CN114469849A - 一种温敏水凝胶封装线粒体的应用 - Google Patents
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Abstract
本发明涉及一种温敏水凝胶封装线粒体的应用，属于生物医药技术领域。本发明提供一种温敏水凝胶封装线粒体在制备预防或治疗心肌损伤的药物上的应用和在制备预防或治疗心肌缺血再灌注损伤的药物上的应用；温敏水凝胶封装线粒体作为体内移植物的应用；温敏水凝胶通过封装线粒体输送至目标区域，在制备预防或治疗性药物上的应用。本发明通过使用亲水性能佳、生物相容性良好的水凝胶作为线粒体移植的载体，从而增加心肌细胞对线粒体的摄取率，改善缺血心肌细胞的能量代谢和机械收缩与舒张功能，延缓心衰的发生。通过本发明能够叠加水凝胶改善损伤心肌力学微环境和线粒体移植的效果，具有重要的临床推广与应用价值。
Description
技术领域
本发明涉及一种温敏水凝胶封装线粒体的应用，属于生物医药技术领域。
背景技术
线粒体是真核细胞获取能量支撑的主要动力工厂，心脏作为高耗能器官，心肌对于线粒体代谢的依赖性尤其显著。心肌缺血及再灌注损伤是临床亟待解决的关键问题，心肌细胞的存活率直接决定了心肌梗死面积和修复后心脏的功能及恶化程度，当前多个研究已证实，结构完整、具有活性的线粒体进行移植能够抑制心肌损伤，改善心脏功能，80％的外源性线粒体会在很短的时间内被心肌细胞迅速吸收内化，改善细胞的呼吸功能，增加ATP的生成，并且经研究显示，移植线粒体最长能在28天后仍然发挥保护作用，但是，线粒体的吸收效率和在损伤心肌的分布仍然受到移植方式的影响，尽管目前一些研究者试图通过特异性分子靶向递送，但其临床效果仍有待检验，线粒体移植的临床转化还有诸多工作需要进行。
新兴的高分子材料水凝胶近年来被广泛应用于医疗领域，是一种绝佳的药物、外泌体、干细胞等物质的载体，同时，水凝胶本身也具有一定的治疗作用。水凝胶是天然的或者人工合成的大分子与水分子的交联网络，新型可注射智能水凝胶具有良好的生物相容性，具有模拟细胞外基质的特性，并且能在一定时间内降解，一般降解产物也没有生物毒性。在心脏疾病领域已有多种形式的水凝胶用于临床治疗，例如正在进行临床试验的VentriGel等。研究表明，水凝胶发挥治疗作用的方式可能是通过打断基质降解，影响力学环境的恶性循环、促进血管新生和细胞归巢等机制发挥了保护作用，而作为载体又能大大提高干细胞、质粒等在心肌局部的移植植入率。水凝胶封装线粒体能够为线粒体提供类细胞及基质环境，并且有研究证实水凝胶封装线粒体可作为人工细胞的制备基础，只是目前尚没有水凝胶作为线粒体移植载体的相关报道。
发明内容
本发明的目的是为解决线粒体如何移植以治疗心肌缺血再灌注损伤的技术问题。
为达到解决上述问题的目的，本发明所采取的技术方案是提供一种温敏水凝胶封装线粒体在制备预防或治疗心肌损伤的药物上的应用。
本发明提供一种温敏水凝胶封装线粒体在制备预防或治疗心肌缺血再灌注损伤的药物上的应用。
本发明提供一种温敏水凝胶封装线粒体在非诊断方法或非治疗方法上的应用。
优选地，所述温敏水凝胶封装线粒体作为体内移植物。
本发明提供一种温敏水凝胶在制备预防或治疗性药物上的应用，所述温敏水凝胶通过封装线粒体输送至目标区域。
相比现有技术，本发明具有如下有益效果：
本发明通过使用亲水性能佳、生物相容性良好的水凝胶作为线粒体移植的载体，从而增加心肌细胞对线粒体的摄取率，改善缺血心肌细胞的能量代谢和机械收缩与舒张功能，延缓心衰的发生。通过本发明能够叠加水凝胶改善损伤心肌力学微环境和线粒体移植的效果，具有重要的临床推广与应用价值。
附图说明
图1为通过超声心动图检测水凝胶封装线粒体移植对缺血再灌注损伤小鼠心功能的改善效果图。
图2为通过Evan‘s blue/TTC染色评估水凝胶封装线粒体移植后缺血再灌注损伤小鼠心脏梗死面积结果图。
图3为通过HE染色评估水凝胶封装线粒体移植对缺血再灌注损伤小鼠心肌炎性细胞浸润和病理结构的影响图。
图4为通过WB评估水凝胶封装线粒体移植对缺血再灌注损伤小鼠心肌凋亡的影响图。
具体实施方式
为使本发明更明显易懂，兹以优选实施例，并配合附图作详细说明如下：
如图1-4所示，本发明所采取的技术方案是提供一种温敏水凝胶封装线粒体在制备预防或治疗心肌损伤的药物上的应用。
本发明提供一种温敏水凝胶封装线粒体在制备预防或治疗心肌缺血再灌注损伤的药物上的应用。
本发明提供一种温敏水凝胶封装线粒体在非诊断方法或非治疗方法上的应用。温敏水凝胶封装线粒体作为体内移植物。
本发明提供一种温敏水凝胶在制备预防或治疗性药物上的应用，所述温敏水凝胶通过封装线粒体输送至目标区域。
实施例
1.用生理盐水将Pluronic F127粉末(Sigma公司，)配制成15％(w/w％)的水凝胶，低温保存。
2.获得C57BL/6小鼠心肌，应用线粒体分离试剂盒(碧云天生物技术有限公司，C3606)将心肌经酶消化、线粒体分离试剂重悬后适当研磨，1000g、4℃离心5分钟，取上清，转移到新的EP管中，继续离心(3500g、4℃、10min)获得纯化线粒体，使用15％w/w的Pluronic F127温敏水凝胶在0-4℃低温环境中重悬，摇床缓慢混匀5-10min，备用。以上皆为无菌操作。
3.制备小鼠的心肌缺血再灌注损伤模型(缺血45min,再灌注24小时)，在再灌注时沿心肌乳头肌方向分3个位点分别注射25ul微升水凝胶封装的线粒体(5-10×104个/ml)；对照组实施假手术：缺血再灌注损伤组只注射相同体积的生理盐水。灌注24小时后行超声心动图检测小鼠心脏功能，Evans blue/TTC染色检测小鼠心肌的梗死面积，HE染色检测心肌组织损伤程度及炎症细胞浸润情况。
实验结果：
如图1所示：为通过超声心动图检测水凝胶封装线粒体移植对缺血再灌注损伤小鼠心功能的改善效果图；图中：IR组代表缺血再灌注损伤模型组；IRFM组代表F127水凝胶封装线粒体进行心肌注射的小鼠治疗组；WT代表实施假手术的对照组；
如图中超声心动图显示水凝胶辅助线粒体移植组的缺血再灌注损伤小鼠心脏左室射血分数显著增加(P＜0.05)。
如图2所示为通过Evan‘s blue/TTC染色评估水凝胶封装线粒体移植后缺血再灌注损伤小鼠心脏梗死面积结果图。图中IR组代表缺血再灌注损伤模型组；IRFM组代表F127水凝胶封装线粒体进行心肌注射的小鼠治疗组；IRM代表实施线粒体进行心肌注射的小鼠治疗组；图中白色为心肌梗死区(不能被Evans blue和TTC染液染色)，红色为心肌缺血区(不能被Evans blue但能被TTC染色)，蓝色为非缺血区(被Evans blue和TTC染液染色)。
图3为通过HE染色评估水凝胶封装线粒体移植对缺血再灌注损伤小鼠心肌炎性细胞浸润和病理结构的影响图。其中WT代表实施假手术的对照组；IR组代表缺血再灌注损伤模型组；IRM代表实施线粒体直接进行心肌注射的小鼠治疗组；IRF代表实施F127水凝胶直接进行心肌注射的小鼠治疗组；IRFM组代表F127水凝胶封装线粒体进行心肌注射的小鼠治疗组；图中HE染色中红色为胞浆染色，蓝色为细胞核，从图中可见损伤模型组大量炎症细胞浸润，水凝胶辅助线粒体移植组IRFM组的炎症细胞浸润显著减轻。
图4为通过WB评估水凝胶封装线粒体移植对缺血再灌注损伤小鼠心肌凋亡的影响图。其中WT代表实施假手术的对照组；IR组代表缺血再灌注损伤模型组；IRM代表实施线粒体直接进行心肌注射的小鼠治疗组；IRF代表实施F127水凝胶直接进行心肌注射的小鼠治疗组；IRFM组代表F127水凝胶封装线粒体进行心肌注射的小鼠治疗组；从图中可见水凝胶辅助线粒体移植组IRFM组的心肌凋亡显著减轻。
以上所述，仅为本发明的较佳实施例，并非对本发明任何形式上和实质上的限制，应当指出，对于本技术领域的普通技术人员，在不脱离本发明的前提下，还将可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。凡熟悉本专业的技术人员，在不脱离本发明的精神和范围的情况下，当可利用以上所揭示的技术内容而做出的些许更动、修饰与演变的等同变化，均为本发明的等效实施例；同时，凡依据本发明的实质技术对上述实施例所作的任何等同变化的更动、修饰与演变，均仍属于本发明的技术方案的范围内。
Claims (5)
1.一种温敏水凝胶封装线粒体在制备预防或治疗心肌损伤的药物上的应用。
2.一种温敏水凝胶封装线粒体在制备预防或治疗心肌缺血再灌注损伤的药物上的应用。
3.一种温敏水凝胶封装线粒体在非诊断方法或非治疗方法上的应用。
4.如权利要求3所述的一种温敏水凝胶封装线粒体在非诊断方法或非治疗方法上的应用，其特征在于，所述温敏水凝胶封装线粒体作为体内移植物。
5.一种温敏水凝胶在制备预防或治疗性药物上的应用，其特征在于，所述温敏水凝胶通过封装线粒体输送至目标区域。
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