CN113925972B - Otub1蛋白用于治疗骨质疏松症的应用 - Google Patents
Otub1蛋白用于治疗骨质疏松症的应用 Download PDFInfo
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Abstract
本发明公开了一种去泛素化酶OTUB1在骨质疏松症治疗中的应用。首次研究OTUB1自身的生理功能,且证明OTUB1在骨质疏松领域具有潜在应用。构建OTUB1特异性敲除小鼠模型,并且证明OTUB1特异性敲除小鼠对骨量有明显影响。此外证明OTUB1敲除小鼠的成骨细胞功能减弱。最终证明OTUB1能够去除FGFR2的泛素链,并维持FGFR2蛋白水平的稳定性,进而调控骨发育。
Description
技术领域
本发明涉及生物医药领域,具体涉及一种OTUB1蛋白用于治疗骨质疏松症的应用。
背景技术
骨质疏松(Osteoporosis)是一种严重危害健康、降低个体生活质量并造成重大经济负担的慢性疾病。调查结果显示,我国约2.1亿人存在低骨量问题,近7000万骨质疏松患者。骨质疏松症已经成为我国50岁以上人群的重要健康问题。以北京为例,40岁及以上人群中81.5%超声检查提示骨健康状况异常,40岁以下人群中76.2%骨健康状况异常。此外,因骨质疏松引起的椎骨、髋骨等骨折发生率约为50%-60%。骨质疏松患者骨折后1年内,患者死于并发症的比例高达20%,致残率高达50%。并且骨质疏松的医疗经济负担巨大,到2025年,全球骨质疏松年度直接费用预计将达到1600亿人民币。骨骼系统的稳态是由骨形成和骨吸收来维持的。在细胞水平上,成骨细胞(负责骨形成)和破骨细胞(负责骨吸收)之间的耦合构成最小的功能单位,两种细胞的分化和功能协调在维持成年骨骼中起着至关重要的作用。因此,深入探究骨细胞的功能的调控机制对于了解骨质疏松的发病机制,寻找骨质疏松的治疗靶点至关重要。
蛋白质稳定性调控中最重要的调控方式是泛素化修饰。泛素化修饰系统中泛素化酶促进蛋白质底物形成泛素链,随后泛素化的蛋白质底物被蛋白酶体降解;去泛素化酶则去除蛋白质底物上的泛素链,避免蛋白质底物被降解,维持蛋白质自身的稳定性。目前已报道的去泛素化酶分为6大类:UCH类、USP类、OTU类、MJD类、PPPDE类和JAMM类,其中OTU类和USP类是研究最广泛的两大类,而OTU类因其切除泛素链的特异性而被大家所熟知。OTUB1(OTU domain ubiquitin aldehyde binding 1)是OTU类中非经典的一个蛋白,它不仅可以像其他OTU类去泛素化酶依赖OTU结构域去除泛素链,还可以在DNA损伤修复中结合UBC13这样的E2来抑制DNA双链损伤后组蛋白的泛素链的形成。类似的,OTUB1还可以结合MDM2协同的E2抑制MDM2对p53 K48型泛素链的降解。深入的研究发现OTUB1不仅抑制E2和E3的相互作用,还可以通过不依赖自身酶活性的方式维持E2 UBE2E1的稳定性。目前报道OTUB1的最主要的功能是调控肿瘤。在肺癌中,OTUB1可以抑制RAS的单泛素化使其能够正常膜定位。在乳腺癌和卵巢癌中,OTUB1去除FOXM1的泛素链并维持FOXM1的稳定性。在前列腺癌中,OTUB1活化RhoA和RhoA介导的前列腺癌的侵袭。此外,OTUB1还可以通过上皮间质转化促进脑胶质瘤、结直肠癌、胃癌和食管癌的侵袭。肿瘤中一些重要的通路也被OTUB1调控:OTUB1通过调控TGFβ通路中的磷酸化Smad2/3的稳定性调控细胞迁移,通过调控mTOR通路中的DEPTOR稳定性调控细胞自噬,通过维持c-IAP1的稳定性调控细胞凋亡。最新的研究还发现OTUB1能促进阿尔兹海默症中关键分子Tau的累积,维持p100的稳定性从而抑制NF-κB的异常激活,调控IL15介导的CD8+ T和NK细胞的活化。总的来说,目前对于OTUB1的研究主要集中在肿瘤领域,关于OTUB1自身的生理功能,尤其是在骨质疏松领域,仍缺乏系统而深入的研究。
发明内容
基于此,本发明首次研究OYUB1自身生理功能在骨质疏松领域的潜在应用价值。
本发明的一方面,提供一种OTUB1基因或OTUB1蛋白在筛选用于治疗或预防骨质疏松疾病的药物靶点的用途。
所述骨代谢疾病为骨生长或骨质的异常增加或减少的相关疾病;优选为骨质疏松症。
本发明的另一方面,提供一种OTUB1基因或OTUB1蛋白作为预防和/或治疗骨代谢疾病治疗靶点、或者作为骨代谢疾病诊断靶点的用途。
所述骨代谢疾病为骨生长或骨质的异常增加或减少的相关疾病;优选为骨质疏松症。
本发明的另一方面,提供一种制备预防或治疗骨生长或骨质的异常降低相关疾病的药物中的用途,所述药物包含可使OTUB1基因或OTUB1蛋白含量或表达量上调的制剂。
本发明的另一方面,提供一种制备预防或治疗成骨细胞分化/矿化相关疾病的药物中的用途,所述药物包含可使OTUB1基因或OTUB1蛋白含量或表达量上调的制剂。
本发明的另一方面,提供一种用于模拟骨发育不良造成的相关疾病的动物模型,将动物的OTUB1基因敲除。
所述的动物模型动物为小鼠。
所述的动物模型主要是:1)OTUB1的表达降低;2)OTUB1特异性敲除小鼠出生比率不变,但是体形较对照组较小;3)骨密度、骨体积分数、骨小梁数目、骨小梁厚度指标在OTUB1敲除后明显下调;4)血清值指标中成骨指标PINP下调,破骨指标CTX不变;5)骨标志物Runx2、Col1a1、Ocn下调;
所述的动物模型的制备方法包括:1)过表达OTUB1后FGFR2蛋白水平上调;2)敲除OTUB1加快FGFR2蛋白水平下调;3)过表达OTUB1延缓FGFR2的蛋白水平下降。
本发明的另一方面,提供一种用于模拟骨发育不良造成的相关疾病的动物模型的制备方法,将动物的OTUB1基因敲除,优选的所述动物为小鼠。
所述的动物模型主要是:1)OTUB1的表达降低;2)OTUB1特异性敲除小鼠出生比率不变,但是体形较对照组较小;3)骨密度、骨体积分数、骨小梁数目、骨小梁厚度指标在OTUB1敲除后明显下调;4)血清值指标中成骨指标PINP下调,破骨指标CTX不变;5)骨标志物Runx2、Col1a1、Ocn下调;
所述的动物模型的制备方法包括:1)过表达OTUB1后FGFR2蛋白水平上调;2)敲除OTUB1加快FGFR2蛋白水平下调;3)过表达OTUB1延缓FGFR2的蛋白水平下降。
本发明的另一方面,提供去泛素化酶OTUB1在制备骨质疏松症预防或治疗药物中的应用, OTUB1去除FGFR2泛素化链;
进一步的,所述骨质疏松症是指骨密度、骨体积分数、骨小梁数目、骨小梁厚度等指标下调;
进一步的,所述FGFR2泛素化链具体为K11和K48泛素链。
所述的应用:1)过表达OTUB1后FGFR2蛋白水平上调;2)敲除OTUB1加快FGFR2蛋白水平下调;3)过表达OTUB1延缓FGFR2的蛋白水平下降。
所述的去泛素化酶OTUB1在制备骨质疏松药物中的应用。
本发明具有的技术效果为:
(1) 本发明首次研究OTUB1自身的生理功能,且证明OTUB1在骨质疏松领域具有潜在应用。
(2) 本发明构建OTUB1特异性敲除小鼠模型,并且证明OTUB1特异性敲除小鼠对骨量有明显影响。
(3) 本发明证明OTUB1敲除小鼠的成骨细胞功能减弱。
(4) 本发明证明OTUB1能够去除FGFR2的泛素链,并维持FGFR2蛋白水平的稳定性,进而调控骨发育。
(5) 本发明构建在成骨细胞中特异性敲除OTUB1的小鼠模型(条件性敲除小鼠模型),同样证明成骨细胞中otub1敲除的小鼠出现多项骨指标明显下调和骨量下降等表型,并明确验证otub1是在成骨细胞中发挥作用影响骨发育。
附图说明
图1 OTUB1敲除小鼠模型构建以及骨结构分析。
(A)OTUB1全身性敲除小鼠模型构建策略;(B)Western blot检测OTUB1敲除小鼠成纤维细胞中OTUB1蛋白水平;(C)野生对照组和OTUB1敲除组小鼠整体拍照;(D)野生对照组和OTUB1敲除组小鼠体重统计;(E)野生对照组和OTUB1敲除组小鼠骨整体结构分析。
图2 OTUB1敲除小鼠的成骨细胞功能减弱。
(A)qPCR方法对骨分化标志物进行检测;(B)qPCR方法对骨增值标志物进行检测;(C)成骨细胞的ALP和ARS染色
图3 OTUB1调控骨发育的机制探究。
(A)OTUB1敲除的成骨细胞中检测FGFR2的蛋白水平;(B)成骨细胞中敲除OTUB1,检测FGFR2蛋白的降解效率;(C)过表达Myc-FGFR2、OTUB1和各种泛素Ub的突变体,检测OTUB1去除FGFR2的泛素化链类型;(D)成骨细胞中敲除OTUB1,检测FGFR2蛋白的泛素化水平。
图4 OTUB1成骨细胞特异性敲除小鼠模型构建以及骨指标和成骨指标的变化。
(A)OTUB1成骨细胞特异性敲除的小鼠构建策略;(B)OTUB1敲除BMSC中OTUB1蛋白的表达;(C)OTUB1成骨细胞特异性敲除小鼠与对照组小鼠体型观察;(D)OTUB1成骨细胞特异性敲除小鼠体重变化;(E)OTUB1成骨细胞特异性敲除小鼠骨骼长度变化;(F)OTUB1成骨细胞特异性敲除小鼠骨受力强度和硬度变化;(G)Micro-CT扫描整根股骨;(H)骨密度(BMD,mg HA/ cm3)、骨体积分数(BV/TV, %)、骨小梁数目(Tb.N, mm)、骨小梁厚度(Tb.Th, mm)指标;(I)OTUB1成骨细胞特异性敲除小鼠血清中P1NP和CTX的表达水平。
具体实施方式
以下通过参考示范性实施例,本发明的目的和功能以及用于实现这些目的和功能的方法将得以阐明。然而,本发明并不受限于以下所公开的示范性实施例;可以通过不同形式来对其加以实现。说明书的实质仅仅是帮助相关领域技术人员综合理解本发明的具体细节。
实施例1 OTUB1敲除小鼠模型的制备和对其骨骼结构进行分析。
分离成纤维细胞检测目的基因表达是确定小鼠模型建立成功与否的关键。委托南京集萃药康公司制备含loxP位点的loxP小鼠,之后将OTUB1基因的2、3号外显子插入loxP位点的loxP小鼠, loxP小鼠通过与全身性表达Cre的工具鼠(CMV-Cre)配繁,最后得到OTUB1全身性敲除小鼠模型(图1A)。
通过以下步骤分离培养成纤维细胞:
1. 取母鼠怀孕13.5天的孕鼠脱臼后75%酒精浸泡数分钟;
2. 取出孕鼠胚胎放在已加满遇冷PBS的12孔板,随后用PBS洗涤3次;
3. 去除胚胎四肢和头部,并留存四肢做基因型鉴定;
4. 将剩余胚胎转移至加了1 mL PBS的12孔板并编号,随后逐个用小剪刀剪碎,每只剪碎时间大于2 min(视胚胎多少决定分成几组解剖,一般3-4只为一组,这样会避免消化时间过久对细胞的损伤);
5. 所有胚胎剪碎后,用移液管吸取剪碎的细胞悬液转移到6孔板,并加入3 mL 胰酶,继续用剪刀剪1 min;
6. 再给每个胚胎加2 mL胰酶,用移液管反复捶打30-40次,37 ℃消化25 min左右直至看到胚胎成粘稠状(消化时间具体参考所取小鼠胚胎发育时间,发育越早消化时间越短);
7. 继续用移液管吹打胚胎20-30次,随后加入6 mL DMEM再吹打20次;
8. 转移到15 mL离心管,1500 rpm离心10 min;
9. 离心完沉淀均在管底方为消化合适,随后10 mL DMEM重悬铺在T25瓶或者10cm盘;
10. 3-4 h后换液,2-3 d内长满用作实验。
在获得成纤维细胞后,通过Western blot 验证OTUB1蛋白的表达水平。Westernblot步骤为:1) 细胞裂解液样品制备:a. 弃掉细胞培养液,用细胞刮刀将细胞刮下来,加入预冷的PBS;b. 2000rpm 3min,随后继续用PBS洗脱1-2次直至细胞培养基清洗干净;c.加入对应的细胞裂解液和loading buffer,沸水中煮10-20 min后5000 rpm离心3 min备用; 2)SDS-PAGE电泳分离蛋白,一般先90-100 V跑20 min后160 V跑40 min左右;3)当上述电泳快完毕时,剪切膜(9 mm/6 mm)和滤纸浸泡入电转缓冲液几分钟;4)按照滤纸-膜-胶的顺序将夹板放入电转槽中,150 mA冰上电转2 h;5)断开电源,取出夹板,将NC膜置于丽春红中染色,切出目的条带,TBST洗脱2-3次;6)牛奶封闭,将NC膜置于含有5%TBST配制的脱脂牛奶的中室温封闭1h;7)封闭后一抗4 ℃过夜,回收一抗,TBST洗膜3次,每次10 min,加入二抗,室温孵育30 min-1 h,TBST洗膜洗膜3次,每次10 min;8)显色反应:ECL发光试剂A.B液等体积混合后加到NC膜上,反应约3 min后吸去发光液,于暗室中曝光,观察western blot结果。实验结果发现OTUB1蛋白表达水平显著下调,提示OTUB1全身性敲除小鼠模型成功建立(图1B)。接着,简单的分析了OTUB1敲除小鼠的体型变化,结果发现OTUB1敲除小鼠体型明显偏小(图1C),且体重降低(图1D)。
最后,对整个小鼠的骨骼进行阿尔辛蓝和茜素红染色,其中茜素红染色是将硬骨染色成红色;阿尔辛蓝染色是将软骨染色成蓝色。具体的阿尔辛蓝和茜素红染色的步骤为:小鼠模型被取出内脏并置于 4℃ 的水中过夜。 将鼠体浸入 67 ℃ 水浴 1 分钟,剥皮,并在乙醇中固定 3 天。 将Alcian blue 8GX(150 mg;Sigma,A5268-10G)加入到 80 mL 95%乙醇和 20 mL冰醋酸的混合物中。 软骨染色 8 - 12 h,在 100% 乙醇中冲洗过夜,然后在2% KOH 中清洗 6 h。 将茜素红 S(50 mg;Sigma)加入 1 L 2% KOH 中,将胚胎浸泡 3 h以复染骨。 骨架在 2% KOH 和 20% 甘油中澄清并储存在 50% 乙醇和 50% 甘油中。结果发现OTUB1敲除小鼠出现明显的颅骨闭合不全、胸骨和指骨中硬骨缺失,但软骨并无明显差别(图1E),最终结果提示OTUB1影响硬骨发育。
实施例2 OTUB1敲除小鼠的成骨细胞功能影响
1. 成骨标志物的检测
硬骨的发育离不开成骨细胞的功能,为此我们检测了OTUB1敲除小鼠的成骨细胞功能。采用手术法分离新生小鼠颅骨骨片,用0.25%胰蛋白酶+0.02%乙二胺四乙酸(EDTA)预消化20~30 min,而后用0.08%胶原酶+0.05%胰蛋白酶+0.004% EDTA的复合酶多次消化(每次10 min)收集细胞,然后接种于细胞培养瓶中进行培养,分离成骨细胞。然后采用qPCR方法对成骨分化的标志物Runx2、Col1a1、Ocn进行检测,具体步骤如下:1. 根据提取RNA的浓度确定逆转录所需要的RNA体积,加入到PCR管中备用;2. 装有RNA的PCR管置于PCR仪中65℃预热5 min,随后置于4 ℃ 使RNA维持较为松散的结构,便于随后的逆转录过程;3. 加入2 μL 5×RT mix、~1 μg RNA以及ddH2O,总10 μL;4. 根据逆转录RNA试剂盒的说明书确定逆转录的具体程序;逆转录之后的cDNA测定浓度,根据具体的需求,确定随后的cDNA的使用,其中cDNA最好能够现配现用,如实在不行可放置于-80℃冰箱保存。5. cDNA随后直接用来做qPCR,具体基因扩增的引物如下:
β-actin:
5′-GTACGCCAACACAGTGCTG-3′(forward)
5′-CGTCATACTCCTGCTTGCTG-3′(reverse)
Otub1:
5′-TGATGGCAACTGCTTCTACCG-3′(forward)
5′-GTCCTCTTTACTCTTGGCAGAC -3′(reverse)
Runx2:
5′-CCCAGCCACCTTTACCTACA-3′(forward)
5′-TATGGAGTGCTGCTGGTCTG-3′(reverse)
Col1a1:
5′-GAGCGGAGAGTACTGGATCG-3′(forward)
5′-GTTAGGGCTGATGTACCAGT-3′ (reverse)
Ocn:
5′-CTCACAGATGCCAAGCCCA-3′(forward)
5′-CAAGGTAGCGCCGGAGTCT-3′ (reverse)
Cyclin D1:
5′-GCGTACCCTGACACCAATCTC-3′(forward)
5′-CTCCTCTTCGCACTTCTGCTC-3′ (reverse)
Cdkn1a:
5′- CCTGGTGATGTCCGACCTG -3′(forward)
5′- CCATGAGCGCATCGCAATC -3′ (reverse)
c-myc:
5′- ATGCCCCTCAACGTGAACTTC -3′(forward)
5′- CGCAACATAGGATGGAGAGCA -3′ (reverse)
结果如图2A所示,成骨分化的标志物Runx2、Col1a1、Ocn均显示出明显的下调。于此同时,我们以同样方法检测成骨细胞的增殖Marker,发现cyclin D、Cdkn1a、c-myc等增殖标志物没有明显变化(图2B)。以上结果提示OTUB1敲除只影响骨分化,不影响骨增殖。
敲除小鼠成骨细胞功能影响
碱性磷酸酶(ALP)是成熟成骨细胞的标志性酶,同时成骨细胞形成的钙结节也是成骨细胞的标记物。因此采用ALP和ARS染色,对成OTUB1敲除后成骨细胞分化能力进行验证。将小鼠成骨细胞以每孔 1 × 105 个细胞的密度接种在 24 孔板中,并在生长培养基中扩增 3 天。 我们通过在分化培养基中培养来诱导成骨细胞分化。 在诱导成骨细胞分化后第 14 天,将细胞用 4% 多聚甲醛固定 15 min,0.1% Triton X-100 固定 10 min,然后用 ALP 检测溶液和 1% 茜素红 S(ARS)染色。ALP和ARS染色结果如图2C所示,与对照组相比,成骨细胞分化能力明显降低。基于上述结果,均证明OTUB1敲除小鼠的成骨细胞分化功能减弱。
实施例3 OTUB1去除FGFR2的泛素链并维持FGFR2蛋白水平的稳定性,进而调控骨发育。
FGFR2(Fibroblast growth factor receptor 2)在骨骼形态发生、发育和生长中不可或缺,是成骨细胞增殖、分化和矿化的重要调控因子,我们通过Western blot筛选发现是OTUB1敲除成骨细胞中FGFR2的蛋白水平出现显著下调(图3A)。
用放线菌酮(CHX)处理实施例2中获得的OTUB1敲除小鼠成骨细胞,抑制细胞内蛋白质合成。我们发现对照组中OTUB1和FGFR2的蛋白水平都有些许下调,但在敲除OTUB1组中可以看到FGFR2的蛋白水平显著下降,这提示敲除OTUB1加快了FGFR2的蛋白水平下降(图3B)。
众所周知,泛素化修饰是经典的调控蛋白水平稳定性的翻译后修饰类型。而泛素链因泛素分子含有1个甲硫氨酸残基和7个赖氨酸残基,可被分为M1 K6 K11 K27 K29 K33K48和K63共8种泛素链,K11和K48修饰是经典的蛋白质降解信号。且OTUB1是一个经典的去泛素化酶,可以去除底物蛋白上的泛素链。鉴于OTUB1调控FGFR2蛋白水平,因此我们推测OTUB1可能通过去除FGFR2的降解型泛素链维持FGFR2蛋白稳定性。泛素化实验操作如下所示:
1. 收集细胞,预冷PBS洗3次后备用;
2. 加500 μl RIPA裂解液(根据预实验结果增加去污剂NP40、SDS的浓度,确保泛素链的干净),超声至澄清,12000 rpm离心10 min;
3. 取上清50 μl作为总细胞裂解液,其余上清加入相应抗体,旋转混合器上4 ℃孵育3 h(如果做的纯内源泛素化实验,可以考虑增加孵育时间和使用protein-A/G-agarose预清除)
4. 加入40 μl 裂解液洗脱过的protein-A/G-agarose,旋转混合器上4 ℃孵育8h或则过夜;随后裂解液洗脱5×8 min(遵循多次洗脱原则,不建议长时间洗脱);
5. 洗脱好的agarose中加入40 μl裂解液和loading buffer,混匀后100 ℃沸水煮15 min,5000 rpm离心3 min,备用;
6.蛋白样品进行免疫印迹检测泛素化水平。
通过泛素化实验,结果显示OTUB1去除FGFR2的K11和K48泛素链(图3C)。于此同时,我们发现OTUB1敲除成骨细胞中FGFR2的泛素化修饰水平显著上调(图3D)。以上结果表明OTUB1去除FGFR2的降解型泛素链,进而维持FGFR2自身的蛋白稳定性,最终调控FGFR2介导的成骨细胞的发育。
实施例4 成骨细胞OTUB1特异性敲除小鼠模型的制备和对骨量的影响。
骨发育是由成骨细胞和破骨细胞共同作用的结果,前述实验已经证明在OTUB1敲除小鼠模型中,OTUB1的敲除对骨发育的影响。为了进一步证明OTUB1在成骨细胞中发挥作用,我们又构建了成骨细胞中OTUB1特异性敲除的小鼠模型。
小鼠OTUB1成骨细胞特异性敲除模型的制备
前面我们证实OTUB1依赖FGFR2调控成骨细胞发育,本实施例中我们同样将loxP小鼠通过与成骨特异性表达Cre的工具鼠(OSX-Cre)配繁(区别于实施例1中的全身性表达Cre的工具鼠(CMV-Cre)配繁),最后得到OTUB1成骨细胞特异性敲除小鼠模型(图4A)。
进一步我们分离敲除小鼠来源的骨髓间充质干细胞(Bone marrow stromalcell,BMSC), 具体步骤如下:
6-8周小鼠脱颈处死后用75 %酒精浸泡3 min消毒;取每只小鼠双侧股骨及胫骨,用纱布将附着的肌肉及其它结缔组织剥离干净,全程注意无菌操作,在6孔板中每孔加入2mL PBS,将剔除干净的股骨和胫骨放入6孔板中浸泡5 min后,再次用纱布或剪刀去除残存的肌肉或结缔组织;用眼科剪剪掉股骨及胫骨两端,用5 mL注射器吸取4 mL PBS冲出骨髓,直至骨干发白;冲出的骨髓置于15 mL离心管中,用移液枪吹打使其充分混匀,1000 rpm离心5 min,弃上清,收集细胞,加入2 mL红细胞裂解液,轻轻吹打混匀后静置5 min;裂解后,离心管中加入4 mL α-MEM完全培养基稀释并终止裂解,1000 rpm 离心5 min,弃上清,收集细胞,观察细胞沉淀是否充分裂解;加入5 mL 含有15 %血清、1 %双抗的α-MEM完全培养基重悬细胞,用70 μm的细胞筛过滤后进行细胞计数,接种细胞于10 cm培养皿中培养,3天后弃掉上清,贴壁细胞即为骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs);连续传代纯化:每2-3天换液,细胞生长接近70 %-80 %密度时,用0.25 %胰酶消化,按照1:3传代,实验使用第三代细胞。
通过Western blot检测发现OTUB1敲除的BMSC细胞中OTUB1蛋白表达水平显著下调,提示OTUB1成骨细胞特异性敲除小鼠模型成功建立(图4B)。同时我们也发现该模型中FGFR2蛋白水平也下调(图4B),同样验证了OTUB1依赖FGFR2调控成骨细胞发育的结论。
成骨细胞特异性敲除小鼠形态学观察。
进一步对OTUB1成骨细胞特异性敲除小鼠(即OTUB1条件性敲除小鼠,即该小鼠成骨细胞中OTUB1特异性敲除,下简称OTUB1条敲小鼠)进行形态学观察,发现OTUB1条敲小鼠体型明显偏小(图4C)。进一步称量其体重发现OTUB1条敲小鼠体重降低(图4D)。同时,将8周小鼠脱颈处死后用取每只小鼠双侧股骨,用纱布将附着的肌肉及其它结缔组织剥离干净,并直尺测量长度进行统计发现OTUB1条敲小鼠骨长度显著下调(图4E)。随后进行三点弯曲实验:为测量肱骨的骨强度,采用小动物骨强度测试仪(YLS-16A,中国济南燕益市)进行三点弯曲试验。 解剖后立即使用三点弯曲试验检查新鲜肱骨。 三点弯曲试验使用两个末端支撑点和一个中心加载点。 从载荷-变形曲线中收集生物力学测量数据。 记录最大载荷(N)。结果发现OTUB1条敲小鼠的骨受力强度和硬度(图4F)显著下调。
扫描与重建分析小鼠模型骨指标变化
首先去除左股骨的软组织,用4 %多聚甲醛溶液固定24 h,转入70 %乙醇溶液后可在4 ℃长期保存。使用Micro-CT扫描整根股骨,参数为220 μA/60 kV,分辨率为9.21 μm,扫描结果如图4G所示;扫描结束后用COBRA Exxim软件重建,从股骨生长板最低点以下0.5 mm处开始,选取0.5 mm厚为感兴趣区域,使用Inveon Research Workplace分析软件计算感兴趣区域的骨指标,包括骨密度(BMD, mg HA/ cm3)、骨小梁数目(Tb.N, mm)、骨体积分数(BV/TV, %)、骨小梁厚度(Tb.Th, mm)。结果如图4H所示,骨密度、骨小梁数目、骨体积分数、骨小梁厚度指标在OTUB1敲除后明显下调。
骨形成标记物和骨吸收标记物的检测
采用ELISA试剂盒检测骨形成标记物1型胶原蛋白末端前肽(N-terminalpropeptide of type 1 collagen, P1NP)和骨吸收标记物C末端肽(C-telopeptide, CTX)的表达水平。酶联免疫吸附实验步骤为:取小鼠血液,在4℃条件下,转速3000 rpm离心10min,取上清至新的离心管中即为血清,血清分装储存在−80°C,结果如图4I,检测血清值指标发现成骨指标PINP下调破骨指标CTX不变。这个结果表明OTUB1敲除小鼠的成骨能力下降,破骨能力不变,总体表现为骨吸收保持不变,但没有新的骨量补充,最终骨量下降。P1NP和CTX是骨代谢最直接的指标之一,代表着骨形成与骨吸收。理论上PINP的表达水平越高,对应的骨形成能力越高。CTX的表达水平越高,对应的骨吸收能力越高。机体的骨形成大于骨吸收,最直接的效应表现为骨量上升。机体的骨吸收小于骨形成,最直接的效应就表现为骨量下降。
由此可见,成骨细胞中OTUB1敲除的小鼠同样出现OTUB1蛋白表达水平显著下调、FGFR2蛋白水平下调;骨受力强度和硬度显著下调;多项骨指标明显下调和骨量下降等表型,与OTUB1全身性敲除小鼠模型结论一致,证明OTUB1是在成骨细胞中发挥作用影响了骨发育,同时再次验证OTUB1通过调节FGFR2表达,在成骨细胞中对骨发育发挥作用。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明技术原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (8)
1.OTUB1蛋白在制备预防或治疗成骨细胞分化相关疾病的药物中的用途,其特征在于,所述OTUBI通过去除FGFR2泛素化链维持FGFR2蛋白稳定性,从而调控成骨细胞分化。
2.根据权利要求1所述的用途,其中OTUB1通过去除FGFR2的K11和K48泛素链从而维持FGFR2蛋白水平稳定。
3.根据权利要求1或2所述的用途,其中骨分化以1型胶原蛋白末端前肽表达量表征的。
4.OTUB1基因或OTUB1蛋白在制备通过去除FGF2泛素化链从而维持FGF2蛋白稳定性的药物中的用途。
5.OTUB1蛋白在制备骨质疏松症预防或治疗药物中的应用,其特征在于,所述OTUBI通过去除FGFR2泛素化链维持FGFR2蛋白稳定性,从而调控成骨细胞分化。
6.根据权利要求5所述的用途,其中OTUB1通过去除FGFR2的K11和K48泛素链从而维持FGFR2蛋白水平稳定。
7.一种动物模型的制备方法,其特征在于,将动物成骨细胞OTUB1特异性敲除,所述动物为小鼠;所述的方法包括如下步骤:
1)制备含loxP位点的loxP小鼠;
2)将OTUB1基因的2、3号外显子插入loxP位点的loxP小鼠;
3)将loxP小鼠通过与成骨特异性表达Cre的工具鼠OSX-Cre配繁。
8.如权利要求7所述的动物模型的制备方法制备获得的动物模型在研究动物骨形成中的应用,其特征在于通过检测该动物模型中1型胶原蛋白末端前肽的表达量来监测动物成骨能力改变。
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