CN114751960B - 多肽及其在骨修复中的应用 - Google Patents
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Abstract
本发明涉及一种多肽及其在骨修复中的应用，属于生物医药技术领域。本发明提供的多肽，其氨基酸序列如SEQ ID NO:1所示。本发明还公开了所述多肽GC16在骨损伤，和/或骨修复中的用途。进一步的，本发明还公开了一种用于骨修复的多肽支架。本发明多肽GC16能够吸引并募集细胞进入骨修复材料，具有加速骨缺损的修复和再生的作用，可作为骨组织工程的功能因子。
Description
技术领域
本发明属于生物医药技术领域，涉及一种可促进骨修复的人工合成多肽GC16。
背景技术
骨缺损是一种常见的临床疾病，《中国骨质疏松白皮书》数据显示，我国每年新增骨损伤患者约300万人，给公众健康带来了巨大负担。骨缺损可能由多种原因引起，包括创伤、感染、肿瘤和衰老等。虽然骨组织具有强大的自我修复和重建再生能力，但尺寸较大的缺损，往往伴随着骨不连、功能障碍、愈合延迟，甚至不愈合等结局。此时，需要特殊的治疗方法进行干预，以恢复受损骨组织的结构和功能。
自体骨移植被认为是修复临界骨缺损的金标准，然而自体骨移植物的应用具有一定的局限性，其受到供体部位发病、供体来源缺乏和感染风险增加等问题的严重限制。异体骨移植(取自其他患者)可部分弥补自体骨的不足，提供一些生长因子，并具有骨诱导特性。但这种方法也面临着来源有限、伦理等一系列问题。目前，采用无机非金属或高分子材料支架的组织工程骨受到广泛关注，通过3D打印等个性化定制的支架材料能够很好的匹配缺损区域，利用其疏松多孔的结构引导细胞血管生成从而实现骨再生。然而组织工程骨高度依赖其搭载的种子细胞和细胞因子，以在体内募集和诱导修复细胞(如骨髓间充质干细胞BMSC)增殖和分化，从而形成新骨组织。目前FDA批准的具有促进新骨形成的药物中，甲状旁腺素(PTH)在高剂量摄入时可致骨肉瘤形成，骨形成蛋白(BMP2)的半衰期短，可引起异位骨形成、骨溶解和局部炎症反应。因而有效且安全的促进骨形成的因子仍有待开发。
发明内容
本发明的目的在于克服上述现有技术的不足之处，提供一种可促进骨修复的人工合成多肽。
为实现上述目的，本发明采用的技术方案为：
本发明提供了一种多肽，所述多肽的氨基酸序列如SEQ ID NO:1所示。
本发明的多肽含有16个氨基酸，氨基酸序列为GPGGDKCKCHGLSGSC，即Gly Pro GlyGly Asp Lys Cys Lys Cys His Gly Leu Ser Gly Ser Cys，发明人将多肽命名为GC16，以下多肽均用此名。
本发明的多肽，其分子量为1505.69Da。
本发明多肽GC16可采用常规的合成方法，如液相分段合成法、固相合成、生物合成法等，作为本发明所述多肽的优选实施方式，所述多肽采用固相多肽合成工艺合成多肽。进一步的，为确保生物安全性，本发明多肽的纯度≥95％。可使用HPLC对产物进行纯化。
本发明多肽可用于骨损伤和/或骨修复。进一步的，适用于广泛的骨缺损适应症，如截断性骨缺损、骨相关创伤、肿瘤骨缺损等，可促进骨缺损修复和/或用于修复骨缺损。
GC16的设计来源于WNT3A配体蛋白上与细胞膜Frizzled受体及LRP5/6共受体结合的识别区段，该蛋白可激活经典Wnt信号通路，激活膜内β-catenin入核，从而启动下游功能基因的转录。大量研究表明经典Wnt信号通路参与调控成骨谱系细胞存活、增殖、分化的各个阶段，其主要功能包括：调控成骨前体细胞成骨向分化，促进成骨细胞增殖及提高成骨细胞和骨细胞的存活率。因此，基于GC16的体内实验数据，我们推测其可发挥类似WNT3A蛋白的作用，激活经典Wnt信号通路所对应的骨修复作用。
发明所述多肽应用的优选实施方式，所述多肽应用的浓度为25～150μg/mL，优选100～150μg/mL，进一步优选为100μg/mL。
作为优选方案，本发明多肽GC16可与组织工程上可接受的载体联用来治疗骨损伤和/或骨修复。进一步的，本发明所述多肽GC16可以以任何形式负载在骨修复材料上，植入骨缺损部位。例如，多肽GC16可以负载于骨水泥中、也可以与骨修复水凝胶联用注射到骨损伤部位、以及可作为种植体表面涂层等。
GC16能够以组织工程支架搭载的作用方式，加速骨缺损区域的修复，促进骨再生。所述组织工程上可接受的载体通常意义上是指组织工程支架，进一步的，可适用于现有所有组织工程支架，包括可生物降解型的骨组织工程支架材料和非生物降解型的骨组织工程支架材料。
进一步地，通过构建大鼠颅骨缺损模型，将GC16植入骨缺损区域，4周及8周后取颅骨组织，使用Micro-CT扫描组织、重建组织三维视图、分析缺损区新生骨相对骨密度，并通过组织切片、HE、Masson染色观察组织形态。通过Micro-CT、HE及Masson染色结果综合分析多肽成骨效果，相比于无效肽，多肽GC16可显著加速颅骨缺损修复，因此，说明其具有作为骨组织工程功能因子治疗骨缺损的潜力。
作为优选方案，多肽药物GC16能够吸引募集细胞进入支架材料内，于4周时促进炎性机化组织和纤维组织形成，完成缺损连接，于8周时促使缺损区由矿化程度较高的纤维组织完成修复，从而加速骨缺损的修复和再生，经验证为骨组织工程的有效功能因子。
进一步的，本发明还公开了一种骨修复组合物，所述骨修复组合物含有治疗有效剂量的多肽GC16和组织工程上可接受的载体。
作为优选方案，本发明还公开了一种多肽支架，其包含多肽GC16。优选地，所述多肽支架为生物陶瓷、金属、碳基和可降解高分子复合材料等。
进一步的，所述可降解高分子复合明胶支架优选为：海藻酸钠、壳聚糖、透明质酸及甲基丙烯酸酐化明胶(GelMA)支架。
作为优选方案，本发明多肽支架中，GC16均匀分散在甲基丙烯酸酐化明胶(GelMA)支架中。
作为优选方案，在多肽支架中，多肽GC16的浓度为25～150μg/mL，优选为100～150μg/mL，进一步优选为100μg/mL。
甲基丙烯酰化明胶(GelMA)是一种光敏性生物材料，其粉末溶于水等液体时，可做到与功能因子均匀混合。GelMA具有极好的操作性,可在光引发剂的作用下迅速交联形成三维结构。GelMA具有良好的生物相容性，其结构上具有细胞黏附位点，可促进细胞的增殖迁移。
GC16修饰的GelMA支架还可在模具辅助下或通过3D打印制备成任意外形，以贴合缺损区形态的。通过改变GelMA的取代度和浓度，可以灵活调整固化后的力学性能，使其具备一定的弹性、强度和支撑性，以恢复缺损骨的结构和部分功能。
本发明的有益效果:
1)本发明提供的多肽类药物GC16能够吸引并募集细胞进入骨修复材料(如支架等)，具有加速骨缺损的修复和再生的作用，可作为骨组织工程的功能因子。
2)本发明的多肽GC16可发挥类似WNT3A重组蛋白的作用，可激活经典Wnt信号通路所对应的骨修复作用。
3)本发明提供的多肽GC16属小分子多肽，制备工艺简单，成本低，产量高，具有较高的转化价值和临床应用前景。
附图说明：
图1为多肽植入大鼠颅骨缺损4周及8周后各组Micro-CT扫描三维重建图。
图2为多肽植入大鼠颅骨缺损4周及8周后各组缺损区新生组织相对骨密度(relative BMD)比较。
图3为多肽植入大鼠颅骨缺损4周及8周后各组HE染色。
图4为多肽植入大鼠颅骨缺损4周及8周后各组Masson染色。
具体实施方式
下面结合实施例对本发明的具体实施方式做进一步的描述，并不因此将本发明限制在所述的实例范围之中。
实施例1多肽载体GelMA水凝胶支架的制备
1.1GelMA制备：2g明胶于60℃溶于10mL PBS后，加入125μL甲基丙烯酸酐(MA)搅拌2小时，加入40mL PBS终止反应，将反应液倒入12-14kDa透析袋，去离子水透析，冻干机冻干，所得粉末即为GelMA。
1.2多肽修饰：2g GelMA冻干粉于60℃溶于10mL PBS中，按照0.1mg/mL的浓度添加多肽，将GC16与GelMA溶液充分混匀，然后加入2.5％光引发剂LAP，再次充分混匀。用移液枪吸取20μL上诉混合物注入聚四氟乙烯定制模具上直径为5mm的圆孔中，紫外灯照射1min，待材料固化后，将材料从孔板上分离下来，放置于冰上待用。对照组使用搭载有无效肽(Control peptide)的GelMA水凝胶支架。其中，GC16采取固相多肽合成工艺合成多肽，使用HPLC对产物进行纯化，合成的多肽纯度为96.25％；无效肽的氨基酸序列为SEQ ID NO:2所示，即CKPLRLSKEEHPLK，无效肽同样采取固相多肽合成工艺合成多肽，使用HPLC对产物进行纯化，合成的多肽纯度为96.25％(下述实施例中，如无特别声明，无效肽均为此氨基酸序列)。
实施例2多肽植入大鼠颅骨缺损后的骨修复效果
2.1动物模型：采用12周龄SD雄性大鼠，每只约320±20g，每组3只。使用2％戊巴比妥(按大鼠体重300g/ml比例注射)进行腹腔注射麻醉，取俯卧位，剃毛刀行头部备皮，碘伏消毒术区，一次性无菌孔巾铺在消毒区域。从鼻骨开始沿头顶部正中线纵行皮肤切口1.5-2.0cm，手术刀柄轻轻分离皮下组织，沿颅骨矢状缝整齐切开骨膜，钝性分离骨膜，充分暴露顶骨、枕骨和部分额骨。颅顶骨中线两侧使用环钻各制备一个直径为5mm的圆形全层骨缺损，植入消毒好的材料。实验组植入搭载有多肽GC16的GelMA支架材料，对照组植入搭载有无效肽的GelMA支架材料。复位皮肤，缝合，再次消毒。
2.2组织取材：4周/8周后处死大鼠，取颅顶骨，4％多聚甲醛浸泡组织于4℃过夜固定，浸泡于PBS中保存。
2.3结果验证：使用Micro-CT对颅骨游离标本进行扫描，扫描条件：70kVp，200μA，精度10μm。使用Micro-CT自带分析软件进行三维重建及相对骨密度分析。
如图1颅骨缺损三维重建图显示：植入材料4周后，无效肽组缺损区几乎无新骨形成，而多肽GC16显著促进缺损区边缘的骨形成，使骨缺损面积显著减少。8周时，GC16更进一步促使新生的修复骨进入缺损中央，而对照组新生骨仅存在于缺损边缘区域。
如图2相对骨密度分析结果显示：材料植入4周后，相比于无效肽对照组，GC16组新生骨的相对骨密度显著提高，而相比于4周，在8周时GC16组相对骨密度进一步提高，且与对照组仍有显著统计学差异。*p＜0.05，**p＜0.01。
综上所述，GC16可加速骨缺损部位的骨修复，并且可提升新生骨的骨密度，可以实现促进骨修复的效果。
实施例3多肽植入大鼠颅骨缺损后缺损区新生组织形态学观察
3.1样品制备：大鼠颅骨组织泡在pH＝7.0浓度为12％的EDTA中，浸泡6周脱钙至骨头足够柔软。
3.2组织切片：组织脱水、石蜡包埋、切成厚度为6μm的组织切片。
3.3结果验证：组织切片在65℃温度下烘烤2小时，二甲苯及梯度乙醇脱蜡水化，HE及Masson染色观察缺损区新生组织的组织学形态。
如图3HE染色结果显示：植入材料4周后，与对照无效肽组相比，多肽GC-16可以很好地吸引募集细胞进入支架材料，缺损区未见明显支架材料存留，缺损由机化的炎性组织以及纤维组织完成连接修复。植入材料8周后，无效肽对照组缺损区中央仍存留大量空白凝胶，纤维组织仅于材料周围完成缺损区连接，在连接组织中见少量成熟骨形成，而GC16组缺损区由矿化程度较高的纤维组织完成修复。
如图4Masson染色结果显示：多肽GC16可募集细胞进入材料内部，4周时骨缺损区由紫红色的机化组织以及部分蓝色纤维组织完成连接，8周时缺损区新生组织为蓝染矿化程度更高的纤维组织完成连接，而对照的无效肽组，4周及8周时缺损区中央均存留大量无细胞长入的支架材料，仅周围由纤维组织完成连接。综合上述组织学染色结果，提示GC16具有募集细胞长入，加速骨缺损修复及新生骨形成的作用。
SEQUENCE LISTING
<110> 四川大学
<120> 多肽及其在骨修复中的应用
<130> GC16
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 16
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Gly Pro Gly Gly Asp Lys Cys Lys Cys His Gly Leu Ser Gly Ser Cys
1 5 10 15
<210> 2
<211> 14
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Cys Lys Pro Leu Arg Leu Ser Lys Glu Glu His Pro Leu Lys
1 5 10
Claims (5)
1.一种多肽，其特征在于：所述多肽的氨基酸序列如SEQ ID NO:1所示。
2.一种骨修复组合物，其特征在于：所述骨修复组合物含有治疗有效剂量的如权利要求1所述的多肽和组织工程上可接受的载体。
3.一种多肽支架，其特征在于：包含权利要求1中所述的多肽。
4.根据权利要求3所述的多肽支架，其特征在于：所述支架为甲基丙烯酸酐化明胶支架；所述多肽的浓度为25~150μg/mL。
5.如权利要求1所述的多肽在制备治疗骨损伤和/或骨修复组合物中的应用。
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