CAJ | 학술논문

目的观察4种常用骨质疏松治疗药物(维生素K2、PTH、活性维生素D3、阿仑膦酸盐)对原代SD大鼠成骨细胞基质gla蛋白(MGP) mRNA表达的影响,探讨MGP在骨质疏松发病中的可能作用.方法采用胰酶-胶原酶序贯消化法获得新生1～3天内SD大鼠颅盖骨成骨细胞,进行原代培养.第二代细胞经Ⅰ型胶原、碱性磷酸酶(ALP)、矿化结节染色进行成骨细胞表型鉴定后给予不同浓度的维生素K2、PTH、活性维生素D3、阿仑膦酸盐干预培养24 h后抽提成骨细胞总RNA,采用荧光实时定量RT-PCR检测成骨细胞MGP基因mRNA的表达.结果 (1)原代成骨细胞Ⅰ型胶原染色呈棕红色,ALP染色显示细胞内棕黑细微颗粒,可形成矿化结节.(2)4种常见骨质疏松药物均上调成骨细胞MGP mRNA的表达,维生素K2浓度为10-5、10-6、10-7 mol/L时成骨细胞MGP mRNA的表达量分别是空白对照组的2.56、2.12、1.57倍,差异均有统计学意义(P＜0.05).PTH(1-34)浓度为10-7、10-8、10-9 mol/L时,升高成骨细胞MGPmRNA的表达量分别是空白对照组的6.78、5.31、2.23倍,差异均有显著性(P＜0.05).活性维生素D3浓度为10-8、10-9和10-m mol/L时成骨细胞MGP mRNA的表达量分别是空白对照组的8.93、6.95、3.47倍,差异均有统计学意义(P＜0.05).(3)阿仑膦酸盐为10-4、10-5、10-6 mol/L时成骨细胞MGP mRNA的表达量是空白对照组的3.47、2.49、1.98倍,差异均有统计学意义(P＜0.05).结论骨质疏松治疗药物维生素K2、PTH、活性维生素D3、阿仑膦酸盐均可促进原代培养SD大鼠成骨细胞MGP mRNA的表达,且呈剂量依赖性.MGP可能为骨质疏松治疗药物作用的共同靶点,参与了骨质疏松的发病机制.
목적 관찰4충상용골질소송치료약물(유생소K2、PTH、활성유생소D3、아륜련산염)대원대SD대서성골세포기질gla단백(MGP) mRNA표체적영향,탐토MGP재골질소송발병중적가능작용.방법 채용이매-효원매서관소화법획득신생1～3천내SD대서로개골성골세포,진행원대배양.제이대세포경Ⅰ형효원、감성린산매(ALP)、광화결절염색진행성골세포표형감정후급여불동농도적유생소K2、PTH、활성유생소D3、아륜련산염간예배양24 h후추제성골세포총RNA,채용형광실시정량RT-PCR검측성골세포MGP기인mRNA적표체.결과 (1)원대성골세포Ⅰ형효원염색정종홍색,ALP염색현시세포내종흑세미과립,가형성광화결절.(2)4충상견골질소송약물균상조성골세포MGP mRNA적표체,유생소K2농도위10-5、10-6、10-7 mol/L시성골세포MGP mRNA적표체량분별시공백대조조적2.56、2.12、1.57배,차이균유통계학의의(P＜0.05).PTH(1-34)농도위10-7、10-8、10-9 mol/L시,승고성골세포MGPmRNA적표체량분별시공백대조조적6.78、5.31、2.23배,차이균유현저성(P＜0.05).활성유생소D3농도위10-8、10-9화10-m mol/L시성골세포MGP mRNA적표체량분별시공백대조조적8.93、6.95、3.47배,차이균유통계학의의(P＜0.05).(3)아륜련산염위10-4、10-5、10-6 mol/L시성골세포MGP mRNA적표체량시공백대조조적3.47、2.49、1.98배,차이균유통계학의의(P＜0.05).결론 골질소송치료약물유생소K2、PTH、활성유생소D3、아륜련산염균가촉진원대배양SD대서성골세포MGP mRNA적표체,차정제량의뢰성.MGP가능위골질소송치료약물작용적공동파점,삼여료골질소송적발병궤제.
Objective To observe the expression of matrix gla protein(MGP) mRNA in primary osteoblasts of Sprague-Dawley (SD) rat in vitro after treatment with anti-osteoporosis agents [vitamin K2,PTH,1,25 (OH)2D3,and alendronate],and to investigate the potential role of MGP in the pathogenesis of osteoporosis.Methods Primary osteoblasts(OBs) were derived from sequential trypsin/collagenase-digested calvaria isolated from newborn SD rat (postnastal day 1-3).OBs of the second generation were identified by Van Gieson collagen staining,alkaline phosphatase(ALP) staining and calcified nodules staining.OBs of the fourth generation were selected to interfere with vitamin K2,PTH,1,25 (OH)2D3,and alendronate,then cultured for 24 h in mediums which contained various concentrations of vitamin K2 (10-7,10-6,and 10-5 mol/L),PTH (10-9,10-8,and 10-7 mol/L),1,25 (OH) 2D3(10-10,10-9,and 10-8mol/L),alendronate(10-6,10-5,and 10-4mol/L).After being cultured for 24 h,total RNA was extracted and examined by real-time quantitative RT-PCR.Results The primary cultured cells had typical morphological characters of osteoblast.van Gieson collagen staining,ALP staining,and calcified nodules staining were all positive.Vitamin K2,PTH,1,25 (OH)2D3,and alendronate could modulate the expression of MGP mRNA in osteoblasts in a dose-dependent fashion.MGP mRNA expressions were 2.56-fold,2.12-fold,and 1.57-fold with 10-5,10-6,and 10-7 mol/L of vitamin K2 treatment,respectively.The expressions were 6.78-fold,5.31-fold,and 2.23-fold with 10-7,10-8,and 10-9mol/L of PTH(1-34) treatment,8.93-fold,6.95-fold,and 3.47-fold with 10-8 10-9,and 10-10mol/L of 1,25 (OH)2D3 treatment,and 3.47-fold,2.49-fold,and 1.98-fold with 10-4,10-5,and 10-6mol/L of alendronate treatment.Conclusion Vitamin K2,PTH,1,25 (OH)2D3,and alendronate all can regulate MGP mRNA expression in calvarial osteoblasts in a dose-dependent manner.MGP seems to be a potent target of anti-osteoporosis agents,and involved in the pathogenesis of osteoporosis.