CN110433156A - 芝麻素在成骨分化中的新应用 - Google Patents
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Abstract
本发明公开了芝麻素在成骨分化中的新应用,涉及骨修复技术。本发明具体公开了芝麻素在制备用于促进MSC往成骨细胞方向分化的促进剂中的应用。本发明的研究发现芝麻素具有新的用途,其可以促进MSC往成骨方向分化,因此,芝麻素可以作为一种新的骨损伤修改药物、成骨分化促进剂、磷酸化促进剂或成骨分化相关基因表达增强剂等,用于促进MSC往成骨方向分化,提高骨形成能力,修复骨损伤。
Description
技术领域
本发明涉及骨修复技术领域,具体而言,涉及芝麻素在成骨分化中的新应用。
背景技术
骨缺损主要包括骨缺损和缺损修复两大部分。骨缺损是临床置疑上的常见病,也是骨科治疗的难题之一。因此,有必要为骨缺损治疗提供相关治疗药物。
发明内容
本发明的目的在于提供芝麻素在成骨分化中的新应用。本发明的研究发现芝麻素具有新的用途,其可以促进MSC往成骨方向分化,因此,芝麻素可以作为一种新的成骨分化促进剂、磷酸化促进剂、成骨分化相关基因表达增强剂等,用于促进MSC往成骨方向分化,提高骨形成能力,修复骨损伤。
本发明是这样实现的:
第一方面,本发明提供了芝麻素在制备用于骨损伤修复药物或用于促进MSC往成骨细胞方向分化的促进剂中的应用。
优选地,芝麻素通过促进调控因子的磷酸化以促进MSC往成骨细胞方向分化;其中,所述调控因子是指调控成骨分化相关基因表达的因子。
我国民族药具有鲜明的地域性和民族传统,主要由藏、蒙、维、傣、壮五种民族药体系为主。藏药是在广泛吸收、融合了中医药学,印度医药学和大食医药学等理论的基础上,通过长期实践所形成的独特的医药体系,是我国较为完整、较有影响的民族药之一。藏药典籍《藏本草》记载,藏药康定五加可用于治疗风湿性关节炎,跌打损伤,小儿筋骨痿软。根据典籍我们判断康定五加中很可能存在一种天然药用化合物,可以促进骨组织生成。前期研究通过一系列化合物结构分析与活性筛选工作,我们在康定五加中发现了一种小分子化合物芝麻素,可显著促进骨组织修复。
其结构式如下:
本发明的研究进一步发现,芝麻素可显著促进骨缺损修复。并进一步深入研究,发现芝麻素具有促进MSC往成骨细胞方向分化或解除MSC沉默状态恢复成骨能力的作用,芝麻素所具有的这两个作用从分子机理水平上解释芝麻素是如何发挥骨缺损修复。本发明的研究发现了芝麻素的新应用,例如将其作为骨损伤修复的药物,或将其作为促进MSC成骨的促进剂,如作为促进MSC往成骨细胞方向分化的促进剂或作为用于解除MSC沉默状态恢复成骨能力的促进剂,本发明为芝麻素更为广阔的应用方向提供了基础。
第二方面,本发明提供了芝麻素在制备用于促进调控因子的磷酸化的磷酸化促进剂中的应用;其中,所述调控因子是指调控成骨分化相关基因表达的因子。
进一步地,在本发明的一些实施方案中,所述调控因子为选自Smad因子和ATK因子。
进一步地,在本发明的一些实施方案中,所述成骨分化相关基因选自Runx2基因、OCN基因、OSX基因、ColA1基因和ALP基因中的任意一种或多种的组合。
进一步地,在本发明的一些实施方案中,所述芝麻素通过促进所述Smad因子的磷酸化进而增强所述成骨分化相关基因的表达以起到促进MSC往成骨细胞方向分化的作用。
进一步地,在本发明的一些实施方案中,所述Smad因子选自Smad1因子、Smad5因子和Smad8因子中的任意一种或几种。
进一步地,在本发明的一些实施方案中,所述芝麻素通过促进所述Smad1因子、所述Smad5因子和所述Smad8因子的磷酸化进而增强所述Runx2基因、所述OCN基因、所述OSX基因、所述ColA1基因和所述ALP基因的表达以起到促进MSC往成骨细胞方向分化的作用。
进一步地,在本发明的一些实施方案中,所述芝麻素与Bmp2联合通过促进所述Smad1因子、所述Smad5因子和所述Smad8因子的磷酸化进而增强所述Runx2基因、所述OCN基因、所述OSX基因、所述ColA1基因和所述ALP基因的表达以起到促进MSC往成骨细胞方向分化的作用。
BMP-Smad信号通路是调控MSC成骨分化的重要通路。在MSC成骨分化BMP-Smad信号通路中,BMP ligand首先与细胞表面BMP受体结合并引发受体复合物磷酸化,磷酸化后的BMP-受体复合物会磷酸化R-Smad(包含Smad1、Smad5和Smad8)。磷酸化后的R-Smad将与Smad4形成复合物,R-Smad-Smad4复合物进入细胞核,促进成骨分化基因表达,促进MSC成骨分化。Smad6/7是Smad通路的负调控因子,负责在激活初期抑制R-Smad的磷酸化。文献报道,激活的BMP-Smad信号通路甚至可直接抑制p16INK4A介导的干细胞沉默。本发明研究发现,芝麻素可显著增强Smad1/5/8磷酸化,并能显著增强下游成骨基因Runx2、OCN、OSX、ColA1、ALP基因的表达,表明芝麻素促进MSC成骨分化主要是通过BMP-Smad信号通路实现的。
进一步地,在本发明的一些实施方案中,所述调控因子为AKT因子,所述成骨分化相关基因为p16INK4A基因。
进一步地,在本发明的一些实施方案中,所述芝麻素通过作为促进AKT因子的磷酸化的磷酸化促进剂,进而抑制所述p16INK4A基因表达,以解除MSC沉默状态,进而起到促进MSC往成骨细胞方向分化的作用。
INK4细胞周期抑制因子p16INK4A位于染色体9P21,可与细胞周期依赖激酶CDK4/6结合并抑制其活性。这会导致细胞周期蛋白cyclin-D不能与CDK4/6结合,抑制CDK4/6介导的细胞膜母细胞瘤抗癌因子Rb磷酸化。因此转录因子E2F无法激活细胞复制所必需的转录因子,细胞复制将停留在G0/G1期,无法进入S期。在许多人类和哺乳动物的衰老组织中,均发现p16INK4A高表达。p16INK4A高表达的MSC含有高活性的β-半乳糖苷酶,这是干细胞衰老的重要特征。使用iRNA敲减p16INK4A,可以逆转MSC的衰老,恢复部分复制能力和分化潜能。以上信息均表明,p16INK4A是MSC老化的重要调控因子。在本发明研究中,我们发现芝麻素可显著降低老年鼠MSC中p16INK4ARNA的表达,解除MSC沉默状态并促进MSC成骨分化。结果表明芝麻素可能主要通过抑制p16INK4A解除老年鼠干细胞沉默状态,而不是通过调节老年鼠MSC成骨-成脂分化方向。
在MSC中,活化的PI3K在细胞质膜上产生第二信使PIP3,可介导PDK1使AKT磷酸化激活。磷酸化的AKT能直接磷酸化PRAS40,使其对mTORC1的抑制作用解除,促进mTORC1磷酸化激活。激活的mTORC1能活化下游效应分子p70S6K。文献表明,抑制AKT、抑制mTOR或抑制p70S6K均可以显著降低p16INK4A的表达。根据以上信息,AKT-mTOR-p70S6K是p16INK4A介导的衰老过程的关键调控通路,抑制通路中任何节点均可以通过降低p16INK4A表达并解除老年鼠MSC沉默状态。在本发明研究中,我们发现芝麻素可降低老年鼠MSC中AKT的磷酸化程度,表明芝麻素可能通过抑制AKT-mTOR-p70S6K信号轴抑制p16INK4A表达。解除MSC沉默状态并促进MSC成骨分化。
第三方面,本发明提供了芝麻素在制备用于增强成骨分化相关基因表达的增强剂中的应用,所述成骨分化相关基因包括Runx2基因、OCN基因、OSX基因、ColA1基因和ALP基因。
进一步地,在本发明的一些实施方案中,所述芝麻素与Bmp2联合在制备用于增强成骨分化相关基因表达的增强剂中的应用。
第四方面,本发明提供了一种增强成骨分化相关基因表达的增强剂,其包括有芝麻素与Bmp2,所述成骨分化相关基因包括Runx2基因、OCN基因、OSX基因、ColA1基因和ALP基因。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对范围的限定,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他相关的附图。
图1为SE对颅骨缺损小鼠的治疗作用。
图2为SE对骨MSC体外促成骨效应。
图3为SE对老年鼠MSC成骨分化信号通路的作用。
图4为本发明研究涉及的主要信号通路。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
以下结合实施例对本发明的特征和性能作进一步的详细描述。
以下实验例所用的方法:
1骨组织μCT检测分析
颈椎脱臼法处死小鼠,取出小鼠胫骨后剔除小鼠胫骨上的多余的肌肉和结蹄组织,4%的多聚甲醛固定24小时后转入到70%的乙醇中4度保存。进行μCT扫描分析,扫描条件为8.96um像素、峰值电压45千伏500uA,X-线球管扫描旋转角度为0.6度(180度扫描范围),每张扫描图片曝光时间为400毫秒。利用高分辨率(10μm)μCT进行小鼠胫骨的成像和量化分析。选取胫骨骺板下1mm至2mm为感兴趣区,分析骨小梁的显微结构。骨小梁结构分析:应用μCT,评价骨重建中骨小梁的定量参数,松质骨骨密度(Bone mineral content)、松质骨骨量(Bone volume/Tissue volume,BV/TV)、骨小梁厚度(Trabecular thickness,Tb.Th)、骨小梁数量(Trabecular number,Tb.N)、骨小梁间距(Trabecular separation,Tb.Sp)、结构模型指数(Structure Model Index,SMI)等静态形态学指标,并进行三维图像重建。
2小鼠骨髓间充干细胞(BMSCs)的分离培养
(1)取8周龄C57小鼠,脱颈椎法处死,75%酒精浸泡15分钟
(2)取C57小鼠的胫骨和股骨,剔除胫骨和股骨上的肌肉和软组织。
(3)减掉股骨和胫骨的骨骺端,用2ml注射器吸取完全培养基(DMEM低糖+10%FBS+1%双抗)冲洗小鼠股骨和胫骨直到小鼠股骨和胫骨颜色变白。
(4)用70微米滤网将骨髓冲洗液过滤去除冲洗液中的骨碎片,将过滤后的冲洗液在1000rpm的条件下离心15分钟,去掉上清,2ml完全培养基重悬,接种到10cm培养皿中静置培养(37℃,5%CO2,饱和湿度)。
(5)静置培养三天后,将培养基吸掉,用PBS轻轻洗掉未贴壁的细胞。更换新的完全培养基。
(6)当细胞克隆生长到一定大小后,将培养基去掉,加入2ml PBS,洗掉残余的培养基,加入2ml 0.25%的胰酶,在37℃消化1分钟,然后将加入2ml完全培养基中和胰酶,将细胞收集到15ml离心管中,1000rpm条件下离心5分钟,去掉上清后加入2ml完全培养基重悬,接种到10cm培养皿中继续传代培养。
3小鼠骨髓间充质干细胞成骨诱导分化
(1)将P3代小鼠骨髓间充质干细胞以1×105个/孔的密度接种于六孔板中。
(2)当细胞生长到愈合度为80%左右时,成骨诱导分化组加入成骨诱导培养基(OM),对照组加入普通完全培养基(BM)。
(3)细胞每3天换液一次,成骨诱导21天后进行茜素红染色检测。
4茜素红(Alizarin Red)染色及定量分析
(1)吸掉细胞培养基,PBS冲洗两次,4%的多聚甲醛固定15分钟后,PBS冲洗两次。
(2)加入茜素红染色液,放入摇床,缓慢摇晃20分钟后,吸掉茜素红染色液,PBS冲洗两次,将非特异性染色洗掉,拍照观察。
(3)将拍照观察后的培养板中加入1ml 0.1N NaOH溶液,室温溶解15分钟。将100微升溶解液加入到96孔板中,在458nm处检测吸光度。
5实时荧光定量PCR检测mRNA的表达
(1)吸掉培养基,PBS冲洗两次,去除残留的培养基,将培养板放置在冰上。
(2)每孔加入1ml Trizol充分裂解细胞后,将细胞裂解液转移到1.5ml EP管中。
(3)每个EP管中加入200微升氯仿,利用振荡器剧烈振荡15秒,室温静置2分钟。
(4)4℃,12000rpm,离心15分钟后,分为3层,上层为水相,下层为有机相。
(5)吸取上清液,并转移到新的EP管中,加入500微升冷的异丙醇,上下颠倒混匀,然后在冰上静置15分钟。
(6)4℃,12000rpm,离心10分钟。
(7)吸掉上清,保留底部沉淀,加入1ml 75%的乙醇(DEPC水配置),轻轻混匀。
(8)4℃,12000rpm,离心10分钟,去掉上清,保留沉淀。
(9)重复第7和8步骤
(10)将EP管放入到超净台中,缓慢吹干,然后加入15微升的DEPC水溶解沉淀。
(11)利用NanoDrop2000检测mRNA的浓度。
(12)每取1μg mRNA进行逆转录反应体系如下:
mRNA | 1μg |
oligodT | 1μl |
5×Reation Buffer | 4μl |
2.5mM dNTP | 8μl |
RevertAid逆转录酶 | 0.5μl |
合计 | 20μl |
反应条件如下:
42℃ | 1hour |
70℃ | 10min |
4℃ | ∞ |
(13)将逆转录完成的cDNA稀释10倍后按照如下实时荧光定量PCR反应体系加样:
cDNA | 1μl |
去离子水 | 3.6μl |
Forward primer | 0.1μl |
Reverse primer | 0.1μl |
SYBR Premix Ex Taq | 5μl |
ROX Reference Dye | 0.2μl |
合计 | 10μl |
反应条件如下:
(14)以GAPDH为内参,以2-ΔΔCt值计算各组之间的相对表达量,PCR引物序列如下:
6 Western blot检测分析
(1)提取细胞总蛋白:
1)吸掉细胞培养基,PBS冲洗两次,加入100微升细胞裂解液。
2)利用细胞刮刀将细胞收集到离心管中,放置在冰上裂解30分钟。
3)4℃,12000rpm,离心15分钟。将上清转移到新的离心管中。
(2)BCA法检测蛋白浓度:
1)将200微升的A液,4微升B液和10微升蛋白裂解液加入到96孔板中。
2)37℃孵育30分钟。
3)利用酶标仪在562nm处检测吸光度,根据实验室原有标准曲线计算蛋白浓度。
4)加入蛋白上样缓冲液,95℃,加热5分钟。
(3)聚丙烯酰胺凝胶的配置
1)用去离子水将玻璃板清洗干净,在烘箱中烘干。
2)用夹子将玻璃板夹紧,加入去离子水,观察是否漏液。
3)按照如下方法配置10%的分离胶:
去离子水 | 4ml |
4×Tris×HCl/SDS,PH8.8 | 2ml |
40%丙烯酰胺 | 2ml |
10%APS | 80μl |
TEMED | 8μl |
4)溶剂配好后,立即将配好的溶剂加入到玻璃板中,然后缓慢加入少量的75%的乙醇。
5)室温下等待分离胶凝固后,吸掉75%的乙醇。按照如下方法配置浓缩胶:
6)溶剂配好后,立即加入到玻璃板中,然后插入1mm的梳子,等浓缩胶凝固后,拔出梳子,准备上样。
(4)蛋白质凝胶电泳
1)取出蛋白质样品,计算上样量,每个样品上样量为40微克。将蛋白质样品加入到凝胶孔中,同时在加入蛋白Marker。
2)将凝胶板放入到电泳槽中加满电泳液,首先在80V恒定电压下进行电泳,当蛋白Marker到达分离胶顶端并开始分离出条带后,将电压调到120V。在120V恒定电压条件下,当溴酚蓝电泳到凝胶板底部后停止电泳。
(5)转膜
1)裁剪合适大小的PVDF膜,在甲醇中浸泡15秒,活化PVDF膜。
2)按照10×transbuffer:甲醇:去离子水=1:2:7的比例配置1×转膜缓冲液
3)将海绵,滤纸,PVDF膜以及凝胶放入到转膜缓冲液中。
4)去掉凝胶多余的部分,从负极到正极依次排列为海绵-滤纸-凝胶-PVDF膜-滤纸-海绵,紧密排列好后,赶走气泡,压紧夹板。
5)将夹板放入转膜槽中,加满转膜液,在300毫安恒定电流条件下转膜100分钟。
6)转膜完成后,将PVDF膜放入到丽春红染色液中浸泡10秒,然后利用TBST冲洗两次,观察蛋白质转膜情况。
7)裁剪目的条带。
(6)抗体孵育与显影
1)利用TBST配置5%BSA封闭液,缓慢摇晃,室温封闭1小时。
2)用5%BSA按照一定比例配置一抗,缓慢摇晃,4℃孵育过夜。
3)回收抗体,TBST洗涤三次,每次15分钟
4)用5%BSA按照一定比例配置二抗,缓慢摇晃,室温封闭1小时。
5)回收抗体,TBST洗涤三次,每次15分钟
6)利用Millipore,ImmobilonTM,Western显影系统进行显影。
7统计学分析
结果以平均值±标准差表示,两组数据利用Student’s-test进行统计分析,多组数据采用One-way ANOVA进行统计分析。P<0.05认为具有显著性差异。
实验例1
SE对颅骨缺损小鼠的治疗作用
实验选取2月龄昆明小鼠作为实验对象,在右侧顶骨钻直径2mm的小孔,缝合2周后开始给药。灌胃SE(50mg/Kg/d)对颅骨缺损小鼠进行治疗,治疗期为1月。实验结束后分离小鼠颅骨,进行Micro-CT检测。结果如图1所示,SE灌胃对模型小鼠颅骨缺损区的愈合有显著的促进作用,缺损区相对骨体积显著回升,且这种治疗作用没有性别选择性。
实验例2
SE对骨MSC体外促成骨效应
从4月龄小鼠股骨分离MSC为Young MSC(图中绿色bars),从4月龄D-gal模型组小鼠股骨分离MSC为Old MSC(图中灰色bars),均传代至P4开始实验。成骨诱导液(osteogenicmedia,OM)为MSC基础培养基+10%血清+Bmp2。各实验组诱导MSC成骨分化12日,实验结束后分别提取细胞总RNA和总蛋白,并对细胞进行茜素红染色。
由图2中A可观察到,在没有成骨诱导液的情况下,SE对MSC没有诱导成骨的作用,但是有成骨诱导液的情况下,SE可显著促进成骨分化,表明SE不能决定MSC成骨分化方向,但是可以增强成骨分化进程。由于本实验使用Bmp2诱导MSC成骨分化,所以SE所强化的成骨分化进程应是Bmp2启动的Smad1/5/8信号通路。图2中B是用溶剂提取茜素红染色每孔的红色染料,使用酶标仪定量分析后的统计图,结果显示相比年轻MSC,老年鼠MSC对成骨诱导液不敏感,成骨能力显著降低。SE可显著增强年轻或老年鼠MSC成骨分化,老年鼠MSC成骨分化能力可以恢复到Young MSC+OM的水平,但是不能恢复到Young MSC+OM+SE的水平。图2中C至G分别是对不同组MSC不同成骨基因的检测,结果发现老年鼠MSC成骨基因受到显著抑制,在成骨诱导液刺激后表达不能显著升高,但是加入SE后,老年鼠MSC成骨基因如Runx2基因、OCN基因、OSX基因、ColA1基因和ALP基因表达显著提升。
实验例3
SE对骨MSC成骨信号的作用
如图3中A所示,SE增强了Smad1/5/8的磷酸化,表明SE可通过Smad通路增强成骨分化。图3中B和C分别是对7A中条带灰度的定量统计,显示SE刺激可使Smad1/5/8磷酸化显著上升(p<0.05)。相当一部分研究表明,老年鼠MSC失去部分成骨能力是因为成骨/成脂分化紊乱,导致成脂能力上升而成骨能力下降。图3D显示,老年鼠MSC中p16INK4A基因表达大幅上升,SE刺激可显著降低其表达,表明SE通过抑制p16INK4A表达,解除老年鼠MSC沉默状态,恢复其成骨能力。图3E显示老年鼠MSC成脂分化标志基因PPARγ显著上升,而SE刺激对PPARγ表达并无显著影响,表明SE可能不是通过调控成骨-成脂分化起效的。接下来我们探索了调控p16INK4A的上游通路的情况,发现SE可显著抑制p16INK4A上游通路AKT的磷酸化水平,表明SE抑制p16INK4A表达是通过抑制上游AKT-mTOR-p70S6K通路实现的。
综上,本发明研究涉及MSC两条通路,通路1:AKT-mTOR-p70S6K-p16INK4A衰老通路;通路2:BMP-Smad成骨分化通路(见图4)。研究发现SE对年轻MSC和老年鼠MSC成骨分化均有促进作用。我们推测:对年轻MSC而言,p16INK4A表达很低,SE可能直接通过BMP-Smad成骨分化通路促进成骨分化。而对于老年鼠MSC而言,SE可能一方面通过抑制p16INK4A表达解除老年鼠MSC沉默状态,一方面通过BMP-Smad成骨分化通路促进成骨分化。本研究涉及的主要信号通路,总结在图4中。
根据研究结果,我们得到以下结论:SE一方面可以通过AKT-mTOR-p70S6K信号轴抑制p16INK4A表达,解除老年鼠MSC沉默状态并恢复成骨能力;一方面可以通过刺激BMP-Smad通路激活,提升MSC成骨基因表达水平,促进MSC成骨分化。通过以上两方面的综合作用,SE可恢复老年鼠MSC的成骨能力,有利于修复骨缺损或骨损伤。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.芝麻素在制备用于骨损伤修复药物或用于促进MSC往成骨细胞方向分化的促进剂中的应用;
优选地,芝麻素通过促进调控因子的磷酸化以促进MSC往成骨细胞方向分化;其中,所述调控因子是指调控成骨分化相关基因表达的因子。
2.芝麻素在制备用于促进调控因子的磷酸化的磷酸化促进剂中的应用;其中,所述调控因子是指调控成骨分化相关基因表达的因子。
3.根据权利要求1或2所述的应用,其特征在于,所述调控因子为选自Smad因子和ATK因子。
4.根据权利要求3所述的应用,其特征在于,所述调控因子为Smad因子,所述成骨分化相关基因选自Runx2基因、OCN基因、OSX基因、ColA1基因和ALP基因中的任意一种或多种的组合。
5.根据权利要求4所述的应用,其特征在于,所述芝麻素通过促进所述Smad因子的磷酸化进而增强所述成骨分化相关基因的表达以起到促进MSC往成骨细胞方向分化的作用。
6.根据权利要求5所述的应用,其特征在于,所述Smad因子选自Smad1因子、Smad5因子和Smad8因子中的任意一种或几种。
7.根据权利要求3所述的应用,其特征在于,所述调控因子为AKT因子,所述成骨分化相关基因为p16INK4A基因。
8.根据权利要求7所述的应用,其特征在于,所述芝麻素通过作为促进AKT因子的磷酸化的磷酸化促进剂,进而抑制所述p16INK4A基因表达,以解除MSC沉默状态,进而起到促进MSC往成骨细胞方向分化的作用。
9.芝麻素在制备用于增强成骨分化相关基因表达的增强剂中的应用,其特征在于,所述成骨分化相关基因包括Runx2基因、OCN基因、OSX基因、ColA1基因和ALP基因。
10.根据权利要求9所述的应用,其特征在于,所述芝麻素与Bmp2联合在制备用于增强成骨分化相关基因表达的增强剂中的应用。
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