CN112402458A - 一种间充质干细胞和外泌体联合制剂在制备心肌梗死药物中的应用 - Google Patents
一种间充质干细胞和外泌体联合制剂在制备心肌梗死药物中的应用 Download PDFInfo
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Abstract
本发明提供了一种间充质干细胞和外泌体联合制剂在制备心肌梗死药物中的应用，间充质干细胞和外泌体联合制剂包括间充质干细胞和外泌体。间充质干细胞在培养过程中会分泌大量的外泌体，间充质干细胞分泌的外泌体能够调节局部组织微环境，促进血管新生，改善局部供血，恢复心室壁厚度、改善心梗大鼠的左心室射血分数，从而达到改善大鼠心脏功能的效果。并且间充质干细胞本身具有一定的治疗心肌梗死效果，外泌体悬液能够为间充质干细胞提供更好的生存环境，能够最大限度地维持细胞的活性和功能。
Description
技术领域
本发明属于生物医药技术领域，尤其涉及一种间充质干细胞和外泌体联合制剂在制备心肌梗死药物中的应用。
背景技术
心肌梗死是冠状动脉急性、持续性缺氧所引起的心肌坏死。临床上多有剧烈而持久的胸骨后疼痛，休息及硝酸酯类药物不能完全缓解，伴有血清心肌酶活性增高及进行性心电图变化，可并发心律失常、休克或心力衰竭。急性心肌梗死是危害人类健康的重大疾病，在发达国家被成为“头号杀手”，是世界范围的主要死亡原因之一。AMI 发病率的增加给个人、家庭和社会带来沉重的负担，控制AMI发病率，提高AMI的救治水平，成为心血管领域的一个重要课题。
间充质干细胞作为一种组织来源的间充质干细胞，是一类具有自我更新和多项分化潜能的多能干细胞，由于其取材方便和不具有免疫排斥反应，是再生医学中一种较为理想的种子细胞。研究显示，干细胞行驶生物学功能的最重要途径是通过分泌大量生物活性因子，进而调节病变组织的机能、促进血管再生、加速组织再生修复等。而大量的生物活性因子的分泌途径主要是通过外泌体，一种生物膜包裹的，直径在40-100 nm不等的分泌小泡。越来越多的研究发现干细胞分泌的外泌体可在急性期减少细胞凋亡、减轻炎症反应、减少心肌梗死面积，后期促进血管生成，抑制纤维化，减轻心肌重构从而改善心肌梗死后的心脏功能。
当前的细胞移植疗法可促进梗死局部血管新生和心肌再生，进而改善心肌梗死后的心功能，而且其在心血管病治疗上的安全性和有效性已得到证实，为受损的心脏修复提供了巨大的希望，但其移植后的低生存率、低定植率、且难以向心血管细胞谱系分化等问题成为制约其疗效的关键因素。
发明内容
针对现有技术存在的问题，本发明提供了一种间充质干细胞和外泌体联合制剂在制备心肌梗死药物中的应用。
为实现上述发明目的，本发明采用下述技术方案予以实现：
一种间充质干细胞和外泌体联合制剂在制备心肌梗死药物中的应用，间充质干细胞和外泌体联合制剂包括间充质干细胞和外泌体。
在治疗心肌梗死方面，间充质干细胞能够迁移到损伤部位分泌大量生物活性因子，进而调节病变组织的机能、促进血管再生、加速组织再生修复。外泌体能够在心肌细胞、心脏成纤维细胞、内皮细胞及心脏干细胞间进行分子信号传导以及生物信号信息的传递，广泛参与了细胞存活、心肌梗死、心室重构及心脏再生等心血管系统的生理和病理过程。所以将二者联合使用，可以提高间充质干细胞的存活，使其更好地发挥有效作用，从而更好的治疗心肌梗死。
优选地，所述外泌体为间充质干细胞的同源外泌体。培养间充质细胞的过程中，通过对间充质干细胞饥饿培养的方法，促进其生物活性物质的分泌，获得同源外泌体，以外泌体的形式回收同源干细胞分泌的生物活性物质回收方式更为简便。
优选地，所述间充质干细胞选自骨髓间充质干细胞、脐带间充质干细胞、脂肪间充质干细胞或牙髓间充质干细胞。
本发明提供一种间充质干细胞和外泌体联合制剂的制备方法，包括如下步骤：
（1）将间充质干细胞诱导培养分化，回收干细胞；
（2）从步骤（1）所述的间充质干细胞中获得同源外泌体，制备成外泌体悬液；
（3）将步骤（1）获得的干细胞重悬于步骤（2）获得的外泌体悬液中，制备成联合制剂。
优选地，所述步骤（1）选择3-5代间充质干细胞在含10%胎牛血清的α-MEM培养基诱导培养，待细胞融合率达到85%～95%时，消化回收细胞。
优选地，所述步骤（2）选择3～5代间充质干细胞在10%胎牛血清的α-MEM培养基诱导培养，待细胞融合率达到70%时，更换不含血清的α-MEM培养基，37℃孵箱中孵育72h后回收培养上清，过滤后通过超速离心法分离纯化得到外泌体，将所述外泌体在2-8℃条件下保存备用。采用0.22μm孔径滤膜过滤培养上清。
将步骤（1）制备好的细胞用生理盐水清洗细胞两次，重悬于已经复温至室温的外泌体悬液中，制备成联合制剂。本发明采用饥饿培养间充质干细胞的方法，在较短时间内就能得到间充质外泌体提取物，通过超速离心的方法浓缩以获得高浓度的间充质干细胞外泌体，将间充质干细胞重悬于浓缩的间充质干细胞外泌体中，大大提高了治疗效果。
优选地，所述超速离心法包括如下步骤：4℃离心机800×g离心5分钟，回收上清；2000×g离心10分钟，回收上清；16000×g离心30分钟，回收上清；150000×g离心90分钟，弃上清，150000×g离心90分钟，弃上清。
优选地，所述步骤（3）中干细胞在外泌体联合制剂中的含量为0.5×106～2×106个/100μL。
相对于现有技术，本发明的有益效果在于：
本发明提供了一种间充质干细胞和外泌体联合制剂在制备心肌梗死药物中的应用，间充质干细胞和外泌体联合制剂包括间充质干细胞和外泌体。间充质干细胞在培养过程中会分泌多种大量的外泌体，间充质干细胞分泌的外泌体能够调节局部组织微环境，促进血管新生，改善局部供血，恢复心室壁厚度、改善心梗大鼠的左心室射血分数，从而达到改善大鼠心脏功能的效果。本发明通过间充质干细胞与外泌体联合作用，所制备的药物能显著提高心梗大鼠的心脏功能，恢复心室壁厚度和节律性运动，改善心梗大鼠的左心室射血分数，显著减少心梗大鼠梗死区域的梗死面积。
附图说明
图1A为本发明实施例中超声仪检测正常大鼠的鉴定结果；
图1B为本发明实施例中超声仪检测心肌梗死模型大鼠的鉴定结果；
图2A为本发明实施例中生理盐水组大鼠的心脏超声检测结果；
图2B为本发明实施例中联合制剂组大鼠的心脏超声检测结果；
图3A为本发明实施例中心梗大鼠治疗前后的左心室射血分数统计图；
图3B为本发明实施例中心梗大鼠治疗前后的短轴缩短率变化统计图；
图4A为本发明实施例中生理盐水组的Masson染色结果；
图4B为本发明实施例中联合制剂组的Masson染色结果。
具体实施方式
下面通过具体实施方式来进一步说明本发明的技术方案。本领域技术人员应该明了，所述实施例仅仅是帮助理解本发明，不应视为对本发明的具体限制。
实施例1
一、脐带间充质干细胞的分离
将获取的新鲜脐带中的多余的脐带保存液弃掉，倒入75%酒精消毒三分钟后取出脐带，放入含有双抗的生理盐水中反复清洗，去掉表面残留酒精。剪掉结扎部分并废弃，将剩余脐带均匀分成小段进行清洗，反复洗净残留血液，去除脐静脉和脐动脉，剥离Wharton胶，将其剪碎至1mm3大小的组织块。根据组织块的量加入适量常规培养基混合均匀后平铺在15cm培养皿中，置于37℃，5% CO2且湿度饱和培养箱内培养24h，每培养皿补加8mL培养液。第5天时补液，第8天时换液，待细胞融合度达到40%-60%以上后，弃掉上清，加生理盐水洗涤两遍后，用0.25%胰酶消化1.5min左右，加终止液吹打收集细胞，离心弃上清后加入新鲜培养基在10cm培养皿中进行传代培养，待细胞融合达80% 进行传代扩增，取第3-5代间充质干细胞进行后续实验。
二、脐带间充质干细胞外泌体的提取
选择生长状态良好的3-5代脐带间充质干细胞，用含体积分数10%胎牛血清的α-MEM培养基培养，待细胞融合率达到约70%时，更换不含血清的α-MEM培养基，37℃孵箱中孵育72h后回收全部培养上清，以0.22μm孔径滤膜过滤，过滤后的培养上清在4℃离心机进行以下梯度离心， 800×g离心5分钟，回收上清；2000×g离心10分钟，回收上清；16000×g离心30分钟，回收上清；150000×g离心90分钟，弃上清，150000×g离心90分钟，弃上清。将沉淀用适量的生理盐水重悬后，作为制备心肌梗死治疗制剂的外泌体悬液，在2-8℃条件下保存备用。
三、脐带间充质干细胞的培养
选择生长状态良好的3-5代脐带间充质干细胞，按照6000～8000/cm2的密度接种于细胞培养瓶中，用含体积分数为10%胎牛血清的α-MEM培养基培养。接种培养2～4天后，细胞融合率达到80～90%，细胞生长状态良好，回收全部细胞，并用生理盐水清洗细胞2～3次，将最终获得的细胞按照2×106的细胞密度重悬于已经复温至18～24℃的200μL外泌体悬液中，制备成联合制剂。
四、SD大鼠的心肌梗死模型的建立及鉴定
选取体重180-200g SD大鼠，用1%戊巴比妥钠（40mg/kg）进行腹腔注射麻醉后，仰卧固定于实验台上，连接动物人工呼吸机，呼吸频率86次/分左右，潮气量15mL，呼吸比设定为1：1。左前胸去毛，消毒铺巾，于胸骨旁第3、4肋骨间隙切开皮肤，钝性分离皮下组织、肌肉，然后将第3根肋骨剪断，并用缝线分离暴露心脏，于左心耳下缘2mm处结扎冠状动脉左前降支，结扎正确完成后可观察到左室前壁及心尖部位发白，然后从内向外依次缝合胸腔，并排出腔内气体以恢复胸腔内负压状态，最后进行造模大鼠的术后护理。SD大鼠造模手术1-2周后，利用超声仪器检测心脏超声的结果鉴定心肌梗死模型是否成功造模。
模型鉴定结果如图1A-图1B所示，图1A为超声仪检测正常大鼠的鉴定结果，图1B为超声仪检测心肌梗死模型大鼠的鉴定结果，可以看出与正常大鼠的鉴定结果相比，造模大鼠的左心室前壁运动功能减弱且室壁变薄，即可确定模型成功。
五、SD大鼠心肌梗死模型的治疗及超声检测。
对经过超声检测确定心肌梗死造模成功的大鼠进行间充质干细胞联合外泌体的心肌内治疗。用1%戊巴比妥钠（40mg/kg）进行腹腔注射麻醉后，仰卧固定于实验台上，连接动物人工呼吸机，呼吸频率86次/分左右，潮气量15ml，呼吸比设定为1：1。左前胸去毛，消毒铺巾，于胸骨旁第3、4肋骨间隙切开皮肤，钝性分离皮下组织、肌肉，然后将第4根肋骨剪断，并用缝线分离暴露心脏，然后在心梗区域边缘取2-3个点进行心肌内注射联合制剂治疗心肌梗死大鼠，每只大鼠的注射剂量为150μL。最后，从内向外依次缝合胸腔，并排出腔内气体，进行治疗大鼠的术后护理，于治疗后不同时间点通过超声检测心梗大鼠的治疗效果。
超声检测结果如图2A-图3B所示，其中图2A 为生理盐水组大鼠的心脏超声检测结果，图2B 为联合制剂组大鼠的心脏超声检测结果；结果显示与生理盐水对照组相比，间充质干细胞联合外泌体制剂治疗心梗大鼠后能够显著改善心室壁厚度和节律性运动；图3A 为生理盐水组组\联合制剂组心梗大鼠治疗前后的左心室射血分数统计图；图3B 为生理盐水组\联合制剂组心梗大鼠治疗前后的短轴缩短率变化统计图；结果显示与生理盐水组相比，间充质干细胞联合外泌体制剂治疗心梗大鼠后能够显著提高左心室射血分数。
六、SD大鼠心脏组织的Masson三色染色
实验周期结束后，大鼠进行安乐死，将心脏进行取材后固定于4%多聚甲醛溶液中固定12h左右，取出心脏组织裁剪出发生梗死的心脏区域，再固定12h后，将固定液弃掉，加入PBS反复清洗至无多聚甲醛固定液残留后，弃掉PBS加入30%的蔗糖脱水至心脏组织沉至管底。然后使用冰冻包埋剂对心脏组织进行包埋、切片。最后，用Weigert 铁苏木素A、B液等比例混合液染片5-10min，流水冲洗；再用1%盐酸酒精分化液分化数秒到数十秒，流水冲洗干净；后用丽春红酸性品红染液染色5-10min，流水冲洗；后用磷钼酸溶液处理5min，倾去玻片上的磷钼酸溶液，用苯胺蓝染液复染90s左右，倾去玻片上的染色液后，用1%冰醋酸处理1min左右，冲洗切片至无蓝色脱出，即可按程序脱水、中性树脂封片，结果用体式显微镜采集。检测结果如图4A和图4B所示，其中图4A为生理盐水组Masson染色结果；图4B为联合制剂组Masson染色结果；由结果可知，与生理盐水对照组相比，联合制剂组治疗心梗大鼠后能够显著减小大鼠梗死区域的梗死面积，由此说明本发明制备的间充质干细胞与外泌体联合制剂可以有效应用于心肌梗死疾病领域。
以上所述，仅为本申请的具体实施方式，但本申请的保护范围并不局限于此，任何在本申请揭露的技术范围内的变化或替换，都应涵盖在本申请的保护范围之内。因此，本申请的保护范围应以所述权利要求的保护范围为准。
Claims (8)
1.一种间充质干细胞和外泌体联合制剂在制备心肌梗死药物中的应用，其特征在于，间充质干细胞和外泌体联合制剂包括间充质干细胞和外泌体。
2.如权利要求1所述的应用，其特征在于，所述外泌体为间充质干细胞的同源外泌体。
3.如权利要求1所述的应用，其特征在于，所述间充质干细胞选自骨髓间充质干细胞、脐带间充质干细胞、脂肪间充质干细胞或牙髓间充质干细胞。
4.一种间充质干细胞和外泌体联合制剂的制备方法，其特征在于，包括如下步骤：
（1）将间充质干细胞诱导培养分化，回收干细胞；
（2）从步骤（1）所述的间充质干细胞中获得同源外泌体，制备成外泌体悬液；
（3）将步骤（1）获得的干细胞重悬于步骤（2）获得的外泌体悬液中，制备成联合制剂。
5.如权利要求4所述的制备方法，其特征在于，所述步骤（1）选择3～5代间充质干细胞在含10%胎牛血清的α-MEM培养基诱导培养，待细胞融合率达到85%～95%时，消化回收细胞。
6.如权利要求4所述的制备方法，其特征在于，所述步骤（2）选择3～5代间充质干细胞在10%胎牛血清的α-MEM培养基诱导培养，待细胞融合率达到70%时，更换不含血清的α-MEM培养基，37℃孵箱中孵育72h后回收培养上清，过滤后通过超速离心法分离纯化得到外泌体。
7.如权利要求6所述的制备方法，其特征在于，所述超速离心法包括如下步骤： 4℃离心机800×g离心5分钟，回收上清；2000×g离心10分钟，回收上清；16000×g离心30分钟，回收上清；150000×g离心90分钟，弃上清，150000×g离心90分钟，弃上清。
8.如权利要求4所述的制备方法，其特征在于，所述步骤（3）中干细胞在联合制剂中的含量为0.5×106～2×106个/100μL。
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| CN114015649A (zh) * | 2021-11-16 | 2022-02-08 | 山西省中医药研究院（山西省中医院） | 一种丹参饮刺激的骨髓间充质干细胞外泌体及其制备方法和应用 |
| CN114015649B (zh) * | 2021-11-16 | 2024-04-16 | 山西省中医药研究院（山西省中医院） | 一种丹参饮刺激的骨髓间充质干细胞外泌体及其制备方法和应用 |
| CN114099534A (zh) * | 2021-11-29 | 2022-03-01 | 苏州大学附属第一医院 | 高表达miR-214的外泌体及其制备方法与应用 |
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