【作者】 王嘉；

【导师】 李锋；

【作者基本信息】 华中科技大学 ， 外科学（骨外）， 2021， 博士

【摘要】 目的:破骨细胞是机体唯一能够吸收骨质的细胞,其生成过多或者骨吸收能力过度增强,被认为是骨质疏松症发生的重要机制。在破骨细胞分化过程中,细胞的糖酵解活性显著提高,并且NF-κB和MAPK信号通路被激活。PFKFB3是一种能够调节细胞糖酵解活性的关键酶。据报道,抑制PFKFB3的表达可以显著降低细胞的糖酵解活性并阻碍NF-κB和MAPK信号通路的激活,因此我们推测其可能参与了破骨细胞的分化调控。本课题旨在探究PFKFB3在破骨细胞分化过程中的作用及潜在分子机制,为骨质疏松症和其它破骨细胞过度激活导致的疾病提供新的干预靶点和治疗思路。方法:1.使用细胞因子RANKL诱导BMMs细胞向破骨细胞定向分化,通过免疫印迹检测PFKFB3在破骨细胞分化过程中的蛋白表达变化情况,通过葡萄糖测定试剂盒和乳酸测定试剂盒检测破骨细胞分化过程中糖酵解活性的变化情况。2.在破骨细胞分化过程中,使用针对PFKFB3的sh RNA腺病毒或者PFKFB3的抑制剂PFK15降低PFKFB3在BMMs细胞中的表达,TRAP染色检测破骨细胞分化情况,F-Actin染色和骨板吸收实验检测破骨细胞执行骨吸收功能的能力,葡萄糖测定试剂盒和乳酸测定试剂盒检测破骨细胞的糖酵解活性,q PCR及免疫印迹检测破骨细胞标志基因的表达情况和NF-κB、MAPK信号通路的激活状况。3.建立卵巢切除小鼠模型,该模型是目前为止模拟绝经后骨质疏松症最常见、最可靠的动物模型。模型建立后,使用PFK15对小鼠进行为期6周的腹腔注射,然后收集小鼠股骨标本,micro-CT扫描检测小鼠骨量变化,骨组织切片染色检测破骨细胞分化情况。4.使用乳酸对PFK15导致的破骨细胞分化受限进行挽救,TRAP染色检测破骨细胞分化情况,免疫印迹检测破骨细胞分化相关标志基因和NF-κB、MAPK信号通路中重要蛋白的表达水平。结果:1.在破骨细胞分化过程中,PFKFB3的蛋白表达水平升高,糖酵解的活性上调,表明PFKFB3可能参与了破骨细胞的分化调控。2.针对PFKFB3的sh RNA腺病毒或者PFKFB3的抑制剂PFK15都可以明显阻碍破骨细胞的分化,减弱破骨细胞执行骨吸收功能的能力并下调破骨细胞标志基因的表达水平,证明PFKFB3对破骨细胞分化以及执行骨吸收功能至关重要。3.PFKFB3的抑制剂PFK15可以部分缓解卵巢切除小鼠的骨量丢失,并在该模型中减少破骨细胞的生成,表明PFKFB3在小鼠体内依然具有调节破骨细胞分化的潜力。4.抑制PFKFB3的表达可以降低NF-κB和MAPK信号通路中重要蛋白分子的磷酸化水平,说明PFKFB3的抑制对破骨细胞分化的阻碍效应可能是NF-κB和MAPK信号通路激活受限所导致的。5.在破骨细胞分化过程中,抑制PFKFB3的表达可以明显减少破骨细胞的葡萄糖消耗和乳酸生成,降低其糖酵解活性。补充乳酸则可以部分挽救PFKFB3抑制导致的破骨细胞分化受限以及NF-κB、MAPK信号通路激活障碍,表明糖酵解参与了PFKFB3所介导的破骨细胞分化调控。结论:在细胞水平上,抑制PFKFB3的表达能够阻碍破骨细胞的分化并减弱破骨细胞的骨吸收功能,糖酵解参与了PFKFB3所介导的破骨细胞分化调控。在动物水平上,PFKFB3的抑制剂PFK15可以部分缓解小鼠卵巢切除导致的骨量丢失。本研究明确了PFKFB3在破骨细胞分化和执行骨吸收功能中的重要作用,为骨质疏松症和其它破骨细胞过度激活导致的疾病提供新的干预靶点和治疗思路。更多还原

【Abstract】 Objective:Osteoclast is the only cell type responsible for bone resorption in human body.An excessive number or overactive function of osteoclasts is considered to be the major pathological mechanism of osteoporosis.The differentiation of osteoclasts is dependent on the collaborative effects of two essential cytokines,M-CSF and RANKL.Stimulation by RANKL during osteoclastogenesis leads to the activation of NF-κB and MAPK signaling cascades and induces a metabolic shift to elevated glycolytic metabolism.PFKFB3 is an important enzyme with the ability to regulate glycolysis.Suppression of PFKFB3 can reduce the cellular glycolytic activity and inhibit the activation of NF-κB and MAPK pathways.We thus speculated that PFKFB3 might participate in the regulation of osteoclastogenesis.The aim of this study is to investigate the role and the underlying mechanism of PFKFB3 during osteoclast differentiation and provide novel targets and potential therapeutic strategies for osteoporosis and other osteoclast-related disorders.Methods:1.During RANKL-induced osteoclast differentiation from BMMs,the protein levels of PFKFB3 were detected by western blotting,and the glycolytic activity in BMMs was measured using the glucose assay kit and the lactate assay kit.2.The adenovirus carrying PFKFB3 sh RNA or PFKFB3 inhibitor PFK15 was used respectively to reduce the expression of PFKFB3 in BMMs,followed by osteoclast differentiation using RANKL stimulation.TRAP staining was performed to evaluate the differentiation of osteoclasts,F-Actin staining and pit formation assay were conducted to assess the function of osteoclasts.Meanwhile,the expression of osteoclast marker genes and the functional status of NF-κB and MAPK signaling were detected by q PCR and western blotting.3.A post-menopausal osteoporosis murine model was established using ovariectomy surgery.The development of osteoporosis in this model was attributed to the lack of estrogen and the excessive activation of osteoclasts.After 6 weeks of intervention by PFK15,the femurs of the mice were collected for micro-CT scanning and histomorphometric analysis.4.Lactate was used to rescue the inhibitory effects of PFK15 on osteoclastogensis.Osteoclast differentiation was evaluated by TRAP staining.The expression of osteoclast marker genes and the functional status of NF-κB and MAPK signaling were detected by q PCR and western blotting.Results:1.During RANKL-induced osteoclastogenesis,both the protein expression of PFKFB3 and the glycolytic activity represented by glucose consumption and lactate production increased,indicating that PFKFB3 might participate in the regulation of osteoclast differentiation.2.Either the adenovirus carrying PFKFB3 sh RNA or PFKFB3 inhibitor PFK15 significantly inhibited osteoclast differentiation,impaired bone resorption function of osteoclasts and reduced the expression of osteoclast marker genes,suggesting that PFKFB3 is a prerequisite for osteoclast differentiation and function.3.PFKFB3 inhibitor PFK15 attenuated ovariectomy-induced bone loss and reduced osteoclast formation in the ovariectomized murine model,indicating that PFKFB3 can also generate its effects on osteoclastogenesis in vivo.4.Inhibition of PFKFB3 repressed the activation of NF-κB and MAPK signaling,suggesting that the inhibitory effects on osteoclastogenesis caused by PFKFB3 inhibition might rely on the suppression of NF-κB and MAPK pathways.5.Inhibition of PFKFB3 reduced the glycolytic activity represented by glucose consumption and lactate production.Administration of lactate partially reversed the repression of osteoclastogenesis caused by PFKFB3 inhibition and abrogated the inhibitory effects of PFK15 on the activation of MAPK and NF-κB pathways.These results revealed that glycolysis participated in PFKFB3-mediated regulation of osteoclastogenesis.Conclusion:In conclusion,our results demonstrated that inhibition of PFKFB3 suppressed osteoclast differentiation and impaired osteoclastic bone resorption in vitro,and prevented ovariectomy-induced bone loss in vivo.All in all,the results of this study clarified the essential role of PFKFB3 during RANKL-induced osteoclastogenesis and indicated that blockage of glycolysis by targeting PFKFB3 represented a potential therapeutic strategy for osteoporosis and other osteoclast-related disorders.更多还原