑制TR-PV6 mRNA的表达,5 mmol/L联合作用组即可明显抑制,随着作用浓度增加TRPV6 mRNA的表达量逐渐减少。结论 p44/42途径阻断剂PD98059能抑制成骨细胞增殖,明显降低成骨细胞TRPV6 mRNA表达,减少钙结节形成。p38途径阻断剂SB203580能抑制成骨细胞增殖,明显降低成骨细胞TRPV6 mRNA表达,减少钙结节形成。PD98059与SB203580在抑制成骨细胞增殖、降低成骨细胞TRPV6 mRNA表达、减少钙结节形成的作用方面存在协同作用。

Objective To study the effects of MAPK on expression of TRPV6 in osteo-blast of rat.Methods Osteoblast extracted from Wistar rat by means of continue digestive enzyme.Based on The proliferation and morphology of osteoblast,Alkaline phosphatase staining and Alizarin red staining of mineralized node through the method of modified Kaplow technology.Groups：1.control group;2.PD group（PD 98059 5 μmol/L;PD98059 10 μmol/L;PD98059 20 μmol/L;3.SB group（SB203580 5 μmol/L;SB203580 10 μmol/L;SB203580 20 μmol/L）;4.PD＋SB group（PD98059 5 μmol/L＋SB203580 5 μmol/L;PD98059 10 μmol/L＋SB203580 10 μmol/L;PD98059 20 μmol/L＋SB203580 20 μmol/L）.MTT for activity and RT-PCR for TRPV6 mRNA of osteoblast had been done.Statistics result：±S,t-test.Results The result suggested that the cell proliferation activity has the decline trend with the rise of PD98059 and SB203580 concentration and the cell possessed lower activity while combination of PD98059 and SB203580 compared with seperated.Bacteria count demonstrated that cell number obviously lower than control when combination of PD98059 and SB203580,P〈0.01;and evidencely lower than the seperated group.The result revealed that,in control group,cell concentrated as fusiform,polygon and cell extention thicking.ALP staining showed that cell number obviously lower than the seperated group when combination of PD98059 and SB203580,the size of cell was small and cell extention narrow,the ALP staining positive granule reduced.The variation became much more obviously with the increase of medicine concentration.Alizarin red staining showed that：compared with control,the number of calcium node has reduced and small in size;In the group of conbination（10 μmol/Land 20 μmol/L）,the number of cell has reduced more evidencely,no calcium nodes.The combination of PD98059 and SB203580 obviously suppressed the expression of TRPV6 mRNA,while the 5 μmol/L group could evidencely suppressed and with the increased of the con-centration,the expression of TRPV6 mRNA obviously reduced.Conclusion The PD98059 that is inhibitor of the p44/42 MAPK pathway,can suppress osteoblast proliferation,obviously reduced osteoblast TRPV6 mRNA and droped the formation of calcium node.The SB203580 that is inhibitor of the p38 MAPK pathway,can suppress osteoblast proliferation,obviously reduced osteoblast TRPV6 mRNA,droped the formation of calcium node.The PD98059 and SB203580 exist synergy in the suppression of osteoblast proliferation,reduced osteoblast TRPV6 mRNA level,droped the formation of calcium node.