“鐵蓄積”在絕經(jīng)后骨質(zhì)疏松癥（Ⅰ型）中作用機(jī)制的相關(guān)研究
發(fā)布時(shí)間：2018-09-18 10:40
【摘要】：第一部分“鐵蓄積”對(duì)去勢(shì)前后小鼠骨代謝的影響及相關(guān)機(jī)制目的:觀察雌激素的有無(wú)對(duì)鐵蓄積小鼠骨量、骨轉(zhuǎn)換指標(biāo)及氧化應(yīng)激的影響。方法:建立高鐵小鼠模型,將小鼠隨機(jī)分為對(duì)照組(con組)、高鐵組(F組)、去勢(shì)組(OVX組)、高鐵去勢(shì)組(F+OVX組)。con組及OVX組注射生理鹽水,余兩組注射枸櫞酸鐵胺(FAC),8周后檢測(cè)小鼠體重,對(duì)肝臟及股骨遠(yuǎn)端行普魯士藍(lán)鐵染色,ELISA檢測(cè)血清Fer、ALP、osteocalcin、CTX、TRAP-5b、MDA、SOD;股骨遠(yuǎn)端Micro-CT三維分析,提取各組小鼠股骨骨髓細(xì)胞進(jìn)行破骨細(xì)胞培養(yǎng),抗酒石酸酸性磷酸酶染色觀察破骨細(xì)胞分化。結(jié)果:各組小鼠體重?zé)o明顯差異。F組及F+OVX組血清鐵蛋白含量明顯升高(P0.05),且肝臟及股骨遠(yuǎn)端鐵染色著色明顯。ELISA結(jié)果示:未去勢(shì)(雌激素存在)的F組與con組相比,CTX、TRAP-5b、MDA、SOD組間無(wú)明顯統(tǒng)計(jì)學(xué)差異(P均0.05),F組ALP及osteocalcin表達(dá)量較con組明顯降低(P均0.05);而去勢(shì)的兩組相比,鐵劑干預(yù)后,MDA、TRAP-5b、CTX明顯升高(P均0.05),SOD、ALP、osteocalcin則降低(P均0.05)。Micro-CT分析表明:雌激素存在時(shí),對(duì)照組與鐵干預(yù)組骨密度及相關(guān)指標(biāo)無(wú)明顯差異(P0.05);雌激素缺乏時(shí),與OVX組比較,F+OVX組骨量下降更為嚴(yán)重(P0.05)。TRAP染色顯示去勢(shì)前各組小鼠破骨細(xì)胞數(shù)無(wú)明顯差異(P0.05);而去勢(shì)后,F+OVX組破骨細(xì)胞數(shù)明顯高于OVX組(P0.05)。結(jié)論:未去勢(shì)時(shí)(有雌激素),FAC對(duì)骨量、骨吸收影響甚微;去勢(shì)后(無(wú)雌激素),FAC能顯著增強(qiáng)破骨活性,使骨量下降。雌激素與鐵對(duì)骨代謝的該種拮抗作用與骨形成無(wú)關(guān),而僅與骨吸收相關(guān)。第二部分鐵與雌二醇的聯(lián)合作用對(duì)成骨細(xì)胞、破骨細(xì)胞生物活性的影響及相關(guān)機(jī)制目的:研究成骨細(xì)胞、破骨細(xì)胞在鐵與雌二醇的共同干預(yù)下,各自生物活性指標(biāo)變化,并探討NF-κB通路在其中的作用。方法:1、成骨細(xì)胞選取小鼠顱骨原代細(xì)胞,分為對(duì)照組,FAC組、E2組、FAC+E2組,各組用相應(yīng)的10μM枸櫞酸鐵胺(FAC)及10n M雌二醇(E2)干預(yù),3d后行堿性磷酸酶(ALP)染色,10d后取培養(yǎng)基上清檢測(cè)ALP活性;熒光定量PCR(q-PCR)檢測(cè)成骨相關(guān)基因差異。2、破骨細(xì)胞選取細(xì)胞系RAW264.7,分組同成骨細(xì)胞,以50μM FAC及10n M E2干預(yù),TRAP染色及噬骨陷窩實(shí)驗(yàn)檢測(cè)破骨細(xì)胞分化差異,q-PCR檢測(cè)破骨相關(guān)基因差異;熒光酶標(biāo)儀檢測(cè)成骨細(xì)胞及破骨細(xì)胞活性氧水平。提取破骨細(xì)胞胞漿及胞核蛋白,Western-Blot檢測(cè)NF-κB通路相關(guān)蛋白表達(dá)量的差異。結(jié)果:1、成骨細(xì)胞ALP檢測(cè)結(jié)果示,無(wú)論有無(wú)雌二醇,FAC均能抑制成骨細(xì)胞ALP活性,基因水平上,FAC顯著下調(diào)SP7、Runx2基因表達(dá)水平(P均0.05),該下調(diào)作用與雌激素?zé)o關(guān)。2、破骨細(xì)胞TRAP染色結(jié)果示:FAC能顯著增加TRAP染色陽(yáng)性細(xì)胞數(shù)(P0.05),而當(dāng)雌二醇存在時(shí),該作用被抑制。噬骨陷窩實(shí)驗(yàn)結(jié)果與之一致。q-PCR結(jié)果:無(wú)雌二醇干預(yù)時(shí),鐵劑能顯著上調(diào)MMP9、Acp5、Ctsk、Calcr基因表達(dá)水平(P均0.05),雌二醇存在時(shí),兩組基因表達(dá)無(wú)明顯統(tǒng)計(jì)學(xué)差異(P均0.05)�；钚匝鮎OS檢測(cè)結(jié)果:在成骨細(xì)胞及破骨細(xì)胞中,FAC均能增加ROS活性(P均0.05),而雌二醇則能下調(diào)活性氧水平(P均0.05)。蛋白水平上,FAC使胞核蛋白p50、p65、胞漿蛋白p IκBα表達(dá)量顯著增加,并下調(diào)胞核蛋白pp65、胞漿蛋白p50、IκBα表達(dá)水平(P均0.05)。結(jié)論:雌二醇與鐵在骨代謝中的拮抗作用可能僅與骨吸收過(guò)程相關(guān),而與骨形成無(wú)關(guān)。僅當(dāng)雌二醇不存在時(shí),鐵才能通過(guò)上調(diào)ROS,進(jìn)而促進(jìn)破骨細(xì)胞分化,該作用可能與NF-κB信號(hào)通路相關(guān)。
[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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R580
本文編號(hào)：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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R580
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 耿力;姚珍薇;楊洪昌;駱建云;韓力力;盧起;;雌激素受體β基因CA重復(fù)序列多態(tài)性與絕經(jīng)后骨質(zhì)疏松癥的關(guān)聯(lián)性研究(英文)[J];遺傳學(xué)報(bào);2007年10期
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