【作者】 赵良功；

【导师】 夏亚一；

【作者基本信息】 兰州大学 ， 骨外， 2015， 博士

【摘要】 目的：随着老龄化社会的到来,骨质疏松成为世界广泛关注的健康问题之一,是绝经后女性和老年人的常见病。针对骨质疏松合理的治疗方案包含药物治疗与运动治疗相结合。但由于应力在成骨过程中作用机制不甚明确,临床医生很难指导患者进行正确地运动治疗。同时,现有药物在治疗骨质疏松的同时还存在增加乳腺癌和子宫内膜癌等患病率的副作用。探索并发现促成骨效率高而副作用小或无副作用的骨质疏松治疗药物非常重要。本论文通过分别研究MAPK家族成员ERK5在流体剪切力促进成骨细胞分化及p38 MAPK在红芪多糖促进成骨过程中不同的调控作用,为临床正确指导患者进行合理运动及成骨减少性疾病治疗药物的开发提供了理论依据。方法：第一部分：对MC3T3-E1成骨细胞加载间歇流体剪切力或间歇性流体剪切力,应用MEK5/ERK5特异性阻断剂BIX02189抑制ERK5的活性,研究比较两种流体剪切力对成骨细胞分化促进作用的强弱以及ERK5在间歇性流体剪切力促进成骨细胞分化过程中的调控作用；第二部分：水提醇沉淀法从中药材红芪中分离得到不同分子量范围的红芪多糖(HPS),进一步纯化得到HPS3d。通过对成骨细胞给予不同浓度HPS3d刺激72小时,并且应用p38 MAPK特异性阻断剂SB203580抑制p38的活性,通过MTT、RT-PCR和Westernblot等方法研究HPS3d促进成骨细胞分化和P38 MAPK对上述过程的调控作用。结果：第一部分：(1)同对照组相比,持续性与间歇性流体剪切力均可以促进ERK5的磷酸化,增强ALP的活性,促进OCN及OPN的蛋白表达。进一步比较发现,加载间歇性流体剪切力的成骨细胞中,ERK5磷酸化水平、ALP的活性OCN、 OPN的蛋白表达量均明显高于加载持续性流体剪切力的成骨细胞。(2)加载间歇性流体剪切力后,成骨细胞中ERK5的磷酸化水平较正常组明显升高,而应用10μM的MEK5/ERK5阻断剂BIX02189孵育2小时后,MEK5/ERK5的活性完全受到抑制。同对照组相比,间歇性流体剪切力可以增强成骨细胞中ALP的活性,增加OPN、OCN和Runx-2的表达。但抑制MEK5/ERK5的活性后,这种促进作用也相应的受到减弱。同时,单独应用BIX02189对ALP的活性,OPN, OCN和Runx-2的表达无明显影响(p>0.05)。第二部分：(1)通过对红芪药材进行提取、分离和纯化,从中获得了组成均一的红芪多糖HPS3d。经过分析发现,HPS3d中多糖含量为94.35%,蛋白质含量为3.4%,糖醛酸含量为13.3%,HPS3d为酸性糖蛋白复合物。元素分析结果O为53.91%,C为38.18%,H为5.66%,S为1.73%,N为0.52%。HPS3d的分子量范围为84.6kDa,空间分子构象为高枝化度结构。结构中含有鼠李糖(5.93%)、阿拉伯糖(39.95%)、葡萄糖(6.76%)、半乳糖(39.95%)及半乳糖醛酸(8.05%)。HPS3d的支链主要由阿拉伯糖构成,而主链由半乳糖与半乳糖醛酸构成。(2)成骨细胞分别经过浓度为0.5mg/ml、1.0mg/ml、2.0mg/ml、4.0mg/ml、 8.0mg/ml、16.0mg/ml及32mg/ml的HPS3d刺激72h后,用MTT法检测细胞活性。结果可见,当HPS3d浓度为0.5-8.0mg/ml时,细胞活性同对照组相比无显著性差异(P>0.05)。(3)成骨细胞分别经过浓度为0.5mg/ml、1.0mg/ml、2.0mg/ml、4.0mg/ml和8.0mg/ml的HPS3d刺激72h后发现,当HPS3d浓度为0.5mg/ml时,ALP活性,OPN、OCN和BSP的]mnRNA表达未受到明显影响。当HPS3d浓度为1.0mg/ml、 2.0mg/ml和4.0mg/ml时,ALP活性,OPN、OCN和BSP的mRNA表达呈浓度依赖性升高。并且当HPS3d浓度为8.0mg/ml时,ALP活性,OPN、OCN和BSP的mRNA表达同4.0mg/ml组比较不再继续升高(P>0.05)。(4)用浓度为0.5-8.0mg/ml的HPS3d刺激成骨细胞胞72h后,Runx-2和Osterix的mRNA表达量自HPS3d浓度为1.0mg/ml时开始呈剂量依赖性升高,并在4.0mg/ml时达到最高,同对照组相比,分别升高了5.2倍和6.6倍。进一步用浓度为2.0mg/ml和4.0mg/mlHPS3d刺激成骨细胞72小时后,Western blot检测Runx-2和Osterix蛋白表达量均明显升高。(5)用浓度为0.5-8.0mg/ml的HPS3d刺激成骨细胞胞72h后,BMP-2的mRNA表达量自HPS3d浓度为1.0mg/ml时开始呈剂量依赖性升高,并在4.0mg/ml时达到最高。进一步用浓度为2.0mg/mI和4.0mg/mIHPS3d刺激成骨细胞72小时后,Western blot检测发现SMAD1/5/8的磷酸化水平较对照组升高。(6)用p38特异性阻断剂SB203580预处理成骨细胞后再经过4.0mg/ml HPS3d刺激72小时后,同对照组相比,经HPS3d单独刺激的成骨细胞中p38MAPK的磷酸化水平升高。p38 MAPK的特异性阻断剂可以抑制上述HPS3d介导的p38 MAPK磷酸化。而HPS3d促进的ALP活性增强、OCN、OPN、BSP、 Runx-2和Osterix的mRNA表达升高在用SB203580后均受到不同程度的抑制。结论1、强度为12dyn/cm2的持续性与间歇性流体剪切力均可以促进MC3T3-E1成骨细胞ERK5的磷酸化,增强ALP的活性,促进OCN及OPN的蛋白表达。进而促进成骨细胞分化。同持续性流体剪切相比,间歇性流体剪切力是一种更为有效的成骨细胞分化促进应力形式。2、抑制MEK5/ERK5的活性后,间歇性流体剪切力促进的成骨细胞中ALP的活性与OPN、OCN表达增加均受到抑制。MEK5/ERK5对流体剪切力促进成骨细胞分化具有调控作用。而成骨细胞分化关键性调控因子Runx-2的表达同时降低,说明除了ERKl/2与p38外,Runx-2的表达还受到MEK5/ERK5的调控。3、最终获得的红芪多糖的主要部分HPS3d为白色絮状物,极易溶于水,为酸性糖蛋白复合物。元素含量O为53.91%,C为38.18%,H为5.66%,S为1.73%,N为0.52%。分子量范围为84.6kDa,分子结构为高枝化度；支链主要由阿拉伯糖构成,而主链由半乳糖与半乳糖醛酸构成。4、适当浓度的HPS3d可以促进ALP活性,使OPN、OCN和BSP的mRNA表达并且呈浓度依赖性升高,进而促进成骨细胞分化；同时HPS3d使BMP-2、SMAD1/5/8、Runx-2和Osterix蛋白和mRNA的表达均升高。说明在HPS3d促进成骨细胞分化的过程中BMP-2/SMAD/Runx-2/Osterix信号通路被激活。同时中低浓度HPS3d对细胞无明显毒性作用。5、HPS3d还可以激活p38 MAPK,在p38的活性受到特异性阻断剂的抑制后,成骨细胞分化相关的多种蛋白表达及Runx-2和Osteri的表达均明显受到抑制,说明p38 MAPK通过Runx-2/Osterix发挥调控HPS3d促进成骨细胞分化的作用。更多还原

【Abstract】 Objective:Along with the aging society developing, Osteoporosis(OP) becomes one of health issues which attract extensive attention worldwide. The rational treatment of OP should include both drug and exercise. Because of the mechanism of mechanotransduction in osteoblasts is still unclear. It is difficulty for the clinicians to guide patients exercise with the correct way. Meanwhile, conventional drug therapies have both pros and cons. For example, long-term use of estrogen therapy increases the possibility of breast and endometrial cancers. It is very important to find and develop new agents which have high recovery efficiency of bone mass without side effects. In current study, we explored the role of the two members of MAPK family, ERK5 in fluid shear stress(FSS) induced osteoblast differentiation and p38 MAPK in Radix Hedysari polysaccharides (HPS) stimulating osteoblast differentiation which would not only be helpful for development of bone tissue engineering but also provide new drug discovery targets for the treatment of OP.Methods:Part Ⅰ:MC3T3-E1 osteoblastic cells were subjected to intermittent FSS and steady FSS respectively. The MEK5/ERK5 pathway was selectively inhibited by BIX02189. Then investigating which shear pattern was more effective for promoting osteoblast differentiation and The MEK5/ERK5 pathway mediates osteoblast differentiation promoted by intermittent FSS;PartⅡ:The crude polysaccharide HPS was isolated from the dried roots of Hedysarum polybotrys by hot-water extract in 45% yield. Then the concentrated supernatant was precipitated with 95% ethanol to obtain three crude polysaccharides accordingly. The purified HPS3d was obtained by anion-exchange column from Radix Hedysari and identified the structural characteristics of HPS3d. The osteoblasts was treated with different concentrations of HPS3d at 72 h of culture and the p38 MAPK was selectively inhibited by SB203580, p38 MAPK regulated HPS3d stimulated osteoblast differentiation was investigated by MTT, RT-PCR and Western blot.Results:PartⅠ:(1) Both steady FSS and intermittent FSS could up-regulate ERK5 phosphorylation, ALP activity and the expression of OPN and OCN compared with the control. Furthermore, the levels of ERK5 phosphorylation, ALP activity and the expression of OPN and OCN were higher in cells treated with intermittent FSS than those in cells treated with steady FSS.(2) Phosphorylation of ERK5 was markedly up-regulated after intermittent FSS in contrast to the unloaded control and was nearly completely blocked after incubation with 10 mM BIX02189 for 2 h. Intermittent FSS up-regulated ALP activity and the expression of OPN, OCN and Runx-2 compared with the control. When osteoblasts were incubated with BIX02189 before exposure to intermittent FSS, these effects were all reduced. However, the ALP activity and expression of OPN, OCN and Runx-2 in unloaded cells were not affected by BIX02189 (p>0.05).Part II:(1) In the present study, the purified HPS3d was obtained by anion-exchange column. It consisted of 94.38% polysaccharide,3.40% protein and 13.30% uronic acid. The molecular weight was measured to be 84.6 kDa. The backbone consisted of galactopyranose and galacturonopyranose, and the side chains were composed of glucopyranose, rhamnopyranose and arabinofuranose. The result of elemental analysis of HPS3d revealed that there were mainly C (38.18%, in mass), H (5.66%), O (53.91%), S (1.73%). The backbone structure of HPS3d mainly contained galactose and galacturonic acid and branch chains mainly contained arabinose.(2) The osteoblasts was treated with different concentrations of HPS3d (control,0.5 to 32 mg/ml) at 72 h of culture. The results showed the viability of osteoblasts was not affected by HPS3d (0.5 to 8.0 mg/ml). Interestingly, higher concentrations(16 and 32 mg/ml) resulted in a decrease in cell viability.(3) The osteoblasts was treated with different concentrations of HPS3d (control,0.5 to 8 mg/ml) at 72 h of culture. The ALP activity and the mRNA expression of OPN, OCN and BSP were not affected when the concentration was 0.5 mg/ml. HPS3d (1.0 to 4.0 mg/ml) concentration-dependently enhanced the ALP activity and the mRNA expression of OPN, OCN and BSP and not continuously enhance when the concentration was8.0 mg/ml (P> 0.05).(4) A significant increase in Runx-2 and Osterix mRNA expression were observed starting at 1.0 mg/ml and reached 5.2 and 6.6-times increase, respectively, at 4.0 mg/ml when compared with control after 72h incubation by HPS3d. The Western blot analysis also showed that the protein expression of Runx-2 and Osterix were significantly unregulated by HPS3d at 2.0 and 4.0 mg/ml.(5) The osteoblasts was treated with different concentrations of HPS3d (control,0.5 to 8 mg/ml) at 72h of culture. The mRNA expression of BMP2 were concentration-dependently enhanced by HPS3d(1.0 to 4.0 mg/ml). The Western blot showed that the phosphorylation level of SMAD1/5/8 were significantly unregulated by HPS3d at 2.0 and 4.0 mg/ml compared with control.(6) The osteoblasts was treated with HPS3d at 72h of culture. Compared with control group, HPS3d could up-regulate the p38 MAPK phosphorylation level and was down-regulated with p38 MAPK selective inhibitor SB203580. Further study showed that the HPS3d induced ALP activity and the mRNA expression of OPN, OCN and BSP were also down-regulated while SB203580 presented.Conclusion:(1) Both 12dyn/cm2 steady FSS and intermittent FSS could stimulate osteoblasts differentiation compared with the control. Furthermore, intermittent FSS is a more intense stimulus than steady FSS for osteoblast differentiation.(2) Besides ERK1/2 and p38, the MEK5/ERK5 pathway also mediates FSS promotes osteoblast differentiation. The MEK5/ERK5 pathway regulates FSS-induced Runx-2 expression, which initiates the transcription of osteogenic genes to promote osteoblast differentiation.(3) The purified HPS3d was obtained by anion-exchangecolumn. HPS3d consisted of 94.38% polysaccharide,3.40% proteinand 13.30% uronic acid. The molecular weight of HPS3d was 84.6 kDa. The backbone of HPS3d mainly consistedof galactopyranose and galacturonopyranose, and that the sidechains were composed of glucopyranose, rhamnopyranose andarabinofuranose.(4) HPS3d up-regulated ALP activity and the expressionof other osteogenic marker genes and stimulated osteoblast differentiation. In addition, HPS3d increased the expression and phosphorylation level of Runx-2, Osterix, BMP-2 and SMAD1/5/8 respectively. These findings suggest that HPS3d stimulates osteoblast differentiation by activation of BMP-2/SMAD/Runx-2/Osteri pathway.(5) HPS3d could activate p38 MAPK. And when p38 MAPK was selectively inhibited, the HPS3d induced osteoblast differentiation was suppressed. Indicated that p38 MAPK regulated the osteoblast differentiation which was induced by HPS3d.更多还原