CN113481292A - 11β-HSD2及表观遗传标记作为关节软骨发育不良及骨关节炎易感早期标志物的用途 - Google Patents
11β-HSD2及表观遗传标记作为关节软骨发育不良及骨关节炎易感早期标志物的用途 Download PDFInfo
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Abstract
本发明公开了11β‑HSD2及其表观遗传标记作为关节软骨发育不良及骨关节炎易感的早期标志物的用途。研究结果表明，与正常对照组相比，宫内发育迟缓(IUGR)新生儿脐带间充质干细胞或整体脐带中的11β‑HSD2启动子区组蛋白乙酰化及表达水平降低；IUGR新生儿脐带间充质干细胞软骨分化能力更低，在高浓度糖皮质激素作用下更容易出现骨关节炎类似的软骨细胞退变表现。因此，通过检测脐带11β‑HSD2基因的表达及表观遗传水平来判断受试者的软骨发育不良及成年后患骨关节炎的风险。为关节软骨发育不良及胎源性成年骨关节炎的早期预警技术提供了研究依据，也为胎源性成年骨关节炎的综合防治提供新思路。
Description
本申请是申请日为2017年12月19日、申请号为201711378099.9、发明名称为《11β-HSD2及表观遗传作为关节软骨发育不良及骨关节炎易感的早期标志物的用途》的分案申请。
技术领域
本发明属于临床医学技术领域，涉及一类2型11β-羟类固醇脱氢酶(11β-hydroxysteroid dehydrogenase type-2，11β-HSD2)基因及其表观遗传作为关节软骨发育不良及成年骨关节炎早期预警的生物标志物。
背景技术
骨关节炎是一种以关节软骨退行性病变为主要病理特征的慢性关节疾病。传统观点认为，骨关节炎为老年退行性疾病。由于其发病率高、治疗难度大，对骨关节炎的诊治进行研究意义重大。宫内发育迟缓(intrauterine growth retardation，IUGR)是指足月儿出生体重小于2.5kg，或胎儿体重低于其孕龄平均体重的两个标准差。大样本流行病学研究发现，低出生体重儿成年后手、髋等多关节骨关节炎易感[1-3]。本室前期研究发现，多种孕期外源物暴露(如咖啡因、尼古丁、乙醇、地塞米松)可致子代大鼠IUGR，且IUGR个体宫内出现软骨发育不良，表现为软骨基质染色变浅、细胞活力下降，且在成年后存在骨关节炎易感现象[4,5]。提示，骨关节炎具有宫内起源。然而，胎源性成年骨关节炎的早期预警及诊治技术匮乏，与胎源性成年骨关节炎易感的分子靶标尚未明确有关。
糖皮质激素(glucocorticoid，GC)是决定宫内胎组织形态、功能成熟的关键因素，母源性GC参与了胎儿早期的生长和发育，并参与多个脏器(如关节软骨)的发育。11β-HSD2通过代谢灭活母源性GC，可有效的调节胎组织局部的活性GC水平及功能[6,7]。我们研究表明，孕期暴露多种外源物(如咖啡因、尼古丁、乙醇)所致的宫内神经内分泌代谢改变(包括下丘脑-垂体-肾上腺轴功能发育抑制和外周糖、脂代谢变化)，与母源性GC过暴露和组织内发育相关重要基因11β-HSD2低表达有关；而11β-HSD2的表达或活性降低，可放大母源性高GC对胎儿生长发育的抑制作用。提示，11β-HSD2也可能在软骨局部表达，在调控软骨局部GC效应中起着重要作用。另有研究发现，运用GC治疗骨关节炎的同时会导致关节软骨基质减少、软骨细胞凋亡增加[8,9]。提示，11β-HSD2可作为关节软骨发育不良及成年骨关节炎易感的分子靶标。然而，目前尚未见任何相关报道。
人华通胶间充质干细胞(human Wharton`s jelly-derived mesenchymal stemcells，WJ-MSCs)是一种多潜能干细胞，可以在体外分化为软骨细胞[10-12]。在WJ-MSCs软骨细胞分化模型上采用IL-1β(interleukin-1β，IL-1β)体外诱导，可建立细胞水平的骨关节炎样模型。在个体发育过程中，间充质干细胞(包括WJ-MSCs)可作为宫内不良环境影响的靶组织，可被编程，即“记忆”早期生命过程中接受的可影响成年后功能的不良刺激[13]。也有研究报道，小于胎龄儿来源的WJ-MSCs中保留有可识别的表观遗传印记[14,15]。值得注意的是，目前各实验室在WJ-MSCs的分离和体外扩增方面尚未达成共识，且尚无一种方法能快速高效分离出均质而符合明确界定的WJ-MSCs，这显然为将来基于高通量筛选开展临床早期预警的实施提供了诸多不便。有报道提示，整体脐带中的表观遗传分析可能用于评估个体出生后肥胖和代谢综合征的易感性[16]。以上研究提示，WJ-MSCs可成为验证胎儿不良发育潜在分子机制的初步筛选系统，其编程印记也可在整体脐带中体现，基于整体脐带的表观遗传印记检测为将来基于大规模新生儿的高通量筛选并预测疾病风险提供了新的思路。
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发明内容
为了克服现有技术存在的不足，本发明的目的是通过检测新生儿脐带间充质干细胞WJ-MSCs和整体脐带中11β-HSD2基因表达及表观遗传的水平，为宫内关节软骨发育不良及胎源性成年骨关节炎易感的早期预警提供研究依据。
本发明的目的通过下述技术方案实现：
本发明第一方面提供新生儿脐带间充质干细胞WJ-MSCs或整体脐带11β-HSD2基因表达及启动子区H3K9ac在制备用于检测早期预警软骨发育不良及胎源性成年骨关节炎易感的试剂盒中的应用。
优选的，所述11β-HSD2基因表达及启动子区H3K9ac在新生儿脐带间充质干细胞WJ-MSCs或整体脐带中的水平下降，则有软骨发育不良及胎源性成年骨关节炎易感的风险。
优选的，所述的试剂盒中包含检测11β-HSD2基因表达的试剂检测11β-HSD2基因启动子区H3K9ac水平的试剂。
优选的，所述检测11β-HSD2基因表达的试剂中包含扩增11β-HSD2的引物对、检测11β-HSD2基因启动子区H3K9ac水平的引物对。
优选的，所述扩增11β-HSD2的引物对序列如SeqID No.1和Seq ID No.2所述。
优选的，所述检测11β-HSD2基因启动子区H3K9ac水平的引物对如Seq ID No.3和Seq ID No.4所述。
本发明第二方面提供一种早期预警软骨发育不良及胎源性成年骨关节炎易感的试剂盒，包含扩增11β-HSD2的引物对、检测11β-HSD2基因启动子区H3K9ac水平的引物对。
优选的，所述扩增11β-HSD2的引物对序列如Seq ID No.1和Seq ID No.2所述。
优选的，所述检测11β-HSD2基因启动子区H3K9ac水平的引物对如Seq ID No.3和Seq IDNo.4所述。
本发明通过研究证实，与对照组相比，孕期咖啡因暴露(prenatal caffeineexposure，PCE)组子代大鼠胎软骨11β-HSD2及软骨标志基因COL2A1和蛋白聚糖(aggrecan，AGAN)的mRNA表达水平降低(P＜0.01)；与对照组相比，PCE胎软骨11β-HSD2启动子区H3K9ac水平明显降低(P＜0.01)，以上变化从宫内持续到出生后。进一步证实，慢性应激后，与对照组相比，PCE组软骨细胞排列更紊乱，软骨细胞数明显减少，Mankin’s评分显著升高，软骨标志基因COL2A1表达显著降低，且比慢性应激前降低更为明显(P＜0.01)。以上提示，PCE组子代大鼠软骨局部11β-HSD2基因低表达和启动子区H3K9ac低水平，并从宫内延续至出生后，引起软骨局部内源性活性GC增多，致使软骨表型基因持续抑制及软骨发育不良，在不良环境-慢性应激诱导下呈现骨关节炎易感。
接着，本发明发明人发现IUGR来源的WJ-MSCs软骨定向分化不良，且在IL-1β处理下软骨基质降解更明显。进一步，比较了IUGR和正常新生儿脐带WJ-MSCs中11β-HSD2及下游基因mRNA表达水平，与对照组相比，IUGR脐带WJ-MSCs 11β-HSD2及下游基因COL2A1和AGANmRNA表达明显降低(P＜0.01)，同时IUGR脐带WJ-MSCs 11β-HSD2启动子区H3K9ac水平明显降低(P＜0.01)。提示，具有11β-HSD2基因低表达和启动子区H3K9ac低水平的WJ-MSCs存在软骨分化不良，且在炎症刺激下更易呈现骨关节炎样细胞反应。
进一步，本发明发明人比较了IUGR患儿和正常新生儿整体脐带中11β-HSD2的基因表达和启动子区H3K9ac水平变化。发现与正常脐带相比，IUGR脐带中11β-HSD2的基因表达和启动子区H3K9ac水平均显著降低。提示，IUGR整体脐带存在与IUGR来源的WJ-MSCs相同的分子改变，即11β-HSD2的基因低表达和启动子区H3K9ac低水平。
综上，新生儿脐带11β-HSD2基因表达及启动子区H3K9ac水平联合起来可以用于关节软骨发育不良及胎源性成年骨关节炎易感的早期预警。
附图说明
图1、对照组和PCE组胎软骨11β-HSD2基因及其启动子区H3K9ac、软骨标志基因二型胶原(collagentype II alpha 1，COL2A1)和蛋白聚糖(aggrecan，AGAN)mRNA表达水平。与对照组相比，*P＜0.05，**P＜0.01。
图2、对照组和PCE组出生后12周软骨11β-HSD2基因及其启动子区H3K9ac及软骨标志基因mRNA表达水平。与对照组相比，*P＜0.05，**P＜0.01。
图3、对照组和PCE组出生后10周给予慢性应激2周后关节软骨基质染色、Mankin’s评分及COL2A1的mRNA表达水平。与对照组相比，**P＜0.01。
图4、WJ-MSCs软骨定向分化和骨关节炎样细胞反应；
图5、IUGR和正常新生儿WJ-MSCs中11β-HSD2基因及其启动子区H3K9ac及软骨标志基因mRNA表达水平。与对照组相比，**P＜0.01。
图6、IUGR和正常新生儿脐带中11β-HSD2基因及其启动子区H3K9ac及软骨标志基因mRNA表达水平。与对照组相比，**P＜0.01。
具体实施方式
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。
SPSS 17和Prism 5.0用于数据分析。定量数据被表示为均值±标准误(mean±S.E.M.)和获得的qRT-PCR独立样本t检验分析。慢性应激后Mankin’s评分及COL2A1的mRNA表达数据采用双因素方差分析。
【实施例1】
对照组和PCE组胎软骨、出生后12周软骨11β-HSD2启动子区H3K9ac、11β-HSD2及软骨标志基因二型胶原(collagentype II alpha 1，COL2A1)和蛋白聚糖(aggrecan，AGAN)mRNA表达水平。
1、实验动物
SPF级健康Wistar大鼠，购自湖北省疾病预防控制中心，动物许可证号：SCXK(鄂)2008-2010。本研究获武汉大学医学部伦理委员会批准，并严格按照国际实验动物保护认证评估机构相关处理准则执行。
实验动物饲养于屏障环境内，温度22～25℃，湿度50％，12小时昼夜交替。
2、动物处理
从湖北省疾控中心购买健康且成年的Wistar大鼠，雌性体重为209±12g，雄性体重为258±17g。每晚6时按照雌雄2:1进行合笼，次晨检查阴栓及精子涂片确定雌鼠是否成功受孕，并记为孕0天(gestation day 0，GD0)。受孕大鼠随机分为2组：对照组、PCE组。于GD9开始，每天上午8-10时，PCE组给予咖啡因(120mg/kg.d)灌胃，对照组灌胃等体积的生理盐水。在GD20，一部分孕鼠经乙醚麻醉并剖宫取胎鼠(n＝8)，将胎鼠下肢离断，保留完整膝关节，左膝冻存保存于-80℃冰箱待用，右膝置于中性甲醛固定，包埋，切片，行苏木精-伊红(hematoxylin-eosin，HE)和甲苯胺蓝染色。另一部分孕鼠(包括对照组、PCE组)自然分娩。以生产日作为出生后0天(postnatal day 0，PD0)，每窝挑选2只雄性仔鼠至出生后：其中1只在12周(postnatal week 12，PW12)处死；另外1只在PW10给予2周冰水游泳刺激后处死，并按照上述方式保存标本(n＝8)。
3、基因表达检测
提取宫内胎鼠软骨、PW12及给予2周冰水游泳刺激的PW12软骨总RNA，逆转录为cDNA，通过qRT-PCR进行定量分析相关基因表达情况。各基因引物序列如表1所示：
引物设计：利用PrimerPremier 5.0及NCBI Blast数据库设计和验证引物。设计好引物序列后，将其交由生工生物工程(上海)股份有限公司合成，并PAGE纯化。将装有新合成引物的EP管7500g离心10分钟，加入管壁上标注体积的分子生物学超纯水，震荡混匀备用。
表1：RT-PCR引物序列
4、染色质免疫共沉淀
在匀浆后的软骨组织用1ml PBS洗涤3次所获取下层细胞沉淀中加入37％的甲醛，室温放置10min进行染色质交联，然后加入2.5mM的甘氨酸室温放置5min终止交联。PBS清洗收集细胞后用超声破碎仪(工作条件为30％功率，2son，1s off)，进行超声破碎5min裂解DNA链。4℃环境下12,000rpm离心10min，取上清收集DNA片段。分装DNA上清液，分别为IgG、Input、H3K9ac三管。除Input外各分装DNA上清液中加入1mg/ml的BSA封闭Protein Gbeads封闭，加入总量为1μg的对应抗体(abcam，USA)，4℃孵育过夜。3000rpm离心1min，收集免疫沉淀物。清洗并离心3次免疫沉淀物，65℃水浴过夜解交联。应用PCR纯化试剂盒(TiangenCo.，Beijing,China)纯化DNA，纯化后的DNA用50μL水溶解，于-20℃保存。每个纯化后的样本针对11β-HSD2启动子区进行qRT-PCR，引物序列如表2所示，组蛋白乙酰化结果以Input为参照。
表2.RT-PCR引物序列
5、结果
对照组、PCE组胎软骨11β-HSD2及软骨标志基因mRNA表达如图1A所示。与对照组相比，PCE胎软骨11β-HSD2及软骨标志基因COL2A1和AGAN的mRNA表达降低(P＜0.05,P＜0.01)。11β-HSD2启动子区H3K9ac水平改变如图1B所示。与对照组相比，PCE胎软骨11β-HSD2启动子区H3K9ac水平明显降低(P＜0.01)。
对照组、PCE组出生后12周软骨11β-HSD2及软骨标志基因mRNA表达水平如图2A、2B所示。与对照组相比，PCE组软骨11β-HSD2及软骨标志基因COL2A1和AGAN的mRNA表达降低(P＜0.05,P＜0.01)，同时11β-HSD2启动子区H3K9ac水平也降低(P＜0.01)。
对照组、PCE组出生后10周给予2周慢性应激后甲苯胺蓝染色(图3A)、Mankin’s评分(图3B)及COL2A1的mRNA表达(图3C)如图3所示。与对照组相比，未接受慢性应激的子代，PCE组关节软骨基质合成不良、软骨细胞排列紊乱，软骨标志基因COL2A1降低。慢性应激后，PCE组软骨细胞排列更紊乱，软骨细胞数明显减少，Mankin’s评分显著升高，软骨标志基因COL2A1显著降低，且比慢性应激前降低更明显(P＜0.01)。
本实施例结果提示，PCE组子代大鼠软骨局部存在11β-HSD2基因低表达和启动子区H3K9ac低水平，并从宫内延续至出生后，引起软骨局部内源性活性GC增多，致使软骨表型基因持续抑制及软骨发育不良，最终在不良环境(如慢性应激)诱导下呈现骨关节炎易感。
【实施例2】
正常对照组和IUGR组新生儿脐带间充质干细胞(Wharton`sjelly-derivedmesenchymal stem cells，WJ-MSCs)11β-HSD2启动子区H3K9ac、11β-HSD2及下游基因mRNA表达水平。
1、脐带间充质干细胞WJ-MSCs培养进行总RNA提取
从武汉大学中南医院妇产科收集新生儿脐带标本。分为2组：正常对照组和IUGR组(n≧8)，培养间充质干细胞后提取总RNA。加入1ml Trizol于匀浆器内，冰上充分匀浆后，转至无RNase 1.5ml EP管中；加入200μl氯仿抽提，剧烈震荡15s，冰上静置10min；4℃12,000×rpm离心15min；小心吸取500μl上层水相移至一新无RNase 1.5ml EP管中，加等体积的异丙醇，颠倒混匀沉淀RNA，20～30℃静置10min；4℃12,000×rpm离心10min，弃上清，加1ml75％乙醇洗涤沉淀，颠倒混匀；4℃9500×rpm离心5min，弃乙醇(重复两次)，室温放置10min晾干；加60℃温育的DEPC处理水20μl；紫外分光光度计测RNA浓度及纯度，-80℃保存。
2、脐带WJ-MSCs向软骨细胞分化后骨关节炎样细胞造模
脐带WJ-MSCs软骨定向分化后骨关节炎易感细胞模型。将第4代WJ-MSCs以普通完全培养基培养24小时，然后加入软骨分化培养基，培养基中含有DMEM/F1295％、Gibco小牛血清5％、1％的100×ITS、100nM地塞米松、10ng/mL TGFβ1、40μg/mL脯氨酸、50μg/mL抗坏血酸、100mg/L丙酮酸、10％青链霉素等，置于37℃恒温培养箱内，维持CO2浓度为5％、湿度为95％进行分化，在诱导21天，定向分化为软骨细胞。IL-1β诱导分化后的细胞骨关节炎造模。将分化培养基换为含重组人白细胞介素-1β(recombinant human interleukin-1β，rhIL-1β)10ng/mL的DMEM/F12的培养基，培养24小时进行骨关节炎造模。
3、新生儿脐带WJ-MSCs中11β-HSD2及下游基因表达检测
细胞培养增殖WJ-MSCs 5天，提取细胞总RNA之后逆转录为cDNA，并通过qRT-PCR进行定量分析相关基因表达情况。各基因引物序列如表1所示。
4、新生儿脐带WJ-MSCs 11β-HSD2启动子区H3K9ac水平检测
将正常对照组和IUGR患儿获取脐带间充质干细胞细胞悬液，进行染色质免疫共沉淀实验，最终获得Input、H3K9ac、IgG不同的IP产物，并通过DNA纯化试剂盒纯化样本，qRT-PCR检测相关指标变化。引物序列如表2所示。
5、实验结果
软骨定向分化后及骨关节炎样细胞造模后，骨关节炎软骨细胞类似表现如图4所示。定向软骨分化后，与正常对照组相比，IUGR来源的WJ-MSCs基质染色显著变浅；进一步在IL-1β处理后，与正常对照组相比，IUGR组分化后的细胞基质染色呈现更差的状态。
IUGR和正常新生儿脐带WJ-MSCs中11β-HSD2及下游基因mRNA表达(图5A)及启动子区H3K9ac水平(图5B)如图5所示。与正常对照组相比，IUGR脐带WJ-MSCs 11β-HSD2及下游基因COL2A1和AGAN mRNA表达明显降低(P＜0.01)，同时IUGR脐带WJ-MSCs 11β-HSD2启动子区H3K9ac水平明显降低(P＜0.01)。
本实施例结果提示，IUGR来源的WJ-MSCs中11β-HSD2基因低表达和启动子区H3K9ac低水平，其特点是WJ-MSCs软骨定向分化不良且分化后的细胞在炎症刺激下更易呈现关节炎样细胞反应。
【实施例3】
正常对照组和IUGR组新生儿脐带11β-HSD2启动子区H3K9ac、11β-HSD2及软骨标志基因二型胶原(collagen type II alpha 1，COL2A1)和蛋白聚糖(aggrecan，AGAN)mRNA表达水平变化。
1、新生儿脐带组织中11β-HSD2表达及软骨标志基因表达检测
从武汉大学中南医院妇产科收集新生儿脐带标本。分为2组：正常对照组和IUGR组(n≧8)。取10cm脐带剪碎加入培养基，含(1mg/mL)透明质酸酶胶原酶I(Invitrogen公司)。37℃匀速摇荡12小时，加PBS液反复吹打稀释，收集稀释液(含组织)，3500rpm离心20min，然后弃上清液和漂浮的脂肪组织，收集组织。加入1ml Trizol于匀浆器内，冰上充分匀浆后，转至无RNase 1.5ml EP管中；加入200μl氯仿抽提，剧烈震荡15s，冰上静置10min；4℃12,000×rpm离心15min；小心吸取500μl上层水相移至一新无RNase 1.5ml EP管中，加等体积的异丙醇，颠倒混匀沉淀RNA，20～30℃静置10min；4℃12,000×rpm离心10min，弃上清，加1ml 75％乙醇洗涤沉淀，颠倒混匀；4℃9500×rpm离心5min，弃乙醇(重复两次)，室温放置10min晾干；加60℃温育的DEPC处理水20μl；紫外分光光度计测RNA浓度及纯度，-80℃保存。提取组织总RNA之后逆转录为cDNA，并通过qRT-PCR进行定量分析相关基因表达情况。各基因引物序列如表1所示。
2、新生儿脐带WJ-MSCs 11β-HSD2启动子区H3K9ac水平检测
将正常和IUGR患儿获取脐带组织，进行染色质免疫共沉淀实验，最终获得Input、H3K9ac、IgG不同的IP产物，并通过DNA纯化试剂盒纯化样本，RT-PCR检测相关指标变化。引物序列如表2所示。
3、结果
IUGR和正常新生儿脐带中11β-HSD2及软骨标志基因mRNA表达(图6A)、启动子区H3K9ac水平(图6B)如图6所示。与正常对照组相比，IUGR脐带中11β-HSD2及软骨标志基因COL2A1、AGAN的mRNA表达明显降低(P＜0.01)，同时IUGR脐带11β-HSD2启动子区H3K9ac水平明显降低(P＜0.01)。
本实施例结果提示，11β-HSD2基因低表达和启动子区H3K9ac低水平也存在于IUGR来源的整体脐带中。
序列表
<110> 武汉大学
<120> 11β-HSD2及表观遗传标记作为关节软骨发育不良及骨关节炎易感早期标志物的用途
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Claims (8)
1.11β-HSD2基因表达及启动子区H3K9ac在制备用于检测早期预警软骨发育不良的试剂盒中的应用，其特征在于，所述11β-HSD2基因表达及启动子区H3K9ac来源于新生儿脐带间充质干细胞WJ-MSCs或整体脐带；
所述11β-HSD2基因表达及启动子区H3K9ac在新生儿脐带间充质干细胞WJ-MSCs或整体脐带中的水平下降，则有软骨发育不良易感的风险。
2.11β-HSD2基因表达及启动子区H3K9ac在制备用于检测早期预警胎源性成年骨关节炎易感的试剂盒中的应用，其特征在于，所述11β-HSD2基因表达及启动子区H3K9ac来源于新生儿脐带间充质干细胞WJ-MSCs或整体脐带；
所述11β-HSD2基因表达及启动子区H3K9ac在新生儿脐带间充质干细胞WJ-MSCs或整体脐带中的水平下降，则有胎源性成年骨关节炎易感的风险。
3.根据权利要求1或2所述的应用，其特征在于，所述的试剂盒中包含检测11β-HSD2基因表达的试剂和检测11β-HSD2基因启动子区H3K9ac水平的试剂。
4.根据权利要求3所述的应用，其特征在于，所述检测11β-HSD2基因表达的试剂中包含扩增11β-HSD2的引物对、检测11β-HSD2基因启动子区H3K9ac水平的引物对。
5.根据权利要求4所述的应用，其特征在于，所述扩增11β-HSD2的引物对序列如Seq IDNo.1和Seq ID No.2所述；所述检测11β-HSD2基因启动子区H3K9ac水平的引物对如Seq IDNo.3和Seq ID No.4所述。
6.一种早期预警软骨发育不良的试剂盒，其特征在于，包含扩增11β-HSD2的引物对、检测11β-HSD2基因启动子区H3K9ac水平的引物对。
7.一种早期预警胎源性成年骨关节炎易感的试剂盒，其特征在于，包含扩增11β-HSD2的引物对、检测11β-HSD2基因启动子区H3K9ac水平的引物对。
8.根据权利要求6或7所述的试剂盒，其特征在于，所述扩增11β-HSD2的引物对序列如Seq ID No.1和Seq ID No.2所述；所述检测11β-HSD2基因启动子区H3K9ac水平的引物对如Seq ID No.3和Seq ID No.4所述。
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