【作者】 罗婷婷；

【导师】 尚政军；

【作者基本信息】 武汉大学 ， 口腔颌面外科学， 2021， 博士

【摘要】 骨肉瘤（OS）是最常见的原发性恶性骨肿瘤,其特征是高侵袭性、进行性骨破坏和极易发生远处转移。骨肉瘤好发于儿童和青少年,其中约5%的骨肉瘤发生于颌骨。以往对于骨肉瘤的研究主要集中于肿瘤细胞,而忽视了微环境中破骨细胞等非肿瘤细胞在肿瘤演进中的作用。近年来研究表明,在骨肉瘤溶骨性破坏中破骨细胞数量较其在正常骨组织明显增多,抑制破骨细胞分化可明显减少骨肉瘤造成的骨破坏,但是骨肉瘤中破骨细胞分化和功能的调控机制仍不明确。因此,探索骨肉瘤微环境中破骨细胞分化机制对于开发靶向肿瘤微环境中破骨细胞的新药物具有重要意义。小细胞外囊泡（s EVs）可以通过传递生物活性物质至受体细胞介导细胞间通讯,参与细胞间信息交流并调节受体细胞状态及功能。研究表明,s EVs是介导恶性肿瘤微环境中间质细胞重编程,进而促进肿瘤进展的重要分子介质。目前,对于s EVs调控破骨细胞的研究较少,其机制也不清楚。本研究旨在探讨骨肉瘤细胞源性s EVs对破骨细胞分化及功能的影响、s EVs中mi RNA介导破骨细胞分化和功能激活的信号机制、萝卜硫素（SFN）对骨肉瘤细胞源性小细胞外囊泡及破骨细胞分化的抑制作用。本文将从以下三个部分进行论述。第一部分:骨肉瘤源性s EVs在破骨细胞分化中的作用研究目的:探讨骨肉瘤细胞来源的s EVs在破骨细胞分化过程中的作用。方法:采用差速超速离心法提取骨肉瘤细胞s EVs,并采用透射电镜（TEM）、动态光散射（DLS）、Western Blot进行鉴定。用s EVs处理RAW264.7细胞,在共聚焦荧光显微镜下观察RAW264.7细胞对s EVs的摄取,并通过CCK-8、Transwell实验检测s EVs对RAW264.7细胞增殖、迁移的的影响;核因子κB受体活化因子配体（RANKL）体外诱导破骨细胞分化后,TRAP染色、罗丹明标记鬼笔环肽染色、破骨细胞功能实验检测破骨细胞的分化及骨吸收功能;q RT-PCR和Western Blot检测破骨细胞功能相关指标组织蛋白酶K（CTSK）、基质金属蛋白酶9（MMP9）表达。结果:TEM、DLS和Western Blot检测结果显示提取的s EVs符合小细胞外囊泡的特征;共聚焦荧光显微镜结果显示RAW264.7细胞能够摄取骨肉瘤细胞来源的s EVs;CCK-8、Transwell实验显示s EVs刺激后的RAW264.7细胞增殖、迁移能力均增强;TRAP染色、罗丹明标记鬼笔环肽染色、破骨细胞功能实验结果显s EVs刺激后破骨细胞的数量增加和功能增强;q RT-PCR和Western Blot结果显示s EVs刺激后CTSK、MMP9的表达升高。结论:骨肉瘤细胞通过分泌并传递s EVs促进破骨细胞的分化及功能。第二部分:骨肉瘤源性s EVs传递mi R-19a-3p促进破骨细胞分化及骨破坏目的:探讨骨肉瘤细胞分泌的s EVs促进破骨细胞分化及骨破坏的机制。方法:通过生物信息学分析得出可能参与骨肉瘤中破骨细胞分化和功能增强的相关基因及靶向mi RNAs。双荧光素酶报告基因实验验证mi R-19a-3p与PTEN的靶向关系。mi RNA oligo（mi R-19a-3p mimics-NC、mimics、inhibitor-NC、inhibitor）转染RAW264.7细胞后,RANKL诱导并进行TRAP染色、罗丹明标记鬼笔环肽染色、破骨细胞功能实验;q RT-PCR和Western Blot检测CTSK、MMP9、PTEN的表达;同时利用Western Blot检测AKT信号通路的激活情况。mi RNA oligo转染骨肉瘤细胞后,提取其s EVs(s EVs ^oligo),刺激培养的RAW264.7细胞,经RANKL诱导后,检测破骨细胞分化、功能及相关分子的表达,采用骨髓单核巨噬细胞（BMMs）重复上述实验进行验证;小鼠皮下注射K7M2细胞建立骨肉瘤模型,检测小鼠血液中s EVs内mi R-19a-3p的含量,micro-CT扫描分析并股骨的骨参数,苏木精-伊红（HE）染色观察并分析骨组织形态,TRAP染色检测骨组织内破骨细胞分布情况。结果:生物信息学分析显示骨肉瘤中破骨细胞分化和功能的增强可能与mi R-19a-3p和PTEN有关。双荧光素酶报告基因实验结果显示,mi R-19a-3p可以靶向结合PTEN。RAW264.7细胞转染mi RNA oligo后进行诱导,结果显示mi R-19a-3p mimics组破骨细胞分化数目增加,骨吸收功能增强,PTEN表达降低,磷酸化AKT表达增加,而mi R-19a-3p inhibitor组呈现相反的结果;此外s EVs ^oligo刺激RAW264.7细胞并进行诱导,结果显示s EVs ^mimics组破骨细胞分化数目增加,骨吸收功能增强,PTEN表达降低,p-AKT表达增加,而s EVs ^inhibitor组结果呈现相反趋势;采用BMMs体外诱导破骨细胞分化的结果与RAW264.7细胞模型一致;骨肉瘤小鼠血液中s EVs内mi R-19a-3p的含量明显高于正常小鼠,micro-CT和HE染色结果显示骨肉瘤小鼠股骨骨量减少,TRAP染色结果显示骨肉瘤小鼠股骨内破骨细胞减少。结论:骨肉瘤细胞可通过分泌s EVs传递mi R-19a-3p,靶向降解PTEN,激活AKT通路,促进破骨细胞的分化及功能。第三部分:SEVs部分参与了SFN对骨肉瘤中破骨细胞分化的调控目的:探讨SFN对骨肉瘤中破骨细胞分化及骨吸收的影响,以及可能的机制。方法:骨肉瘤细胞经SFN刺激后,提取其分泌的s EVs,刺激RANKL诱导的RAW264.7细胞,检测破骨细胞分化、功能水平及相关分子的表达。CCK8检测SFN对RAW264.7细胞和BMMs增殖的影响。RANKL诱导RAW264.7细胞和BMMs形成破骨细胞,建立破骨细胞体外诱导模型。TRAP染色、罗丹明标记鬼笔环肽染色检测破骨细胞的数量及大小。TEM观察细胞内自噬小体。用不同浓度的SFN处理破骨细胞前体细胞,并诱导其形成破骨细胞,探索SFN作用的合适浓度。检测不同时间点SFN刺激对破骨细胞分化的影响,探索SFN发挥作用的阶段。用雷帕霉素（RAP）激活自噬通路后,检测SFN对破骨细胞分化的影响。利用q RT-PCR和Western blot检测CTSK、MMP9、自噬相关分子Atg5-Atg12、Beclin1、LC3的表达。利用Western blot检测JNK通路的激活情况。JNK通路的激活剂茴香霉素（Anisomycin）、抑制剂SP600125预处理RAW264.7细胞后加入RANKL进行诱导,Western Blot检测Atg5-Atg12、Beclin1、LC3的表达。小鼠颅骨中部皮下注射LPS建立骨破坏模型,SFN腹腔注射给药。Micro-CT、HE染色检测小鼠颅骨骨组织形态及骨破坏情况。TRAP染色检测骨组织内破骨细胞的分布。免疫组化、免疫荧光染色检测骨组织内CTSK、LC3的分布情况。结果:QRT-PCR显示SFN刺激骨肉瘤细胞后,提取的s EVs中mi R-19a-3p减少,用SFN-s EVs刺激RAW264.7细胞并进行诱导,破骨细胞分化明显减少。此外,SFN可以直接抑制破骨细胞的分化,且呈剂量依赖性。SFN浓度为2.5μM时,可以有效抑制破骨细胞分化且对细胞的增殖影响较小。在破骨细胞诱导早期加入SFN时,可以明显抑制其形成。用RAP激活自噬通路后,SFN对破骨细胞分化的抑制作用减弱。QRT-PCR和Western blot结果显示SFN抑制破骨细胞内CTSK、MMP9、Atg5-Atg12、Beclin1、LC3的表达,而使用RAP处理细胞后,SFN抑制效果降低。Western blot结果显示SFN抑制破骨细胞内磷酸化JNK（p-JNK）的表达,且呈剂量依赖性;Anisomycin激活JNK信号后,自噬相关蛋白表达增加;而SP600125阻断JNK信号后,自噬相关蛋白表达降低。Micro-CT、HE染色结果显示,皮下注射LPS后小鼠颅骨骨破坏显著,腹腔注射SFN可以明显减少骨破坏。TRAP染色结果显示SFN减弱了LPS诱导的破骨细胞数量增加的作用。免疫组化、免疫荧光染色结果显示LPS组小鼠颅骨骨组织内CTSK、LC3表达增加,同时腹腔注射SFN后其表达减少。结论:SFN可以抑制骨肉瘤细胞分泌的s EVs中mi R-19a-3p的表达,间接抑制破骨细胞的分化;此外,SFN可以阻断JNK信号,抑制自噬通路,直接抑制破骨细胞的分化及骨吸收功能。综上所述,我们的研究表明:1.骨肉瘤细胞分泌s EVs,可以促进破骨细胞的分化及功能。2.骨肉瘤细胞通过s EVs传递mi R-19a-3p至破骨细胞前体细胞,靶向降解磷酸酶张力同源物（PTEN）,进而激活AKT信号促进破骨细胞的分化及骨吸收功能。此外体内实验验证了骨肉瘤小鼠血液中s EVs内mi R-19a-3p含量增高,而股骨骨量减少,破骨细胞数量增加。3.SFN可以抑制骨肉瘤细胞来源s EVs中mi R-19a-3p的含量,间接抑制破骨细胞的分化及功能;此外,SFN能抑制自噬通路,进而直接抑制破骨细胞分化。因此,我们认为s EVs参与了骨肉瘤中破骨细胞的分化,s EVs中mi R-19a-3p或将成为预防和治疗骨肉瘤骨破坏及肿瘤进展的新靶标。更多还原

【Abstract】 Osteosarcoma（OS）is the most common primary bone cancer characterized by highly aggressive,progressive bone destruction,and high incidence of distant metastasis.OS most often occur in children and adolescents,with about 5%of osteosarcomas occurring in the jaw.Previous studies on OS mainly focused on tumor cells,while ignoring the effect of non-tumor cells such as osteoclasts in the microenvironment on tumor progression.Recent studies have shown that the number of osteoclasts is significantly higher in osteolytic destruction of OS than normal bone tissue,and inhibition of osteoclastogenesis can significantly reduce the bone destruction caused by OS.However,mechanisms governing osteoclastogenesis in OS are still unclear.Therefore,it is of great significance to explore the mechanism of osteoclast formation in OS microenvironment for the development of new drugs targeting osteoclasts in the tumor microenvironment.Small extracellular vesicles（s EVs）could mediate cellular communications by delivering bioactive molecules to recipient cells,participating in cellular information exchange and regulating the status and function of recipient cells.Studies have shown that s EVs are important molecular mediators that mediate the reprogramming of mesenchymal cells in the microenvironment of malignant tumors and thus promote tumor progression.At present,there are few studies on the regulation of s EVs on osteoclasts,and the mechanism is not clear.The aim of this study was to investigate the effect of OS cell-derived s EVs on osteoclast formation and function,the mechanism underlying the regulation of osteoclast formation and function by mi RNA delivered by OS cells’s EVs,and the inhibitory effect of sulforaphane（SFN）on the level of mi RNA in OS cell-derived s EVs and osteoclast formation.In this paper,we will discuss from the following three parts.Part 1.Study on the role of Osteosarcoma-derived s EVs in osteoclastogenesisObjective:To investigate the role of s EVs secreted by OS cells in osteoclastogenesis and bone destruction.Methods:SEVs were extracted from OS cells by differential ultracentrifugation and identified by transmission electron microscopy（TEM）,dynamic light scattering（DLS）and Western Blot.RAW264.7 cells were stimulated by s EVs,and the uptake of s EVs by RAW264.7 cells was observed by confocal microscopy.The effects of s EVs on the proliferation and migration of RAW264.7 cells were detected by CCK-8 and Transwell assay.For the osteoclastogenesis assay,RAW264.7 cells were induced by nuclear factor kappa B receptor activating factor ligand（RANKL）in vitro.Osteoclastogenesis and bone resorption were detected by TRAP staining,TRITC-phalloidin staining,and resorption pit assay.QRT-PCR and Western Blot were used to detect the expression of cathepsin K（CTSK）and matrix metalloproteinase-9（MMP9）.Results:The results of TEM,DLS and Western Blot showed that the extracted s EVs were consistent with the characteristics of small extracellular vesicles.Confocal microscopy results showed that s EVs were absorbed by RAW264.7 cells.CCK-8 and Transwell assay showed that s EVs promoted the proliferation and migration of RAW264.7 cells.TRAP staining,TRITC-phalloidin staining,and resorption pit assay revealed that osteoclast formation and function were enhanced with the treatment of s EVs.QRT-PCR and Western Blot results showed that the treatment of s EVs increased the expression of CTSK and MMP9.Conclusion:s EVs secreted by OS cells could promote the formation and function of osteoclast.Part 2.Osteosarcoma-derived s EVs promote osteoclast differentiation and bone destruction through the delivery of mi R-19a-3pObjective:To investigate the mechanism of s EVs derived from OS cells promoting osteoclastogenesis and bone destruction.Methods:Bioinformatics analysis was applied to predict the related genes and targeted mi RNAs involved in osteoclastogenesis in OS.The dual luciferase reporter gene assay was used to verify the target binding of mi R-19a-3p with PTEN.RAW264.7 cells were transfected with mi RNA Oligo（mi R-19a-3p mimics-NC,mimics,inhibitor-NC,and inhibitor）,and then induced with RANKL.Osteoclast formation and function were detected by TRAP staining,TRITC-phalloidin staining,and resorption pit assay.The expressions of CTSK,MMP9 and PTEN were detected by q RT-PCR and Western Blot.The activation of Akt signaling pathway was detected by Western Blot.After transfecting OS cells with mi RNA oligo,s EVs(s EVs ^oligo)was extracted.RAW264.7 cells were treated with s EVs ^oligoand induced with RANKL,and then osteoclast formation,function and expression of related molecules were detected.Results were verified by bone marrow macrophages（BMMs）.Mice were subcutaneously injected with K7M2 cells to establish OS model.The levels of mi R-19a-3p in s EVs were detected in the blood of mice.The bone parameters of femur were analyzed by micro-CT scanning,hematoxylin-eosin（HE）staining.TRAP staining detected the distribution of osteoclasts in femur.Results:Bioinformatics analysis showed that mi R-19a-3p and PTEN may be involved in the enhancement of osteoclast formation and function in OS.Dual luciferase reporter gene assay revealed the target binding of mi R-19a-3p with PTEN.RAW264.7 cells were transfected with mi RNA Oligo and induced with RANKL.Results showed that the osteoclast formation,function,the expression of PTEN and phosphorylated Akt（p-Akt）were increased in mi R-19a-3p mimics group,while the results were opposite in mi R-19a-3p inhibitor group.In addition,RAW264.7 cells were stimulated by s EVs ^oligoand induced with RANKL.The results showed that the number of osteoclasts was increased,the bone resorption function was enhanced,the expression of PTEN was decreased and the expression of p-Akt was increased in the s EVs ^mimicsgroup,while the s EVs ^inhibitor group showed the opposite results.The results were verified by BMMS in vitro.The level of mi R-19a-3p in s EVs derived from OS mice blood was significantly higher than normal mice.Micro-CT and HE staining results displayed the osteopenia in femur of OS mice.TRAP staining results showed that the number of osteoclasts in OS mice was reduced.Conclusion:Mi R-19a-3p delivery via OS cell-derived s EVs promotes osteoclast formation and bone destruction through PTEN degradation and further activating PI3K/AKT signaling pathway.Part 3.SEVs are partly involved in regulation of SFN on osteoclast formation in OSObjective:To investigate the effect of SFN on osteoclastogenesis in OS and related mechanism.Methods:OS cells were stimulated by SFN and s EVs were extracted.RAW264.7 cells were treated with SFN-s EVs and induced with RANKL,and then osteoclast formation,function and expression of related molecules were detected.The effect of SFN on RAW264.7 cells and BMMS proliferation were measured by CCK8.For osteoclastogenesis,RAW264.7 cells and BMMs were induced with RANKL.The number and size of osteoclast were detected by TRAP staining and TRITC-phalloidin staining.The autophagosomes were observed by TEM.The pre-osteoclast cells were exposed to various concentrations of SFN at different time points to explore the effective concentration and the stage at which SFN mainly affected osteoclastogenesis.After the autophagy pathway was activated with rapamycin（RAP）,the effect of SFN on osteoclast formation was detected.QRT-PCR and Western blot were conducted to detect the expressions of CTSK,MMP9,autophagy-related molecules Atg5-Atg12,Beclin1 and light chain 3（LC3）.The JNK signaling was detected by Western blot.RAW264.7 cells were pretreated with anisomycin,the JNK signaling activator,and SP600125,the JNK signaling inhibitor,and then induced with RANKL.The expressions of Atg5-Atg12,Beclin1 and LC3 were detected by Western Blot.The bone destruction model was established by subcutaneous injection of LPS in the middle of the calvaria of mice,and SFN was intraperitoneally injected.Micro-CT and HE staining were used to detect the bone tissue morphology and the bone parameters of mice calvaria.Trap staining was used to detect the distribution of osteoclasts in mice calvaria.The expression and distribution of CTSK and LC3 in bone were detected by immunohistochemistry（IHC）and immunofluorescence（IF）staining.Results:QRT-PCR showed the decrease of mi R-19a-3p in SFN-s EVs.RAW264.7 cells were stimulated by SFN-s EVs and induced with RANKL,and the results showed that the number of osteoclasts was decreased significantly.SFN inhibited osteoclast formation in a dose-dependent manner.Combination with the results of osteoclastogenesis and CCK8 assay,SFN at 2.5μM effectively inhibited the formation of osteoclasts with little effect on the proliferation of pre-osteoclasts.Besides,SFN significantly inhibited osteoclast formation at the early period.The activation of autophagy by RAP almost reversed the SFN-elicited anti-osteoclastogenesis.The results of q RT-PCR and Western blot showed that SFN inhibited the expressions of CTSK,MMP9,Atg5-Atg12,Beclin1 and LC3,while RAP treatment decreased this inhibition effect.Furthermore,Western blot analysis revealed that SFN inhibited phosphorylated JNK（p-JNK）expression in a dose-dependent manner.The JNK activator anisomycin significantly promoted autophagy,whereas the inhibitor SP600125markedly suppressed autophagic activation in pre-osteoclasts.Micro-CT and HE staining results showed that the calvaria destruction was significant after subcutaneous injection of LPS,and SFN could protect against LPS-induced calvarial erosion.Trap staining results showed that SFN attenuated the effect of LPS-induced osteoclast increase.The results of IHC and IF staining showed that the expressions of CTSK and LC3 were increased in mice calvaria in the LPS group,while they were decreased after intraperitoneal injection of SFN.Conclusion:SFN indirectly inhibits osteoclastogenesis by decreasing the level of s EVs’mi R-19a-3p.Besides,SFN directly inhibits osteoclastogenesis through the suppression of the autophagic pathway and the JNK signaling participates in this process.Above all,our study shows that:1.SEVs secreted by OS cells could promote the formation and function of osteoclasts.2.Mi R-19a-3p delivery via OS cell-derived s EVs promotes osteoclast formation and bone destruction through phosphatase and tensin homolog（PTEN）degradation and further activating phosphatidylinositol 3-kinase（PI3K）/proteinkinaseB（AKT）signalingpathway.Inaddition,in vivoexperimentsshowedthe increased level of s EVs’mi R-19a-3p in blood of OS mice,and OS mice exhibitedosteopenia with increased number of osteoclasts.Herein,we demonstrated that blood s EVs’mi R-19a-3p derived from OS could be considered as a crucial factor in osteoclastogenesisand bone destruction,and we clarified the associated mechanism in vitro study.3.S FNcould inhibit osteoclastogenesis in OS by decreasing the level of s EVs’mi R-19a-3p derivedfrom OS cells and directly suppressing autophagic pathway of pre-osteoclast.Therefore,webelieve that OS cells’s EVs are involved in regulation of osteoclastogenesis,and mi R-19a-3p in s EVs may become a new target for the prevention and treatment of OS bonedestruction and tumor progression.更多还原