技术领域Technical field
本发明涉及动物模型构建技术领域，具体涉及一种父源性骨关节炎的动物模型构建方法、干预靶标及其应用。The invention relates to the technical field of animal model construction, and specifically relates to a method for constructing an animal model of paternal osteoarthritis, an intervention target and its application.
背景技术Background technique
骨关节炎是一种好发于老年人的慢性退行性关节疾病，主要以关节软骨退变、骨赘形成、软骨下骨硬化和滑膜炎症为特点[1]。骨关节炎也是引起老年人关节疼痛、慢性致残的首要原因，其患病率随年龄递增，65岁以上人群甚至已高达50％[2,3]。伊朗学者Safiri及其同事通过分析2017年全球疾病负担研究中关于1990年到2017年全球多个国家和地区的骨关节炎负担，发现2017年骨关节炎的年发病率比1990年增加了8.2％[4]。因此，当前迫切需要明了驱动骨关节炎发生的病理生理机制，以便实现早期预防及诊断，从而筛选出更优的个体化治疗手段。传统观点认为，骨关节炎属于老年退行性疾病，主要由机械及年龄相关因素引起的关节软骨退变所致。然而，多项大样本流行病学调查显示，低出生体重患儿成年后，手、膝和髋等关节部位的骨关节炎发生率显著增加[5-8]。其中，澳大利亚学者Hussain等2015年对3604名年龄大于40岁且诊断为髋、膝骨关节炎的病例进行了临床回顾性调查发现，低出生体重者患髋关节骨关节炎的风险是正常体重者的2.04倍[7]。该学者2018年的另一项研究证实，低出生体重与成年髋关节骨关节炎之间存在明确的因果关系[8]。提示，低出生体重与成年骨关节炎的发生关系密切。本室前期系列研究已证实，孕期母体咖啡因暴露所致的低出生体重子代出现关节软骨发育不良及成年后骨关节炎易感[9]。然而，父体不良环境对子代关节软骨发育及其远期骨关节炎易感的危害尚不清楚。Osteoarthritis is a chronic degenerative joint disease that commonly occurs in the elderly and is mainly characterized by articular cartilage degeneration, osteophyte formation, subchondral bone sclerosis and synovial inflammation[1] . Osteoarthritis is also the primary cause of joint pain and chronic disability in the elderly. Its prevalence increases with age, and has even reached as high as 50% in people over 65 years old[2,3] . Iranian scholar Safiri and colleagues analyzed the burden of osteoarthritis in many countries and regions around the world from 1990 to 2017 in the 2017 Global Burden of Disease Study and found that the annual incidence of osteoarthritis in 2017 increased by 8.2% compared with 1990.[4] . Therefore, there is an urgent need to understand the pathophysiological mechanisms that drive the occurrence of osteoarthritis in order to achieve early prevention and diagnosis, and to select better personalized treatments. The traditional view is that osteoarthritis is a degenerative disease of the elderly, mainly caused by the degeneration of articular cartilage caused by mechanical and age-related factors. However, multiple large-sample epidemiological surveys have shown that the incidence of osteoarthritis in joints such as hands, knees, and hips is significantly increased in children with low birth weight as adults[5 -8]. Among them, Australian scholar Hussain et al. conducted a clinical retrospective investigation in 2015 on 3,604 cases of hip and knee osteoarthritis diagnosed with hip and knee osteoarthritis. They found that people with low birth weight had a higher risk of hip osteoarthritis than those with normal weight. 2.04 times[7] . Another study by this scholar in 2018 confirmed that there is a clear causal relationship between low birth weight and adult hip osteoarthritis[8] . It is suggested that low birth weight is closely related to the occurrence of adult osteoarthritis. Previous series of studies in our laboratory have confirmed that offspring with low birth weight caused by maternal caffeine exposure during pregnancy develop articular cartilage dysplasia and are susceptible to osteoarthritis in adulthood[9] . However, the harm of adverse paternal environment on offspring's articular cartilage development and long-term osteoarthritis susceptibility remains unclear.
软骨形成是胚胎发育和脊椎动物骨骼修复中高度协调的事件[10-12]。在胚胎软骨形成过程中，间充质干细胞凝集分化为前软骨细胞团，并进一步形成由早期软骨细胞组成的软骨原基。随后，软骨原基中的软骨细胞增殖，并分泌大量的细胞外基质成分来促进软骨发育[13]。关节软骨是一种无神经、血管以及淋巴管的结缔组织，其退行性改变参与了骨关节炎的发生。软骨细胞是关节软骨中唯一的细胞类型，其在维持关节软骨稳态中扮演重要的角色。本发明前期的系列研究显示，母体孕期暴露于不良环境因素可以致子代关节软骨发育抑制并延续到出生后[14,15]。提示，软骨发育异常与骨关节炎的发生紧密相关。Chondrogenesis is a highly coordinated event in embryonic development and vertebrate bone repair[10-12] . During embryonic chondrogenesis, mesenchymal stem cells aggregate and differentiate into prechondrocyte clusters and further form cartilage primordia composed of early chondrocytes. Subsequently, chondrocytes in the cartilage primordium proliferate and secrete a large amount of extracellular matrix components to promote cartilage development[13] . Articular cartilage is a connective tissue without nerves, blood vessels and lymphatic vessels, and its degenerative changes are involved in the occurrence of osteoarthritis. Chondrocytes are the only cell type in articular cartilage and play an important role in maintaining articular cartilage homeostasis. A series of early studies of this invention showed that maternal exposure to adverse environmental factors during pregnancy can inhibit the development of articular cartilage in offspring and continue to after birth[14,15] . It is suggested that cartilage dysplasia is closely related to the occurrence of osteoarthritis.
众多的研究表明，表观遗传学是不良环境因素对疾病影响的一个重要机制，其可以从父母传给子代，并引起子代表型的改变[16-19]。DNA甲基化是一种主要的表观遗传修饰，其参与了生物体内的多种调控过程，包括基因表达的调控、基因组印记和X染色体失活等[20,21]。DNA甲基化对环境因素特别敏感，由不良环境因素引起的DNA甲基化改变会导致子代发育缺陷，并引起多种与发育缺陷相关的疾病[22-25]。研究表明，在世代间的表观遗传传递过程中，精子DNA甲基化组要比卵母细胞DNA甲基化组更重要[16,18,26]。如斑马鱼的精子DNA甲基化组在斑马鱼早期胚胎发生过程中遗传和维持，并可促进胚胎发生[27]；营养缺乏可改变父体精子DNA甲基化组，并有助于代谢性疾病在子代中的遗传[18]。在本研究中，本发明发现，父体孕前咖啡因暴露(paternal pre-pregnant caffeine exposure，PPCE)的精子Plagl1启动子高甲基化。此外，在PPCE雄性子代大鼠软骨中检测到印记基因Plagl1启动子多个位点高甲基化。由此，本发明推断，Plagl1启动子高甲基化可能在PPCE雄性子代软骨表型改变中发挥重要的调控作用。Numerous studies have shown that epigenetics is an important mechanism for the impact of adverse environmental factors on diseases. It can be passed from parents to offspring and cause changes in the offspring's phenotype[16 -19]. DNA methylation is a major epigenetic modification that is involved in various regulatory processes in organisms, including the regulation of gene expression, genomic imprinting, and X chromosome inactivation[20,21] . DNA methylation is particularly sensitive to environmental factors. DNA methylation changes caused by adverse environmental factors can lead to developmental defects in offspring and cause a variety of diseases related to developmental defects[22-25] . Studies have shown that in the process of epigenetic transmission between generations, the sperm DNA methylome is more important than the oocyte DNA methylome[16, 18, 26] . For example, the sperm DNA methylome of zebrafish is inherited and maintained during early embryogenesis of zebrafish and can promote embryogenesis[27] ; nutritional deficiency can change the father's sperm DNA methylome and contribute to metabolic Inheritance of disease in offspring[18] . In this study, the present invention found that the sperm Plagl1 promoter was hypermethylated in paternal pre-pregnant caffeine exposure (PPCE). In addition, hypermethylation of multiple sites in the promoter of the imprinted gene Plagl1 was detected in the cartilage of PPCE male offspring rats. Therefore, the present invention infers that Plagl1 promoter hypermethylation may play an important regulatory role in the cartilage phenotype changes of PPCE male offspring.
已知印记基因Plagl1通常是以其编码的锌指蛋白Zac1发挥转录调控作用。印记基因Plagl1及其编码的锌指蛋白Zac1具有良好的同源性，在人和啮齿类动物中的功能表现一致，并优先表达于父本等位基因。既往研究发现，由Plagl1编码的锌指蛋白Zac1通过调控功能基因表达，在小鼠发育和体外软骨形成过程中发挥着重要的作用[28]。在本研究中，本发明发现，PPCE雄性子代大鼠软骨中Plagl1的表达持续降低，且从宫内延续到出生后。同时，在体外胎软骨细胞培养过程中，敲低Plagl1可观察到软骨基质合成障碍，并且发现TGFβ信号通路相关组份TGFβR1表达的抑制。而通过过表达Plagl1可增加软骨基质含量，激活TGFβ信号通路。此外，在整体水平给予PPCE雄性子代大鼠腺相关病毒过表达Plagl1可以改善PPCE雄性子代软骨质量低下，并激活TGFβ信号通路。提示，Plagl1通过TGFβR1调控TGFβ信号通路功能影响PPCE雄性子代大鼠关节软骨发育。以上内容表明，软骨细胞中Plagl1可能作为父源性骨关节炎易感的治疗靶标，为父源性骨关节炎易感的早期防治应用提供干预靶标和实验依据。It is known that the imprinted gene Plagl1 usually plays a transcriptional regulatory role by encoding the zinc finger protein Zac1. The imprinted gene Plagl1 and its encoded zinc finger protein Zac1 have good homology, have consistent functional performance in humans and rodents, and are preferentially expressed in the paternal allele. Previous studies have found that the zinc finger protein Zac1 encoded by Plagl1 plays an important role in mouse development and in vitro cartilage formation by regulating functional gene expression[28] . In this study, the present invention found that the expression of Plagl1 in the cartilage of PPCE male offspring rats continued to decrease, and continued from intrauterine to postnatal. At the same time, during in vitro fetal chondrocyte culture, knockdown of Plagl1 observed impairment of cartilage matrix synthesis, and inhibition of the expression of TGFβR1, a component of the TGFβ signaling pathway. Overexpression of Plagl1 can increase cartilage matrix content and activate the TGFβ signaling pathway. In addition, giving PPCE male offspring rats adeno-associated virus overexpression of Plagl1 at the overall level can improve the poor cartilage quality of PPCE male offspring and activate the TGFβ signaling pathway. It is suggested that Plagl1 affects the articular cartilage development of PPCE male offspring rats by regulating the function of TGFβ signaling pathway through TGFβR1. The above content shows that Plagl1 in chondrocytes may serve as a therapeutic target for paternal osteoarthritis susceptibility, providing an intervention target and experimental basis for the early prevention and treatment of paternal osteoarthritis susceptibility.
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发明内容Contents of the invention
为了克服现有技术存在的不足，本发明的目的在于建立一种父源性骨关节炎动物模型，进一步确定印记基因Plagl1的启动子区DNA甲基化水平及其表达与父源性成年骨关节炎易感间的相互关系，为父源性骨关节炎易感的早期防治应用提供干预靶标和实验依据，即提供一种基于印记基因Plagl1作为父源性骨关节炎易感的干预靶标及其应用。In order to overcome the shortcomings of the existing technology, the purpose of the present invention is to establish an animal model of paternal osteoarthritis and further determine the DNA methylation level of the promoter region of the imprinted gene Plagl1 and its expression in relation to paternal adult bone joints. The relationship between susceptibility to inflammation provides an intervention target and experimental basis for the early prevention and treatment of paternal osteoarthritis susceptibility, that is, an intervention target based on the imprinted gene Plagl1 as a susceptibility to paternal osteoarthritis and its application.
本发明的目的通过下述技术方案实现：The object of the present invention is achieved through the following technical solutions:
第一方面，本发明提供一种父源性骨关节炎动物模型的构建方法，其特征在于：包括下列步骤：In a first aspect, the present invention provides a method for constructing an animal model of paternal osteoarthritis, which is characterized in that it includes the following steps:
S1：选取健康的6周龄成年雄性啮齿类动物，交配前每日给予60mg/kg咖啡因胃內灌注2月，自由饮食；S1: Healthy 6-week-old adult male rodents were selected and given intragastric infusion of 60 mg/kg caffeine daily for 2 months before mating, and they were allowed to eat freely;
S2：之后把咖啡因暴露的雄性成年啮齿类动物与正常成年雌性啮齿类动物交配，获得子代，以生产日作为出生后0天，出生后1天时选取窝仔数为12～14只的窝仔，调整每窝雄、雌性仔鼠各6只进行哺乳喂养；S2: Then, the caffeine-exposed male adult rodents were mated with normal adult female rodents to obtain offspring. The day of birth was taken as day 0 after birth, and litters with a litter size of 12 to 14 were selected on day 1 after birth. pups, adjust each litter of 6 male and 6 female pups for lactation;
S3：仔鼠出生后4周断奶并雄、雌分笼，部分继续正常饮食饲养至8周；S3: The pups are weaned 4 weeks after birth and placed in separate cages for males and females, and some continue to be fed a normal diet until 8 weeks;
S4：部分子代饲养至出生后12周检测关节软骨质量，以及进行长距离跑步造模后最终得到父源性骨关节炎动物模型。S4: Some of the offspring were raised until 12 weeks after birth to detect the quality of articular cartilage, and the paternal osteoarthritis animal model was finally obtained after long-distance running modeling.
作为优选方案，所述步骤S1中，啮齿类动物为SPF级Wistar、SD大鼠或昆明种小鼠。As a preferred version, in step S1, the rodent is an SPF grade Wistar, SD rat or Kunming mouse.
进一步地，所述步骤S3中，正常饮食是配方与《中华人民共和国国家标准GB14924.3-2001》规定的小鼠、大鼠配方饲料相同。Further, in step S3, the normal diet has the same formula as the formula feed for mice and rats specified in the "National Standard of the People's Republic of China GB14924.3-2001".
第二方面，本发明提供一种基于印记基因Plagl1作为父源性骨关节炎易感的干预靶标。In the second aspect, the present invention provides an intervention target based on the imprinted gene Plagl1 as a susceptibility to paternal osteoarthritis.
作为优选方案，所述扩增Plagl1基因的引物对序列如SEQ ID No.1和SEQ ID No.2所示；所述检测Plagl1基因启动子区DNA甲基化水平的引物对如SEQ ID No.3和SEQ IDNo.4所示。As a preferred embodiment, the sequence of the primer pair for amplifying the Plagl1 gene is as shown in SEQ ID No. 1 and SEQ ID No. 2; the sequence of the primer pair for detecting the DNA methylation level in the promoter region of the Plagl1 gene is as shown in SEQ ID No. 3 and SEQ IDNo.4.
第三方面，本发明提供一种父源性骨关节炎动物模型在筛选父源性骨关节炎的干预靶标中的应用，其特征在于：所述父源性骨关节炎动物模型由上述任一方法构建得到，所述动物模型中印记基因Plagl1的启动子区甲基化水平增加，伴随着Plagl1表达降低。优选地，所述父源性骨关节炎的干预靶标为干预Plagl1表达后能改善父源性骨关节炎样表型。In a third aspect, the present invention provides an application of an animal model of paternal osteoarthritis in screening intervention targets for paternal osteoarthritis, characterized in that: the animal model of paternal osteoarthritis is composed of any of the above The method was constructed to show that in the animal model, the methylation level of the promoter region of the imprinted gene Plagl1 increased, accompanied by a decrease in Plagl1 expression. Preferably, the intervention target for paternal osteoarthritis is to improve the paternal osteoarthritis-like phenotype after interfering with Plagl1 expression.
本发明的技术原理及研究过程如下：The technical principle and research process of the present invention are as follows:
本发明通过研究证实，与对照组相比，PPCE组父体精子Plagl1启动子区DNA甲基化水平显著增加，同时胎软骨中Plagl1表达降低，基因启动子区DNA甲基化水平增加，并持续到出生后。提示，PPCE组父体精子与胎软骨中Plagl1 DNA甲基化水平具有一致性。The present invention has confirmed through research that compared with the control group, the DNA methylation level in the Plagl1 promoter region of the father's sperm in the PPCE group increased significantly, while the expression of Plagl1 in fetal cartilage decreased, and the DNA methylation level in the gene promoter region increased and continued. to after birth. It was suggested that the Plagl1 DNA methylation levels in the paternal sperm and fetal cartilage in the PPCE group were consistent.
接着，本发明人发现PPCE组及对照组在给予长距离跑步进行骨关节炎造模后，PPCE组相较于对照组，其雄性子代骨关节炎改良Mankin评分增加，并且存在骨关节炎易感性增加的现象。而软骨中Plagl1在出生后的持续低表达，很可能是介导软骨发育不良的主要机制，从而引起子代成年骨关节炎易感性增加。即PPCE组胎软骨中Plagl1表达降低、甲基化水平增加，出生后骨关节炎发生风险更高，有较好的一致性。Next, the inventor found that after the PPCE group and the control group were given long-distance running for osteoarthritis modeling, the male offspring of the PPCE group had an increased osteoarthritis modified Mankin score compared with the control group, and were prone to osteoarthritis. The phenomenon of increased sensibility. The sustained low expression of Plagl1 in cartilage after birth is likely to be the main mechanism mediating chondrodysplasia, thereby causing the offspring to increase their susceptibility to adult osteoarthritis. That is, the expression of Plagl1 in fetal cartilage in the PPCE group was reduced, the methylation level was increased, and the risk of postnatal osteoarthritis was higher, with good consistency.
综上，Plagl1可为父源性骨关节炎的早期干预靶标并用于早期防治父源性骨关节炎的应用。In summary, Plagl1 can be an early intervention target for paternal osteoarthritis and be used for early prevention and treatment of paternal osteoarthritis.
本发明相对于现有技术具有如下的优点及效果：Compared with the existing technology, the present invention has the following advantages and effects:
1、本发明的技术构思贴近日常生活且较为新颖，选取了咖啡因这种日常暴露的外源物，使得模型构建更具备广泛适用性。1. The technical concept of the present invention is close to daily life and relatively novel. It selects caffeine, an exogenous substance that is exposed daily, to make the model construction more widely applicable.
2、本发明的动物造模方法简单可靠，能够模拟父体不良环境暴露所致子代骨关节炎易感的现象。本模型指标稳定性好，可重复性强，为父源性骨关节炎的构建提供了一种可靠的方法。2. The animal modeling method of the present invention is simple and reliable, and can simulate the susceptibility of offspring to osteoarthritis caused by adverse environmental exposure of the father. This model has good index stability and strong reproducibility, providing a reliable method for the construction of paternal osteoarthritis.
3、本发明为父体不良环境暴露动物模型，与母体孕期不良环境暴露动物模型具有显著的区别。本模型为PPCE模型，尚无相关动物模型的构建方法及骨关节炎方面的应用。3. The present invention is an animal model of maternal adverse environmental exposure, which is significantly different from the maternal adverse environmental exposure animal model during pregnancy. This model is a PPCE model, and there are no relevant animal model construction methods and applications in osteoarthritis.
4、基于本发明构建的父源性骨关节炎疾病模型，可用于指导男性合理备孕和临床合理用药、探讨父源性骨关节炎的发生发展机制、早期干预靶标中的应用。4. The paternal osteoarthritis disease model constructed based on the present invention can be used to guide men in rational preparation for pregnancy and clinical rational use of drugs, explore the occurrence and development mechanism of paternal osteoarthritis, and be used in early intervention targets.
附图说明Description of the drawings
图1.PPCE雄性成年子代大鼠长距离跑步后软骨表型的改变。Figure 1. Changes in cartilage phenotype in PPCE male adult offspring rats after long-distance running.
图中：A.软骨组织H&E染色；B.软骨组织番红O-固绿染色；C.软骨的Mankin’s评分。In the picture: A. H&E staining of cartilage tissue; B. Safranin O-fast green staining of cartilage tissue; C. Mankin’s score of cartilage.
图2.PPCE雄性子代宫内及出生后早期关节软骨基质含量改变。Figure 2. Changes in articular cartilage matrix content in PPCE male offspring in utero and early postnatal period.
图中：A.胎鼠软骨组织番红固绿染色；B.胎鼠软骨组织的Col2a和ACAN的mRNA表达；C.出生后6周(PW6)和出生后12周(PW12)软骨组织番红O-固绿染色；D.PW6和PW12软骨组织的Col2a和ACAN的mRNA表达。In the picture: A. Safranin fast green staining of fetal mouse cartilage tissue; B. Col2a and ACAN mRNA expression in fetal mouse cartilage tissue; C. Safranin cartilage tissue at 6 weeks after birth (PW6) and 12 weeks after birth (PW12) O-fast green staining; D. mRNA expression of Col2a and ACAN in PW6 and PW12 cartilage tissues.
图3.PPCE可致父体精子及子代软骨Plagl1启动子高甲基化及低表达。Figure 3. PPCE can cause hypermethylation and low expression of Plagl1 promoter in paternal sperm and offspring cartilage.
图中：A.精子中印记基因Plagl1的甲基化；B.胎鼠软骨中印记基因Plagl1的甲基化；C.出生后12周(PW12)软骨中印记基因Plagl1的甲基化；D.出生前、后软骨组织中Plagl1的mRNA表达检测；E.出生前、后软骨组织中Plagl1的蛋白免疫组化检测；F.出生前、后软骨组织中Plagl1的蛋白免疫组化定量分析值。In the picture: A. Methylation of imprinted gene Plagl1 in sperm; B. Methylation of imprinted gene Plagl1 in fetal mouse cartilage; C. Methylation of imprinted gene Plagl1 in cartilage at 12 weeks after birth (PW12); D. Detection of Plagl1 mRNA expression in prenatal and postnatal cartilage tissue; E. Immunohistochemical detection of Plagl1 protein in prenatal and postnatal cartilage tissue; F. Quantitative immunohistochemical analysis value of Plagl1 protein in prenatal and postnatal cartilage tissue.
图4.动物水平过表达Plagl1可改善PPCE雄性子代关节软骨基质含量降低。Figure 4. Overexpression of Plagl1 at the animal level can improve the decrease in articular cartilage matrix content in male offspring of PPCE.
图中：A.软骨组织番红固绿染色；B.软骨组织的Col2a1和ACAN的mRNA表达；C.软骨组织中Col2a1的蛋白免疫组化检测；D.软骨组织中Col2a1的蛋白免疫组化定量分析值。In the picture: A. Safranin-fast green staining of cartilage tissue; B. mRNA expression of Col2a1 and ACAN in cartilage tissue; C. Immunohistochemical detection of Col2a1 protein in cartilage tissue; D. Immunohistochemical quantification of Col2a1 protein in cartilage tissue Analytical value.
具体实施方式Detailed ways
通过以下详细说明结合附图可以进一步理解本发明的特点和优点。所提供的实施例仅是对本发明方法的说明，而不以任何方式限制本发明揭示的其余内容。The features and advantages of the present invention can be further understood from the following detailed description in conjunction with the accompanying drawings. The provided embodiments are only illustrative of the method of the present invention and do not limit the remaining contents disclosed in the present invention in any way.
【实施例1】本发明父源性骨关节炎动物模型的构建[Example 1] Construction of paternal osteoarthritis animal model of the present invention
1、动物和处理1. Animals and handling
SPF级Wistar大鼠(年龄：6周；体重：200-220g)购自于湖北省疾病预防控制中心。所有的动物均在标准条件下(室温：18-22℃；湿度：40％-60％；光照周期：12h)适应性喂养2周。随后，将雄性Wister大鼠随机分为两组：对照组(Control-F0，生理盐水灌胃)和PPCE组(PPCE-F0,咖啡因灌胃：60mg/kg.d)。干预结束之后，将雄性Wistar大鼠和正常雌性大鼠每日于18：00按照1：2的比例进行合笼交配。次日6：00，将合笼交配后的雌性大鼠行阴道涂片镜检。如果涂片在镜下检查发现存在精子，证明该雌鼠受孕成功，并将雌鼠标记为孕0天(gestational day 0，GD0)。在GD20时，将部分孕鼠在2％异氟烷麻醉下安乐处死。剥离胎鼠，记录胎仔数，将胎仔数8-16只纳入到研究中。对于胎鼠膝关节软骨取材，本发明将胎鼠置于冰上，用镊子对其双下肢进行分离。随机选取右下肢固定到4％多聚甲醛中，后续进行脱水、包埋、切片和染色，将左下肢放置于-80℃冰箱，待后续提取RNA进行基因表达检测。对照组和PPCE组剩下的孕鼠保留到正常分娩(n＝8)。随后根据出生时间，将每窝中雄性子代随机分为4批，分别标记为出生后6周(postnatal week 6,PW6)、PW12、PW26和PW32周。对于PW26周雄性子代大鼠，本发明通过长距离跑步造模的方式进行骨关节炎造模。分组如下：Control-F1、PPCE-F1、Control-F1(Running)和PPCE-F1(Running)。长距离跑步造模方式如下：子代大鼠放置于小动物跑步机，10°倾斜，速度15m/min，持续45min，每天一次，持续6周之后获取仔鼠关节软骨组织待测。SPF grade Wistar rats (age: 6 weeks; weight: 200-220g) were purchased from Hubei Provincial Center for Disease Control and Prevention. All animals were adaptively fed for 2 weeks under standard conditions (room temperature: 18-22°C; humidity: 40%-60%; photoperiod: 12h). Subsequently, male Wister rats were randomly divided into two groups: control group (Control-F0, intragastric administration of normal saline) and PPCE group (PPCE-F0, intragastric administration of caffeine: 60 mg/kg.d). After the intervention, male Wistar rats and normal female rats were caged and mated in a ratio of 1:2 every day at 18:00. At 6:00 the next day, vaginal smears were performed on the female rats after mating. If the smear shows the presence of sperm under the microscope, it proves that the female mouse was successfully conceived and the female mouse is marked as gestational day 0 (GD0). At GD20, some pregnant mice were euthanized under 2% isoflurane anesthesia. The fetal rats were dissected, the number of litters was recorded, and the number of litters 8-16 were included in the study. For the extraction of fetal rat knee joint cartilage, the present invention places the fetal rat on ice and separates its lower limbs with tweezers. The right lower limb was randomly selected and fixed in 4% paraformaldehyde, followed by dehydration, embedding, sectioning and staining. The left lower limb was placed in a -80°C refrigerator for subsequent extraction of RNA for gene expression detection. The remaining pregnant rats in the control group and PPCE group were retained until normal delivery (n=8). Then, the male offspring in each litter were randomly divided into 4 batches according to their birth time, which were marked as postnatal week 6 (PW6), PW12, PW26 and PW32 weeks respectively. For PW26-week-old male offspring rats, the present invention uses long-distance running to model osteoarthritis. The groups are as follows: Control-F1, PPCE-F1, Control-F1(Running), and PPCE-F1(Running). The long-distance running modeling method is as follows: offspring rats are placed on a small animal treadmill with a 10° inclination and a speed of 15m/min for 45 minutes, once a day for 6 weeks. After 6 weeks, the articular cartilage tissue of the offspring rats is harvested for testing.
2、软骨组织番红固绿染色2. Safranin-fast green staining of cartilage tissue
石蜡切片脱蜡至水，将组织切片放入固绿染液中5min，自来水洗去多余的染液直至软骨部呈无色，随后用苏木素分化液浸泡8s并用自来水稍洗。随后，将软骨组织切片放入番红染液中10s，并将切片依次放入4缸无水乙醇快速脱水。待用干净的环保型透明剂透明5min后，用中性树胶封片，并在显微镜下观察。Dewax the paraffin sections to water, put the tissue sections into fast green staining solution for 5 minutes, wash away excess dyeing solution with tap water until the cartilage becomes colorless, then soak in hematoxylin differentiation solution for 8 s and wash briefly with tap water. Subsequently, the cartilage tissue sections were placed in safranin staining solution for 10 s, and the sections were placed in 4 cylinders of absolute ethanol for rapid dehydration. After being transparent for 5 minutes with a clean environmentally friendly transparent agent, seal the slide with neutral gum and observe it under a microscope.
3、软骨组织HE染色3. HE staining of cartilage tissue
石蜡切片脱蜡至水，苏木素染色5min，自来水迅速洗去多余苏木素，再置于1％盐酸酒精10s，自来水稍洗，1％稀氨水返蓝10s，自来水洗5min，沥干切片的水，最后用伊红染液染色3min，并浸入梯度乙醇(80％，95％，100％)中脱水各2min。用两缸干净的环保型透明剂各透明5min后，将切片自然晾干，再用中性树胶封片，显微镜下观察拍照。Dewax the paraffin sections to water, stain with hematoxylin for 5 minutes, quickly wash away excess hematoxylin with tap water, then place in 1% hydrochloric acid alcohol for 10 seconds, wash briefly with tap water, return to blue with 1% dilute ammonia water for 10 seconds, wash with tap water for 5 minutes, drain the water from the sections, and finally Stain with eosin staining solution for 3 minutes, and then immerse in gradient ethanol (80%, 95%, 100%) for dehydration for 2 minutes each. Use two bottles of clean environmentally friendly transparent agent to clear the sections for 5 minutes each, dry the sections naturally, seal them with neutral gum, and observe and take pictures under a microscope.
4、基因表达检测4. Gene expression detection
从NCBI网站(http：//www.ncbi.nlm.nih.gov)下载目的基因和管家基因的mRNA序列，应用primer5.0设计上下游引物，并在NCBI网站上行核苷酸Blast同源比对，确证扩增片段的特异性。将大鼠软骨用于总RNA提取，之后逆转录为cDNA，并通过RT-PCR进行定量分析相关基因表达情况。各基因引物序列如下表1所示。Download the mRNA sequences of the target gene and housekeeping gene from the NCBI website (http://www.ncbi.nlm.nih.gov), use primer5.0 to design upstream and downstream primers, and perform nucleotide Blast homology alignment on the NCBI website , confirm the specificity of the amplified fragments. Rat cartilage was used for total RNA extraction, and then reverse transcribed into cDNA, and the expression of related genes was quantitatively analyzed by RT-PCR. The primer sequences of each gene are shown in Table 1 below.
表1.qRT-PCR引物序列及退火温度。Table 1. qRT-PCR primer sequences and annealing temperatures.
5、数据处理及分析5. Data processing and analysis
SPSS 20(SPSS Science Inc.,Chicago,Illinois)和GraphPad Prism 6.01(Graph Pad Software,La Jolla,CA,USA)软件用于数据处理及分析。以平均值±标准误(Mean±S.E.M.)来表示定量数据。采用Student’s t-test来进行两组之间的均值比较。采用Mann-Whitney检验对改良Mankin评分进行分析；采用Two-way ANOVA检验用于PPCE组在ACLT前后免疫组化数据的分析。将P<0.05定义为具有统计学上的显著性差异。SPSS 20 (SPSS Science Inc., Chicago, Illinois) and GraphPad Prism 6.01 (Graph Pad Software, La Jolla, CA, USA) software were used for data processing and analysis. Quantitative data are expressed as mean ± S.E.M. Student’s t-test was used to compare means between two groups. The Mann-Whitney test was used to analyze the modified Mankin score; the Two-way ANOVA test was used to analyze the immunohistochemical data of the PPCE group before and after ACLT. P<0.05 was defined as a statistically significant difference.
6.实验结果6.Experimental results
6.1PPCE雄性成年子代大鼠长距离跑步后软骨表型的改变6.1 Changes in cartilage phenotype of PPCE male adult offspring rats after long-distance running
为了探究PPCE对雄性成年子代大鼠骨关节炎易感的影响，通过长距离跑步这种方式对成年雄性子代大鼠进行骨关节炎造模来评估软骨表型的改变。HE染色的结果显示，对照组子代大鼠关节软骨表面完整；而长距离跑步后造模组关节软骨表面粗糙不平，且PPCE-F1(Running)组软骨层结构明显紊乱(图1中A)。番红-固绿染色的结果发现，与Control-F1组相比，PPCE-F1组关节软骨番红着色稍变浅(图1中B)。在长距离跑步造模后，Control-F1和PPCE-F1组关节软骨面均出现了不同程度的破坏，而与Control-F1组相比，PPCE-F1组关节软骨病理评分显著增加(图1中C)。以上结果表明，PPCE可致雄性成年子代大鼠对骨关节炎的易感性增加。In order to explore the effect of PPCE on the susceptibility of male adult offspring rats to osteoarthritis, long-distance running was used to create an osteoarthritis model in adult male offspring rats to evaluate the changes in cartilage phenotype. The results of HE staining showed that the articular cartilage surface of offspring rats in the control group was intact; while the articular cartilage surface of the model group after long-distance running was rough and uneven, and the cartilage layer structure of the PPCE-F1 (Running) group was obviously disordered (A in Figure 1) . The results of safranin-fast green staining showed that compared with the Control-F1 group, the safranin staining of articular cartilage in the PPCE-F1 group was slightly lighter (B in Figure 1). After long-distance running modeling, the articular cartilage surfaces of the Control-F1 and PPCE-F1 groups were damaged to varying degrees. Compared with the Control-F1 group, the pathological score of the articular cartilage in the PPCE-F1 group was significantly increased (Figure 1 C). The above results indicate that PPCE can increase the susceptibility of male adult offspring rats to osteoarthritis.
6.2PPCE雄性子代宫内及出生后早期关节软骨基质含量改变6.2 Changes in articular cartilage matrix content in PPCE male offspring in utero and early postnatal period
为了进一步探究PPCE所致的雄性成年子代大鼠骨关节炎易感是否与宫内及出生后早期关节软骨发育不良有关。首先，本发明通过番红-固绿染色观察了PPCE-F1雄性子代宫内关节软骨形态及基质含量的变化。结果发现，与Control-F1组相比，PPCE-F1组番红着色较浅(图2中A)。进一步，本发明通过qRT-PCR检测了软骨基质相关的基因Col2a1和ACANmRNA的表达。结果显示，与Control-F1组相比，PPCE-F1组Col2a1和ACAN mRNA表达均显著降低(图2中B)。此外，本发明通过组织免疫荧光染色检测了关节软骨Col2a1的蛋白表达。结果显示，与Control-F1组相比，PPCE-F1组荧光强度较弱，提示蛋白表达降低。为了进一步探究PPCE对雄性子代大鼠出生后的软骨基质含量的变化，本发明进行了番红-固绿染色来检测软骨基质含量的改变。结果显示，与Control-F1组相比，PPCE-F1组雄性大鼠在PW6、PW12关节软骨番红着色均较浅，提示关节软骨基质含量降低(图2中C)。进一步，本发明通过qRT-PCR检测了软骨基质相关基因Col2a1和ACAN mRNA的表达。结果发现，与Control-F1组相比，PPCE-F1组雄性大鼠在PW6、PW12关节软骨Col2a1和ACAN mRNA的表达均显著降低(图2中D)。此外，本发明通过免疫组织化学染色检测了Col2a1的蛋白表达，结果显示，与Control-F1组相比，PPCE-F1组雄性大鼠在PW6、PW12关节软骨蛋白表达显著降低(图2中D)。以上结果表明，PPCE可致雄性子代大鼠关节软骨发育不良从宫内延续到出生后。To further explore whether the susceptibility to osteoarthritis in male adult offspring rats caused by PPCE is related to intrauterine and early postnatal articular cartilage dysplasia. First, the present invention observed the changes in intrauterine articular cartilage morphology and matrix content of PPCE-F1 male offspring through safranin-fast green staining. The results showed that compared with the Control-F1 group, the safranin coloration of the PPCE-F1 group was lighter (A in Figure 2). Furthermore, the present invention detected the expression of cartilage matrix-related genes Col2a1 and ACANmRNA through qRT-PCR. The results showed that compared with the Control-F1 group, the expression of Col2a1 and ACAN mRNA in the PPCE-F1 group was significantly reduced (Figure 2, B). In addition, the present invention detected the protein expression of Col2a1 in articular cartilage through tissue immunofluorescence staining. The results showed that compared with the Control-F1 group, the fluorescence intensity of the PPCE-F1 group was weaker, indicating reduced protein expression. In order to further explore the changes in cartilage matrix content of PPCE on male offspring rats after birth, the present invention performed safranin-fast green staining to detect changes in cartilage matrix content. The results showed that compared with the Control-F1 group, the safranin staining of the articular cartilage of male rats in the PPCE-F1 group was lighter at PW6 and PW12, indicating that the articular cartilage matrix content was reduced (C in Figure 2). Furthermore, the present invention detected the expression of cartilage matrix-related genes Col2a1 and ACAN mRNA through qRT-PCR. The results showed that compared with the Control-F1 group, the expression of Col2a1 and ACAN mRNA in the articular cartilage of PW6 and PW12 male rats in the PPCE-F1 group was significantly reduced (D in Figure 2). In addition, the present invention detected the protein expression of Col2a1 through immunohistochemical staining. The results showed that compared with the Control-F1 group, the expression of cartilage protein in the PW6 and PW12 joints of male rats in the PPCE-F1 group was significantly reduced (D in Figure 2) . The above results indicate that PPCE can cause articular cartilage dysplasia in male offspring rats from intrauterine to postnatal.
综上所述：PPCE可导致子代关节软骨基质合成障碍，从而引起子代大鼠骨关节炎易感。说明本方法是建立父源性骨关节炎动物模型的有效方法，可用于研究父源性骨关节炎的发生机制，基于此进一步指导应用于父体孕前不良环境所致骨关节炎的早期干预靶标发现及防治策略中的应用。In summary: PPCE can cause impairment of articular cartilage matrix synthesis in offspring, thereby making offspring rats susceptible to osteoarthritis. It shows that this method is an effective method to establish an animal model of paternal osteoarthritis, and can be used to study the mechanism of paternal osteoarthritis. Based on this, further guidance can be applied to the early intervention targets of osteoarthritis caused by the adverse environment of the paternal body before pregnancy. Application in detection and prevention strategies.
【实施例2】本发明父源性骨关节炎的干预靶标确证和应用[Example 2] Confirmation and application of intervention targets for paternal osteoarthritis of the present invention
1、动物和处理1. Animals and handling
SPF级Wistar大鼠(年龄：6周；体重：200-220g)购自于湖北省疾病预防控制中心。所有的动物均在标准条件下(室温：18-22℃；湿度：40％-60％；光照周期：12h)适应性喂养2周。随后，将雄性Wister大鼠随机分为两组：对照组(Control-F0,生理盐水灌胃)和PPCE组(PPCE-F0,咖啡因灌胃：60mg/kg.d)。干预结束之后，将雄性Wistar大鼠和正常雌性大鼠每日于18：00按照1：2的比例进行合笼交配。次日6：00，将合笼交配后的雌性大鼠行阴道涂片镜检。如果涂片在镜下检查发现存在精子，证明该雌鼠受孕成功，并将雌鼠标记为孕0天(gestational day 0，GD0)。在GD20时，将部分孕鼠在2％异氟烷麻醉下安乐处死。剥离胎鼠，记录胎仔数，将胎仔数8-16只纳入到研究中。对于胎鼠膝关节软骨取材，本发明将胎鼠置于冰上，用镊子对其双下肢进行分离。随机选取右下肢固定到4％多聚甲醛中，后续进行脱水、包埋、切片和染色，将左下肢放置于-80℃冰箱，待后续提取RNA进行基因表达检测。对照组和PPCE组剩下的孕鼠保留到正常分娩(n＝8)。随后根据出生时间，将每窝中雄性子代随机分为4批，分别标记为出生后6周(postnatal week,PW)、PW12、PW26和PW32周。对于PW26周雄性子代大鼠，本发明通过长距离跑步造模的方式进行骨关节炎造模。分组如下：Control-F1、PPCE-F1、Control-F1(Running)和PPCE-F1(Running)。长距离跑步造模方式如下：子代大鼠放置于小动物跑步机，10°倾斜，速度15m/min，持续45min，每天一次，持续6周之后获取仔鼠关节软骨组织待测。SPF grade Wistar rats (age: 6 weeks; weight: 200-220g) were purchased from Hubei Provincial Center for Disease Control and Prevention. All animals were adaptively fed for 2 weeks under standard conditions (room temperature: 18-22°C; humidity: 40%-60%; photoperiod: 12h). Subsequently, male Wister rats were randomly divided into two groups: control group (Control-F0, intragastric administration of normal saline) and PPCE group (PPCE-F0, intragastric administration of caffeine: 60 mg/kg.d). After the intervention, male Wistar rats and normal female rats were caged and mated in a ratio of 1:2 every day at 18:00. At 6:00 the next day, vaginal smears were performed on the female rats after mating. If the smear shows the presence of sperm under the microscope, it proves that the female mouse was successfully conceived and the female mouse is marked as gestational day 0 (GD0). At GD20, some pregnant mice were euthanized under 2% isoflurane anesthesia. The fetal rats were dissected, the number of litters was recorded, and the number of litters 8-16 were included in the study. For the extraction of fetal rat knee joint cartilage, the present invention places the fetal rat on ice and separates its lower limbs with tweezers. The right lower limb was randomly selected and fixed in 4% paraformaldehyde, followed by dehydration, embedding, sectioning and staining. The left lower limb was placed in a -80°C refrigerator for subsequent extraction of RNA for gene expression detection. The remaining pregnant rats in the control group and PPCE group were retained until normal delivery (n=8). Then, the male offspring in each litter were randomly divided into 4 batches according to their birth time, which were marked as postnatal week (PW), PW12, PW26 and PW32 weeks respectively. For PW26-week-old male offspring rats, the present invention uses long-distance running to model osteoarthritis. The groups are as follows: Control-F1, PPCE-F1, Control-F1(Running), and PPCE-F1(Running). The long-distance running modeling method is as follows: offspring rats are placed on a small animal treadmill with a 10° inclination and a speed of 15m/min for 45 minutes, once a day for 6 weeks. After 6 weeks, the articular cartilage tissue of the offspring rats is harvested for testing.
2、精子全基因组甲基化测序分析及软骨Plagl1启动子甲基化检测2. Sperm whole genome methylation sequencing analysis and cartilage Plagl1 promoter methylation detection
取F0代精子进行全基因甲基化测序，测序委托北京诺禾致源科技股份有限公司完成。取PPCE雄性子代GD20和PW12软骨样本进行基因启动子甲基化测序分析，分析委托上海天昊生物科技有限公司完成。F0 generation sperm were collected for whole-gene methylation sequencing, which was completed by Beijing Novogene Technology Co., Ltd. Cartilage samples of GD20 and PW12 male offspring of PPCE were taken for gene promoter methylation sequencing analysis. The analysis was entrusted to Shanghai Tianhao Biotechnology Co., Ltd. to complete.
3、软骨Plagl1免疫组化和定量分析3. Immunohistochemistry and quantitative analysis of cartilage Plagl1
免疫组化过程使用SP方法，根据试剂商提供的步骤完成。石蜡包埋组织被切成5μm厚度，染色用兔抗-Plagl1单克隆抗体。每个软骨切片分区统计检测(n＝4)。使用光学显微镜成像，通过Image-Pro Plus6.0软件对结果进行定量分析。The immunohistochemistry process uses the SP method and is completed according to the steps provided by the reagent supplier. Paraffin-embedded tissues were sectioned at 5 μm thickness and stained with rabbit anti-Plagl1 monoclonal antibody. Statistical testing of each cartilage section partition (n=4). Optical microscopy imaging was used, and the results were quantitatively analyzed by Image-Pro Plus6.0 software.
4、Plagl1过表达4. Plagl1 overexpression
分别往250μl无血清培养基中加入5μl pcDNA3.1(+)Plagl1过表达质粒和5μlpcDNA3.1(+)-GFP NC质粒。待混匀静置10min后，向上述液体中各加入250μl含有8μllipo3000的无血清培养基，吹打混匀后静置15min。最后向细胞培养板中各加入500μl混合转染试剂，补充1.5ml全培养基，待转染8h后，换成2ml全培养基进行培养24h。最后收细胞提RNA或者蛋白进行后续实验。Add 5 μl of pcDNA3.1(+)Plagl1 overexpression plasmid and 5 μl of pcDNA3.1(+)-GFP NC plasmid to 250 μl of serum-free medium respectively. After mixing and letting stand for 10 minutes, add 250 μl of serum-free medium containing 8 μllipo3000 to each of the above liquids, mix by pipetting and let stand for 15 minutes. Finally, add 500 μl of mixed transfection reagent to each cell culture plate and supplement 1.5 ml of complete culture medium. After 8 hours of transfection, replace with 2 ml of complete culture medium and culture for 24 hours. Finally, the cells were collected to extract RNA or protein for subsequent experiments.
5、免疫细胞荧光染色5. Fluorescence staining of immune cells
细胞接种于六孔板中进行爬片处理，待进行上述转染步骤后,用4％的多聚甲醛进行细胞固定15min。用PBS洗涤3次，随后向每孔中加入1ml 0.1％ Triton X 100进行细胞破膜15min。待用PBS进行洗涤之后，加入3％的BSA室温封闭30min。向每孔的爬片中分别滴加100μl稀释后的一抗(Col2a1)置于4℃孵育过夜。第二天，回收一抗，加入CY3标记的荧光二抗，常温孵育1h。待用PBS洗涤后，加入DAPI溶液复染核。最后用抗荧光淬灭剂封片之后，置于荧光显微镜下观察。The cells were seeded in a six-well plate and processed for climbing. After the above-mentioned transfection steps, the cells were fixed with 4% paraformaldehyde for 15 minutes. Wash three times with PBS, and then add 1 ml of 0.1% Triton X 100 to each well for cell membrane rupture for 15 min. After washing with PBS, add 3% BSA and block at room temperature for 30 minutes. Add 100 μl of diluted primary antibody (Col2a1) to the slide in each well and incubate at 4°C overnight. The next day, recover the primary antibody, add CY3-labeled fluorescent secondary antibody, and incubate at room temperature for 1 hour. After washing with PBS, add DAPI solution to counterstain the nuclei. Finally, the slides were sealed with anti-fluorescence quenching agent and observed under a fluorescence microscope.
6、腺相关病毒关节腔注射6. Adeno-associated virus intra-articular injection
在PW4时，分别从对照组和PPCE组的每一窝子代中随机选取两只雄性大鼠，按照以下分组分为四组(n＝8/组)：Control-F1(ssAAV.NC)、Control-F1(ssAAV.Plagl1)、PPCE-F1(ssAAV.NC)和PPCE-F1(ssAAV.Plagl1)。在PW8周时，Control-F1(ssAAV.Plagl1)和PPCE-F1(ssAAV.Plagl1)中的每只雄性大鼠以关节腔注射的方式给予Plagl1过表达腺相关病毒。Control-F1(ssAAV.NC)和PPCE-F1(ssAAV.NC)中的每只大鼠以关节腔注射的方式给予阴性对照腺相关病毒。在PW12时，以麻醉的方式安乐死大鼠，取右膝关节固定后进行后续脱钙、包埋、切片和染色；取左膝关节冻存于-80℃深低温冰箱进行后续基因表达检测。At PW4, two male rats were randomly selected from each litter of the control group and PPCE group and divided into four groups (n=8/group) according to the following groups: Control-F1 (ssAAV.NC), Control-F1(ssAAV.Plagl1), PPCE-F1(ssAAV.NC), and PPCE-F1(ssAAV.Plagl1). At PW8 weeks, each male rat in Control-F1 (ssAAV.Plagl1) and PPCE-F1 (ssAAV.Plagl1) was given Plagl1 overexpression adeno-associated virus by intra-articular injection. Each rat in Control-F1(ssAAV.NC) and PPCE-F1(ssAAV.NC) was given negative control adeno-associated virus by intra-articular injection. At PW12, the rats were euthanized under anesthesia, and the right knee joint was removed and fixed for subsequent decalcification, embedding, sectioning, and staining. The left knee joint was frozen and stored in a -80°C deep-freezing refrigerator for subsequent gene expression detection.
7、数据处理及分析7. Data processing and analysis
SPSS 20(SPSS Science Inc.,Chicago,Illinois)和GraphPad Prism 6.01(Graph Pad Software,La Jolla,CA,USA)软件用于数据处理及分析。以平均值±标准误(Mean±S.E.M.)来表示定量数据。采用Student’s t-test来进行两组之间的均值比较。采用Mann-Whitney检验对改良Mankin评分进行分析；采用Two-way ANOVA检验用于PPCE组在ACLT前后免疫组化数据的分析。将P<0.05定义为具有统计学上的显著性差异。SPSS 20 (SPSS Science Inc., Chicago, Illinois) and GraphPad Prism 6.01 (Graph Pad Software, La Jolla, CA, USA) software were used for data processing and analysis. Quantitative data are expressed as mean ± S.E.M. Student’s t-test was used to compare means between two groups. The Mann-Whitney test was used to analyze the modified Mankin score; the Two-way ANOVA test was used to analyze the immunohistochemical data of the PPCE group before and after ACLT. P<0.05 was defined as a statistically significant difference.
8.实验结果8.Experimental results
8.1PPCE可致父体精子及子代软骨Plagl1启动子高甲基化及低表达8.1PPCE can cause hypermethylation and low expression of Plagl1 promoter in paternal sperm and offspring cartilage
为了进一步探究精子表观遗传重编程改变在PPCE父体-子代获得性表型中的作用，本发明首先对父体(F0)精子进行了全基因组DNA甲基化测序分析，通过使用IGV_2.8.9软件筛选发现，与Control-F0组相比，PPCE-F0组精子中Plagl1基因启动子甲基化水平增加(图3中A)。此外，对雄性子代GD20、PW12关节软骨中Plagl1基因启动子甲基化检测发现，与Control-F1组相比，PPCE-F1组雄性大鼠关节软骨中Plagl1基因启动子多个位点的甲基化水平显著增加(图3中B和C)。以上结果表明，PPCE所致的父体精子Plagl1基因启动子高甲基化能够逃逸精子表观遗传重编程，在雄性子代关节软骨中产生代际遗传效应。其次，在动物水平通过RT-qPCR检测发现，与Control-F1相比，PPCE-F1组雄性大鼠GD20、PW6和PW12关节软骨Plagl1 mRNA的表达均显著降低(图3中D)。免疫组织化学染色发现，与Control-F1相比，PPCE-F1组雄性大鼠GD20、PW6和PW12关节软骨中Plagl1阳性细胞数显著降低，提示蛋白表达降低(图3中E和F)。以上结果表明，PPCE所致的雄性子代大鼠关节软骨Plagl1启动子高甲基化可致其低表达，并且从宫内持续到出生后。In order to further explore the role of sperm epigenetic reprogramming changes in the father-offspring acquired phenotype of PPCE, the present invention first performed whole-genome DNA methylation sequencing analysis on father (F0) sperm, using IGV_2. 8.9 software screening found that compared with the Control-F0 group, the methylation level of the Plagl1 gene promoter in the sperm of the PPCE-F0 group increased (A in Figure 3). In addition, the detection of Plagl1 gene promoter methylation in the articular cartilage of male offspring GD20 and PW12 found that compared with the Control-F1 group, the methylation of multiple sites of the Plagl1 gene promoter in the articular cartilage of male rats in the PPCE-F1 group was higher. The sylation level increased significantly (Fig. 3, B and C). The above results indicate that hypermethylation of the Plagl1 gene promoter in paternal sperm caused by PPCE can escape sperm epigenetic reprogramming and produce intergenerational genetic effects in the articular cartilage of male offspring. Secondly, RT-qPCR detection at the animal level found that compared with Control-F1, the expression of Plagl1 mRNA in GD20, PW6 and PW12 articular cartilage of male rats in the PPCE-F1 group was significantly reduced (Figure 3, D). Immunohistochemical staining found that compared with Control-F1, the number of Plagl1-positive cells in the articular cartilage of GD20, PW6, and PW12 male rats in the PPCE-F1 group was significantly reduced, indicating reduced protein expression (Figure 3, E and F). The above results indicate that PPCE-induced hypermethylation of the Plagl1 promoter in the articular cartilage of male offspring rats can lead to low expression, which lasts from intrauterine to postnatal.
8.2动物水平过表达Plagl1可改善PPCE雄性子代关节软骨基质含量降低8.2 Overexpression of Plagl1 at the animal level can improve the decrease in articular cartilage matrix content in male offspring of PPCE
鉴于以上结果，Plagl1启动子高甲基化所致的Plagl1低表达可能参与PPCE雄性子代大鼠关节软骨基质含量降低。因此，在动物水平，通过构建腺相关病毒载体Plagl1(ssAAV.Plagl1)并通过关节腔注射，来探究关节软骨过表达Plagl1是否可逆转PPCE所致雄性子代软骨基质合成不良。通过番红-固绿染色发现，与PPCE(ssAAV.NC)组相比，PPCE(ssAAV.Plagl1)组关节软骨番红着色较深，提示基质含量增加(图4中A)；qRT-PCR检测发现，与PPCE(ssAAV.NC)组相比，PPCE(ssAAV.Plagl1)组关节软骨Col2a1和ACAN mRNA表达显著增加(图4中B)；进一步通过免疫组织化学染色检测发现，与PPCE(ssAAV.NC)组相比，PPCE(ssAAV.Plagl1)组关节软骨Col2a1的蛋白表达显著增加(图4中C和D)。以上结果表明，过表达Plagl1可改善PPCE所致雄性子代关节软骨基质含量降低。In view of the above results, the low expression of Plagl1 caused by hypermethylation of the Plagl1 promoter may be involved in the reduction of articular cartilage matrix content in PPCE male offspring rats. Therefore, at the animal level, we constructed an adeno-associated virus vector Plagl1 (ssAAV.Plagl1) and injected it into the joint cavity to explore whether overexpression of Plagl1 in articular cartilage could reverse the poor cartilage matrix synthesis in male offspring caused by PPCE. Through safranin-fast green staining, it was found that compared with the PPCE (ssAAV.NC) group, the articular cartilage in the PPCE (ssAAV.Plagl1) group had darker safranin staining, indicating an increase in matrix content (A in Figure 4); qRT-PCR detection It was found that compared with the PPCE(ssAAV.NC) group, the expression of Col2a1 and ACAN mRNA in articular cartilage in the PPCE(ssAAV.Plagl1) group was significantly increased (Figure 4, B); further immunohistochemical staining revealed that the expression was significantly higher than that of the PPCE(ssAAV.Plagl1) group (Figure 4, B). Compared with the NC) group, the protein expression of Col2a1 in articular cartilage in the PPCE (ssAAV.Plagl1) group was significantly increased (Figure 4, C and D). The above results indicate that overexpression of Plagl1 can improve the decrease in articular cartilage matrix content in male offspring caused by PPCE.
本实例结果提示，PPCE可导致胎软骨细胞中Plagl1表达降低甲基化水平增加，并且介导了PPCE子代大鼠出生后骨关节炎易感，而且Plagl1可为父源性骨关节炎易感的早期干预靶标，基于此进一步指导应用于父体孕前不良环境所致骨关节炎的防治策略中的应用。The results of this example suggest that PPCE can reduce the expression of Plagl1 and increase the methylation level in fetal chondrocytes, and mediate the susceptibility of PPCE offspring to osteoarthritis after birth, and Plagl1 can be susceptible to paternal osteoarthritis. Early intervention targets, based on this, further guide the application of prevention and treatment strategies for osteoarthritis caused by the father's adverse environment before pregnancy.
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