CN113425731B - 一种增效干细胞治疗心梗的药物及其应用 - Google Patents
一种增效干细胞治疗心梗的药物及其应用 Download PDFInfo
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Abstract
本发明公开了一种增效干细胞治疗心梗的药物及其应用,具体为2‑脱氧‑D‑葡萄糖在制备增效干细胞治疗心梗的药物中的应用,动物实验证实,注射2‑脱氧‑D‑葡萄糖后,增加了注射间充质干细胞的存活率,增强间充质干细胞的治疗效果。
Description
技术领域
本发明属于药物技术,涉及一种增效干细胞治疗心梗的药物及其应用,具体为2-脱氧-D-葡萄糖在制备增效干细胞治疗心梗的药物中的应用。
背景技术
心肌梗死(MI)是一种严重危害人类健康的心血管疾病,随着我国人民生活水平的不断提高,缺血性心肌梗死的发病率也在不断上升。缺血性心肌梗死会导致心肌细胞坏死和瘢痕形成,进而影响心脏功能。目前药物或器械治疗大多只能缓解症状,但却不能逆转心脏组织损伤。心脏移植虽能彻底改善心脏状态,但因供体来源稀缺、免疫排斥以及昂贵的治疗费用等因素,在临床上很难广泛应用。
干细胞是一系列未分化的细胞,具有分化成特定细胞类型的能力。干细胞按来源可分为胚胎干细胞(Embryonic stem cells, ESC)和成体干细胞。后者包括造血干细胞(Hematopoietic stem cells, HSCs)和间充质干细胞(Mesenchymal stem cells, MSCs)。间充质干细胞几乎存在于人体所有具有特定干细胞龛位的组织中。骨髓来源的间充质干细胞(BM-MSCs)是第一个被发现的间充质干细胞,也是目前最多的用于心梗治疗的干细胞。但是现有技术表明,仅仅依靠干细胞治疗心梗,效果还需改善。
发明内容
2-脱氧-D-葡萄糖(2-DG)是一种葡萄糖类似物,具有干扰病毒特异性糖蛋白的合成、抑制疱疹单病毒、RAN和DNA包膜病毒、癌症细胞增殖等功效;但迄今为止尚未见2-脱氧-D-葡萄糖在心肌梗死方面的研究报道,尤其未见2-DG与干细胞作用关系的研究。
本发明公开了一种利用2-脱氧-D-葡萄糖改善心梗后干细胞治疗效果的新用途。MSCs治疗心梗的最大问题是移植细胞在心梗部位的滞留率低、存活性差。本发明公开了2-脱氧-D-葡萄糖对心肌梗死后间充质干细胞的治疗效果有明显提升。
本发明采用如下技术方案:
2-脱氧-D-葡萄糖在制备增效干细胞治疗心梗的药物中的应用。
2-脱氧-D-葡萄糖在制备治疗心梗的药物中的应用。
2-脱氧-D-葡萄糖和干细胞在制备增效治疗心梗的药物中的应用。
一种增效治疗心梗的药物,其活性成分为2-脱氧-D-葡萄糖和干细胞。还公开了该增效治疗心梗的药物在制备治疗心梗的药物中的应用。
本发明涉及的2-DG在制备预防或治疗心梗药物或保健品中的应用,具体的是2-DG可用于制备心梗保护药物、保健品或者食品,实现2-脱氧-D-葡萄糖尤其是增效干细胞,特别是间充质干细胞改善心梗后心功能、减缓心梗后心室扩张、减小心梗面积的技术效果。
本发明公开的制备的药物,具体的给药方式可采取腹腔注射等,2-脱氧-D-葡萄糖的给药量为300~800mg/kg/天,比如500 mg/kg/天。
考虑到MSCs的移植成功率,MI局部心肌内移植可能比静脉注射或冠状动脉内移植更为优越,但无论采用何种移植途径,最终目的仍然是优化策略,以增强注射的MSCs的存活率。现有技术下,由于MI导致心脏部位环境的复杂,使得现有间充质干细胞注射治疗心梗的效果欠佳,研究者采用各种方法以提高其对心梗的治疗效果,比如体外采用细胞因子培养间充质干细胞后回输等,此方法操作复杂。心肌梗死是一种严重危害人类健康的心血管疾病,随着我国人民生活水平的不断提高,缺血性心肌梗死的发病率也在不断上升。本发明首次采用2-脱氧-D-葡萄糖改善心梗后心功能,尤其是与间充质干细胞结合,增加干细胞治疗心梗的效果,减小心梗面积,从而可以预防或者治疗心梗。
附图说明
图1为2-DG改善MSCs治疗心梗后小鼠的心功能;
图2为2-DG改善MSCs治疗心梗后小鼠的心室重构;
图3为2-DG明显增加移植MSCs存活。
具体实施方式
以下所列实施例,仅为帮助本领域技术人员更全面理解本发明,但不以任何方式限制本发明。本发明涉及的原料试剂以及建模、测试都是本领域常规技术。
所使用的主要材料和来源分别如下:
C57BL/6J小鼠(昭衍(苏州)新药研究中心提供,此实验由苏州大学伦理委员会批准);小动物呼吸机(上海奥尔科特生物科技有限公司,上海);手术器械(六六视觉公司,苏州);缝合针线(上海浦东金环医疗用品有限公司,上海);小动物超声影像系统(VisualSonics Vevo 2100);间充质干细胞(MSCs),采用现有技术从8周雄性C57BL/6J小鼠股骨骨髓中提取,常规培养第六代以后用于实验;2-脱氧-D-葡萄糖(2-DG,苏州天可贸易有限公司,苏州),溶于生理盐水(浓度为31.25mg/mL),备用。
小鼠心肌梗死模型的建立
选用25g左右的C57BL/6J雄性小鼠为实验对象,采用左冠状动脉前降支结扎法制作心梗模型。腹腔注射麻醉后,经口气管插管,接空气呼吸机,呼吸频率110次/min,潮气量2.5ml,吸呼比1:1.3。右侧卧位,左胸纵切口切开外层皮肤,剥离胸大肌,第三、四肋间横切口开胸,暴露心脏,用镊子撕开心包膜。借助手术显微镜可见左冠状动脉大致走行。在左心耳下缘约1.5mm处,将冠状动脉前降支(LAD) 连同少量心肌组织一起结扎,进针深度约1mm,宽度控制在3 mm以内;逐层关胸。
假手术组仅穿过LAD下方不打结,其余同模型组。
结扎后肉眼可见结扎处至心尖变白,1 周后取左心室组织进行心脏组织染色,可看到明显的纤维化,证明心梗模型建立成功。
MSCs组在结扎LAD后当即在心梗边缘区分三点注射提取的MSCs,注射细胞总数量为5x105/20 μl PBS;2-DG组仅在心梗建立前6h、心梗术后第1天、术后第2天腹腔注射2-DG(500 mg/kg/天);MSCs+2-DG组在心梗建立前6h、心梗术后第1天、术后第2天腹腔注射2-DG(500 mg/kg/天),其余同MSCs组;对照组(生理盐水组)小鼠在结扎LAD后注射等量无菌生理盐水作为对照;三组喂养方式一致,然后进行心梗后心功能、心梗面积检测。
实施例1 有效改善心梗后心功能
心脏超声检测心梗后心功能。小鼠麻醉(方法同前),脱毛后左侧卧位,将心脏超声诊断仪探头置于心前壁,于乳头肌水平取左室二维短轴观,同时记录M型扫描,连续3个心动周期测量左室射血分数(EF)、缩短分数(FS)和左室舒张末径(LVEDD)和左室收缩末径(LVESD)。实验结果见图1,* P < 0.05;# P < 0.05。
实施例2 有效改善心室重构
心脏重量Masson染色:术后28天处死小鼠,测量小鼠体重(body weight,BW)、心脏重量(heart weight,HW);计算HW/BW。Masson染色:术后28天处死小鼠,取左心室组织进行心脏组织染色,观察治疗效果。按常规Masson染色步骤进行,在普通光学显微镜下观察并拍照。采用图像分析软件Image J分析各部分面积,计算心梗面积/心脏面积。实验结果见图2,*P < 0.05,**P < 0.01。
实施例3 增加间充质干细胞存活
制备CM-DiI染色的间充质干细胞,利用CM-DiI的红色荧光特性观察移植间充质干细胞的存活率。具体的,配置CM-DiI染色工作液:CM-DiI染料储存液(赛默飞世尔)浓度为1mg/mL,取4uL储存液加到2mL PBS中,使终浓度为2ug/mL。取拟注射的MSCs(500000个),常规离心后用CM-DiL染色工作液重悬,37℃下孵育5min,再放入 4℃下放置 15 分钟,然后用PBS彻底清洗3遍,加入20uL PBS重悬用于心梗后心肌内注射。
术后第3天取心脏制备冰冻切片,做肌钙蛋白的组织切片免疫荧光,绿色标记肌钙蛋白,红色标记CM-DiI,蓝色表示DAPI标记细胞核;术后第3天取心脏消化制备单细胞悬液,流式检测CM-DiI阳性细胞比例和数量。实验结果见图3,*P<0.05。
【结论】上述实验的结果综合证明:2-DG能有效提高MSCs改善心梗的功能。
以上所述仅是本发明的优选实施方式,应当指出:对于本技术领域的普通技术人员来说,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,这些改进和润饰也应视为本发明的保护范围。
Claims (5)
1.2-脱氧-D-葡萄糖在制备增效间充质干细胞治疗心梗的药物中的应用;所述药物为注射制剂。
2.根据权利要求1所述的应用,其特征在于,2-脱氧-D-葡萄糖改善心梗后间充质干细胞治疗的心功能与心室重构。
3.2-脱氧-D-葡萄糖和间充质干细胞在制备增效治疗心梗的药物中的应用,其特征在于,所述药物为注射制剂。
4.2-脱氧-D-葡萄糖在制备增加间充质干细胞在心梗部位存活率的药物中的应用,其特征在于,所述药物为注射制剂。
5.一种药物在制备治疗心梗的药物中的应用,其特征在于,所述药物的活性成分为2-脱氧-D-葡萄糖和间充质干细胞;所述药物为注射制剂。
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