【作者】 黄鹂；

【导师】 邹淑娟；

【作者基本信息】 四川大学 ， 口腔医学（专业学位）， 2021， 博士

【摘要】 绝经期骨质疏松是由于雌激素水平下降所导致的全身性骨代谢性疾病,骨代谢呈高转换型,骨吸收多于骨形成,导致骨量丢失。正畸牙移动的生理学基础之一是牙槽骨的可重塑性,即张力侧由成骨细胞主导的成骨与压力侧由破骨细胞主导的破骨,二者处于动态平衡。作为骨代谢活跃部位,牙槽骨的重塑过程也可受到骨质疏松的影响,活跃的破骨活动可使正畸牙过程中发生牙槽骨吸收,导致“黑三角”出现,严重时牙齿还可能出现松动脱落等并发症。社会的不断发展使人们维护口腔健康的意识逐渐提高,同时对面部美观的要求也更高。那么当越来越多的绝经期骨质疏松患者加入正畸矫治队伍之后,正畸并发症的出现对于临床医生而言是一项巨大的挑战。如何提高这类患者正畸牙移动过程中牙槽骨的重塑过程,防治正畸治疗相关并发症从而为其提供安全有效的正畸治疗是临床中亟待解决的重要问题。本课题前期研究表明,氯化锂连续14天灌胃可促进大鼠正畸牙保持阶段张力侧成骨,推测其促进成骨的作用机制可能是激活了Wnt信号通路。近年来研究证实自噬参与骨代谢,与骨质疏松症的发生发展紧密相连,而氯化锂作为自噬诱导剂可调控骨代谢从而减缓骨质疏松诱导的骨吸收。自噬是真核生物细胞内的一种自我保护机制,其可将受损的细胞器等大分子物质包裹并行降解,从而为细胞提供能量。近年来越来越多的研究发现自噬与凋亡之间的关系极其紧密,二者同时参与了细胞内复杂的信号调控。糖原合成酶激酶-3(GSK-3β)是凋亡信号通路PI3K/AKT/GSK-3β下游分子,该通路受到被抑制时,GSK-3β活性增加,从而诱导细胞凋亡。氯化锂是GSK-3β抑制剂,研究表明其可抑制细胞凋亡水平,从而调控细胞代谢过程。那么氯化锂能否通过调控细胞内自噬及凋亡水平从而影响细胞内成骨活动,以此对骨质疏松患者正畸牙移动过程中的牙槽骨重塑产生积极的影响,让该类患者获得安全有效的正畸牙移动?作用机制是什么?本研究针对这两个问题通过设计体内及体外实验进行初步研究和探讨。目的:建立骨质疏松小鼠正畸牙移动模型,并给予氯化锂灌胃探究其对骨质疏松状态小鼠正畸牙移动重牙槽骨重塑的影响,并对其可能的作用机制进行初步探究。方法:总共购买77只7周龄,体重16～18g的雌性C57BL/6小鼠,先适应性喂养一周,待其充分适应饲养环境。体内实验:随机挑选32只小鼠分成2组:Sham(假手术)组和OVX(去卵巢术)组。OVX组去除双侧卵巢,Sham组仅去除卵巢周围与之等大的脂肪组织。8周后两组各随机选取4只小鼠处死并收样进行Micro-CT扫描,用以验证该小鼠骨质疏松模型是否建立成功。剩余24只小鼠进行正畸牙移动,牙移动3、7、14天。探究骨质疏松对小鼠牙移动过程牙槽骨重塑的影响。为了探究Li Cl对牙移动过程中牙槽骨兴趣区域内自噬、凋亡及骨代谢活动的影响,将45只小鼠随机分成三组:Sham组,OVX组及OVX+Li Cl组(去卵巢术+氯化锂灌胃),于正畸牙移动当天开始对OVX+Li Cl组小鼠连续14天200mg/kg剂量Li Cl灌胃,OVX组和Sham组小鼠用Li Cl的溶液(PBS)灌胃。实验研究方法包括:Micro-CT、H&E染色、ALP、TRAP染色,免疫组织化学(IHC)染色、TUNEL(Td T-mediated DUTP nick end labeling,原位末端转移酶标记技术)染色。体外实验:从Sham组和OVX组各选取四只小鼠处死,提取股骨及胫骨内骨髓间充质干细胞(BMSCs),通过CCK-8、ALP染色及茜素红染色、流式细胞术(FCM)、透射电镜(TEM)、逆转录-聚合酶链反应(Reverse Transcription-Polymerase Chain Reaction,RT-PCR)及蛋白质印迹法(Western blot,WB)探究Li Cl对骨质疏松BMSCs生物学行为的影响。为了探究Li Cl促进骨质疏松BMSCs成骨的分子生物学机制,使用自噬早期抑制剂三甲基腺嘌呤(3-Methyladenine,3-MA)抑制Li Cl的作用,通过细胞免疫荧光(ICC)、RT-PCR及WB检测细胞自噬、凋亡及成骨相关标记物的表达变化。最后加入自噬晚期抑制剂氯喹(Chloroquine,CQ)阻断自噬引发自噬潮,检测自噬相关蛋白表达,进一步验证Li Cl促进自噬的作用机制。结果:1.去卵巢术后8周,与Sham组相比,OVX组小鼠的体重增加更为显著,股骨头骨小梁参数(BMD、BV/TV)及牙槽骨骨小梁参数(BMD、Tb.N及BV/TV)明显降低,说明骨质疏松模型成功建立。2.在10g作用力下,相比较于Sham组,OVX组小鼠牙移动距离更大,压力侧TRAP阳性细胞数明显增多,右上颌第一磨牙周围牙槽骨吸收面积显著增大。说明牙移动过程中,骨质疏松诱导了牙槽骨中更为活跃的破骨活动。3.200mg/kg Li Cl连续14天灌胃减少了骨质疏松小鼠正畸牙移动过程中压力侧TRAP阳性细胞数,张力侧ALP阳性表达增强,Runx2阳性表达下降,同时OPG/RANKL比值上升。自噬标记物(LC3及Beclin-1)阳性表达升高,P62阳性表达及TUNEL阳性细胞数下降,说明Li Cl使牙槽骨兴趣区域细胞自噬水平升高、凋亡下降,成骨活动增强。4.5 m M Li Cl为作用于BMSCs的最适浓度,其可提高骨质疏松BMSCs的自噬(LC3、BECLIN-1及ATG5等)及成骨相关标志物(ALP、RUNX2及BMP2等)的表达,抑制凋亡相关标志物(GSK-3β、BAX/BCL-2)的表达。然而,Li Cl对骨质疏松BMSCs的这一作用可被自噬抑制剂3-MA抑制,导致细胞自噬水平及成骨活性降低而凋亡水平上升。提示Li Cl对骨质疏松BMSCs促进成骨的作用机制可能是通过上调细胞自噬水平实现,凋亡可能参与其中。5.通过使用自噬晚期抑制剂氯喹(CQ)阻断自噬累积自噬潮,发现Li Cl处理之后的骨质疏松BMSCs LC3及P62蛋白表达进一步增加,说明自噬体发生了更多的累积,提示Li Cl可能在自噬早期促进了骨质疏松BMSCs的自噬活性。结论:综上所述,骨质疏松小鼠的牙移动过程中牙槽骨兴趣区域内破骨活动增强,伴随着牙移动速率增快,骨量下降。而Li Cl的运用使得牙槽骨内自噬活动水平提高,凋亡水平下降;压力侧牙槽骨吸收减少,张力侧成骨增加。5m M为Li Cl作用于BMSCs的最适浓度,使骨质疏松BMSCs自噬能力提高,凋亡水平下降,成骨向分化能力增强,而该作用可在一定程度上被3-MA抑制,说明Li Cl促进骨质疏松BMSCs成骨的机制可能是在自噬早期通过促进自噬活性来实现,凋亡也可能参与其中。本实验通过使用Li Cl促进骨质疏松小鼠正畸牙移动过程中的骨重塑效应拟防治绝经期骨质疏松正畸患者牙移动过程中的并发症,为其接受安全有效的治疗提供新的研究数据基础及临床用药选择。更多还原

【Abstract】 Menopausal osteoporosis is a systemic bone metabolic disease caused by the decrease of estrogen level.Estrogen deficiency can lead to high turnover of bone metabolism,bone resorption is more than bone formation,leading to bone loss.One of the physiological bases of orthodontic tooth movement is the remodeling of alveolar bone,that is,osteogenesis in the tensile side and osteoclastic activity in the pressure side,those two are in dynamic balance.As an active part of bone metabolism,the normal remodeling process of alveolar bone can also be affected by osteoporosis.Active osteoclastic activity can lead to alveolar bone resorption during the orthodontic process,resulting in the appearance of "black triangle".In severe cases,teeth may loose and fall off.With the continuous development of society,people’s awareness of maintaining oral health is gradually improved,and the requirements of facial beauty are also higher.So when more and more postmenopausal osteoporosis patients join the orthodontic team,the emergence of orthodontic complications is a great challenge for clinicians.How to improve the process of alveolar bone remodeling during orthodontic tooth movement and prevent the complications related to orthodontic treatment,so as to provide safe and effective orthodontic treatment is an important problem to be solved in clinic.Our previous project has shown that lithium chloride can promote osteogenesis in the tension side during the retention phase orthodontic tooth movement in rats.It is speculated that the mechanism of osteogenesis is the activation of Wnt signaling pathway.In recent years,it has been confirmed that autophagy is involved in bone metabolism and closely related to the occurrence and development of osteoporosis.Lithium chloride,as an autophagy inducer,can regulate bone metabolism and slow down osteoporosis-induced bone resorption.Autophagy is a kind of self-protection mechanism in eukaryotic cells,which can package and degrade damaged organelles and other macromolecules,thus providing energy for cells.More and more studies have found that autophagy and apoptosis are closely related,both of which are involved in complex intracellular signal regulation.GSK-3β is the downstream molecule of apoptosis signal pathway PI3K/AKT/GSK-3β.When the pathway is inhibited,GSK-3β activity is up-regulated,leading to the increase of apoptosis level.Lithium chloride is a GSK-3β inhibitor,studies have shown that it can inhibit the level of apoptosis,thereby regulating the metabolic process of cells.Whether Li Cl could affect the osteogenic activity by regulating the level of autophagy and apoptosis,so as to have a positive impact on alveolar bone remodeling in osteoporotic patients during orthodontic tooth movement,and make these patients obtain safe and effective treatment? If possible,what is the mechanism? In order to solve these two problems,we designed in vivo and in vitro experiments.Objective: To investigate the effects of lithium chloride on orthodontic tooth movement and alveolar bone remodeling in osteoporotic mice and related molecular mechanisms.Methods: A total of 77 7-week-old female C57BL/6 mice weighing 16-18 g were purchased and fed adaptively for one week until they were fully adapted to the environment.In vivo experiment: 32 mice were randomly divided into Sham and OVX groups.Bilateral ovaries were removed in OVX group,while in Sham group,only the adipose tissue around the ovaries was removed.After 8 weeks,four mice were randomly killed respectively and taken for micro-CT scan to verify the success of the osteoporosis model.The remaining 24 mice were moved to investigate the effect of osteoporosis on alveolar bone remodeling during tooth movement.In order to investigate the effects of Li Cl on autophagy,apoptosis and bone metabolism in alveolar bone during tooth movement,45 mice were randomly divided into three groups: Sham group,OVX group and OVX + Li Cl group(ovariectomized + Li Cl gavage).On the day of orthodontic tooth movement,mice in OVX + Li Cl group were gavaged with 200mg/kg Li Cl for 14 consecutive days,while mice in OVX group and Sham group were gavaged with Li Cl solution(PBS).The experimental methods included micro-CT,H&E staining,ALP and TRAP staining,IHC staining and TUNEL staining.In vitro experiment: four mice in Sham group and OVX group were killed respectively and bone marrow mesenchymal stem cells(BMSCs)were extracted.The effects of Li Cl on the biological behavior of BMSCs were investigated by CCK-8,ALP staining,alizarin red staining,flow cytometry(FCM),transmission electron microscopy(TEM),RT-PCR and Western blot.In order to explore the molecular biological mechanism of Li Cl promoting osteogenesis of osteoporotic BMSCs,3-methyladenine(3-MA),an early autophagy inhibitor was used to inhibit the effect of Li Cl,apoptosis-and osteogenesis-related markers were detected by immunofluorescence,RT-PCR and Western blot.Finally,chloroquine(CQ),a late autophagy inhibitor,was added to accumulate the autophagy flux and detect the expression of autophagy-related protein,so as to further verify the mechanism of promoting autophagy by Li Cl.Results1.Eight weeks after ovariectomy,compared with Sham group,OVX group had more significant weight gain,and the trabecular parameters of femoral head(BMD,BV/TV)and alveolar bone(BMD,TB.N and BV/TV)were significantly decreased,which indicated that the osteoporosis model was successfully established.2.Compared with Sham group,the tooth movement distance of OVX group was larger,the number of TRAP positive cells on the pressure side was significantly increased,and the resorption area of alveolar bone around the right maxillary first molar was significantly increased.Those indicated that osteoporosis induces more active osteoclastic activity during tooth movement.3.200mg/kg Li Cl-gavage for 14 days reduced the number of TRAP positive cells on the pressure side,increased the expression of ALP and the ratio of OPG/RANKL on the tensile side,and decreased the Runx2 expression.Meanwhile,the expression of autophagy markers(LC3 and Beclin-1)were increased,the number of TUNEL positive cells was decreased,which indicating that Li Cl increased the autophagy level,decreased apoptosis and enhanced osteogenic activity of cells in the region of interest of alveolar bone.4.5 m M Li Cl was the optimal concentration for osteoporotic BMSCs,which could increase the expression of autophagy(LC3,Beclin-1 and ATG5,etc.),and osteogenesis related markers(ALP,Runx2 and BMP2,etc.),inhibit the expression of apoptosis related markers(GSK-3β and ratio of BAX/BCL-2).However,the effect of Li Cl on osteoporotic BMSCs can be inhibited by 3-MA,resulting in the decrease of autophagy and osteogenic activity and the increase of apoptosis.It is suggested that the mechanism of Li Cl in promoting osteogenesis of osteoporotic BMSCs may be through upregulating the level of autophagy,apoptosis may be involved in it.5.By using chloroquine(CQ),a late autophagy inhibitor,to accumulate the autophagy flux,we found that LC3-II and P62 proteins were increased further after Li Cl treatment,which indicating that autophagosomes accumulated more,suggesting that Li Cl might promote autophagy activity of osteoporotic BMSCs in the early stage.Conclusion:In conclusion,osteoclastic activity in the region of interest of alveolar bone increased during tooth movement in osteoporotic mice,accompanied by faster tooth movement rate and decreased bone mass.The use of Li Cl increased the level of autophagy and decreased the level of apoptosis in alveolar bone;meanwhile,the alveolar bone resorption on the pressure side decreased,and the osteogenesis on the tension side increased.5 m M was the optimal concentration of Li Cl on BMSCs,which can improve the autophagy ability of osteoporotic BMSCs,reduce the level of apoptosis and enhance the osteogenic differentiation ability.The effect of Li Cl can be inhibited by 3-MA,which indicated that the mechanism of Li Cl promoting osteogenesis of osteoporotic BMSCs may be realized by promoting autophagy activity in the early stage,and apoptosis may also participate in it.In this study,we used Li Cl to promote bone remodeling in the process of orthodontic tooth movement in osteoporosis mice,to prevent and treat the complications during the orthodontic tooth movement process of postmenopausal osteoporosis patients,and to provide new research data basis and clinical drug selection to receive safe and effective treatment.更多还原