【作者】 李斌；

【导师】 陈廖斌；

【作者基本信息】 武汉大学 ， 外科学 骨科， 2019， 博士

【摘要】 骨关节炎(osteoarthritis,OA)是一种以关节软骨退行性变为主要病理特征的慢性关节疾病,是中老年人关节疼痛的最常见原因。然而,迄今有关OA的病因及发病机制仍不清楚。近来的研究表明,OA与代谢综合征密切相关。越来越多的学者认为OA属于代谢综合征的范畴。大样本流行病学调查数据显示,宫内生长迟缓(intrauterine growth retardation,IUGR)成年后手、腰及髋部OA发生率明显增加。本团队前期系列动物实验表明,孕期不良环境(摄食限制、咖啡因、尼古丁、乙醇暴露)可引起子代大鼠IUGR、成年后代谢综合征及OA易感性增加。这些研究表明,OA存在宫内发育起源。有研究表明,软骨局部也存在包括胆固醇合成、摄取、流出和转化的系列代谢过程来维持局部胆固醇含量的稳定。病理情况下,成人OA患者软骨局部存在胆固醇摄取、合成、输出和转化过程异常失衡,伴随局部胆固醇异常蓄积现象。蓄积的胆固醇可通过多途径损伤软骨细胞,降低软骨质量。另有研究表明,高胆固醇血症与OA的发生有明显相关性,已成为OA发生的独立危险因素。同时,大量流行病学提示,高胆固醇血症也具有宫内起源。本室研究证实,孕期外源物(如咖啡因、尼古丁和乙醇)暴露可引起IUGR子代在高脂饮食下出现高胆固醇血症,关节软骨胆固醇蓄积。以上表明,软骨局部与血循环胆固醇代谢紊乱均可能参与胎源性OA的发生。合成类糖皮质激素(glucocorticoids,GCs)如地塞米松,临床上被广泛用于早产相关妊娠疾病。大量研究表明,产前使用地塞米松可导致新生儿体重减轻,多器官发育毒性,以及成年期对各种慢性疾病的易感性增加。孕期地塞米松暴露(prenatal dexamethasone exposure,PDE)可引起子代IUGR和多组织发育不良。“宫内编程(intrauterine programming)”是指宫内胎儿遭受恶劣环境,可导致胎儿的器官发育和功能发生变化,并可以持续到出生后。有研究报道,地塞米松可调控肝细胞中胆固醇代谢相关基因的表达。以上提示,PDE可能通过宫内编程引起IUGR子代软骨局部胆固醇代谢异常改变,从而导致成年后软骨局部胆固醇蓄积及OA易感增加。流行病学表明,胎源性OA在雄性个体中较为易感。因此,本课题选取雄性子代作为研究对象,进行如下工作:(1)采用PDE所致雄性子代IUGR模型、木瓜蛋白酶关节腔注射诱导OA模型和软骨细胞复合海藻酸钠微球三维培养模型,证实PDE所致雄性子代软骨胆固醇蓄积,进而引起软骨质量低下,不良环境下OA易感性增加;(2)进一步观察PDE雄性子代大鼠宫内时期关节软骨发育质量、宫内及出生后血胆固醇水平及软骨局部胆固醇代谢通路表达变化,以探讨PDE所致雄性子代大鼠成年后软骨胆固醇蓄积的宫内编程机制;(3)在原代胎鼠软骨细胞上,探讨地塞米松对胎软骨细胞中胆固醇27羟化酶(cholesterol 27-hydroxylase,CYP27A1)表达的影响及分子机制。本课题的实施将有助于我们更全面地了解PDE所致子代胆固醇蓄积、OA易感性增加及其宫内编程机制,同时也为进一步探索胎源性OA的早期预防和治疗提供指导作用。第一部分孕期地塞米松暴露可致雄性子代大鼠成年后关节软骨胆固醇蓄积及骨关节炎易感目的:利用PDE所致雄性子代大鼠IUGR模型、木瓜蛋白酶膝关节腔注射诱导OA模型及软骨细胞复合海藻酸钠微球三维培养体系,探究PDE是否可导致雄性子代成年后软骨胆固醇蓄积,进而引起软骨质量低下及OA易感增加现象。方法:宫内:Wistar母鼠自然受孕后,随机分为对照组和PDE组。自孕期天数(gestational day,GD)9起至GD20,PDE组皮下注射地塞米松0.2 mg/kg.d,对照组给予等量生理盐水。在GD20时,采用苯巴比妥麻醉孕鼠,剖腹取出雄性胎鼠。记录胎鼠的体重和性别,取胎血和双下肢股骨标本。出生后:随机留取部分孕鼠让其自然生产,保留每窝仔鼠数在8-15只的新生鼠,四周断奶,给予正常饮食至出生后8周龄(postnatal week,PW8)。在PW8时,通过将木瓜蛋白酶注射到膝关节腔中来建立OA模型。对照组和PDE组大鼠第1天、第4天、第7天、第10天和第13天通过左膝关节注射4%木瓜蛋白酶溶液100μl;同时,在上述时间点给大鼠右膝注射相同量的生理盐水。在PW12,通过乙醚麻醉处死大鼠,获取血和下肢膝关节。体外:分离并增殖雄性成年软骨细胞,复合海藻酸钠微球建立三维培养体系,给与不同浓度(5、15、45μg/ml)的胆固醇处理24h,收获上清液和细胞,检测上清液糖胺聚糖(glycosaminoglycan,GAG)含量、细胞活力、凋亡、基质合成及降解酶的基因表达变化。结果:(1)木瓜蛋白酶刺激前,PDE组软骨基质番红染色和Col2a1免疫组化染色较对照组轻度减弱;木瓜蛋白酶刺激后,PDE组软骨表面出现明显的毛糙,软骨基质结构紊乱,软骨厚度变薄,基质染色变浅,软骨细胞数明显减少,且PDE组改良Mankin’s评分较对照组升高更显著,Col2a1光密度值显著低于相应的对照组。(2)木瓜蛋白酶刺激前,PDE组软骨组织中TUNEL阳性细胞数较对照组明显增加;Col2a1和Aggrecan的m RNA表达显著降低,基质降解酶MMP3和MMP13的表达均显著升高。(3)木瓜蛋白酶刺激前,PDE组软骨胆固醇含量较对照组明显升高;(4)体外三维培养条件下,不同浓度胆固醇组中成年软骨细胞活力较对照组显著降低,凋亡相关基因的m RNA表达异常(Bax表达升高、Bcl-2表达降低及Bax/Bcl-2表达比升高),微球切片中番红O染色变浅,细胞数显著降低,上清液GAG含量下降,Col2a1和Aggrecan的m RNA表达降低,MMP3和MMP13的m RNA表达升高。结论:PDE可引起成年雄性子代大鼠软骨胆固醇蓄积,导致软骨质量低下;进一步,在膝关节腔内注射木瓜蛋白酶后,OA的易感性增加。第二部分孕期地塞米松暴露所致雄性子代大鼠成年后胆固醇蓄积的宫内编程机制目的:通过整体动物实验,观察PDE雄性子代大鼠宫内时期关节软骨发育质量、宫内及出生后血胆固醇水平及软骨局部胆固醇代谢通路表达变化,以探讨PDE所致雄性子代大鼠成年后软骨胆固醇蓄积的宫内编程机制。方法:同第一部分。结果:(1)PDE组胎鼠体重较对照组明显下降,IUGR率显著升高;(2)胎软骨质量:PDE组胎鼠关节软骨区番红O染色减弱,光密度分析其GAG含量显著降低;关节软骨细胞排列紊乱且数量减少;Col2a1免疫组化染色变浅,光密度分析其含量减少;Col2a1的m RNA表达显著降低;(3)软骨胆固醇代谢通路m RNA表达:宫内,PDE组雄性子代软骨局部胆固醇摄取相关基因SR-B1的m RNA表达无明显变化,LDLR的m RNA表达显著降低;胆固醇合成相关基因HMGCR的m RNA表达明显降低;胆固醇输出相关基因ABCA1、ABCG1和Apo A1的m RNA表达无明显变化;胆固醇转化相关基因CYP27A1和CH25H的m RNA表达显著降低。出生后12周,胆固醇摄取相关基因SR-B1和LDLR的m RNA表达显著升高,胆固醇合成相关基因SREBP-2和HMGCR的m RNA表达无明显变化,胆固醇合成相关基因ABCA1、ABCG1和Apo A1的m RNA表达显著升高,胆固醇转化相关基因CYP27A1的m RNA表达显著降低,CH25H的m RNA表达无明显变化。(4)CYP27A1的蛋白表达:免疫荧光染色及半定量结果表明,PDE所致雄性子代胎软骨及出生后软骨CYP27A1的蛋白表达较对照组均显著降低;(5)表遗传酶及CYP27A1组蛋白乙酰化修饰:宫内,PDE组雄性子代软骨局部糖皮质激素受体(GR)和组蛋白去乙酰化酶1(HDAC1)的m RNA表达显著升高,CYP27A1启动子区组蛋白3赖氨酸位点9乙酰化(H3K9ac)和H3K27ac水平较对照组显著降低;出生后12周,CYP27A1启动子区H3K9ac水平显著降低,H3K27ac水平无明显变化。结论:PDE一方面抑制雄性子代大鼠胎软骨发育,降低软骨局部CYP27A1的H3K9ac和表达水平(低功能编程);另一方面,导致子代大鼠成年后软骨局部胆固醇摄取功能增强,两者共同作用导致子代成年后软骨局部胆固醇蓄积,进而导致软骨质量低下及OA易感性增加。第三部分地塞米松对原代胎软骨细胞CYP27A1表达的影响及分子机制目的:利用原代雄性大鼠胎软骨细胞,探讨地塞米松对CYP27A1表达的影响及分子机制。方法:取大鼠胎关节软骨组织,分离培养获得原代胎软骨细胞,分别进行以下处理:(1)500 nM地塞米松处理胎软骨细胞不同时间(1、2、3、4、5天)或用不同浓度(0、20、100、500 nM)地塞米松处理胎软骨细胞3天,MTS法检测细胞活力;(2)给予胎软骨细胞不同浓度(0、20、100、500 nM)地塞米松处理;或给与胎软骨细胞糖皮质激素受体(GR)拮抗剂RU486预处理2小时后,再加入500nM地塞米松;或HDAC1拮抗剂TSA联合500 nM地塞米松,连续处理72h后收集细胞。检测相关指标变化。结果:(1)500 nM地塞米松给药1～3天后细胞活力无明显改变,但给药4天和5天后,细胞活力明显降低;不同浓度(0、20、100、500 nM)地塞米松处理胎软骨细胞3天后,细胞活力无明显变化;(2)地塞米松处理的胎软骨细胞中,CYP27A1的m RNA和蛋白表达较对照组均显著降低;(3)地塞米松作用下,GR活化入核增多,结合到CYP27A1启动子区的GR显著增加,GR和HDAC1的表达增强,与GR结合的HADC1显著增多;(4)地塞米松可升高HDAC1的m RNA表达,降低CYP27A1的启动子区H3K9ac水平,RU486可取消地塞米松对HDAC1的促表达作用,RU486或TSA可取消地塞米松对CYP27A1的H3K9ac和表达的抑制作用。结论:本部分研究证实地塞米松可促进GR和HDAC1的表达,促进GR入核增加,招募更多的HDAC1形成复合物并结合到CYP27A1启动子区,引起CYP27A启动子区H3K9ac水平降低,最终导致CYP27A1的表达降低。更多还原

【Abstract】 Osteoarthritis(OA)is chronic joint disease featured with degenerative articular cartilage,which is the most common cause of joint pain in middle-aged and elderly people.However,the etiology and pathogenesis of OA are still unclear.Recent studies have revealed that OA was closely involved in metabolic syndrome(MS).More and more scholars believed that OA belonged to the category of MS.Large-scale epidemiological survey results suggested,people with intrauterine growth retardation(IUGR)had an increased incidence of MS.Recent epidemiological data also showed that the incidence of hand OA was considerably higher in men with low birth weight,and a similar trend was observed in women.Previous studies in our laboratory also showed that adverse factors exposure during pregnancy(dietary restriction,caffeine,nicotine,and ethanol)could lead to IUGR and increased susceptibility to MS and OA in offspring rats.All these studies indicate OA has fetal origin.Studies have shown that biological processes including cholesterol synthesis,uptake,outflow and transformation also exist in the cartilage to maintain the physiological homeostasis of local cholesterol metabolism.Under pathological conditions,abnormal imbalance of cholesterol uptake,synthesis,output and transformation occured in the local cartilage of adult patients with OA,accompanied with abnormal accumulation of cholesterol.Accumulation of cholesterol could damage chondrocytes through multiple ways and reduce the cartilage quality.Other studies have shown that hypercholesterolemia is closely correlated with the occurrence of OA and has become an independent risk factor for OA.At the same time,a large number of epidemiology data suggest that hypercholesterolemia also has intrauterine origin.Our previous study confirmed that prenatal exposure to adverse factors(e.g.,caffeine,nicotine and ethanol)could cause hypercholesterolemia and accumulation of articular cartilage cholesterol in IUGR offspring on a high-fat diet after birth.The above indicated that the disorder of blood circulation and cartilage cholesterol metabolism may be involved in the occurrence of fetal originated OA.Synthetic glucocorticoid(GC),such as dexamethasone,is widely used clinically for the premature pregnancy related diseases.Numerous studies have shown that prenatal use of dexamethasone could lead to low birth weight of newborns,multiple organ developmental toxicity and susceptibility to a variety of chronic diseases in adulthood.Dexamethasone is known to regulate the expression of a series of genes related to cholesterol metabolism in hepatocytes.These results suggest that prenatal dexamethasone exposure may affect local cholesterol metabolism in fetal cartilage."Intrauterine programming" refers to the adverse environment encountered by the fetus during the intrauterine period,which can cause changes in the development and function of the fetal organs and can last until after birth.Previous studies in our laboratory have found prenatal dexamethasone exposure(PDE)can cause IUGR and multiple organ dysplasia in offspring.These results suggest that PDE might induce abnormal changes of local cholesterol metabolism in cartilage of IUGR offspring through intrauterine programming,which mediated local cholesterol accumulation and OA susceptibility in adult.Epidemiology showed that fetal originated OA was more susceptible in males.Therefore,this project selects male offspring as the research object.The following work is proposed in this study:(1)IUGR model in male offspring rats induced by PDE,OA model injected with articular cavities papain and three-dimensional culture model of adult chondrocytes with sodium alginate microspheres are utilized to verify the cholesterol accumulation and low quality of cartilage in offspring caused by PDE and OA susceptibility in adverse environment;(2)The changes of articular cartilage development quality and local cholesterol metabolism pathway expression in intrauterine and postnatal cartilage of PDE offspring rats are further observed to explore the intrauterine programming mechanism of cartilage cholesterol accumulation induced by PDE in adult male offspring rats.(3)Based on primary fetal chondrocytes,the effect of dexamethasone on CYP27A1 expression of fetal chondrocytes and its molecular mechanism are investigated.The implementation of this project will help us to have a more comprehensive understanding of cholesterol accumulation,OA susceptibility and its intrauterine programming mechanism induced by PDE,which is better understand fetal originated OA,and also playes a guiding role in the further exploration of early prevention and treatment of OA.Part One Prenatal dexamethasone exposure induces articular cartilage cholesterol accumulation and osteoarthritis susceptibility in male offspring adult ratsObjective: IUGR rat model induced by PDE,OA model induced by papain injection into the knee cavity and the three-dimensional culture model of adult chondrocytes combined with sodium alginate microspheres,were utilized to observe cholesterol accumulation,low cartilage quality and OA susceptibility in male offspring induced by PDE.Methods: In utero: Wistar maternal rats were randomly divided into Control group and PDE group after natural conception.From gestational day(GD)9 to GD20,0.2mg /kg.d dexamethasone was injected subcutaneously in the PDE group,and equal volume normal saline was given to the control group.On GD20,pregnant mice were anesthetized with phenobarbital and removed by laparotomy.Fetal weight and sex were recorded.Male fetal blood and femur specimens of both lower limbs were collected.After birth: some pregnant mice were randomly retained and allowed to give birth naturally.Newborn mice with 8-15 pups per litter were kept and weaned for 4 weeks.Normal diet was given until 8 weeks after birth(postnatal week,PW8).At the time of PW8,the knee joint was injected with papain to make OA model.The rats in the control group and the PDE group were injected with 4% papain solution 100 μl in the left knees joint at the 1st,4th,7th,10 th and 13 th day of the modeling respectively.At the same time,the right knees were injected with normal saline at the above time points.In PW12,rats were sacrificed under ether anesthesia,and blood was taken from the lower limbs and knee joints.In vitro: adult chondrocytes were isolated and proliferated,and sodium alginate microspheres were combined to establish a three-dimensional culture system.Cholesterol was treated with different concentrations(0,5,15,45 g/ml).The supernatant and cells were harvested.Results:(1)Before papain stimulation,cartilage matrix and Col2a1 immunohistochemical staining was slightly weakened in PDE group compared with the control group.After stimulation by papain,cartilage surface of PDE group showed obvious roughness,cartilage matrix structure disorder,cartilage thickness became thinner,matrix staining became lighter,and chondrocyte number decreased significantly.In addition,modified Mankin’s score of PDE group increased more considerably than that of control group,and optical density value of Col2a1 was considerably lower than that of corresponding control group.(2)Before papain stimulation,the number of Tunel positive cells in the PDE group was considerably higher than that in the control group.The m RNA expressions of Col2a1 and Aggrecan were considerably decreased,and the expressions of matrix degrading enzymes MMP3 and MMP13 were considerably increased.(3)Before the stimulation of papain,the cholesterol content in the PDE group was considerably higher than that in the control group.(4)In vitro three-dimensional culture condition,different concentration of cholesterol in the group of adult cartilage cell vitality than the control group considerably reduced,m RNA expression of apoptosis related gene abnormality(increased Bax,reduced the Bcl-2 and higher ratio of Bax/Bcl-2),microspheres section by safranin O stain becomes shallow,cells was considerably reduced,supernatant GAG content decreased,Col2a1 and the expression of aggrecan lower,the expressions of MMP3 and MMP13 were increased.Conclusion: PDE caused cholesterol accumulation in cartilage of adult male offspring rats,resulting in low quality of cartilage,which showed inreased susceptibility to OA after stimulation with papain in the knee joint cavity.Part Two Intrauterine programming mechanism of cholesterol accumulation in cartilage of male offspring rats induced by prenatal dexamethasone exposureObjective: The changes of articular chondrogenesis quality in intrauterine period and the expression changes of local cholesterol metabolism pathway in intrauterine and postnatal cartilage of PDE offspring rats were observed so as to explore the intrauterine programming mechanism of PDE induced cartilage cholesterol accumulation in adult male offspring rats.Methods: Same with the part one.Results:(1)the fetal weight of PDE group was considerably lower than that of the control group,and the IUGR rate was considerably increased.(2)fetal cartilage quality: safranin-o staining was weakened in the articular cartilage area of fetal rats in PDE group,and the content of GAG was considerably reduced by optical density analysis.The thickness of articular cartilage became thinner and the number of cells was decreased.Col2a1 immunohistochemical staining became lighter,and its content was reduced by optical density analysis.The m RNA expressions of Col2a1 and aggrecan were lower than those of the control group.(3)cartilage cholesterol metabolism pathway: In utero,the m RNA expression of SR-B1 in PDE group’s offspring local cholesterol uptake related genes had no significant change,but the m RNA expression of LDLR was considerably reduced.The m RNA expression of cholesterol synthesis related genes HMGCR was considerably decreased.The m RNA expression of cholesterol eflux-related genes ABCA1,ABCG1 and Apo A-1 showed no significant changes.The m RNA expression of cholesterol transformation related genes CYP27A1 and cholesterol hydroxylase CH25 H were considerably reduced.At week 12,m RNA expressions of SR-B1 and LDLR were considerably increased.m RNA expressions of SREBP-2 and HMGCR were not considerably changed,m RNA expressions of ABCA1,ABCG1 and Apo A1 were considerably increased,m RNA expression of CYP27A1 was considerably decreased,and m RNA expression of CH25 H had no changes.(4)epigenetic modification changes: In utero,the m RNA expressions of GR and HDAC1 were increased in the fetal cartilage in the PDE group.The H3K9 ac and H3K27 ac levels of promoter region in CYP27A1 in the PDE group were considerably lower than those in the control group.At PW12,the level of H3K9 ac in the promoter region of CYP27A1 decreased significantly,and there was no significant change in the level of H3K27 ac.Conclusion: PDE,on one hand,inhibited fetal chondrogenesis in offspring rats,reduced the H3K9 ac level and expression of CYP27A1 in local cartilage(low-function programming).On the other hand,the uptake of cholesterol in adult cartilage was increased by PDE.The combined effect of the above caused the local cholesterol accumulation in the cartilage of the offspring in adulthood,which further leads to low cartilage quality and increased susceptibility to OA.Part Three Effect of dexamethasone on CYP27A1 expression in primary fetal chondrocytes and its molecular mechanismObjective: Effects of dexamethasone on CYP27A1 expression of fetal chondrocytes and the molecular mechanism were investigated.Methods: Fetal articular cartilage were obtained to isolate the original generation of fetal cartilage cells,then the following treatments were listed as follows:(1)fetal cartilage cells were treated with 500 n M dexamethasone for different time points(1,2,3,4,5 days)or with different concentrations(0,20,100,500 n M)dexamethasone for 3 days,and the MTS method was used to detect cell activity;(2)fetal chondrocytes were treated with dexamethasone at different concentrations(0,20,100,500 n M),Or the fetal chondrocytes were pretreated with GR antagonist RU486(5 μM)for 2 hours,and then treated with dexamethasone at 500 n M;or the cells were co-treated with TSA and dexamethasone.After continuous treatment for 72 h,cells were collected.Results:(1)Cell viability was not considerably changed after 1～3 days of administration of dexamethasone at 500 n M,but considerably decreased after 4 and 5 days of administration.Fetal chondrocytes treated with dexamethasone at different concentrations(0,20,100,500 n M)showed no significant changes in cell viability after 3 days.(2)in the fetal chondrocytes of dexamethasone group,the m RNA and protein expression of CYP27A1 were considerably lower than those of the control group.(3)Under the effect of dexamethasone,the expression of GR and HDAC1 increased,activation of GR into the nucleus increased,and then GR recruited more HDAC1 to form a complex,which binded to the CYP27A1 promoter region.(4)Dexamethasone could increase the m RNA expression of HDAC1 and reduce the level of H3K9 ac in the promoter region of CYP27A1.RU486 could cancel the promoting effect of dexamethasone on HDAC1 expression.RU486 or TSA could cancel the inhibitory effect of dexamethasone on the level and expression of H3K9 ac in CYP27A1.Conclusion: this part confirmed that dexamethasone could activate GR and promote GR into the nucleus,and then GR recruited more HDAC1 to form a complex,which binded to the CYP27A1 promoter region,causing a decrease in the level of H3K9 ac in the CYP27 A promoter region,and thereby leading to a decrease in the expression of CYP27A1.更多还原