【作者】 朱晓峰；

【导师】 张荣华；

【作者基本信息】 暨南大学 ， 中西医结合临床， 2009， 博士

【摘要】 目的:淫羊藿素为补肾强骨中药淫羊藿的主要成分淫羊藿苷在动物体内代谢后脱去糖基的活性成分,可能是淫羊藿口服吸收后主要发挥药理活性的成分,对其研究更符合淫羊藿临床多口服给药的用药方式。目前其对成骨细胞作用机理的研究尚未见报道。本研究观察淫羊藿素对MC3T3-ElSubclone14前体成骨细胞株增殖、分化的影响,以及观察它对与分化相关的雌激素受体信号通路、p38MAPK信号通路、BMPs/smads信号通路的影响,明确淫羊藿素作用于成骨细胞而影响骨代谢的具体靶点,对于评价淫羊藿治疗骨质疏松等骨代谢疾病的有效性提供细胞分子学依据。方法:1.MC3T3-ElSubclone14前体成骨细胞株的培养及活性观察。2.运用MTT法检测淫羊藿素对MC3T3-ElSubclone14的细胞毒性,确定实验用的药物剂量。3.应用WST-8细胞活力方法、BrdU流式细胞仪方法检测淫羊藿素对MC3T3-ElSubclone14细胞增殖的影响。4.应用pNPP方法检测淫羊藿素对MC3T3-ElSubclone14细胞碱性磷酸酶活性的影响,运用双抗夹心ELISA方法检测淫羊藿素对MC3T3-ElSubclone14细胞Ⅰ型胶原和骨钙素的影响,运用茜素红染色计数矿化结节的方法评价淫羊藿素对MC3T3-ElSubclone14细胞矿化的影响。5.运用ER受体阻断剂ICI182780阻断ER受体信号后,检测淫羊藿素对MC3T3-ElSubclone14细胞碱性磷酸酶活性、Ⅰ型胶原和骨钙素的影响,评价ER受体信号在淫羊藿素对MC3T3-ElSubclone14细胞分化过程中的作用。6.运用p38MAPK受体阻断剂SB203580阻断p38MAPK信号后,检测淫羊藿素对MC3T3-ElSubclone14细胞碱性磷酸酶活性、Ⅰ型胶原和骨钙素的影响,评价p38MAPK信号在淫羊藿素对MC3T3-ElSubclone14细胞分化过程中的作用。7.实时荧光PCR检测淫羊藿素对BMP-2、4、7mRNA的表达。运用BMPs受体阻断剂Noggin阻断BMPs/Smads信号后,检测淫羊藿素对MC3T3-ElSubclone14细胞表达碱性磷酸酶活性、Ⅰ型胶原和骨钙素的影响,评价BMPs/Smads信号在淫羊藿素对MC3T3-ElSubclone14细胞分化过程中的作用。8.Western-blot检测ER受体阻断剂ICI182780阻断ER受体信号后,淫羊藿素对p38和Smad1/5/8的蛋白磷酸化情况,评价它们和ER受体信号的关系。结果:1.MC3T3-ElSubclone14前体成骨细胞株,在具有分化条件的培养基中可以向成骨细胞分化。2.淫羊藿素对MC3T3-ElSubclone14细胞毒性的检测发现,IC50浓度为10^0.154mol/L,10^-5.93mol/L浓度以下则对细胞基本上无抑制作用,因此实验药物浓度选择10^-8、10^-7、10^-6mol/L 0.01μM、0.1μM、1μM)为梯度浓度。3.WST-8和BrdU流式细胞仪检测发现,淫羊藿素0.01μM、0.1μM、1μM浓度组的MC3T3-ElSubclone14的细胞活力和细胞增殖指数与对照组比较,在统计学上无显著差异（P＞0.05）。4.在作用48或72小时后,淫羊藿素0.01μM、0.1μM、1μM浓度组MC3T3-ElSubclone14细胞的碱性磷酸酶、Ⅰ型胶原和骨钙素与对照组比较,在统计学上有显著差异（P＜0.01或0.05）;淫羊藿素作用10天或14天后,各组的矿化结节计数和对照组比较,在统计学上有显著差异（P＜0.01）。5.运用ER受体阻断剂ICI182780阻断ER受体信号后,1μM浓度的淫羊藿素促进MC3T3-ElSubclone14细胞分化的特性明显下降,和单纯应用淫羊藿素组在统计学上有显著差异（P＜0.01）;运用p38MAPK受体阻断剂SB203580阻断p38MAPK信号后,1μM浓度的淫羊藿素促进MC3T3-ElSubclone14细胞分化的特性明显下降,和单纯应用淫羊藿素组在统计学上有显著差异（P＜0.01）;运用BMPs受体阻断剂Noggin阻断BMPs/Smads信号后,1μM浓度的淫羊藿素促进MC3T3-ElSubclone14细胞分化的特性明显下降,和单纯应用淫羊藿素组在统计学上有显著差异（P＜0.01）。6.淫羊藿素可以提高BMP-2mRNA的表达,并随着浓度的增高而增高,和对照组比较,在统计学上有明显差异（P＜0.01或0.05）,1μM浓度的淫羊藿素可以提高BMP-4mRNA的表达（P＜0.05）,但对BMP-7mRNA无作用（P＞0.01）。7.Western-blot检测发现运用ER受体阻断剂ICI182780阻断ER受体信号后,p38和Smad1/5/8的磷酸化明显减弱。结论:1.一定浓度（0.01μM、0.1μM、1μM）的ICT对MC3T3-ElSubclone14细胞增殖无明显促进作用。2.一定浓度（0.01μM、0.1μM、1μM）的ICT可以促进MC3T3-ElSubclone14细胞分化,促进其矿化。3.ICT促进MC3T3-ElSubclone14细胞分化和ER受体信号通路、p38MAPK信号通路、BMPs/Smads信号通路密切相关。4.在ICT促进MC3T3-ElSubclone14细胞分化的过程中,ER受体信号通路在p38MAPK信号通路和BMPs/Smads信号通路的上游。5.实验结果提示ICT在MC3T3-ElSubclone14细胞分化过程中所显示的主要作用可能和它的类雌激素样活性有关。更多还原

【Abstract】 Objective:Epimedium grandiflorum Morr,a traditional Chinese herb,was effective to treatment many diseases of bone.Icariin,a principal flavonoid glycoside in Epimedium grandiflorum Morr,is metabolized to Icaritin in vivo.Icaritin may have beneficial effects on bone mass.but its role and mechanism in osteoblasts is unclear.This study is to observe the effects of icaritin on the proliferation and differentiation of MC3T3-E1Subclonel4,a pre-osteoblasts cell line,and observe the effects on ER, p38MAPK and BMPs/Smads signaling pathways which were closely related to differentiation of osteoblasts.To define targets of icaritin on osteoblasts,which can provide cellular and molecular evidences for Epimedium grandiflorum Morr treating osteoporosis and other bone metabolic diseases.Methods:1.MC3T3-E1Subclonel4 was cultured and its biological activity was observed.2.MTT method was used to observe cytotoxicity of Icaritin,and to determine the experimental concentration of Icaritin in the Medium.3.WST-8 method and BrdU method were used to observe cell activity and proliferation of MC3T3-E1Subclonel4 cells after treating with different concentration of Icaritin.4.pNPP method was used to observe ALP activity of MC3T3-E1Subclonel4 cells after treating with different concentration of Icaritin.ELISA kit was used to observe TypeⅠcollagen and osteocalcin activity of MC3T3-E1Subclonel4 cells after treating with different concentration of Icaritin.Alizarin dyeing was used to observe mineralization of MC3T3-E1Subclonel4 cells after treating with different concentration of Icaritin.5.After ER Signaling pathways was blocked by ICI182780,p38MAPK Signaling pathways was blocked by SB203580 respectively,ALP,TypeⅠcollagen and osteocalcin activity of MC3T3-E 1Subclonel4 cell were observed after treating with Icaritin.6.Real-time PCR was used to observe the mRNA relative levels of BMP-2、4、7 after treating with Icaritin.After BMPs/Smads Signaling pathways was blocked by noggin,ALP,Type I collagen and osteocalcin activity of MC3T3-E1Subclonel4 cells were observed after treating with Icaritin.7.Western-blot was used to observe p38 and Smadl/5/8 protein phosphorylation after ER Signaling pathways was blocked by ICI182780 after treating with Icaritin.Results:1.MC3T3-E1Subclonel4 cells was induced successfully into osteoblasts in medium of inducing conditions.2.IC50 of Icaritin’s concentration in medium in the cytotoxicity test was 10^0.154 mol/L.Less than the concentration of 10^-5.93mol/L,Icaritin can’t inhibit the growth of MC3T3-E 1Subclone 14 cells.3.Cell activity and Proliferation index of MC3T3-E1Subclonel4 cells of Icaritin （0.01μM、0.1μM、1μM） groups have no different with control group after 48 and 72hours（P＞0.05）.4.ALP,TypeⅠcollagen and osteocalcin activity oflcaritin（0.01μM、0.1μM、1μM） groups were higher than control group after 48 or 72hours（P＜0.01or 0.05）. Mineralized nodus weren higher control group after 10 or 14days（P＜0.01）.5.ALP,TypeⅠcollagen and osteocalcin activity of MC3T3-E1Subclone 14 cell of experimental group（Icaritin+blocker） were less than Icaritin group after ER Signaling pathways was blocked by ICI182780,p38MAPK Signaling pathways was blocked by SB203580 respectively（P＜0.01）.6.The mRNA levels of BMP-2 and BMP- 4 of Icaritin group was higher than control group（P＜0.01 or 0.05）,but the mRNA levels of BMP-7 of all groups have no significant difference（P＞0.05）.7.ALP,TypeⅠcollagen and osteocalcin activity of MC3T3-E1Subclonel4 cell of experimental group（Icaritin+blocker） were less than Icaritin group after BMPs/Smads Signaling pathways was blocked by noggin（P＜0.01 or 0.05）.8.p38 and Smadl/5/8 protein phosphorylation of icaritin -induced decreased significantly after ER Signaling pathways was blocked by ICI182780.Conclusions:1.Icaritin（0.01μM、0.1μM、1μM） can’t increase activity and proliferation index of MC3T3-E 1Subclone 14 cells.2.Icaritin（0.01μM、0.1μM、1μM） can increase differentiation and mineralization of MC3T3-E1Subclone 14 cells.3.Icaritin’s role in the promotion of MC3T3-E1Subclonel4 cells differentiation is closely related to ER,p38MAPK and BMPs/Smads Signaling pathways.4.In the differentiation process,ER Signaling pathways may be take place upstream of p38MAPK and BMPs/Smads Signaling pathways.5.Icaritin can increase differentiation of MC3T3-E 1Subclone 14 cells may be for its estrogen-like activity.更多还原