CN118021995A - 多肽在制备用于预防和/或治疗骨肉瘤的药物中的应用 - Google Patents
多肽在制备用于预防和/或治疗骨肉瘤的药物中的应用 Download PDFInfo
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Abstract
本发明属于生物医药技术领域,具体涉及一种多肽在制备用于预防和/或治疗骨肉瘤的药物中的应用。多肽的氨基酸序列如SEQ ID NO:1所示;该多肽为小分子多肽与细胞穿膜肽接连所形成的嵌合肽。本发明通过精心设计得到了一种小分子多肽,通过本实施例的实验结果显示,该小分子多肽可以抑制SPICE1与USP10蛋白相互作用,并且能够抑制骨肉瘤细胞增殖,促进骨肉瘤细胞凋亡,这对于提高骨肉瘤的治疗效果、改善患者的预后具有重大的现实意义。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种多肽在制备用于预防和/或治疗骨肉瘤的药物中的应用。
背景技术
关于骨肉瘤的发生和发展的分子调控机制一直属于研发重点,积极寻找新的治疗靶点对于提高骨肉瘤的治疗效果、改善患者的预后具有重大的现实意义。
在现有中国专利申请号为202211389701X,发明名称为一种骨肉瘤分子标记物及其应用中已经明确证明SPICE1是骨肉瘤的潜在新治疗靶点,发现SPICE1在骨肉瘤组织中高表达,敲低该蛋白后,能够抑制骨肉瘤增殖并促进骨肉瘤细胞凋亡。然而SPICE1是一个大分子蛋白,其具体的活性区域尚未被确定,因此关于SPICE1蛋白的具体作用机制需要进一步研究。
发明内容
本发明的目的是解决现有技术的不足,提供一种多肽在制备用于预防和/或治疗骨肉瘤的药物中的应用,本发明通过模拟SPICE1的活性区域,合成了外源性多肽,用以抑制SPICE1与USP10的结合,从而抑制骨肉瘤的进展,具体采用以下的技术方案:
在本发明的第一方面,提供了一种多肽在制备用于预防和/或治疗骨肉瘤的药物中的应用,其多肽的氨基酸序列如SEQ ID NO:1所示;该多肽为小分子多肽与细胞穿膜肽接连所形成的嵌合肽。
SEQ ID NO:1:YGRKKRRQRRRTAEILRLREENA。
上述多肽的结构式为:Biotin-YGRKKRRQRRR-TAEILRLREENA-FITC。
上述多肽分子量为4.05 KDa。
需要说明的是,该小分子多肽为SPICE1蛋白质的氨基酸片段的415到426位氨基酸片段。
作为进一步优选的实施方式,上述药物还包括药学上可接受的载体或赋形剂。该赋形剂为阿拉伯胶、糖浆、羊毛脂、淀粉中的至少一种。
作为进一步优选的实施方式,上述药物的给药途径为口服、透皮、肌肉、皮下或静脉注射。
作为进一步优选的实施方式,上述药物为片剂、丸剂、颗粒剂、胶囊剂或注射剂。
在本发明的第二方面,还提供了一种预防和/或治疗骨肉瘤的药物,该药物包括上述的多肽。
作为进一步优选的实施方式,上述多肽是药物中唯一或主要的有效成分。该药物为液体剂型或冻干粉剂。
本发明的有益效果为:
本发明通过精心设计得到了一种小分子多肽,通过本实施例的实验结果显示,该小分子多肽可以抑制SPICE1与USP10蛋白相互作用,并且能够抑制骨肉瘤细胞增殖,促进骨肉瘤细胞凋亡,这对于提高骨肉瘤的治疗效果、改善患者的预后具有重大的现实意义。
附图说明
图1所示为穿膜肽修饰的SPICE1氨基酸片段的质谱图;
图2所示为穿膜肽修饰的SPICE1氨基酸片段抑制细胞内SPICE1与USP10的结合图;A:骨肉瘤细胞HOS细胞;B:骨肉瘤细胞143B细胞;
图3所示为CCK8实验证实穿膜肽修饰的SPICE1氨基酸片段抑制骨肉瘤细胞增殖图;A:骨肉瘤细胞HOS细胞;B:骨肉瘤细胞143B细胞;
图4所示为克隆形成实验证实穿膜肽修饰的SPICE1氨基酸片段抑制骨肉瘤细胞增殖图;A:骨肉瘤HOS细胞及143B细胞各组克隆集落分布情况;B:各组克隆集落数统计图;
图5所示为采用流式细胞仪检测穿膜肽修饰的SPICE1氨基酸片段对骨肉瘤细胞凋亡图;A:骨肉瘤HOS细胞及143B细胞各组凋亡情况;B:各组细胞凋亡统计图。
具体实施方式
以下将结合实施例和附图对本发明的构思、具体结构及产生的技术效果进行清楚、完整的描述,以充分地理解本发明的目的、方案和效果。需要说明的是,在不冲突的情况下,本申请中的实施例及实施例中的特征可以相互组合。
实施例1
一种可以应用在制备预防和/或治疗骨肉瘤的药物中多肽的合成方法,其具体包括以下步骤:
步骤1:根据多肽的序列选择树脂合成,选用Rik Amide AMResim,量为1克;
步骤2:取树脂放到接肽瓶中,先用6克/毫升的DME(二甲醚)溶液泡10分钟左右,然后加含20%六氢吡烷的DME溶夜(6克/毫升),在室温下放在摇床上,振5分钟左右,再抽干,再加含20%六氢吡烷的DME溶液(6克/毫升),在室温下放在摇床上,振荡15分钟,再抽干,用DME洗树脂8次;
步骤3:根据计算的重量将Fmoc保护的氨基酸及缩合剂加到接肽瓶中,再加含有6克/毫升的DME溶夜及碱试剂的树脂溶液,然后在室温下放在摇床上,振荡60分钟,再抽干;取20克树脂,放在试管里加1毫升Kaiser试剂,在水浴下100℃加热5分钟,树脂的颜色如果无色表明反应完全,如果有蓝色,淡紫色则说明反应不完全,重新再加Fmoc保护的氨基酸及缩合剂到接肽瓶中,再加含有6克/毫升的DME溶液及碱试剂的树脂溶液,然后再重复反应,直到颜色变为无色;
步骤4:加含20%六氢吡烷的DME溶夜(6克/毫升),密封好,在室温下放在摇床上,振荡5分钟左右,再抽干,再反应,在室温下放在摇床上,振荡15分钟左右,再抽干,用DME洗树脂三次,无水甲醇洗一次,二氯甲烷洗一次,抽干;
步骤5:根据序列依次合成下一个氨基酸,重复第3步及第4步的操作过程;
步骤6:多肽序列合成完成后,用乙醚洗树脂6次,然后抽干,干燥;
步骤7:按照三氟乙酸:水:对甲苯酚:二硫醇 92.5:2.5:2.5:2.5(体积比)配切割试剂,然后把多肽树脂加到溶液中,密封好在室温振荡4小时,过滤,用三氟乙酸洗,滤液中加无水乙醇,析出沉淀固体用乙醚洗若干次,然后抽干;得到多肽的粗品;
步骤8:利用Merrifield发展的固相多肽合成方法合成多肽,合成方向是从多肽的C端开始向多肽的N端方向合成。其基本原理为:先将所要合成肽链的C末端氨基酸的羧基以共价键结合的方式与不溶性的高分子树脂相连,然后以此结合在固相载体上的氨基酸的氨基经脱去氨基保护基并同下一个氨基酸的羧基在缩合剂的参与下发生缩合反应,形成酰胺键接长肽链。重复(缩合→洗涤→去保护→中和及洗涤→下一轮缩合)操作,达到所要合成的肽链长度,最后将肽链从树脂上裂解下来,经过纯化冻干等处理,即得所要的多肽(穿膜肽修饰的SPICE1氨基酸片段),其多肽的结构为Biotin-YGRKKRRQRRR-TAEILRLREENA-FITC。其中Biotin-FITC是用FITC标记的生物素,指的是将生物素(Biotin)与荧光异硫氰酸酯(FITC)结合的化合物。FITC是一种常用的荧光染料,常用于生物研究中标记蛋白质、抗体、核酸等生物大分子,以进行可视化和检测。YGRKKRRQRRR源自人免疫缺陷病毒的转录反式激活因子,是一种穿透细胞的肽,可以增加异源蛋白质的产量和溶解度。而细胞穿膜肽是一种可以穿透细胞膜进入细胞,并能够保持其自身生物活性的类信号肽的短肽物质。
对上述合成的多肽进行高效液相色谱分析,其步骤如下:
配制缓冲液:准备0.1%三氟乙酸的100%乙腈(A液)和0.1%三氟乙酸100%水(B液)各4升。将C18柱安装在HPLC系统上。调节柱子,将5%A液和95%B液混合,以40 mL/min的流速冲洗该柱15min,然后用50%混合液平衡柱子(含有0.1%TFA)15min,然后将合成的多肽装入柱子;5%~40%的A液和95%~60%B液线性梯度洗脱多肽25min,流速为1.0 mL/min,紫外检测波长为220nm。结果见下表:
对上述合成的多肽进行质谱分析,其步骤如下:
取多肽样品和基质(4-羟基-α-肉桂酸)溶解在含有1%三氟乙酸的甲基氰溶液中(甲基氰和 1%三氟乙酸的体积比为1:1);然后取0.5μL的该溶液,置于金属样品盘中,室温下自然风干,然后进行质谱分析,测其分子量质谱结果如图1所示;并用标准的胰岛素B肽进行校准。
本实施例还对设计得到的多肽进行无序突变,得到了另外一种多肽(命名为SCpep),将该种用于后续实验中作为对照组多肽。其SCpep的结构式为:Biotin-YGRKKRRQRRR-TAAILRLRAANA-FITC;其序列如SEQ ID NO:2所示:YGRKKRRQRRRTAAILRLRAANA。
实施例2
本实施例采用免疫共沉淀实验检测多肽对USP10相互作用的影响
将准备好转染了穿膜肽修饰的SPICE1的氨基酸片段的骨肉瘤HOS细胞及143B细胞,用移液器吸至培养基,用PBS(磷酸盐缓冲液)洗2遍,每个10cm 培养皿加入500μL CO-IP缓冲液冰上裂解15min,然后用细胞刮刮取并转移至新1.5mL Ep管,再冰上裂解45min,13000rpm×15min、4℃离心,将上清转移合并至新的1.5ml Ep管。取50 μL上清液作为Input组,剩余上清液分别加入1 μg 兔免疫球蛋白和SPICE1一抗,将EP管封口后插入4℃冰箱垂直混悬仪中旋转结合过夜;结合结束后分别加入40 μL预先润洗的蛋白质 A/G磁珠,封口后插入4℃冰箱垂直混悬仪中旋转孵育2 h;随后将样品洗杂3次,加入30 μL 上样缓冲液,100℃煮10 min,进行Western Blotting实验;其实验结果如图2所示,且由图2可知,穿膜肽修饰的SPICE1氨基酸片段(多肽)可以与USP10进行结合,从而达到抑制细胞内SPICE1与USP10的结合目的。
实施例3
本实施例采用CCK8法检测多肽对骨肉瘤增殖的影响
将处于对数生长期的骨肉瘤细胞HOS细胞及143B细胞传代至96孔板中,细胞数量为2000个/孔,分别加入PBS以及穿膜肽修饰对照(对照组)和穿膜肽修饰的SPICE1的氨基酸片段(实验组),放置于37℃,5% CO2培养箱中培养。细胞贴壁后,将10 μL CCK8试剂加入到标记为第0天的每孔中,轻轻晃动96孔板,将培养基和CCK8试剂摇匀后放入细胞培养箱继续培养。在培养箱内反应3 h后,将多功能酶标仪设置到450 nm波长检测OD值,并记录数据。用同样方法检测1-4天数据;其实验结果如图3所示,且由图3可知,本发明设计的多肽可以抑制骨肉瘤HOS细胞及143B细胞的增殖。
实施例4
本实施例采用克隆形成方法检测多肽对骨肉瘤增殖的影响
将处于对数生长期的骨肉瘤细胞HOS细胞及143B细胞传代至6孔板中,细胞数量为1000个/孔,分别加入PBS以及穿膜肽修饰对照(对照组)和穿膜肽修饰的SPICE1的氨基酸片段(实验组),放置于37℃,5% CO2培养箱中培养7-14天。当观察到肉眼可见的克隆时,终止培养。除去培养基,加4% 多聚甲醛溶液37℃固定10 min。除去固定液后,经PBS洗涤3次后,加入0.2%的结晶紫染色30 min。PBS多次洗去染色液至PBS澄清后置于空气干燥。将6孔板倒置于专业扫描仪进行扫描。使用Image J软件去除背景并对单位面积(1cm2)的结晶紫染色克隆数进行计算;其实验结果如图4所示,且由图4可知,本发明设计的多肽可以抑制骨肉瘤HOS细胞及143B细胞的增殖。
实施例5
本实施例采用流式细胞仪检测多肽对骨肉瘤细胞凋亡的影响
将处于对数生长期的骨肉瘤细胞HOS细胞及143B细胞传代至6孔板中,细胞密度为40%左右,分别加入PBS以及穿膜肽修饰对照(对照组)和穿膜肽修饰的SPICE1的氨基酸片段(实验组),放置于37℃,5% CO2培养箱中培养48小时。使用凋亡试剂为Annexin V-FITC/PI双染细胞凋亡检测试剂盒(BestBio贝博),步骤如下:消化后离心收集细胞,弃培养基;用预冷PBS洗涤细胞2次;用400 μL 1×上样缓冲液重悬细胞,浓度大约为1×106cells/mL;在细胞悬浮液中加入5 μL Annexin V-FITC,轻轻混匀后于4 ℃避光条件下孵育15分钟;加入10μL PI后轻轻混匀于4 ℃避光条件下孵育 5 分钟;使用流式细胞仪检测。实验结果如图5所示,且由图5可知,本发明设计的多肽可以促进骨肉瘤HOS细胞及143B细胞凋亡。
综上所述,本发明设计的多肽可以通过抑制SPICE1-USP10蛋白-蛋白相互作用,从而抑制肿瘤细胞增殖。
尽管本发明的描述已经相当详尽且特别对几个所述实施例进行了描述,但其并非旨在局限于任何这些细节或实施例或任何特殊实施例,而是应当将其视作是通过参考所附权利要求考虑到现有技术为这些权利要求提供广义的可能性解释,从而有效地涵盖本发明的预定范围。此外,上文以发明人可预见的实施例对本发明进行描述,其目的是为了提供有用的描述,而那些目前尚未预见的对本发明的非实质性改动仍可代表本发明的等效改动。
Claims (10)
1.一种多肽在制备用于预防和/或治疗骨肉瘤的药物中的应用,其特征在于,所述多肽的氨基酸序列如SEQ ID NO:1所示;所述多肽为小分子多肽与细胞穿膜肽接连所形成的嵌合肽。
2.根据权利要求1所述的应用,其特征在于,所述多肽的结构式为:Biotin-YGRKKRRQRRR-TAEILRLREENA-FITC。
3.根据权利要求1所述的应用,其特征在于,所述多肽分子量为4.05 KDa。
4.根据权利要求1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或赋形剂。
5.根据权利要求4所述的应用,其特征在于,所述赋形剂为阿拉伯胶、糖浆、羊毛脂、淀粉中的至少一种。
6.根据权利要求1所述的应用,其特征在于,所述药物的给药途径为口服、透皮、肌肉、皮下或静脉注射。
7.根据权利要求1所述的应用,其特征在于,所述药物为片剂、丸剂、颗粒剂、胶囊剂或注射剂。
8.一种预防和/或治疗骨肉瘤的药物,其特征在于,包含权利要求1所述的多肽。
9.根据权利要求8所述的药物,其特征在于,所述多肽是药物中唯一或主要的有效成分。
10.根据权利要求9所述的药物,其特征在于,所述药物为液体剂型或冻干粉剂。
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