CN102321574A - Xbp1s，一种新型促进软骨修复的转录因子 - Google Patents
Xbp1s，一种新型促进软骨修复的转录因子 Download PDFInfo
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Abstract
我们申请的发明内容是：转录因子XBP1S具有诱导软骨细胞分化、促进软骨修复的生物学功能。我们首次发现转录因子XBP1S可以促进和诱导软骨细胞的分化，促进骨质增长，修复受损的关节，增加骨关节腔中软骨的密度，从而促进形成关节软骨组织，达到预防和治疗关节炎的作用。XBP1S的这一生物学功能的发现具有创造性和创新性，为此，我们特申请专利保护。
Description
技术领域
本发明专利涉及一种新型的诱导软骨细胞分化，促进软骨修复的转录因子剪接型X盒结合蛋白1(X-box binding protein 1 spliced，XBP1S)，此发明属于医药卫生领域，可以直接应用于骨外科或创伤修复等临床和基础医学领域。
转录因子XBP1S作为环磷酸腺苷反应元件结合蛋白/激活转录因子(cyclic-AMP response element binding protein/activating transcription factor，CREB/ATF)家族的一个重要成员，结构上属于亮氨酸拉链蛋白家族。基因结构分析显示XBP1S是一个序列特异的DNA结合蛋白，具有DNA识别结构域、转录活性结构域和结合其它蛋白结构域三个功能域。XBP1S是真核细胞在内质网应激(ER Stress，ERS)状态下激活的一种转录因子，ERS介导的未折叠蛋白反应(Unfolded Protein Response，UPR)直接指导和参与ERS状态下内质网中异常蛋白质的降解和再折叠，维持内质网内环境的稳定，实现对细胞的保护。其中转录因子XBP1S介导的一条UPR信号通路inositol-requiring enzyme1(IRE1)-XBP1是决定细胞最终命运的关键通路，也是促进细胞分化的关键途径。
我们首先在软骨细胞分化的不同时相检测了XBP1S的转录与表达，同时运用免疫组织化学技术在不同胎龄小鼠骨生长板软骨细胞内检测了XBP1S的表达，我们发现XBP1S在软骨细胞分化的增殖期和肥大期均有表达。同时，我们利用基因芯片技术，在软骨细胞中筛选出XBP1S反式激活的一系列差异表达基因。在得到的2,986个差异表达基因中(筛选标准：实验组与对照组荧光信号强弱的比值fold change 2 fold为差异表达基因)，包括1701个表达明显上调的基因；979个表达明显下调的基因。在这些XBP1S反式激活表达上调的基因中，包括诱导软骨细胞分化的骨形态发生蛋白BMP2和生长因子GEP，软骨细胞中XBP1S可以明显上调BMP2和GEP的表达，XBP1S上调BMP2和GEP表达的倍数分别是3.39和3.17；同时，我们利用realtime PCR和免疫印迹等研究方法也发现转录因子XBP1S在软骨细胞中可以明显上调BMP2和GEP的转录与表达(基金编号：No.31040019，2011年12月结题)。GEP，又名Progranulin(PGRN)，属于自分泌生长因子，分子量68.5KD，在合成之后要进行糖基化修饰，通过分泌到细胞外，与靶细胞表面特异受体结合发挥其生物学作用。我们研究结果显示BMP2可以明显上调GEP的转录和表达(见说明书附图Fig.1A)；重组GEP蛋白可以明显促进软骨细胞的分化(见说明书附图Fig.1B，1C)；而运用RNAi技术抑制GEP的表达可 以显著抑制BMP2诱导的软骨细胞分化(见说明书附图Fig.1D，1E)，因此，GEP是BMP2诱导产生的一种可以促进软骨细胞分化的生长因子，并且BMP2诱导的软骨细胞分化依赖于GEP的存在。2011年3月10日，我们在《SCIENCE》上率先公布了生长因子progranulin(PGRN)的受体，这一令人振奋的研究成果很好的解释了PGRN在软骨发育中的多项生物学功能：诱导分化、促进修复和抗炎。PGRN，又名Granulin-Epithelin Precursor(GEP)，作为骨形态发生蛋白2(Bone Morphogenetic Protein2，BMP2)的下游分子参与诱导软骨细胞的分化；而转录因子XBP1S在软骨细胞中明显上调BMP2和GEP，我们的研究结果显示XBP1S可以通过上调GEP、BMP2诱导软骨细胞分化，促进软骨的修复。这一发明可以直接应用于骨外科或创伤修复等临床和基础医学领域，有助于探索与解析软骨发育新的调控机制、为开发参与软骨生成与修复的生物制剂提供靶分子。
背景技术
目前，临床使用的大多数药物治疗不能有效促进关节软骨缺损的愈合；自体软骨来源又非常有限，软骨移植手术也相应受到限制。因此，研发促进软骨修复的生物制剂是非常有意义的。我们在前期研究中发现转录因子XBP1S可以诱导软骨细胞的分化，促进软骨修复。正对XBP1S的这一生物学功能，同时也为了后续的进一步深入研究，我们进一步对其基因序列进行改造，在XBP1S起始密码子ATG后面增加信号肽(singal peptide)序列，以确保XBP1S的真核表达与蛋白质的分离纯化。
相关背景技术如下：
首先，进入互联网美国国家图书馆网站http://www.ncbi.nlm.nih.gov，根据GenBank提供的XBP1S mRNA全序列XBP1S(AB076384)，用引物设计软件NTI vector设计引物，上游引物加入BamH I酶切位点，下游引物加入HindIII酶切位点，目的基因扩增长度1128bp，构建XBP1S腺病毒载体Ad-XBP1S。同样的方法设计内参GAPDH引物，扩增长度542bp。酶切电泳与测序结果显示构建正确，免疫印迹检测所构建病毒可以正确表达(见说明书附图1、2)。
其次，我们利用基因芯片技术，在软骨细胞中筛选出XBP1S反式激活的一系列差异表达基因。在得到的2,986个差异表达基因中(筛选标准：实验组与对照组荧光信号强弱的比值fold change 2 fold为差异表达基因)，包括1701个表达明显上调的基因；979个表达明显下调的基因。在这些XBP1S反式激活表达上调的基因中，包括诱导软骨细胞分化的骨形态发生蛋白BMP2和生长因子GEP，软骨细胞中XBP1S可以明显上 调BMP2和GEP的表达，XBP1S上调BMP2和GEP表达的倍数分别是3.39和3.17；同时，我们利用realtime PCR和免疫印迹等研究方法也发现转录因子XBP1S在软骨细胞中可以明显上调BMP2和GEP的转录与表达(相应结果见说明书附图3)。
第三，我们运用不同类型干细胞(C3H10T1/2，ATDC5)的体外微团培养，在软骨细胞分化的不同时期，采用real-time PCR及免疫印迹等方法检测了XBP1S的转录与表达；同时运用免疫组织化学技术在不同胎龄小鼠骨生长板软骨细胞内检测了XBP1S的表达，我们发现XBP1S在软骨细胞分化的增殖期和肥大期均有表达，并且随着软骨发育的不断成熟，XBP1S表达逐渐增加，在新生小鼠软骨发育的肥大期XBP1S表达达到峰值(相应结果见说明书附图4)。
第四，构建si-XBP1S腺病毒载体，与前面构建好的XBP1S腺病毒载体Ad-XBP1S分别转染干细胞C3H10T1/2和ATDC5，结合干细胞微团培养、小鼠趾骨体外诱导培养等方法，检测XBP1S与软骨细胞分化的关系，结果显示，XBP1S参与诱导软骨细胞的分化，通过上调软骨细胞肥大期标志基因X型胶原(Collagen type X，ColX)的表达，促进软骨细胞的分化；通过免疫组化等相关技术检测XBP1S Knockdown(KD)小鼠趾骨发现XBP1S促进软骨细胞肥大、矿化与内骨生长，促进软骨修复(相应结果见说明书附图5、6、7)。
总之，真核细胞中XBP1S具有调控细胞的生长、增殖、分化或者凋亡的能力，是决定细胞最终命运的调节器。我们进一步通过动物实验证实，转录因子XBP1S可以促进和诱导软骨细胞的分化，促进骨质增长，修复受损的关节，增加骨关节腔中软骨的密度，从而促进形成关节软骨组织，达到预防和治疗关节炎的作用。
发明内容
人类XBP1S基因位于22号染色体(22q12.1)上，在成人组织中广泛存在和表达，是新近发现的一种与蛋白质折叠和内质网构建相关的蛋白质。XBP1S基因结构分析显示XBP1S是一个序列特异的DNA结合蛋白，具有DNA识别结构域、转录活性结构域和结合其他蛋白结构域三个功能域。我们在前期研究中发现转录因子XBP1S可以诱导软骨细胞的分化，促进软骨修复，并进一步在动物实验中证实了XBP1S的这一生物学功能。
转录因子XBP1S的基因序列如下：
ATG gtggtggtg gcagccgcgc cgaacccggc cgacgggacc cctaaagttc tgcttctgtc ggggcagccc
gcctccgccg ccggagcccc ggccggccag gccctgccgc tcatggtgcc agcccagaga ggggccagcc
cggaggcagc gagcgggggg ctgccccagg cgcgcaagcg acagcgcctc acgcacctga gccccgagga
gaaggcgctg aggaggaaac tgaaaaacag agtagcagct cagactgcca gagatcgaaa gaaggctcga atgagtgagc
tggaacagca agtggtagat ttagaagaag agaaccaaaa acttttgcta gaaaatcagc ttttacgaga gaaaactcat
ggccttgtag ttgagaacca ggagttaaga cagcgcttgg ggatggatgc cctggttgct gaagaggagg cggaagccaa
ggggaatgaa gtgaggccag tggccgggtc tgctgagtcc gcagcaggtg caggcccagt tgtcacccct ccagaacatc
tccccatgga ttctggcggt attgactctt cagattcaga gtctgatatc ctgttgggca ttctggacaa cttggaccca
gtcatgttct tcaaatgccc ttccccagag cctgccagcc tggaggagct cccagaggtc tacccagaag gacccagttc
cttaccagcc tccctttctc tgtcagtggg gacgtcatca gccaagctgg aagccattaa tgaactaatt cgttttgacc
acatatatac caagccccta gtcttagaga taccctctga gacagagagc caagctaatg tggtagtgaa aatcgaggaa
gcacctctca gcccctcaga gaatgatcac cctgaattca ttgtctcagt gaaggaagaa cctgtagaag atgacctcgt
tccggagctg ggtatctcaa atctgctttc atccagccac tgcccaaagc catcttcctg cctactggat gcttacagtg
actgtggata cgggggttcc ctttccccat tcagtgacat gtcctctctg cttggtgtaa accattcttg ggaggacact
tttgccaatg aactctttcc ccagctgatt agtgtc TAA
XBP1S氨基酸序列如下：
mvvvaaapnp adgtpkvlll sgqpasaaga pagqalplmv paqrgaspea asgglpqark rqrlthlspe ekalrrklkn
rvaaqtardr kkarmseleq qvvdleeenq klllenqllr ekthglvven qelrqrlgmd alvaeeeaea kgnevrpvag
saesaagagp vvtppehlpm dsggidssds esdillgild nldpvmffkc pspepaslee lpevypegps slpaslslsv
gtssakleai nelirfdhiy tkplvleips etesqanvvv kieeaplsps endhpefivs vkeepveddl vpelgisnll
ssshcpkpss clldaysdcg yggslspfsd mssllgvnhs wedtfanelf pqlisv
我们首次发现转录因子XBP1S可以促进和诱导软骨细胞的分化，促进骨质增长，修复受损的关节软骨，增加骨关节腔中软骨的密度，从而促进形成关节软骨组织，达到预防和治疗关节炎的作用。XBP1S的这一生物学功能的发现具有创造性和创新性。
我们的发明内容是：XBP1S诱导软骨细胞分化、促进软骨修复的生物学功能。
我们申请对转录因子XBP1S上述两项特定的内容进行专利保护。根据中华人民共和国国家知识产权局发布的”专利审查指南(2010)”第三部分”进入国家阶段的国际申请的审查”部分的规定(P302)：如果在申请的发明中，某蛋白质已知而其氨基酸序列是未知的，那么只要本领域技术人员在该申请提交时可以容易地确定其氨基酸序列，编码该蛋白质的基因发明就不具有创造性。但是，如果该基因具有特定的碱基序列，而且与其他编码所述蛋白质的、具有不同碱基序列的基因相比，具有本领域技术人员预料不 到的效果，则该基因的发明具有创造性。
说明书附图说明
图1是XBP1S的PCR扩增结果与真核表达载体酶切电泳图。A.XBP1S PCR扩增结果；B.pcDNA3.1(-)-XBP1S酶切电泳结果.
图2是XBP1S测序结果图.
图3是软骨细胞中XBP1S反式激活基因的差异表达谱分析.
图4是软骨发育不同时期XBP1S的表达结果.BMP2诱导微团培养干细胞C3H10T1/2，A.real-time检测软骨细胞分化不同时间点XBP1S与ColX的mRNA水平；B.免疫印迹检测软骨细胞分化不同时间点XBP1S与ColX的表达；C.免疫组化检测XBP1S在小鼠软骨生长板增殖期与肥大期均有表达.
图5是靶向XBP1S的两条siRNA的测序结果.
图6是siXBP1S导致小鼠软骨发育迟缓的检测结果.A.siXBP1S小鼠(KD)中XBP1S表达降低；B.Safranin O检测KD小鼠中软骨生长板发育迟缓；C，D，E.原位杂交检测KD小鼠中软骨生长板ColII(C)/ColX(D)/Alp(E)表达明显降低.
图7是XBP1S促进软骨细胞肥大、矿化与内骨生长，促进软骨修复的检测分析结果.A，B.Safranin O/Fast Green染色显示Ad-XBP1S诱导培养15.5天胎鼠趾骨的发育；C，D.Alizarin red/Alcian blue染色显示Ad-XBP1S诱导培养15.5天胎鼠趾骨发育的矿化与内骨生长；E，F.Ad-XBP1S促进损伤关节软骨的修复与量化分析.
转录因子XBP1S的基因序列如下：
ATG gtggtggtg gcagccgcgc cgaacccggc cgacgggacc cctaaagttc tgcttctgtc ggggcagccc gcctccgccg ccggagcccc ggccggccag gccctgccgc tcatggtgcc agcccagaga ggggccagcc cggaggcagc gagcgggggg ctgccccagg cgcgcaagcg acagcgcctc acgcacctga gccccgagga gaaggcgctg aggaggaaac tgaaaaacag agtagcagct cagactgcca gagatcgaaa gaaggctcga atgagtgagc tggaacagca agtggtagat ttagaagaag agaaccaaaa acttttgcta gaaaatcagc ttttacgaga gaaaactcat ggccttgtag ttgagaacca ggagttaaga cagcgcttgg ggatggatgc cctggttgct gaagaggagg cggaagccaa ggggaatgaa gtgaggccag tggccgggtc tgctgagtcc gcagcaggtg caggcccagt tgtcacccct ccagaacatc tccccatgga ttctggcggt attgactctt cagattcaga gtctgatatc ctgttgggca ttctggacaa cttggaccca gtcatgttct tcaaatgccc ttccccagag cctgccagcc tggaggagct cccagaggtc tacccagaag gacccagttc cttaccagcc tccctttctc tgtcagtggg gacgtcatca gccaagctgg aagccattaa tgaactaatt cgttttgacc acatatatac caagccccta gtcttagaga taccctctga gacagagagc caagctaatg tggtagtgaa aatcgaggaa gcacctctca gcccctcaga gaatgatcac cctgaattca ttgtctcagt gaaggaagaa cctgtagaag atgacctcgt tccggagctg ggtatctcaa atctgctttc atccagccac tgcccaaagc catcttcctg cctactggat gcttacagtg actgtggata cgggggttcc ctttccccat tcagtgacat gtcctctctg cttggtgtaa accattcttg ggaggacact tttgccaatg aactctttcc ccagctgatt agtgtc TAA
转录因子XBP1S的氨基酸序列如下：
mvvvaaapnp adgtpkvlll sgqpasaaga pagqalplmv paqrgaspea asgglpqark rqrlthlspe ekalrrklkn rvaaqtardr kkarmseleq qvvdleeenq klllenqllr ekthglvven qelrqrlgmd alvaeeeaea kgnevrpvag saesaagagp vvtppehlpm dsggidssds esdillgild nldpvmffkc pspepaslee lpevypegps slpaslslsv gtssakleai nelirfdhiy tkplvleips etesqanvvv kieeaplsps endhpefivs vkeepveddl vpelgisnll ssshcpkpss clldaysdcg yggslspfsd mssllgvnhs wedtfanelf pqlisv
Claims (3)
1.一种转录因子，人类X-box binding proteinl spliced(XBP1S)，具有新型、独特的诱导软骨细胞分化、促进软骨修复的生物学功能。
2.根据权利要求1所述的转录因子XBP1S，其生物学功能特征如下：首先，Ad-XBP1S腺病毒颗粒可以明显上调软骨细胞分化各标志基因II型胶原(ColII)、X型胶原(ColX)的表达；促进软骨细胞的肥大、矿化以及软骨内骨化；促进软骨的修复。
其次，与正常小鼠相比，XBP1S knockdown(KD)小鼠骨发育迟缓、骨生长板增殖缓慢、软骨内骨化延迟；同时，XBP1S KD小鼠的软骨发育相关标志基因ColII、ColX及碱性磷酸酶(Alkaline phosphatase，ALP)的表达均明显降低。
3.根据权利要求1所述的转录因子XBP1S，其所具有的新型、独特的生物学特征是：促进和诱导软骨细胞的分化，促进软骨修复。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105531378A (zh) * | 2013-09-12 | 2016-04-27 | 拉什大学医学中心 | 用于表征可动关节软组织损伤后细胞修复反应的转录组学指数 |
| CN106093393A (zh) * | 2016-06-02 | 2016-11-09 | 滨州医学院 | 一种基于XBP1和XBP1s表达水平的检测胃癌组织的试剂盒 |
| CN106148507A (zh) * | 2016-04-22 | 2016-11-23 | 同济大学苏州研究院 | 一种用于检测人源性X盒结合蛋白1s基因表达水平的引物组合及其应用 |
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| FENGJIN GUO ET AL.: "XBP1U inhibits the XBP1S-mediated upregulation of the iNOS gene expression in mammalian ER stress response", 《CELLULAR SIGNALLING》 * |
| 林建炜: "转录因子XBP1s与细胞增殖及凋亡的实验研究", 《中国优秀硕士学位论文全文数据库》 * |
| 林建炜等: "转录因子XBPlS对人肝癌HepG2细胞增殖及凋亡的影响", 《肿瘤》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105531378A (zh) * | 2013-09-12 | 2016-04-27 | 拉什大学医学中心 | 用于表征可动关节软组织损伤后细胞修复反应的转录组学指数 |
| CN105531378B (zh) * | 2013-09-12 | 2021-04-09 | 拉什大学医学中心 | 用于表征可动关节软组织损伤后细胞修复反应的转录组学指数 |
| CN106148507A (zh) * | 2016-04-22 | 2016-11-23 | 同济大学苏州研究院 | 一种用于检测人源性X盒结合蛋白1s基因表达水平的引物组合及其应用 |
| CN106093393A (zh) * | 2016-06-02 | 2016-11-09 | 滨州医学院 | 一种基于XBP1和XBP1s表达水平的检测胃癌组织的试剂盒 |
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