本文選題：骨質(zhì)疏松 + 新橙皮苷��； 參考：《廣西醫(yī)科大學(xué)》2015年博士論文

【摘要】：骨質(zhì)疏松癥(osteoporosis)是一種全身性骨骼疾病,它的特點為骨組織微結(jié)構(gòu)破壞,骨量減少,骨強(qiáng)度下降,骨脆性增加。骨質(zhì)疏松性骨折在骨質(zhì)疏松癥患者中極易發(fā)生,即在受到輕微的創(chuàng)傷或日�；顒又屑纯砂l(fā)生骨折,容易出現(xiàn)嚴(yán)重后果,給家庭和社會造成負(fù)擔(dān)。骨重建是骨生成和骨代謝最重要的過程,由成骨細(xì)胞所致的骨形成和破骨細(xì)胞所致的骨吸收兩種作用之間的平衡進(jìn)行調(diào)節(jié)。長期和過度的骨吸收打破這種平衡,出現(xiàn)骨吸收增多引起的疾病,如骨質(zhì)疏松癥。破骨細(xì)胞過度的骨吸收在骨質(zhì)疏松癥中起關(guān)鍵作用。因此,抑制破骨細(xì)胞的形成或活性可以作為治療骨質(zhì)疏松的一個有效途徑。破骨細(xì)胞由骨髓巨噬細(xì)胞(BMM)分化而來,核因子-κB受體活化因子配體/核因子-κB受體活化因子(RANKL/RANK)信號通路與破骨細(xì)胞的分化和成熟密切相關(guān)。核因子-κB (NF-κB)屬于二聚體轉(zhuǎn)錄因子,是細(xì)胞增殖和分化的重要調(diào)節(jié)因子,與IκB-α抑制蛋白結(jié)合并保持在無活性狀態(tài)。外部刺激使IκB-α蛋白降解,激活NF-κB并進(jìn)入細(xì)胞核,與相應(yīng)基因的啟動子和增強(qiáng)子區(qū)的KB序列結(jié)合啟動轉(zhuǎn)錄�；罨疶細(xì)胞核因子細(xì)胞質(zhì)1(NFATcl)介導(dǎo)由基因表達(dá)程序?qū)е碌募?xì)胞融合和破骨細(xì)胞分化,是破骨細(xì)胞分化的主轉(zhuǎn)錄因子。NF-κB和NFAT的活化相互影響,NF-κB和NFAT的表達(dá)量能反映RANKL/RANK信號通路激活情況�？咕剖崴嵝粤姿崦�5b(TRACP5b)來源于破骨細(xì)胞,是破骨細(xì)胞和骨吸收的標(biāo)記物,能將骨基質(zhì)降解產(chǎn)物從吸收陷窩轉(zhuǎn)移到功能性分泌區(qū)域,破壞有機(jī)骨基質(zhì)成分。組織蛋白酶K (Cathepsin K)對Ⅰ型和Ⅱ型膠原均有切割作用,是骨生理和病理降解中主要的溶膠原蛋白酶,在破骨細(xì)胞中大量表達(dá),對骨吸收中起到特殊作用。細(xì)胞內(nèi)鈣(Ca2+)振蕩是興奮和非興奮細(xì)胞信號傳導(dǎo)中的普遍存在的模式。RANKL可引起Ca2+振蕩,導(dǎo)致鈣調(diào)磷酸酶介導(dǎo)的NFATc1激活,觸發(fā)破骨細(xì)胞的分化過程中持續(xù)的TNFATcl依賴的轉(zhuǎn)錄程序,促進(jìn)破骨細(xì)胞分化。目前認(rèn)為去卵巢(OVX)骨丟失小鼠模型與人類的情況相似,可以用于研究產(chǎn)生骨丟失的原因并進(jìn)行干預(yù),適合用于預(yù)防骨質(zhì)疏松潛在治療藥物的評價。去卵巢小鼠卵巢切除后骨礦物質(zhì)密度降低和骨微結(jié)構(gòu)受損,非常適合作為絕經(jīng)后骨質(zhì)疏松癥模型來進(jìn)行研究。新橙皮苷是枳實的提取物,屬于黃酮類化合物,是天然的抗氧化劑,具有抗炎,抗腫瘤和保護(hù)心血管系統(tǒng)的作用。但其在抑制骨質(zhì)疏松作用方面,尚未見有相關(guān)研究。在本組實驗中,我們進(jìn)行體外實驗,通過一系列實驗分析經(jīng)過新橙皮苷對RANKL誘導(dǎo)的破骨細(xì)胞分化的抑制作用。包括通過劑量依賴性實驗觀察破骨細(xì)胞分化的數(shù)量；用流式細(xì)胞儀對經(jīng)過新橙皮苷處理的Raw264.7細(xì)胞進(jìn)行細(xì)胞凋亡分析；通過qPCR實驗對破骨細(xì)胞表達(dá)的TRACP及Cathepsin K進(jìn)行測定；通過Luciferase和western blot實驗對 RANKL/RANK信號通路中的NF-κB、NFAT和IκB-α進(jìn)行分析；用羥基磷灰石檢測破骨細(xì)胞的骨吸收作用；通過鈣振蕩實驗對破骨細(xì)胞的分化進(jìn)行分析。我們還進(jìn)行了體內(nèi)實驗,觀察新橙皮苷對去卵巢小鼠模型(OVX)骨質(zhì)影響情況,結(jié)果分別用骨體積分?jǐn)?shù)(BV/TV)、骨小梁厚度(Tb.Th)、骨小梁數(shù)(Tb.N)和骨小梁分離度(Tb.Sp)這四個參數(shù)進(jìn)行分析。本實驗包括如下兩個部分第一部分 新橙皮苷對RANKL誘導(dǎo)的破骨細(xì)胞分化的影響目的：研究新橙皮苷對體外RANKL誘導(dǎo)的破骨細(xì)胞的增殖分化、調(diào)節(jié)因子 NF-κB、NFAT、IκB-α、TRACP、Cathepsin K以及破骨細(xì)胞骨吸收作用和鈣振蕩的影響,探討新橙皮苷對破骨細(xì)胞增殖分化以及對破骨細(xì)胞在骨代謝過程中一系列調(diào)節(jié)因子的作用機(jī)制。方法：在體外,BMM細(xì)胞用RANKL誘導(dǎo),加入不同濃度的新橙皮苷進(jìn)行培養(yǎng),用TRAP染色,在光學(xué)顯微鏡下觀察不同濃度新橙皮苷培養(yǎng)的TRAP染色陽性的破骨細(xì)胞并計數(shù)；RAW264.7細(xì)胞用RANKL誘導(dǎo),并加入不同濃度的新橙皮苷培養(yǎng),用流式細(xì)胞儀進(jìn)行細(xì)胞凋亡分析,觀察健康細(xì)胞的百分率；BMM細(xì)胞用RANKL誘導(dǎo),加入10μM新橙皮苷培養(yǎng),用qPCR實驗對破骨細(xì)胞的TRACP及 Cathepsin K表達(dá)量進(jìn)行測定；BMM細(xì)胞在含有羥基磷灰石涂層的培養(yǎng)基中用RANKL誘導(dǎo),加入10μM新橙皮苷培養(yǎng),觀察羥基磷灰石涂層的吸收面積并用ImageJ軟件分析；經(jīng)p-NF-κB-TA-Luc轉(zhuǎn)染的P3K-Luc細(xì)胞和p-NFAT-TA-Luc轉(zhuǎn)染的Raw264.7細(xì)胞用RANKL誘導(dǎo),加入不同濃度的新橙皮苷培養(yǎng),通過Luciferase實驗,用BMG Polar Star Optima熒光閱讀器檢測NF-κB和NFAT的表達(dá)量;BMM細(xì)胞用RANKL誘導(dǎo),加入10μM的新橙皮苷培養(yǎng),通過western blot實驗對Iκ8-α和NFAT的表達(dá)量分別進(jìn)行短時間和長時間檢測,用ImageJ軟件進(jìn)行分析；BMM細(xì)胞用RANKL誘導(dǎo),加入10μM新橙皮苷培養(yǎng),通過倒置熒光顯微鏡對細(xì)胞鈣振蕩的平均峰值高度進(jìn)行觀察,并用Nikon Basic Research軟件進(jìn)行分析。結(jié)果：新橙皮苷對破骨細(xì)胞分化均有抑制作用,且濃度越大,對破骨細(xì)胞分化的抑制作用越大；在不同濃度的新橙皮苷作用下,細(xì)胞健康率均保持于90%以上的高水平,而壞死率和凋亡率均維持在低水平,不隨新橙皮苷濃度的增加而增加；新橙皮苷均能抑制TRACP 及 Cathepsin K的表達(dá)；新橙皮苷能抑制破骨細(xì)胞對羥基磷灰石涂層的吸收作用；新橙皮苷能抑制NF-κB 和 NFAT的表達(dá),且具有濃度依賴性；新橙皮苷能抑制IκB-α的降解；新橙皮苷對破骨細(xì)胞鈣振蕩有抑制作用。結(jié)論：新橙皮苷能從多方面抑制破骨細(xì)胞的分化,抑制破骨細(xì)胞在骨代謝中的作用,是安全有效的破骨細(xì)胞抑制劑。第二部分新橙皮苷對去卵巢小鼠骨質(zhì)疏松的影響目的：研究新橙皮苷對去卵巢小鼠骨質(zhì)疏松模型的骨體積分?jǐn)?shù)、骨小梁厚度、骨小梁數(shù)和骨小梁分離度的影響,探討新橙皮苷對骨質(zhì)疏松的治療作用。方法：6周齡C57BL6J雌性小鼠30只,隨機(jī)分為假手術(shù)組,模型組,雌激素組,新橙皮苷低劑量組和新橙皮苷高劑量組,每組6只。每只小鼠稱重,予10%水合氯醛腹腔內(nèi)注射麻醉,假手術(shù)組僅切開下腹部兩側(cè)的腹部皮膚和肌層,不摘除卵巢即分層關(guān)閉腹腔。其余各組在腹腔兩側(cè)找到卵巢后,予以結(jié)扎并完整切除,分層關(guān)閉腹腔。術(shù)后各組予慶大霉素抗炎,予水和食物喂養(yǎng)觀察1周后開始隔天按小鼠體重腹腔注射給藥。假手術(shù)組和模型組予生理鹽水,雌激素組予0.1mg/kg雌激素,新橙皮苷低劑量和高劑量組分別予3mg/kg 和 6mg/kg新橙皮苷。7周后,每只小鼠予乙醚麻醉,取單側(cè)脛骨浸泡于4%FPA溶液中,20-24小時后取出,1XPBS溶液沖洗2次,用1XPBS侵泡脛骨保存于1.5m1試管中,行Micro-CT分析,對比觀察各組骨體積分?jǐn)?shù)、骨小梁厚度、骨小梁數(shù)和骨小梁分離度的變化。結(jié)果：新橙皮苷組增加去卵巢骨質(zhì)疏松小鼠的骨體積分?jǐn)?shù),基本恢復(fù)到正常水平,效果與雌激素組相當(dāng),且有濃度依賴性,濃度越大,骨體積分?jǐn)?shù)越明顯；各組的骨小梁厚度無明顯變化；新橙皮苷高劑量組能減少去卵巢骨質(zhì)疏松小鼠的骨小梁分離度,且基本恢復(fù)正常水平,效果與雌激素相當(dāng)；新橙皮苷能增加去卵巢骨質(zhì)疏松小鼠的骨小梁數(shù),有濃度依賴性,高劑量的新橙皮苷與雌激素的效果相當(dāng),使骨小梁數(shù)基本恢復(fù)正常水平。結(jié)論：新橙皮苷能抑制去卵巢小鼠骨質(zhì)疏松的發(fā)展,是治療骨質(zhì)疏松癥的潛在藥物。
[Abstract]:Osteoporosis (osteoporosis) is a systemic skeletal disease characterized by microstructural destruction of bone tissue, reduced bone mass, reduced bone strength, and increased bone brittleness. Osteoporotic fractures are very prone to occur in patients with osteoporosis, that is, fractures may occur in mild trauma or daily activities, and severe consequences are likely to occur. Family and society make burdens. Bone reconstruction is the most important process of bone formation and bone metabolism. The balance between the two effects of osteogenesis caused by osteoblasts and osteoclast caused by osteoclast is regulated. Long and excessive bone resorption breaks this balance and causes diseases caused by increasing bone absorption, such as osteoporosis. Osteoclast. Excessive bone resorption plays a key role in osteoporosis. Therefore, inhibition of osteoclast formation or activity can be an effective way to treat osteoporosis. Osteoclasts are differentiated from bone marrow macrophages (BMM), and nuclear factor kappa B receptor activator ligand / nuclear factor kappa B receptor activating factor (RANKL/RANK) signaling pathway It is closely related to the differentiation and maturation of osteoclasts. Nuclear factor kappa B (NF- kappa B) belongs to the two polymer transcription factor, which is an important regulator of cell proliferation and differentiation, which combines with I kappa B- alpha suppressor and remains inactive. External stimuli degrade the I kappa B- alpha protein, activate NF- kappa B and enter the nucleus, and increase the promoter and increase of the corresponding genes. The KB sequence of the hadron region combined with the activation of transcription. Activated T nuclear factor cytoplasm 1 (NFATcl) mediates the cell fusion and osteoclast differentiation caused by the gene expression program, which is the activation interaction of the main transcription factor.NF- kappa B and NFAT of the osteoclast differentiation. The expression of NF- kappa B and NFAT can reflect the activation of RANKL/RANK signaling pathway. The acid phosphatase 5b (TRACP5b) is derived from osteoclasts and is a marker of osteoclast and bone absorption. It can transfer the degradation products of bone matrix from the absorption lacunar to the functional secretory region and destroy the organic matrix components. Cathepsin K (Cathepsin K) has a cutting effect on type I and type II collagen, which is the bone physiological and pathological degradation. The main sol-gel protease, expressed in osteoclasts, plays a special role in bone resorption. Intracellular calcium (Ca2+) oscillation is a common pattern in the signal transduction of excitatory and non excitatory cells,.RANKL can cause Ca2+ oscillation, leading to calcineurin mediated NFATc1 activation, triggering a sustained TN in the differentiation of osteoclasts. The FATcl - dependent transcriptional program promotes osteoclast differentiation. It is believed that the mouse model of the ovariectomized (OVX) bone loss is similar to the human condition, and can be used to study the cause of bone loss and to intervene, suitable for the evaluation of the potential treatment drugs for osteoporosis. Bone microstructures are damaged and are very suitable for postmenopausal osteoporosis models. Neohesperidin is an extract of Fructus aurantii, which belongs to the flavonoids, is a natural antioxidant, and has the effect of anti-inflammatory, anti-tumor and protection of the cardiovascular system. However, it has not been studied in this group. In the experiment, we conducted an in vitro experiment to analyze the inhibitory effect of new hesperidin on RANKL induced osteoclast differentiation through a series of experiments, including the number of osteoclast differentiation through a dose dependent experiment, and the apoptosis analysis of Raw264.7 cells treated by new hesperidin by flow cytometry; through qPCR TRACP and Cathepsin K expressed in osteoclasts were measured, and NF- kappa B, NFAT and I kappa B- alpha in RANKL/RANK signaling pathway were analyzed by Luciferase and Western blot; the bone resorption of osteoclasts was detected by hydroxyapatite; the differentiation of osteoclasts was analyzed by calcium oscillation test. We also carried out the analysis of osteoclast differentiation. In vivo, the effect of Neo hesperidin on the ovariectomized mouse model (OVX) was observed. The results were analyzed by four parameters: bone volume fraction (BV/TV), bone small Liang Houdu (Tb.Th), bone trabecular number (Tb.N) and bone trabecular separation (Tb.Sp). The first part of the experiment included the following two parts: the first part of new hesperidin to RANKL induced osteoclast Objective: To investigate the effect of Neo hesperidin on the proliferation and differentiation of osteoclasts induced by RANKL in vitro, the effects of regulatory factor NF- kappa B, NFAT, I kappa B- a, TRACP, Cathepsin K, osteoclast absorption and calcium oscillation, and to explore the proliferation and differentiation of osteoclasts and a series of osteoclasts in bone metabolism. Methods: in vitro, in vitro, BMM cells were induced by RANKL and cultured with different concentrations of new hesperidin. TRAP staining was used to observe the TRAP staining positive osteoclasts cultured with different concentrations of new hesperidin under the optical microscope. RAW264.7 cells were induced by RANKL and added to different concentrations of new orange peel. The percentage of healthy cells was observed by flow cytometry, and the percentage of healthy cells was observed. BMM cells were induced by RANKL and cultured with 10 M new hesperidin. The expression of TRACP and Cathepsin K in osteoclasts was measured by qPCR experiment. BMM cells were induced by RANKL in the culture medium containing hydroxyapatite coating and added to 10 micron M. The absorption area of hydroxyapatite coating was observed and analyzed by ImageJ software. The P3K-Luc cells transfected by p-NF- kappa B-TA-Luc and Raw264.7 cells transfected with p-NFAT-TA-Luc were induced by RANKL and added to the culture of new hesperidin in different concentrations. The expression of T; BMM cells were induced by RANKL and added to the culture of 10 M new hesperidin. The expression of I kappa 8- A and NFAT were detected in a short and long time by Western blot experiment. The BMM cells were analyzed by ImageJ software. The BMM cells were induced by RANKL and added 10 mu new hesperidin, and the cell calcium oscillations were flat by inverted fluorescence microscope. The peak height was observed and analyzed with Nikon Basic Research software. Results: new hesperidin had inhibitory effect on osteoclast differentiation, and the greater the concentration, the greater the inhibition effect on osteoclast differentiation; the cell health rate remained at a high level above 90% under the action of different concentrations of new hesperidin, and the necrosis rate and the rate of necrosis were observed. The apoptosis rate remained at a low level and did not increase with the increase of the concentration of new hesperidin, and Neo hesperidin could inhibit the expression of TRACP and Cathepsin K; neo hesperidin could inhibit the absorption of osteoclast to hydroxyapatite coating; neo hesperidin could inhibit the expression of NF- kappa B and NFAT, and was dependent on the concentration of new hesperidin. The degradation of I kappa B- alpha and the inhibitory effect of Neo hesperidin on osteoclast calcium oscillation. Conclusion: Neo hesperidin can inhibit the differentiation of osteoclast from many aspects and inhibit the role of osteoclast in bone metabolism. It is a safe and effective osteoclast inhibitor. The aim of the study is to study the effect of the second part of new hesperidin on osteoporosis in ovariectomized mice. The effect of Neo hesperidin on bone volume fraction, bone trabecular thickness, bone trabecular number and bone trabecular separation in ovariectomized mice was studied. Methods: 30 C57BL6J female mice of 6 weeks old were randomly divided into sham operation group, model group, estrogen group, new hesperidin low dose group and new orange. A high dose of peridoside, each group of 6. Each mouse was weighed and injected with 10% chloral chloral intraperitoneal injection. The sham operation group only cut the abdominal skin and muscle layer on both sides of the lower abdomen, and did not remove the ovaries and closed the abdominal cavity. The other groups were ligated and completely removed and closed the abdominal cavity after the ovaries were found on both sides of the abdominal cavity. Mycophentin was injected with water and food for 1 weeks after 1 weeks. The sham operation group and the model group were given physiological saline, estrogen group was given estrogen, the low dose and high dose group of new hesperidin were given 3mg/kg and 6mg/kg new hesperidin.7 weeks respectively, each mouse was given eether anesthesia for unilateral tibia soaking. In 4%FPA solution, 20-24 hours after removal, 1XPBS solution flushed 2 times, 1XPBS invaded tibia in 1.5m1 test tube, Micro-CT analysis, compare the volume fraction of bone, bone trabecular thickness, bone trabecular number and bone trabecular separation. Results: new hesperidin group increased bone volume fraction of ovariectomized osteoporosis mice, basic Restore to the normal level, the effect is equivalent to the estrogen group, and the concentration dependence, the greater the concentration, the more obvious bone volume, the thickness of bone trabecula in each group has no obvious change; the high dose group of new hesperidin can reduce the bone trabecular separation degree of ovariectomized mice, and basically restore normal level, the effect is equivalent to the estrogens; new orange Peridoside can increase the number of bone trabeculae in ovariectomized mice and have a concentration dependence. The high dose of new hesperidin has the same effect as estrogen, making the number of bone trabeculae basically restored to normal level. Conclusion: Neo hesperidin can inhibit the development of osteoporosis in ovariectomized mice and be a potential drug for the treatment of osteoporosis.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R580

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