葛根素对大鼠成骨细胞OPG和RANKL mRNA表达影响的研究
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摘要
研究目的
本课题通过建立大鼠成骨细胞体外培养实验模型,在细胞和分子水平上观察葛根素对大鼠成骨细胞增殖、分化及基因表达的影响,探讨葛根素防治绝经后骨质疏松症的作用机制,为葛根素应用于临床防治绝经后骨质疏松症提供实验依据。
方法
1.采用多次酶消化法分离新生24h内大鼠颅盖骨进行大鼠原代成骨细胞体外培养,通过相差显微镜观察成骨细胞形态,并用碱性磷酸酶染色方法对所获得的细胞进行鉴定。
2.采用MTT法观察葛根素对大鼠成骨细胞的影响
3.采用RT-PCR的方法观察葛根素对大鼠体外培养成骨细胞OPG和RANKL mRNA表达的影响
结果
1.新生大鼠成骨细胞体外培养模型建立成功,证实所培养细胞具有体内成骨细胞的形态学和生物学特性。
2.不同浓度的葛根素(10-4μmol/L～100μmol/L)作用于大鼠成骨细胞24h和48h均表现出显著的促增殖作用；培养72h及96h,实验结果显示各浓度组均无明显的增殖作用。
3.葛根素可明显地促进大鼠成骨细胞OPG mRNA的表达,抑制RANKL mRNA的表达。
结论
1.不同浓度的葛根素作用于大鼠成骨细胞表现出明显的增殖作用,其增殖的大小与葛根素浓度及作用时间有密切的关联。
2.葛根素可以上调OPG基因的表达,下调RANKL基因的表达,从而调节骨吸收,这可能是葛根素防治绝经后骨质疏松症的重要机制之一。
3.葛根素可以促进大鼠成骨细胞的增殖及增强OPG/RANKL mRNA表达的水平,这可能是葛根素防治绝经后骨质疏松症的作用机制,为葛根素的应用提供了实验依据,而且为发掘新的抗骨质疏松药物提供了线索。
Objective
This subject, by establishing experimental model of rat's cultured osteoblast in vitro, investigates the effects of Puerarin on the proliferation and differentiation and gene expression of rat osteoblast in cell and molecular level,in order to approach the mechanism of antiosteoporotic effects of Puerarin on postmenopausal osteoporosis and provide the experimental evidences for its preventing and treating postmenopausal osteoporosis in clinic.
Methods
1.The osteoblasts are isolated from newborn rats within 24 hours by digesting rat's calvaria repeatedly,and collected and cultured in vitro. Morphologic changes of osteoblasts are observed.Rat osteoblasts are identified by the alkaline phosphatase staining
2.To observe the effects of Puerarin on the proliferation of rat osteoblasts by using MTT method
3.To explore the effects of Puerarin on the expressions of osteoprotegerin (OPG) and receptor activator of nuclear factor-icB ligand(RANKL) by RT-PCR in rat osteoblasts cultured in vitro.
Results
1.Experimental model of rat's cultured osteoblast in vitro is established.The cells possesses the morphological and biological properties of osteoblast.
2.All of the concerntrations are promoted by the effect of Puerarin with different concentrations on the rat osteoblasts for 24 hours and 48 hours;After cultivated 72 hours and 96 hours,all of the concentrations has no effects on proliferation
3.Puerarin can increase OPG mRNA levels remarkably and decrease RANKL mRNA levels in the rat osteoblasts cultured in vitro
Conclusions
1.Puerarin with different concentrations can promote the proliferation of osteoblasts remarkably.and the mount of proliferation has intimate relations with concentration and time
2.Puerarin can upregulate OPG expression and downregulate RANKL expression in rat osteoblasts,which may explain the mechanism of antiosteoporotic effects of Puerarin in preventing and treating postmenopausal osteoporosis.
3.Puerarin can promote the proliferation of osteoblasts and the expressions of osteoprotegerin (OPG) and receptor activator of nuclear factor-KB ligand(RANKL) mRNA,which may be the mechanism of the effects of Puerarin on postmenopausal osteoporosis.Our study not only provides the experimental evidences for the application of Puerarin,also gives a new clue on the development of the antiosteoporosis drugs.
本课题通过建立大鼠成骨细胞体外培养实验模型,在细胞和分子水平上观察葛根素对大鼠成骨细胞增殖、分化及基因表达的影响,探讨葛根素防治绝经后骨质疏松症的作用机制,为葛根素应用于临床防治绝经后骨质疏松症提供实验依据。
方法
1.采用多次酶消化法分离新生24h内大鼠颅盖骨进行大鼠原代成骨细胞体外培养,通过相差显微镜观察成骨细胞形态,并用碱性磷酸酶染色方法对所获得的细胞进行鉴定。
2.采用MTT法观察葛根素对大鼠成骨细胞的影响
3.采用RT-PCR的方法观察葛根素对大鼠体外培养成骨细胞OPG和RANKL mRNA表达的影响
结果
1.新生大鼠成骨细胞体外培养模型建立成功,证实所培养细胞具有体内成骨细胞的形态学和生物学特性。
2.不同浓度的葛根素(10-4μmol/L～100μmol/L)作用于大鼠成骨细胞24h和48h均表现出显著的促增殖作用；培养72h及96h,实验结果显示各浓度组均无明显的增殖作用。
3.葛根素可明显地促进大鼠成骨细胞OPG mRNA的表达,抑制RANKL mRNA的表达。
结论
1.不同浓度的葛根素作用于大鼠成骨细胞表现出明显的增殖作用,其增殖的大小与葛根素浓度及作用时间有密切的关联。
2.葛根素可以上调OPG基因的表达,下调RANKL基因的表达,从而调节骨吸收,这可能是葛根素防治绝经后骨质疏松症的重要机制之一。
3.葛根素可以促进大鼠成骨细胞的增殖及增强OPG/RANKL mRNA表达的水平,这可能是葛根素防治绝经后骨质疏松症的作用机制,为葛根素的应用提供了实验依据,而且为发掘新的抗骨质疏松药物提供了线索。
Objective
This subject, by establishing experimental model of rat's cultured osteoblast in vitro, investigates the effects of Puerarin on the proliferation and differentiation and gene expression of rat osteoblast in cell and molecular level,in order to approach the mechanism of antiosteoporotic effects of Puerarin on postmenopausal osteoporosis and provide the experimental evidences for its preventing and treating postmenopausal osteoporosis in clinic.
Methods
1.The osteoblasts are isolated from newborn rats within 24 hours by digesting rat's calvaria repeatedly,and collected and cultured in vitro. Morphologic changes of osteoblasts are observed.Rat osteoblasts are identified by the alkaline phosphatase staining
2.To observe the effects of Puerarin on the proliferation of rat osteoblasts by using MTT method
3.To explore the effects of Puerarin on the expressions of osteoprotegerin (OPG) and receptor activator of nuclear factor-icB ligand(RANKL) by RT-PCR in rat osteoblasts cultured in vitro.
Results
1.Experimental model of rat's cultured osteoblast in vitro is established.The cells possesses the morphological and biological properties of osteoblast.
2.All of the concerntrations are promoted by the effect of Puerarin with different concentrations on the rat osteoblasts for 24 hours and 48 hours;After cultivated 72 hours and 96 hours,all of the concentrations has no effects on proliferation
3.Puerarin can increase OPG mRNA levels remarkably and decrease RANKL mRNA levels in the rat osteoblasts cultured in vitro
Conclusions
1.Puerarin with different concentrations can promote the proliferation of osteoblasts remarkably.and the mount of proliferation has intimate relations with concentration and time
2.Puerarin can upregulate OPG expression and downregulate RANKL expression in rat osteoblasts,which may explain the mechanism of antiosteoporotic effects of Puerarin in preventing and treating postmenopausal osteoporosis.
3.Puerarin can promote the proliferation of osteoblasts and the expressions of osteoprotegerin (OPG) and receptor activator of nuclear factor-KB ligand(RANKL) mRNA,which may be the mechanism of the effects of Puerarin on postmenopausal osteoporosis.Our study not only provides the experimental evidences for the application of Puerarin,also gives a new clue on the development of the antiosteoporosis drugs.
引文
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