CN114622019A - Birc5基因作为人骨髓间充质干细胞成骨分化抑制剂的应用 - Google Patents
Birc5基因作为人骨髓间充质干细胞成骨分化抑制剂的应用 Download PDFInfo
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Abstract
本发明公开了BIRC5基因作为人骨髓间充质干细胞成骨分化抑制剂的应用,本发明还设计了用于检测人骨髓间充质干细胞中BIRC5基因表达量的试剂盒。本发明首次证明了BIRC5基因在人骨髓间充质干细胞中的作用,且证明BIRC5基因能够有效抑制人骨髓间充质干细胞的成骨分化,因此,BIRC5基因能够作为人骨髓间充质干细胞成骨分化抑制剂,进而来抑制人骨髓间充质干细胞成骨分化。
Description
技术领域
本发明属于人骨髓间充质干细胞成骨分化抑制剂技术领域,具体涉及BIRC5基因作为人骨髓间充质干细胞成骨分化抑制剂的应用。
背景技术
骨、软骨、肌腱等组织缺损修复具有重大临床需求,但因缺乏理想修复供体,目前治疗方法存在创伤大、供体严重不足等弊端。因此,对于这类骨疾病的最佳治疗方式是采用组织工程骨。成骨支架、种子细胞及高效的骨分化诱导因子是组织工程目前的研究热点。人骨髓间充质干细胞(hBMSCs)是一种多能成体干细胞,是组织工程中重要的种子细胞来源。研究hBMSCs的成骨分化生物学效应及其具体的分子机制可为骨质疏松症、骨肿瘤及骨折等疾病提供新的治疗靶点。
发明内容
本发明的目的是提供了BIRC5基因作为人骨髓间充质干细胞成骨分化抑制剂的应用,其中BIRC5是蛋白编码基因,该基因是凋亡抑制剂(IAP)基因家族的成员,该家族编码可防止细胞凋亡的负调节蛋白,本发明通过实验证实BIRC5基因与hBMSCs的成骨分化现象呈负相关,且BIRC5基因与人骨髓间充质干细胞成骨分化的标志性基因表达也呈负相关;同时本发明设计了BIRC5基因(Homo sapiens Gene ID:332)表达量检测的试剂盒。
本发明为实现上述目的采用如下技术,BIRC5基因作为人骨髓间充质干细胞成骨分化抑制剂的应用。
进一步限定,所述BIRC5基因与人骨髓间充质干细胞成骨分化现象呈负相关,且BIRC5基因与人骨髓间充质干细胞成骨分化的标志性基因表达也呈负相关。
进一步限定,用于检测人骨髓间充质干细胞中BIRC5基因表达量的试剂盒,其特征在于包含特异性扩增BIRC5基因的引物;
引物中正向引物序列为:
5'- GGACCACCGCATCTCTACATT-3';
引物中反向引物序列为:
5'- TTTCCTTTGCATGGGGTCGT-3'。
本发明与现有技术相比具有以下优点和有益效果:本发明首次证明了BIRC5基因在人骨髓间充质干细胞中的作用,且证明BIRC5基因能够有效抑制人骨髓间充质干细胞的成骨分化,因此,BIRC5基因能够作为人骨髓间充质干细胞成骨分化抑制剂,进而来抑制人骨髓间充质干细胞成骨分化。
附图说明
图1是hBMSCs诱导的成骨细胞中,BIRC5基因表达显著下调;
图2是hBMSCs中运用慢病毒质粒转染高表达BIRC5基因;
图3是与BIRC5基因可能发生的蛋白和转录调控网络;
图4是数据集GSE80614中,与BIRC5基因正相关和负相关的基因表达热图;
图5是BIRC5基因的GO和KEGG信号富集分析;
图6是BIRC5基因可抑制hBMSCs的成骨分化,并且与成骨分化标志基因的表达呈负相关;
图7是BIRC5基因可抑制hBMSCs的成骨分化现象;
图8是BIRC5基因影响了hBMSCs中ALP含量,进而可影响hBMSCs成骨分化;
图9是BIRC5基因影响hBMSCs钙沉积,进而可影响其成骨分化。
具体实施方式
结合附图详细描述本发明的技术方案。
实施例1
本实施例用于说明在人骨髓间充质干细胞(hBMSCs)诱导的成骨细胞中,BIRC5基因表达降低。
A、数据下载和整理。利用NCBI网站的GEO数据库的3个人骨髓来源间充质干细胞成骨分化数据集GSE80614、GSE12266和GSE18043进行分析成骨分化后的差异基因。选取至少有两个数据集中表达差异的基因为靶基因,并进行生物信息学分析。如图1中A所示,BIRC5基因在GSE80614和GSE12266中呈现低表达状态。****p <0.0001,*p <0.05,NA P>=0.05。
B、qPCR验证hBMSCs成骨诱导7天(OS-7d)后,与未诱导的细胞(control)相比较BIRC5基因表达显著降低。***p <0.001。
实施例2
本实施例用于说明在人骨髓间充质干细胞(hBMSCs)中运用慢病毒质粒转染高表达BIRC5基因。
A、运用PLV-N-Flag载体,构建慢病毒过表达BIRC5质粒,并与293细胞中包装后转染hBMSCs。
B、qPCR检测BIRC5过表达效率。**p <0.001。
实施例3
本实施例用于说明与BIRC5基因可能发生的蛋白和转录调控网络。
利用string网站构建BIRC5的蛋白互作PPI网络,Cytoscape软件进行可视化作图。使用数据库RegNetwork(http://www.regnetworkweb.org/)预测BIRC5基因基因上游的miRNA和转录因子,构建靶基因调控网络。图3是BIRC5基因可能发生的蛋白和转录调控网络,图中三角形表示miRNA,正方形表示转录因子。
实施例4
本实施例用于说明与BIRC5基因正相关和负相关的基因表达热图。
利用R语言软件包,使用GSE80614中的成骨诱导组(OS)数据进行BIRC5基因与所有基因的相关性分析,并分别展示正相关和负相关的基因表达热图。如图4所示,(A)GSE80614中与BIRC5基因正相关表达热图;(B)GSE80614中与BIRC5基因负相关的基因表达热图。
实施例5
本实施例用于验证BIRC5基因的GO和KEGG富集分析。
利用R软件包进行BIRC5基因的GO和KEGG分析。可见BIRC5基因可富集于细胞发育、细胞周期以及细胞外刺激等信号***。如图5所示,其中图5中A为GO分析的波浪图;图5中B为KEGG分析的气泡图。图5说明BIRC5基因的GO和KEGG信号富集分析,为研究其调控成骨分化的分子机制提供理论基础。
实施例6
本实施例用于说明BIRC5基因可抑制hBMSCs的成骨分化,并且与成骨分化标志基因的表达呈负相关。
利用慢病毒过表达BIRC5质粒转染hBMSCs,qPCR实验方法验证成骨分化标志性基因Runx2、OPN、OSX和OCN基因的表达。如图6所示,过表达BIRC5基因后,Runx2、OPN、OSX和OCN基因的表达显著降下调。其中control为对照组,OS-7d是成骨诱导7天组;V- BIRC5是过表达BIRC5组。*p<0.05,**p<0.01,***p<0.001。图6说明BIRC5基因可抑制hBMSCs的成骨分化,并且与成骨分化标志基因表达呈负相关。
实施例7
本实施例用于说明BIRC5基因可抑制hBMSCs的成骨分化现象。
利用ALP活性检测试剂盒和钙含量检测试剂盒检测hBMSCs对照组、OS-7d组和过表达BIRC5(B-V)的ALP活性和钙沉积情况。结果如图7所示,图7中A说明过表达BIRC5基因,与诱导组相比较,可相对降低ALP活性,**p<0.01。图7中B说明过表达BIRC5基因,与诱导组相比较,可降低细胞钙的沉积,***p<0.001。图7说明BIRC5基因参与调控hBMSCs的成骨分化程度,可抑制成骨分化作用。
实施例8
本实施例用于说明BIRC5基因抑制了hBMSCs的成骨分化作用。
利用ALP染色检测试剂盒,检测对照组、OS-7d组和过表达BIRC5组(V-BIRC5)组中ALP的含量。如图8所示,通过显色反应表明过表达BIRC5基因减弱了ALP的染色强度。图8说明BIRC5基因影响了hBMSCs中ALP含量,进而可影响hBMSCs成骨分化。
实施例9
本实施例用于说明过表达BIRC5基因减少hBMSCs钙沉积,进而可抑制其成骨分化作用。
利用茜素红(ARS)染色的实验方法,检测对照组、成骨诱导10天(OS-10d)组和过表达BIRC5组(V- BIRC5)组中钙沉积情况。如图9所示,过表达BIRC5基因减弱了ARS的染色强度。图9说明书BIRC5基因影响了hBMSCs钙沉积,进而可影响其成骨分化。
表1 荧光定量RT-PCR引物序列
以上显示和描述了本发明的基本原理,主要特征和优点,在不脱离本发明精神和范围的前提下,本发明还有各种变化和改进,这些变化和改进都落入要求保护的本发明的范围。
SEQUENCE LISTING
<110> 新乡医学院
<120> BIRC5基因作为人骨髓间充质干细胞成骨分化抑制剂的应用
<130> 2022
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213> 人工序列(artificial sequence)
<400> 1
ggaccaccgc atctctacat t 21
<210> 2
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 2
tttcctttgc atggggtcgt 20
序列表
<110> 新乡医学院
<120> BIRC5基因作为人骨髓间充质干细胞成骨分化抑制剂的应用
<130> 2022
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> 人工序列(artificial sequence)
<400> 1
ggaccaccgc atctctacat t 21
<210> 2
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 2
tttcctttgc atggggtcgt 20
Claims (3)
1.BIRC5基因作为人骨髓间充质干细胞成骨分化抑制剂的应用。
2.根据权利要求1所述的应用,其特征在于:所述BIRC5基因与人骨髓间充质干细胞成骨分化现象呈负相关,且BIRC5基因与人骨髓间充质干细胞成骨分化的标志性基因表达也呈负相关。
3.一种用于检测人骨髓间充质干细胞中BIRC5基因表达量的试剂盒,其特征在于包含特异性扩增BIRC5基因的引物;
引物中正向引物序列为:
5'- GGACCACCGCATCTCTACATT-3';
引物中反向引物序列为:
5'- TTTCCTTTGCATGGGGTCGT-3'。
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