“铁蓄积”在绝经后骨质疏松症（Ⅰ型）中作用机制的相关研究
发布时间：2018-09-18 10:40
【摘要】：第一部分“铁蓄积”对去势前后小鼠骨代谢的影响及相关机制目的:观察雌激素的有无对铁蓄积小鼠骨量、骨转换指标及氧化应激的影响。方法:建立高铁小鼠模型,将小鼠随机分为对照组(con组)、高铁组(F组)、去势组(OVX组)、高铁去势组(F+OVX组)。con组及OVX组注射生理盐水,余两组注射枸橼酸铁胺(FAC),8周后检测小鼠体重,对肝脏及股骨远端行普鲁士蓝铁染色,ELISA检测血清Fer、ALP、osteocalcin、CTX、TRAP-5b、MDA、SOD;股骨远端Micro-CT三维分析,提取各组小鼠股骨骨髓细胞进行破骨细胞培养,抗酒石酸酸性磷酸酶染色观察破骨细胞分化。结果:各组小鼠体重无明显差异。F组及F+OVX组血清铁蛋白含量明显升高(P0.05),且肝脏及股骨远端铁染色着色明显。ELISA结果示:未去势(雌激素存在)的F组与con组相比,CTX、TRAP-5b、MDA、SOD组间无明显统计学差异(P均0.05),F组ALP及osteocalcin表达量较con组明显降低(P均0.05);而去势的两组相比,铁剂干预后,MDA、TRAP-5b、CTX明显升高(P均0.05),SOD、ALP、osteocalcin则降低(P均0.05)。Micro-CT分析表明:雌激素存在时,对照组与铁干预组骨密度及相关指标无明显差异(P0.05);雌激素缺乏时,与OVX组比较,F+OVX组骨量下降更为严重(P0.05)。TRAP染色显示去势前各组小鼠破骨细胞数无明显差异(P0.05);而去势后,F+OVX组破骨细胞数明显高于OVX组(P0.05)。结论:未去势时(有雌激素),FAC对骨量、骨吸收影响甚微;去势后(无雌激素),FAC能显著增强破骨活性,使骨量下降。雌激素与铁对骨代谢的该种拮抗作用与骨形成无关,而仅与骨吸收相关。第二部分铁与雌二醇的联合作用对成骨细胞、破骨细胞生物活性的影响及相关机制目的:研究成骨细胞、破骨细胞在铁与雌二醇的共同干预下,各自生物活性指标变化,并探讨NF-κB通路在其中的作用。方法:1、成骨细胞选取小鼠颅骨原代细胞,分为对照组,FAC组、E2组、FAC+E2组,各组用相应的10μM枸橼酸铁胺(FAC)及10n M雌二醇(E2)干预,3d后行碱性磷酸酶(ALP)染色,10d后取培养基上清检测ALP活性;荧光定量PCR(q-PCR)检测成骨相关基因差异。2、破骨细胞选取细胞系RAW264.7,分组同成骨细胞,以50μM FAC及10n M E2干预,TRAP染色及噬骨陷窝实验检测破骨细胞分化差异,q-PCR检测破骨相关基因差异;荧光酶标仪检测成骨细胞及破骨细胞活性氧水平。提取破骨细胞胞浆及胞核蛋白,Western-Blot检测NF-κB通路相关蛋白表达量的差异。结果:1、成骨细胞ALP检测结果示,无论有无雌二醇,FAC均能抑制成骨细胞ALP活性,基因水平上,FAC显著下调SP7、Runx2基因表达水平(P均0.05),该下调作用与雌激素无关。2、破骨细胞TRAP染色结果示:FAC能显著增加TRAP染色阳性细胞数(P0.05),而当雌二醇存在时,该作用被抑制。噬骨陷窝实验结果与之一致。q-PCR结果:无雌二醇干预时,铁剂能显著上调MMP9、Acp5、Ctsk、Calcr基因表达水平(P均0.05),雌二醇存在时,两组基因表达无明显统计学差异(P均0.05)。活性氧ROS检测结果:在成骨细胞及破骨细胞中,FAC均能增加ROS活性(P均0.05),而雌二醇则能下调活性氧水平(P均0.05)。蛋白水平上,FAC使胞核蛋白p50、p65、胞浆蛋白p IκBα表达量显著增加,并下调胞核蛋白pp65、胞浆蛋白p50、IκBα表达水平(P均0.05)。结论:雌二醇与铁在骨代谢中的拮抗作用可能仅与骨吸收过程相关,而与骨形成无关。仅当雌二醇不存在时,铁才能通过上调ROS,进而促进破骨细胞分化,该作用可能与NF-κB信号通路相关。
[Abstract]:Objective: To observe the effect of estrogen on bone mass, bone turnover index and oxidative stress in mice with iron accumulation. Methods: A high-iron mice model was established and randomly divided into control group (con group), high-iron group (F group), ovariectomy group (OVX group), high-iron castration group (F group). Con group and OVX group were injected with normal saline, and the other two groups were injected with ferric citrate (FAC). After 8 weeks, the weight of mice was measured. The liver and distal femur were stained with Prussian blue iron, the serum levels of Fer, ALP, osteocalcin, CTX, TRAP-5b, MDA and SOD were detected by ELISA. Results: There was no significant difference in body weight among the groups. The serum ferritin content in F group and F + OVX group increased significantly (P 0.05), and the iron staining in liver and distal femur was obvious. The results of ELISA showed that compared with con group, the levels of CTX, TRAP-5b, MDA and SOD were significantly higher in F group and F + OVX group. The expression of ALP and osteocalcin in F group was significantly lower than that in con group (P 0.05), while MDA, TRAP-5b and CTX were significantly higher (P 0.05) and SOD, ALP and osteocalcin were significantly lower (P 0.05) in castrated group than those in control group (P 0.05). There was no significant difference in the number of osteoclasts between the groups before ovariectomy (P 0.05), but after ovariectomy, the number of osteoclasts in the F + OVX group was significantly higher than that in the OVX group (P 0.05). After ovariectomy (without estrogen), FAC significantly enhanced osteoclast activity and decreased bone mass. The antagonistic effect of estrogen and iron on bone metabolism was not related to bone formation, but only related to bone resorption. Methods: 1. Osteoblasts were divided into control group, FAC group, E2 group, FAC + E2 group. Each group was treated with the corresponding 10 mu M ferric citrate (FAC) and 10N M estradiol (E2) for 3 days. Alkaline phosphatase (ALP) staining, 10 days after taking the supernatant of culture medium to detect ALP activity; fluorescence quantitative PCR (q-PCR) detection of osteogenesis-related gene differences. 2, osteoclasts selected cell line RAW264.7, grouped into osteoblasts, 50 mu FAC and 10N M E2 intervention, TRAP staining and osteophagocytic lacunae test to detect osteoclast differentiation differences, q-PCR detection of osteoclast-related. The expression of cytoplasmic and nuclear proteins in osteoclasts was extracted and the expression of NF-kappa B pathway related proteins was detected by Western-Blot. Results: 1. The results of osteoblast ALP detection showed that FAC could inhibit the activity of ALP in osteoblasts with or without estradiol. The down-regulation of SP7 and Runx2 gene expression was not related to estrogen (P 0.05). 2. TRAP staining of osteoclasts showed that FAC could significantly increase the number of TRAP-positive cells (P 0.05), but the effect was inhibited in the presence of estradiol. The expression of MMP9, Acp5, Ctsk and Calcr genes was regulated by FAC (all P 0.05). There was no significant difference between the two groups in the presence of estradiol (all P 0.05). ROS assay showed that FAC increased ROS activity in osteoblasts and osteoclasts (all P 0.05), while estradiol decreased ROS level (all P 0.05). Conclusion: The antagonism of estradiol and iron in bone metabolism may be only related to bone resorption process, but not to bone formation. Only when estradiol does not exist, iron can up-regulate ROS and thus promote bone formation. The role of osteoclast differentiation may be related to the NF- kappa B signaling pathway.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R580
本文编号：2247644
[Abstract]:Objective: To observe the effect of estrogen on bone mass, bone turnover index and oxidative stress in mice with iron accumulation. Methods: A high-iron mice model was established and randomly divided into control group (con group), high-iron group (F group), ovariectomy group (OVX group), high-iron castration group (F group). Con group and OVX group were injected with normal saline, and the other two groups were injected with ferric citrate (FAC). After 8 weeks, the weight of mice was measured. The liver and distal femur were stained with Prussian blue iron, the serum levels of Fer, ALP, osteocalcin, CTX, TRAP-5b, MDA and SOD were detected by ELISA. Results: There was no significant difference in body weight among the groups. The serum ferritin content in F group and F + OVX group increased significantly (P 0.05), and the iron staining in liver and distal femur was obvious. The results of ELISA showed that compared with con group, the levels of CTX, TRAP-5b, MDA and SOD were significantly higher in F group and F + OVX group. The expression of ALP and osteocalcin in F group was significantly lower than that in con group (P 0.05), while MDA, TRAP-5b and CTX were significantly higher (P 0.05) and SOD, ALP and osteocalcin were significantly lower (P 0.05) in castrated group than those in control group (P 0.05). There was no significant difference in the number of osteoclasts between the groups before ovariectomy (P 0.05), but after ovariectomy, the number of osteoclasts in the F + OVX group was significantly higher than that in the OVX group (P 0.05). After ovariectomy (without estrogen), FAC significantly enhanced osteoclast activity and decreased bone mass. The antagonistic effect of estrogen and iron on bone metabolism was not related to bone formation, but only related to bone resorption. Methods: 1. Osteoblasts were divided into control group, FAC group, E2 group, FAC + E2 group. Each group was treated with the corresponding 10 mu M ferric citrate (FAC) and 10N M estradiol (E2) for 3 days. Alkaline phosphatase (ALP) staining, 10 days after taking the supernatant of culture medium to detect ALP activity; fluorescence quantitative PCR (q-PCR) detection of osteogenesis-related gene differences. 2, osteoclasts selected cell line RAW264.7, grouped into osteoblasts, 50 mu FAC and 10N M E2 intervention, TRAP staining and osteophagocytic lacunae test to detect osteoclast differentiation differences, q-PCR detection of osteoclast-related. The expression of cytoplasmic and nuclear proteins in osteoclasts was extracted and the expression of NF-kappa B pathway related proteins was detected by Western-Blot. Results: 1. The results of osteoblast ALP detection showed that FAC could inhibit the activity of ALP in osteoblasts with or without estradiol. The down-regulation of SP7 and Runx2 gene expression was not related to estrogen (P 0.05). 2. TRAP staining of osteoclasts showed that FAC could significantly increase the number of TRAP-positive cells (P 0.05), but the effect was inhibited in the presence of estradiol. The expression of MMP9, Acp5, Ctsk and Calcr genes was regulated by FAC (all P 0.05). There was no significant difference between the two groups in the presence of estradiol (all P 0.05). ROS assay showed that FAC increased ROS activity in osteoblasts and osteoclasts (all P 0.05), while estradiol decreased ROS level (all P 0.05). Conclusion: The antagonism of estradiol and iron in bone metabolism may be only related to bone resorption process, but not to bone formation. Only when estradiol does not exist, iron can up-regulate ROS and thus promote bone formation. The role of osteoclast differentiation may be related to the NF- kappa B signaling pathway.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R580
【参考文献】
相关期刊论文 前1条
1 耿力;姚珍薇;杨洪昌;骆建云;韩力力;卢起;;雌激素受体β基因CA重复序列多态性与绝经后骨质疏松症的关联性研究(英文)[J];遗传学报;2007年10期
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