CN113797314B - Cst多肽在制备股骨头坏死治疗药物中的应用 - Google Patents
Cst多肽在制备股骨头坏死治疗药物中的应用 Download PDFInfo
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Abstract
本发明公开了CST多肽在制备股骨头坏死治疗药物中的应用，尤其是制备激素诱导性股骨头坏死治疗药物中的应用，特别针对制备减轻激素诱导股骨头坏死塌陷药物中的应用。功能多肽CST在股骨头坏死中发挥保护性作用。体外细胞实验表明，CST可拮抗糖皮质激素导致的成骨细胞凋亡过程。同时，动物模型显示，CST可抑制股骨头坏死塌陷发生，改善病情，且长期应用未见明显毒副作用，可用于股骨头坏死等领域，具有广阔的应用价值和市场前景。
Description
技术领域
本发明涉及医学领域，具体涉及CST多肽在制备股骨头坏死治疗药物中的应用。
背景技术
目前，股骨头坏死在临床上多见，而且目前趋于年轻化。据统计，仅我国，股骨头坏死患者就超过500万人。目前针对股骨头坏死无特效药，临床应用的药物效果不佳，如患者病情严重，则需要进行髋关节手术，行髓心减压或髋关节置换手术[1]。然而手术本身存在相关风险及并发症，有其自身的限制因素。随着发病患者不断增多，股骨头坏死的治疗已经对社会及家庭造成巨大负担。
目前临床使用的治疗药物多针对具体症状，而对股骨头坏死本身进行保护及改善的相关药物种类极少，患者选择余地小。同时，目前使用相关药物疗效有限，且长期应用有一定副作用，所以在一定程度上限制了使用。
近期研究提示，CST多肽在细胞凋亡抑制过程中存在潜在保护功能[2]。我们通过前期实验，对CST多肽治疗进行研究，发现其对股骨头坏死过程具有改善功能，可能为长期使用从而减少股骨头坏死发病提供潜在方法。
[1]Li Q,Liao W,Fu G,Liao J,Zhang R,Li M,Yang Y,Ma Y,Zheng M,ZhengQ.Combining autologous bone marrow buffy coat and angioconductive bioceramicrod grafting with advanced core decompression improves short-term outcomes inearly avascular necrosis of the femoral head:a prospective,randomized,comparative study.Stem Cell Res Ther.2021Jun 19；12(1):354.doi:10.1186/s13287-021-02436-0
[2]Zhao Y,Qiu C,Wang W,Peng J,Cheng X,Shangguan Y,Xu M,Li J,Qu R,ChenX,Jia S,Luo D,Liu L,Li P,Guo F,Vasilev K,Liu L,Hayball J,Dong S,Pan X,Li Y,Guo L,Cheng L,Li W.Cortistatin protects against intervertebral discdegeneration through targeting mitochondrial ROS-dependent NLRP3 inflammasomeactivation.Theranostics.2020May 27；10(15):7015-7033.doi:10.7150/thno.45359.eCollection 2020.Theranostics.2020.PMID:32550919
发明内容
本发明的目的是提供一种CST多肽为有效成分的骨保护性制剂在制备股骨头坏死疾病治疗药物中的应用。
CST多肽作为抗凋亡及促进成骨活性的成分，具有改善成骨细胞代谢，减轻股骨头坏死病情严重程度的作用，且体外实验未显示CST多肽有细胞毒性作用，药物长期使用更加安全。
本发明的技术方案是：
Cortistatin(纯度98.4％，CAS:186901-48-4,其氨基酸序列为Pro-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Ser-Ser-Cys-Lys(Disulfide bridge:Cys2-Cys13))(吉尔生化有限公司)其分子量：1720.03。
CST多肽具有良好的维持细胞代谢稳定及抑制细胞凋亡功能，提纯工艺简单，价格低廉，这也是其相较于其他股骨头坏死保护性分子的优势所在。同时，目前对于其纯度，已经达到98％以上，纯度符合药物制备要求。
本发明提供CST多肽在制备股骨头坏死治疗药物中的应用，尤其的，在应用机理上，其作为成骨细胞保护因子及凋亡拮抗分子，阻断成骨细胞过度凋亡功能实现。
本发明所述药物，还包括药学上可接受的载体、助剂或稀释剂。
所述药物的形式选自如下之一：喷雾、气溶胶、溶液、洗液、凝胶、片剂、胶囊、软膏、糊剂、乳剂和悬浮液。
优选的，所述药物的形式优选皮下、肌肉注射针剂、局部凝胶。
另外，公知的，CST多肽包含多种分子亚型，本发明的CST多肽即包括其各种亚型，特别的，优选CST-14、CST-17及其类似物、衍生物等。
通过体外细胞实验及体内动物模型实验结果表明，CST多肽治疗可拮抗成骨细胞过度凋亡及代谢障碍，并在股骨头坏死性疾病中发挥保护性作用。其功能成分CST属于细胞代谢紊乱及凋亡的拮抗分子,可通过现有技术直接提取纯化，从而降低成本。另外，动物模型中，CST多肽的长期应用未见明显毒副作用，可用于股骨头坏死性疾病等领域。
因此其在制备股骨头坏死性疾病治疗药物中具有可预期的应用，具有广阔的市场前景。
附图说明
图1是CST治疗在大鼠股骨头坏死模型中的股骨头外观治疗效果图像
图2是CST治疗在大鼠股骨头坏死模型中的X线治疗效果图像
图3是CST治疗在大鼠股骨头坏死模型中的CT治疗效果图像
图4是CST治疗在大鼠股骨头坏死模型中的基于CT结果的统计学分析
图5是CST治疗在大鼠股骨头坏死模型中的HE染色图像
图6是CST治疗在大鼠股骨头坏死模型中基于HE组织学染色图像的统计学分析
图7是CST治疗在大鼠股骨头坏死模型中的CD31免疫组化染色图像
图8是CST治疗对于地塞米松诱导的成骨细胞过度凋亡的拮抗效应。
具体实施方式
以下实施例中涉及的实验动物、试剂、培养基和缓冲液的来源：
皮质抑素(Cortistatin,分子量：1720.03)(吉尔生化有限公司)。
SD雄性大鼠，(山东大学动物中心)
PBS缓冲液，(碧云天生物试剂公司)
Col2抗体，MMP13抗体，ADAMTS-5抗体(Thermo Fisher，Pierce)
RIPA细胞蛋白质提取裂解液(Thermo Fisher，Pierce)
蛋白酶抑制剂(北京索莱宝科技有限公司)
BCA蛋白定量试剂盒(上海炎熙生物科技有限公司)
Complete EDTA-Free(罗氏生物医药)
二甲苯(国药集团化学试剂有限公司)
中性树胶(上海泰坦科技股份有限公司)
浓盐酸(国药集团化学试剂有限公司)
伊红(上海泰坦科技股份有限公司)
苏木素(上海泰坦科技股份有限公司)
甲醇(国药集团化学试剂有限公司)
柠檬酸盐缓冲液(0.01M，PH＝6.0)(生工生物工程上海股份有限公司)
10％NGS(生工生物工程上海股份有限公司)
过氧化氢(H2O2)(国药集团化学试剂有限公司)
BSA(生工生物工程上海股份有限公司)
无水乙醇(国药集团化学试剂有限公司)
细胞计数仪购自美国Thermo Fisher公司
显微镜购自上海蔡康光学仪器有限公司
离心机购自济南欧莱博医疗器械有限公司
电子天平购自济南欧莱博医疗器械有限公司
酶联免疫检测仪(酶标仪)购自北京美华仪科技有限公司
制冰机购自济南欧莱博医疗器械有限公司
超纯水系统购自济南欧莱博医疗器械有限公司
涡旋混合器购自济南欧莱博医疗器械有限公司
1.股骨头坏死大鼠模型构建
在8周龄野生型SD雄性大鼠中建立糖皮质激素诱导股骨头坏死模型(共21只)。空白组的SD大鼠(7只)不做任何处理。剩余14只大鼠进行甲强龙(40毫克/千克体重)腹膜内注射，第一周每天注射一次甲强龙，后每周3次甲强龙注射连续5周。阳性对照无治疗，实验组行CST多肽(250微克/千克体重)腹膜内注射治疗，从第一周开始每周3次。实验结束后，将所有组的小鼠用过量的10％水合氯醛(山东大学齐鲁医院)安乐死，收集股骨头标本，对其外观进行拍照比较坏死严重程度，并用于X线及CT检测，分析骨质改变的程度。
图1-4为本部分实验的结果，图1为诱导治疗后的股骨头外观，图2为X线图像，图3为CT三维重建图像，图4为基于CT的BV/TV百分比分析，均显示大鼠的股骨头坏死在CST多肽治疗下有明显缓解。
2.组织切片制备
将来自所有组的大鼠股骨头组织固定在10％福尔马林中，至少在室温下固定72小时。50％乙醇(60分钟)、70％乙醇(60分钟)、85％乙醇(60分钟)、95％乙醇(60分钟)、100％乙醇(30分钟)、100％乙醇(30分钟)依次进行组织脱水；经过乙醇和二甲苯(60分钟)、二甲苯(60分钟)依次处理；然后用二甲苯和石蜡(60分钟)，石蜡(80分钟)透明；将组织放入盒中，用石蜡填充，然后将其放在石蜡包埋机的冷台上。将嵌入的组织石蜡块置于切片机上并切片，组织厚度约4μm；将含有组织的石蜡片轻轻涂抹在42℃的水中。完全展平后，用干净的玻璃片轻轻拨起切片；将切片放在载玻片上，编号，并在68℃的烤箱中烘烤至少6小时。
3.苏木精/伊红染色
切片常规脂溶性溶剂脱蜡至水(二甲苯两次，15分钟/每次；100％酒精5分钟；95％酒精5分钟；75％酒精5分钟；50％酒精5分钟)，然后使用苏木精染色液染色5分钟，用清水冲洗干净后，继续使用伊红染色液染色5分钟，清水冲洗干净后脱水(50％酒精5分钟；75％酒精5分钟；95％酒精5分钟；100％酒精5分钟；二甲苯两次，15分钟/每次)，后等切片晾干后使用中性树胶封片，放在光学显微镜下观察，并使用Image J进行组织学评分分析。
图5-6为本部分实验的结果图，图5为HE组织学染色，图6为基于HE染色的空白陷窝百分比计数，表明CST多肽的治疗减轻了大鼠股骨头坏死模型中股骨头组织的骨破坏程度。
4.免疫组织化学染色
组织切片脱蜡并水合。即连续二甲苯8分钟、二甲苯8分钟、二甲苯8分钟、无水乙醇8分钟、无水乙醇8分钟、95％乙醇8分钟、80％乙醇8分钟和75％乙醇8分钟。在70％乙醇8分钟后，将切片用水冲洗4次，每次5分钟；接下来，将脱蜡和水合的切片置于3％过氧化氢溶液中并在37℃下反应20分钟以阻断内源性过氧化物酶。每次用双蒸水洗涤4次，每次5分钟，修复抗原。将柠檬酸盐缓冲液置于金属加热器中并煮沸。煮沸15分钟，关闭电源并保持15分钟。自然冷却至室温；然后每次洗涤PBS 5次，每次5分钟，擦拭周围组织，加入5％山羊血清，阻断非特异性抗原，在室温下反应1小时，然后将CD31抗体逐滴添加至组织切片，并在4℃下在湿盒中孵育过夜；第二天，取出切片，在37℃培养箱中孵育1小时，用PBS冲洗5次，每15分钟，加入增强的辣根过氧化物酶偶联二抗，室温孵育2小时。用PBS清洗多余的二抗(5次，每次5分钟)；滴加新鲜制备的DAB着色溶液，在光学显微镜下观察，并呈现棕黄色，并用PBS洗涤以停止显色；接下来，进行苏木精复染。将有色切片置修饰的苏木精染色中并染色5分钟。在光学显微镜下观察染色。然后用0.2％盐酸分离切片，并用流水冲洗切片。最后，切片通过70％酒精10分钟、75％酒精8分钟、80％酒精8分钟、90％酒精8分钟、无水乙醇8分钟、无水乙醇8分钟、二甲苯8分钟和二甲苯8分钟依次浸洗。脱水2分钟后，擦拭组织周围的组织并擦拭，滴加中性凝胶，将盖玻片置于光学显微镜下观察。
图7为本部分实验的结果图，通过血管内皮特异性的CD31免疫组化染色，表明CST多肽的使用减轻了激素所致股骨头坏死模型中大鼠骨质中血供的破坏。
5.MC3T3-E1细胞培养
MC3T3-E1细胞为成骨细胞稳定细胞系，在DMEM/F-12培养基(Hyclone，Thermo CO)中加入10％的牛血清(FBS，gibco，USA)、1％100u/ml青霉素和100mg/ml链霉素(Hyclone，USA)，于ph7.2同步培养MC3T3-E1细胞(95％空气，5％co2,37°c)。培养基每3天更换一次，细胞达到80-90％汇合时传代。第二代或第三代细胞用于指定的实验。
6.Western Blot
体外培养及刺激后的各组MC3T3-E1细胞，置于冰上，经过处理后用冰水洗涤。收集后加入裂解缓冲液(p0013c，beyotime biotechnology)中提取蛋白质，将培养的髓核细胞然后加入RIPA裂解缓冲液(p0013c，beyotime biotechnology)，收集了每个样品的总蛋白质。在负载缓冲液中的蛋白质在100℃下加热10min(thermofisher)。采用10％SDS-PAGE凝胶(beyontime biotechnology)进行蛋白质电泳(每道30g)，电泳后将蛋白质电泳转移到硝化纤维素膜上。用吐温20(10mm tris-hcl，ph8.0；150mm nacl；0.5％tween 20)在5％脱脂干奶中阻隔2h，与特异性初级抗体(Bax，Bcl-2，β-Tubulin)在37℃下孵育1h，加入PBS洗涤3次后在4℃下过夜，加入辣根过氧化物酶二级抗体(稀释1:2000)，室温孵育1h。将膜用钝性镊子取出，用PBS冲洗至少三次。每膜加1ml工作液(p0018s，Beiotime Biotechnology)并检测(Amersham生命科学，艾林顿，伊利诺伊州，美国)。使用Image J软件进行条带灰度值统计学分析。
图8为本实验结果图，通过使用Western blot，观察地塞米松刺激所导致的凋亡标记物水平异常，而CST多肽则减轻了成骨细胞在地塞米松刺激下的凋亡过程。
7.统计学分析
所有数据均以至少三次独立实验的平均数士标准差表示。对于两组数据的统计学分析采用配对t检验多于两组数据的统计学分析采用单因素方差分析。
通过上述实验，证实CST多肽能够直接拮抗糖皮质激素介导的成骨细胞功能紊乱及过度凋亡，并且对于甲强龙诱导的股骨头坏死动物模型有确切疗效，同时有效促使股骨头内环境稳态。另外，CST多肽便于成骨细胞吸收利用,填补目前国内股骨头坏死防治药物的空白。
此外，细胞实验显示，CST多肽可以减轻糖皮质激素刺激所造成的成骨细胞代谢紊乱及凋亡过程，在股骨头坏死过程中发挥保护作用。
更为重要的是，CST多肽提纯工艺简单，制备方法纯熟，可明显降低成本，从而降低患者经济负担。
以上对本发明的一个实施例进行了详细说明，但所述内容仅为本发明的较佳实施例，不能被认为用于限定本发明的实施范围。凡依本发明申请范围所作的均等变化与改进等，均应仍归属于本发明的专利涵盖范围之内。
Claims (5)
1.CST多肽在制备激素诱导的股骨头坏死治疗药物中的应用，其特征在于：CST作为成骨细胞保护性分子，通过抑制成骨细胞异常凋亡，改善细胞代谢稳态实现；所述CST的氨基酸序列为Pro-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Ser-Ser-Cys-Lys(Disulfidebridge:Cys2-Cys13)。
2.CST多肽在制备减轻激素诱导股骨头坏死塌陷药物中的应用，其特征在于：所述CST的氨基酸序列为Pro-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Ser-Ser-Cys-Lys(Disulfide bridge:Cys2-Cys13)。
3.如权利要求1至2任一项所述的应用，其特征在于：所述药物还包括药学上可接受的载体、助剂或稀释剂。
4.如权利要求1至2任一项所述的应用，其特征在于：所述药物的形式选自如下之一：喷雾、气溶胶、溶液、洗液、凝胶、胶囊、片剂、糊剂、乳剂和悬浮液。
5.如权利要求4所述的应用，其特征在于：所述药物的形式为皮下、肌肉注射针剂、局部凝胶。
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| CN111388651A (zh) * | 2020-05-09 | 2020-07-10 | 山东大学齐鲁医院 | Cst-14在制备骨质疏松症治疗药物中的应用 |
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| CN111388651A (zh) * | 2020-05-09 | 2020-07-10 | 山东大学齐鲁医院 | Cst-14在制备骨质疏松症治疗药物中的应用 |
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