發(fā)布時(shí)間：2018-11-28 09:57

【摘要】：目的探討淫羊藿苷(icariin,ICA)抑制破骨分化的量效和時(shí)效關(guān)系,探究淫羊藿苷在單核細(xì)胞向破骨細(xì)胞分化的過程中對(duì)絲裂原活化蛋白激酶(mitogen-activated protein kinases,MAPKs)信號(hào)通路的影響,以了解淫羊藿苷對(duì)破骨細(xì)胞分化抑制過程的分子生物學(xué)機(jī)制,為淫羊藿苷進(jìn)一步應(yīng)用于臨床提供理論基礎(chǔ)。方法本研究分為兩個(gè)部分進(jìn)行。一淫羊藿苷抑制破骨細(xì)胞分化的量效關(guān)系研究:按照不同的ICA藥物濃度將RAW264.7細(xì)胞分為空白對(duì)照組(等量培養(yǎng)基對(duì)照)、模型對(duì)照組(50ng/ml RANKL)、ICA四個(gè)濃度梯度組(10-7/10-6/10-5/10-4mol/L ICA+50ng/ml RANKL)。TRAP染色觀察破骨細(xì)胞形態(tài)數(shù)目。同時(shí)通過western blot檢測(cè)關(guān)鍵蛋白的表達(dá)和磷酸化水平。二淫羊藿苷抑制破骨細(xì)胞分化的時(shí)效關(guān)系研究:設(shè)置對(duì)照溶劑處理組與淫羊藿苷處理組(ICA濃度為10-5mol/L),每組分別在誘導(dǎo)12、24、36、48、60、72小時(shí)后進(jìn)行TRAP染色觀察破骨細(xì)胞形態(tài)數(shù)目,并通過western blot檢測(cè)關(guān)鍵蛋白的表達(dá)和磷酸化水平。結(jié)果一RAW264.7誘導(dǎo)培養(yǎng)48小時(shí)后可見含3個(gè)以上細(xì)胞核的多核細(xì)胞。Trap染色顯示隨著ICA濃度的升高,破骨細(xì)胞數(shù)量減少,破骨細(xì)胞形態(tài)小,胞核數(shù)目較少。模型對(duì)照組與空白對(duì)照組相比,p38、JNK、ERK磷酸化水平升高,而P38、JNK、ERK表達(dá)水平無統(tǒng)計(jì)學(xué)差異。與不給于ICA的模型對(duì)照組相比,各ICA處理組的P38、JNK、ERK磷酸化水平隨濃度的升高而降低,其抑制作用呈現(xiàn)濃度依賴性,而P38、JNK、ERK表達(dá)水平無統(tǒng)計(jì)學(xué)差異。當(dāng)ICA濃度達(dá)到10-5mol/L時(shí),對(duì)單核細(xì)胞破骨分化的抑制作用達(dá)到最佳。二溶劑對(duì)照組在誘導(dǎo)12小時(shí)后即可見TRAP陽性的破骨細(xì)胞,且隨著誘導(dǎo)時(shí)間的增長(zhǎng),破骨細(xì)胞數(shù)量逐漸增多。與相同誘導(dǎo)時(shí)間的溶劑對(duì)照組相比,ICA組破骨細(xì)胞數(shù)量極少,且細(xì)胞形態(tài)較小。相同誘導(dǎo)時(shí)間的溶劑對(duì)照組和ICA處理組之間P38、JNK、ERK的磷酸化水平均有差異,P38、JNK、ERK表達(dá)水平無統(tǒng)計(jì)學(xué)差異。溶劑對(duì)照組的P38、JNK、ERK的磷酸化水平隨誘導(dǎo)時(shí)間的增加而逐漸升高,而P38、JNK、ERK表達(dá)無統(tǒng)計(jì)學(xué)差異。ICA處理組的各誘導(dǎo)時(shí)間點(diǎn)間兩兩比較,P38、JNK、ERK的磷酸化水平和蛋白表達(dá)水平均無統(tǒng)計(jì)學(xué)差異。結(jié)論淫羊藿苷對(duì)單核細(xì)胞破骨分化具有穩(wěn)定的抑制作用,且抑制作用效果隨淫羊藿苷濃度的升高而增強(qiáng)。淫羊藿苷對(duì)單核細(xì)胞破骨分化時(shí)MAPK信號(hào)通路中關(guān)鍵蛋白p38、ERK和JNK的表達(dá)沒有影響,但是可以顯著抑制其磷酸化水平,且抑制作用與濃度正相關(guān)。淫羊藿苷體外抑制破骨分化過程中MAPK通路的最佳濃度為10-5mol/L。淫羊藿苷對(duì)破骨分化的抑制作用自12小時(shí)起已經(jīng)達(dá)到穩(wěn)定水平。
[Abstract]:Objective to investigate the dose-effect and time-effect relationship of icariin (icariin,ICA) in inhibiting osteoclast differentiation, and to explore the effect of icariin on mitogen-activated protein kinase (mitogen-activated protein kinases,MAPKs) signaling pathway during monocyte differentiation into osteoclasts. In order to understand the molecular biological mechanism of icariin inhibiting osteoclast differentiation, it provides a theoretical basis for the further application of icariin in clinical practice. Methods the study was divided into two parts. Study on the dose-effect relationship of icariin in inhibiting osteoclast differentiation: RAW264.7 cells were divided into blank control group (equal medium control) and model control group (50ng/ml RANKL),) according to different concentrations of ICA. The number of osteoclasts was observed by 10-7/10-6/10-5/10-4mol/L ICA 50ng/ml RANKL). TRAP staining in four concentration gradient groups of ICA. At the same time, the expression and phosphorylation of key proteins were detected by western blot. The effect of icariin on osteoclast differentiation: the control group and icariin treated group (ICA concentration was 10-5mol/L) were used to observe the number of osteoclasts by TRAP staining. The expression and phosphorylation of key proteins were detected by western blot. Results Mononuclear cells containing more than 3 nuclei were found in a RAW264.7 induction culture for 48 hours. Trap staining showed that with the increase of ICA concentration, the number of osteoclasts decreased, the morphology of osteoclasts was small, and the number of nuclei was less. Compared with the control group, the phosphorylation level of p38 JNKK ERK in the model control group was higher than that in the control group, but there was no significant difference in the expression level of P38 + JNK-ERK between the model control group and the control group. Compared with the model control group without ICA, the phosphorylation level of P38 JNKERK decreased with the increase of ICA concentration, and the inhibitory effect was concentration-dependent, but there was no significant difference in the expression level of P38 JNKERK. When the concentration of ICA reached 10-5mol/L, the inhibitory effect on osteoclast differentiation of monocyte reached the best. TRAP positive osteoclasts were found in the solvent control group 12 hours after induction, and the number of osteoclasts increased with the increase of induction time. Compared with the solvent control group for the same induction time, the number of osteoclasts in ICA group was very small and the morphology of osteoclasts was smaller. The phosphorylation level of P38 JNK-ERK was different between the solvent control group and the ICA treatment group, but there was no significant difference in the expression level of P38 JNK-ERK between the same induction time group and the ICA treatment group. The phosphorylation level of P38 JNK-ERK in the solvent control group increased gradually with the increase of induction time, but there was no significant difference in the expression of P38 JNK-ERK. There was no significant difference in phosphorylation level and protein expression level of ERK. Conclusion Icariin has a stable inhibitory effect on osteoclast differentiation of monocytes, and the inhibitory effect is enhanced with the increase of icariin concentration. Icariin had no effect on the expression of p38 ERK and JNK in the MAPK signaling pathway during osteoclast differentiation, but could significantly inhibit its phosphorylation level, and the inhibitory effect was positively correlated with the concentration. The best concentration of Icariin in inhibiting osteoclast differentiation in vitro was 10-5 mol / L 路L ~ (-1) 路L ~ (-1). The inhibitory effect of icariin on osteoclast differentiation has reached a stable level since 12 hours.
【學(xué)位授予單位】：首都醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R783.5

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