CN116392576A - 一种gip受体激动剂在制备治疗脊髓损伤修复靶点药物中的应用 - Google Patents
一种gip受体激动剂在制备治疗脊髓损伤修复靶点药物中的应用 Download PDFInfo
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Abstract
本发明涉及生物技术及神经损伤修复技术领域,尤其涉及一种GIP受体激动剂在制备治疗脊髓损伤修复靶点药物中的应用,通过体外培养远代脊髓运动神经元,使用多种GIPR激动剂激活GIP/GIPR信号,显著促进脊髓运动神经元轴突生长,保护神经元降低氧化应激损伤;通过建立大鼠脊髓损伤模型,GIPR激动剂给药处理,减少脊髓损伤后氧化应激水平和细胞凋亡,减少胶质疤痕的形成。本发明首次证实GIP/GIPR信号是在脊髓损伤修复过程中的重要作用,使用GIP/GIPR信号激活可以减少脊髓损伤后氧化应激水平和细胞凋亡,减少胶质疤痕的形成为脊髓损伤修复提供适宜的再生环境,同时促进脊髓神经元轴突延伸和神经干细胞增殖进而有效修复脊髓损伤,可作为治疗靶点造福脊髓损伤患者。
Description
技术领域
本发明涉及生物技术及神经损伤修复技术领域,尤其涉及一种GIP受体激动剂在制备治疗脊髓损伤修复靶点药物中的应用。
背景技术
中枢神经系统中发生的损伤的再生非常困难,尤其是脊髓损伤(SCI)。SCI后的复杂病理事件通常会限制损伤部位神经组织的再生,并经常导致运动和感觉功能的不可逆转丧失。脊髓损伤不仅验证影响患者的生理和心理健康,还会给患者造成巨大的经济压力。据统计,全球SCI患者人数每年在25到50万人,仅中国就有超过百万的SCI患者。因此,脊髓损伤治疗策略的研发具有重要的社会意义。脊髓损伤分为原发性和继发性。原发性脊髓损伤通常是指直接或间接暴力导致脊髓组织发生不可逆转的破坏,原发性损伤通常难以预防干预。目前对于脊髓损伤的治疗策略主要集中在继发性脊髓损伤的病理过程进行有效干预。继发性脊髓损伤指在原发损伤发生后,为缓解应激状态下出现的电解质紊乱、组织水肿、氧化应激、凋亡等病理生理学变化导致组织损伤进一步恶化。现今临床上针对脊髓损伤的治疗方法存在广泛争议,缺乏有效的治疗方法,由于胶质瘢痕的形成、轴突生长抑制因子的存在、神经营养因子分泌的不足以及神经元内在生长状态等因素的影响,神经损伤后轴突再生比较困难。因此需找合适的治疗策略和靶点,在脊髓损伤后提供事宜的再生环境,提高神经内在活力就显得十分重要了。
葡萄糖依赖性促胰岛素肽(GIP)由肠上皮K细胞合成并分泌。GIP的分泌主要受营养物质的调节,而GIP的主要作用是葡萄糖依赖性刺激胰腺b细胞的胰岛素分泌。有趣的是,最近的研究发现在神经发育以及神经再生方面GIP同样发挥作用。研究表明:除了主要的肠道功能外,GIP及其受体GIPR在哺乳动物脑和脊髓中均有表达,文献指出脊髓损伤后GIP和GIPR的表达均显著上调。在GIPR缺失的小鼠中观察到坐骨神经夹伤后轴突再生受损,这提示GIP/GIPR信号的激活在自发神经再生的条件下起着有益的作用。本申请人发现目前尚未有报道GIP/GIPR信号在脊髓损伤中的应用。
发明内容
本发明的目的是为了解决现有技术中存在的缺点,而提出的一种GIP受体激动剂在制备治疗脊髓损伤修复靶点药物中的应用,通过体外培养远代脊髓运动神经元,使用多种GIPR激动剂激活GIP/GIPR信号,可以显著促进脊髓运动神经元轴突生长,保护神经元降低氧化应激损伤。
为了实现上述目的,本发明采用了如下技术方案:
一种GIP受体激动剂在制备治疗脊髓损伤修复靶点药物中的应用,通过体外培养远代脊髓运动神经元,使用多种GIPR激动剂激活GIP/GIPR信号,显著促进脊髓运动神经元轴突生长,保护神经元降低氧化应激损伤。
优选地,所述GIP受体激动剂包括天然GIP多肽(如GIP1-42、GIP1-30)、GIP修饰肽(如D-Ala2-GIP)、GIP模拟肽(如Tirzepatide)、以及其他可使GIPR激活的小分子化合物等。
优选地,在培养的神经干细胞中激活GIP/GIPR信号能够促进神经干细胞增殖。
优选地,通过建立大鼠脊髓损伤模型,GIPR激动剂给药处理,减少脊髓损伤后氧化应激水平和细胞凋亡,减少胶质疤痕的形成,促进损伤后脊髓神经元轴突延伸和神经干细胞增殖。
优选地,所述GIPR激动剂给药处理为联合生物材料进行局部缓释,也可以进行整体用药(如静脉注射),均能达到类似的治疗效果。
与现有技术相比,本发明具有以下有益效果:
本发明通过大量研究首次证实GIP/GIPR信号是在脊髓损伤修复过程中的重要作用,使用GIP/GIPR信号激活可以减少脊髓损伤后氧化应激水平和细胞凋亡,减少胶质疤痕的形成为脊髓损伤修复提供适宜的再生环境,同时促进脊髓神经元轴突延伸和神经干细胞增殖进而有效修复脊髓损伤,可作为治疗靶点造福脊髓损伤患者。
附图说明
图1为本发明中GIP处理促进脊髓运动神经元轴突延伸示意图;
图2为本发明中在体实验显示GIP促进脊髓损伤后轴突再生示意图;
图3为本发明中GIP/GIPR信号激活促进神经干细胞增殖示意图;
图4为本发明中GIP对神经干细胞成球个数及直径的影响示意图;
图5为本发明中在体实验中GIP信号激活增加神经干细胞增殖示意图;
图6为本发明中GIP/GIPR信号激活保护氧化应激状态下的神经元示意图;
图7为本发明中在体实验显示GIP处理显著降低脊髓损伤后氧化应激水平示意图;
图8为本发明中GIP/GIPR信号激活能够减少神经元凋亡示意图;
图9为本发明中GIP信号激活减少脊髓损伤后胶质瘢痕面积示意图;
图10为本发明中GIP处理促进脊髓损伤后功能恢复示意图。
具体实施方式
下面结合附图将对本发明实施例中的技术方案进行清楚、完整地描述,以使本领域的技术人员能够更好的理解本发明的优点和特征,从而对本发明的保护范围做出更为清楚的界定。本发明所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例,基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1:GIP/GIPR信号激活促进脊髓神经元轴突生长
1.脊髓运动神经元体外培养
使用SD大鼠进行脊髓运动神经元原代培养,将出生后1天大鼠处死后取出脊髓,剥离脊膜和血管,剪碎后胰酶消化,多聚赖氨酸包被培养基后使用Neurobasal完全培养基(50mL体系,NeuroBasalMedium:48mL;B27(50×):2%;P-S:1%;Glutamax(100×):1%)进行培养,免疫荧光染色ChAT检测运动神经元纯度。
2.体外脊髓神经元损伤模型建立
将培养的原代脊髓运动神经元用0.125%的胰酶进行消化模拟损伤过程,接种至培养皿后使用不同浓度的GIPR激动剂GIP1-30(100nM–1μM)处理损伤后的脊髓运动神经元,24小时后固定细胞。
3.免疫荧光染色统计轴突长度
免疫荧光染色Tuj1,拍照统计运动神经元的轴突长度,结果如图1所示,GIP刺激后脊髓运动神经元的轴突长度显著增加,这说明GIP/GIPR信号激活促进了脊髓运动神经元轴突生长。
4.脊髓损伤模型的建立
将体重180-220g的雄性成年大鼠进行麻醉,胸背部备皮,沿T10棘突上沿切口,钝性剥离T9右侧椎板,固定周围组织肌肉,咬除棘突椎板及右侧关节突,于后正中沟插入刀片,横切至中央管,此时大鼠一侧下肢出现痉挛抽搐然后瘫痪视为造模成功。
5.给药处理
损伤后隔天进行尾静脉注射(10-100nM/kg)GIP修饰肽(D-Ala2-GIP)直至14天,对照组使用生理盐水进行注射。
6.免疫荧光染色
收集实验组和对照组脊髓组织,固定后进行冰冻切片,免疫荧光染色,结果显示(图2),D-Ala2-GIP组有更多的神经元轴突延伸至损伤区,这说明GIP/GIPR信号的激活促进损伤后脊髓神经元的轴突再生。
实施例2:GIP/GIPR信号激活促进神经干细胞增殖
1.神经干细胞的原代培养
SD孕鼠(E14d)麻醉处死后取出胎鼠,于体视显微镜下找准大脑部位并剥膜,剔除海马等组织,将大脑皮层放入预先准备好的3mL完全培养基(50mL体系,DMEM/F12:NeuroBasalMedium(1:1)各加23.75mL;B27(50×):2%;N2(100×):2%;EGF:1μg;bFGF:1μg;P-S:1%;Glutamax(100×):1%中,轻吹打30下,过200目筛网。吹打60下,稀释到完全培养基于37摄氏度培养,待细胞成球后进行消化传代。
2.EdU染色检测干细胞增殖状况
GIP1-30处理干细胞,24h后EdU检测干细胞增殖情况,结果显示与对照组相比GIP刺激能够促进神经干细胞增殖(图3)。
3.检测GIP处理后干细胞成球情况
显微镜明场拍摄GIP处理24h后的神经干细胞成球情况,统计成球数量以及成球直径,与对照组相比,GIPR激活增加干细胞成球数量以及成球大小(图4)。
4.脊髓损伤模型的建立
将体重180-220g的雄性成年大鼠进行麻醉,胸背部备皮,沿T10棘突上沿切口,钝性剥离T9右侧椎板,固定周围组织肌肉,咬除棘突椎板及右侧关节突,于后正中沟插入刀片,横切至中央管,此时大鼠一侧下肢出现痉挛抽搐然后瘫痪视为造模成功。
5.给药处理
损伤后隔天进行尾静脉注射(10-100nM/kg)GIP修饰肽(D-Ala2-GIP),对照组使用生理盐水进行注射。
6.免疫荧光染色
3天后收集实验组和对照组脊髓组织,固定后进行冰冻切片,免疫荧光染色Sox2,结果显示(图5),D-Ala2-GIP组显示出更多的Sox2阳性信号,这提示GIP/GIPR信号的激活促进脊髓神经干细胞的增殖。
实施例3:GIP/GIPR信号激活减少脊髓损伤导致的氧化应激和细胞凋亡
1.脊髓运动神经元体外培养
使用SD大鼠进行脊髓运动神经元原代培养,将出生后1天大鼠处死后取出脊髓,剥离脊膜和血管,剪碎后胰酶消化,多聚赖氨酸包被培养基后使用Neurobasal完全培养基(50mL体系,NeuroBasalMedium:48mL;B27(50×):2%;P-S:1%;Glutamax(100×):1%)进行培养,免疫荧光染色ChAT检测运动神经元纯度。
2.体外脊髓神经元氧化应激模型建立及GIP处理
将培养的原代脊髓运动神经元用100μM过氧化氢处理3小时建立神经元氧化应激模型,去除过氧化氢后加入GIPR激动剂GIP1-30(100nM–1μM)处理损伤后的脊髓运动神经元,24小时后收集细胞。
3.检测细胞凋亡及氧化应激水平
使用活性氧荧光探针DCFH-DA标记ROS,拍照统计实验组与对照组神经元氧化应激水平,结果显示(图6),GIP刺激降低了神经元氧化应激水平。Western blot结果显示,GIP处理组Bax/Bcl2比值下降,说明GIP/GIPR信号激活能够减少神经元凋亡(图7)。
4.脊髓损伤模型的建立
将体重180-220g的雄性成年大鼠进行麻醉,胸背部备皮,沿T10棘突上沿切口,钝性剥离T9右侧椎板,固定周围组织肌肉,咬除棘突椎板及右侧关节突,于后正中沟插入刀片,横切至中央管,此时大鼠一侧下肢出现痉挛抽搐然后瘫痪视为造模成功。
5.给药处理
损伤后隔天进行尾静脉注射(10-100nM/kg)GIP修饰肽(D-Ala2-GIP),对照组使用生理盐水进行注射,假手术组仅打开椎板不对脊髓进行损伤。
6.免疫荧光标记ROS
损伤3天后收集各组脊髓组织,直接进行冰冻切片,使用荧光探针标记ROS,结果显示(图8),脊髓损伤后实验组有更低的氧化应激水平。
实施例4:尾静脉注射GIP受体激动剂促进脊髓损伤后的功能恢复
1.脊髓半横断模型的建立
将体重180-220g的雄性成年大鼠进行麻醉,胸背部备皮,沿T10棘突上沿切口,钝性剥离T9右侧椎板,固定周围组织肌肉,咬除棘突椎板及右侧关节突,于后正中沟插入刀片,横切至中央管,此时大鼠一侧下肢出现痉挛抽搐然后瘫痪是为造模成功。
2.尾静脉注射GIP
实验动物分为三组,对照组,假手术组及实验组,实验组在手术后的时间段内隔天尾静脉注射50nM/kgD-Ala2-GIP,对照组注射相同体积的生理盐水,假手术组仅剥离椎板,不做半横断处理。术后继续观察至2周。
3.行为学检测脊髓损伤恢复状况
在术后1天,3天,7天及14天对各组大鼠进行BBB(Basso,Beattie&Bresnahanlocomotorratingscale)功能评分以判断脊髓损伤恢复状况,结果显示(图9)GIP/GIPR信号激活显著促进了脊髓损伤的恢复。
4.免疫荧光染色
收集术后三天各组脊髓组织,固定后制备冰冻切片,免疫荧光染色观察胶质疤痕大小,结果显示(图10)GIP/GIPR信号激活显著减小胶质疤痕面积,利于脊髓损伤恢复。
实施例5:局部缓释GIP受体激动剂促进脊髓损伤后的功能恢复
1.脊髓半横断模型的建立
将体重180-220g的雄性成年大鼠进行麻醉,胸背部备皮,沿T10棘突上沿切口,钝性剥离T9右侧椎板,固定周围组织肌肉,咬除棘突椎板及右侧关节突,于后正中沟插入刀片,横切至中央管,去除右侧约3mm脊髓组织。
2.GIP联合水凝胶治疗
将D-Ala2-GIP与水凝胶材料(例如光固化海藻酸钠)混合后注入脊髓缺损部位,紫外照射固化后逐层缝合肌肉与皮肤,对照组使用单纯水凝胶材料进行治疗。
3.行为学检测脊髓损伤恢复状况
在术后1天,3天,7天及14天对各组大鼠进行BBB(Basso,Beattie&Bresnahanlocomotorratingscale)功能评分以判断脊髓损伤恢复状况,结果显示GIP联合水凝胶组显著促进了受损轴突的再生和干细胞增殖,减少氧化应激和细胞凋亡,减少胶质疤痕,进而加速脊髓损伤的恢复。
综上所述,本发明通过大量研究首次证实GIP/GIPR信号是在脊髓损伤修复过程中的重要作用,使用GIP/GIPR信号激活可以减少脊髓损伤后氧化应激水平和细胞凋亡,减少胶质疤痕的形成为脊髓损伤修复提供适宜的再生环境,同时促进脊髓神经元轴突延伸和神经干细胞增殖进而有效修复脊髓损伤,可作为治疗靶点造福脊髓损伤患者。
本发明中披露的说明和实践,对于本技术领域的普通技术人员来说,都是易于思考和理解的,且在不脱离本发明原理的前提下,还可以做出若干改进和润饰。因此,在不偏离本发明精神的基础上所做的修改或改进,也应视为本发明的保护范围。
Claims (5)
1.一种GIP受体激动剂在制备治疗脊髓损伤修复靶点药物中的应用,其特征在于,通过体外培养远代脊髓运动神经元,使用多种GIPR激动剂激活GIP/GIPR信号,显著促进脊髓运动神经元轴突生长,保护神经元降低氧化应激损伤。
2.根据权利要求1所述的应用,其特征在于,所述GIP受体激动剂包括天然GIP多肽、GIP修饰肽和GIP模拟肽。
3.根据权利要求1所述的应用,其特征在于,在培养的神经干细胞中激活GIP/GIPR信号能够促进神经干细胞增殖。
4.根据权利要求1所述的应用,其特征在于,通过建立大鼠脊髓损伤模型,GIPR激动剂给药处理,减少脊髓损伤后氧化应激水平和细胞凋亡,减少胶质疤痕的形成,促进损伤后脊髓神经元轴突延伸和神经干细胞增殖。
5.根据权利要求4所述的应用,其特征在于,所述GIPR激动剂给药处理为联合生物材料进行局部缓释或整体用药。
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