CN113925878A - 一种细胞外囊泡在治疗神经损伤性勃起功能障碍中的应用 - Google Patents
一种细胞外囊泡在治疗神经损伤性勃起功能障碍中的应用 Download PDFInfo
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Abstract
本发明公开了一种细胞外囊泡在治疗神经损伤性勃起功能障碍中的应用。本发明首次提供了从褪黑素预处理离体的人间充质干细胞中提取得到的细胞外囊泡在治疗神经损伤性勃起功能障碍中的应用,本发明将所述的细胞外囊泡溶于磷酸盐缓冲液中得到浓度为5×1010个颗粒/毫升的注射剂。动物模型实验表明,与未进行褪黑素预处理的人间充质干细胞来源的细胞外囊泡相比,本发明的褪黑素预处理人间充质干细胞中提取的细胞外囊泡促进血管内皮修复和神经再生的功效更强,能够更好地促进勃起功能恢复,本发明解决了难治性勃起功能障碍患者阴茎血管内皮和神经损伤的技术问题,为治疗勃起功能障碍提供了一种安全、有效、使用方便的治疗药物制剂。
Description
技术领域
本发明涉及一种细胞外囊泡在治疗神经损伤性勃起功能障碍中的应用,属于生物医药技术领域。
背景技术
目前勃起功能障碍的一线治疗手段是口服磷酸二酯酶-5抑制剂,例如他达拉非、西地那非等。该类型药物主要结构为非选择性或者选择性磷酸二酯酶-5抑制剂,治疗勃起功能障碍的原理为通过抑制阴茎磷酸二酯酶-5活性,通过减少降解来增加环磷酸鸟苷浓度。目前勃起功能障碍的临床一线治疗药物磷酸二酯酶-5抑制剂发挥作用依赖于完整的血管内皮,不能逆转血管内皮和神经损伤,伤,对于糖尿病导致的勃起功能障碍、前列腺癌术后勃起功能障碍等临床治疗效果不确切。作为勃起功能障碍临床二线治疗手段,阴茎海绵体注射血管活性药物、真空负压吸引或低能量冲击波治疗依旧无法彻底修复阴茎血管内皮和神经损伤。利用人间充质干细胞阴茎海绵体注射的方式可以促进阴茎血管内皮和神经损伤修复,然而,不仅面临着免疫排斥、成瘤风险和伦理限制,而且操作繁琐,需要细胞储存复苏设备。
细胞外囊泡(extracellular vesicles)是细胞在增殖过程中释放入细胞外空间的囊状结构。根据直径大小,可以分为:外泌体(exosome,直径:50-100nm)及微颗粒(microparticle,直径:100-1000nm)。细胞外囊泡作为载体,运载着包括信号蛋白、脂质、核酸等成分,能够被远处的目标细胞所摄取内吞,从而能改变靶细胞的生物学行为。
发明内容
本发明所要解决的技术问题是:现有技术中用于治疗勃起功能障碍的药物存在副作用大,效果不佳等问题。
为了解决上述技术问题,本发明提供了一种细胞外囊泡在制备治疗神经损伤性勃起功能障碍的药物中的应用,所述的细胞外囊泡来源于褪黑素预处理离体的人间充质干细胞后提取得到的细胞外囊泡。
优选地,所述褪黑素的浓度为1μM。
优选地,所述的人间充质干细胞包括脂肪来源间充质干细胞、骨髓来源间充质干细胞、脐带间充质干细胞、胎盘间充质干细胞和尿源性间充质干细胞中的至少一种。
优选地,所述的药物包括注射剂。
优选地,所述注射剂为细胞外囊泡溶于溶剂后制得,所述溶剂包括磷酸盐缓冲液,所述磷酸盐缓冲液的pH值为7.2~7.4。
优选地,所述的注射剂中细胞外囊泡的浓度为5×1010个颗粒/毫升。
与现有技术相比,本发明的有益效果在于:
1.本发明通过阴茎海绵体注射褪黑素预处理人间充质干细胞后的细胞外囊泡解决难治性勃起功能障碍患者阴茎血管内皮和神经损伤的技术问题,为治疗勃起功能障碍提供安全、有效、使用方便的治疗药物制剂;且与单纯人间充质干细胞的细胞外囊泡相比,褪黑素预处理人间充质干细胞后提取的细胞外囊泡促进血管内皮修复和神经再生的功效更强,更好的改善勃起功能;
2.本发明中褪黑素预处理人间充质干细胞后提取的细胞外囊泡保留了促进血管内皮和神经修复的有效成分,制备成制剂后,只需冷藏保存,且阴茎海绵体注射操作简便,克服了利用间充质干细胞阴茎海绵体注射的方式存在的免疫排斥、成瘤风险、操作繁琐等缺陷。
附图说明
图1为褪黑素预处理人脂肪间充质干细胞后的细胞外囊泡电镜图,标尺为100nm;
图2为褪黑素预处理人脂肪间充质干细胞后提取的细胞外囊泡的粒径分布图;
图3为褪黑素预处理人脂肪间充质干细胞后提取的细胞外囊泡治疗神经损伤性勃起功能障碍大鼠4周后勃起功能恢复情况,其中,A为正常大鼠(Sham)的海绵体内压和动脉压记录曲线,B为磷酸盐缓冲液(PBS)对照组的海绵体内压和动脉压记录曲线,C为注射未经褪黑素预处理的人脂肪间充质干细胞的细胞外囊泡组(EVs)的海绵体内压和动脉压记录曲线,D为注射褪黑素预处理人脂肪间充质干细胞后的细胞外囊泡组(MT-EVs)的海绵体内压和动脉压记录曲线,E为四组海绵体内压比平均动脉压比值对比,***表示经过统计学分析,两组间(注射PBS组与注射未经褪黑素预处理人脂肪间充质干细胞后的细胞外囊泡组)具有显著性差异,P值小于0.001,**表示经过统计学分析,两组间(注射未经褪黑素预处理的人脂肪间充质干细胞的细胞外囊泡组与注射褪黑素预处理人脂肪间充质干细胞后的细胞外囊泡组)具有显著性差异,P值小于0.01;
图4为褪黑素预处理人脂肪间充质干细胞后提取的细胞外囊泡治疗神经损伤性勃起功能障碍大鼠4周后海绵体内皮细胞eNOS的免疫荧光染色,其中,A为正常大鼠(Sham)对照组,B为磷酸盐缓冲液(PBS)对照组,C为注射未经褪黑素预处理的人脂肪间充质干细胞的细胞外囊泡组(EVs),D为注射褪黑素预处理人脂肪间充质干细胞后的细胞外囊泡组(MT-EVs),E为四组海绵体内皮细胞eNOS表达水平的统计学分析,**表示两组间具有显著性差异,P值小于0.01,*表示两组间具有显著性差异,P值小于0.05;
图5为褪黑素预处理人脂肪间充质干细胞后提取的细胞外囊泡治疗神经损伤性勃起功能障碍大鼠4周后阴茎背神经nNOS的免疫荧光染色,其中,A为正常大鼠(Sham)对照组,B为磷酸盐缓冲液(PBS)对照组,C为注射未经褪黑素预处理的人脂肪间充质干细胞的细胞外囊泡组(EVs),D为注射褪黑素预处理人脂肪间充质干细胞后的细胞外囊泡组(MT-EVs),E为四组阴茎背神经nNOS表达水平的统计学分析,***表示两组间具有显著性差异,P值小于0.001,**表示两组间具有显著性差异,P值小于0.01。
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。
实施例1
一种用于治疗勃起功能障碍的细胞外囊泡:
1、褪黑素预处理人间充质干细胞后的细胞外囊泡的提取:
从人自体组织或者细胞库获得P4代脂肪来源间充质干细胞在临床级无血清培养基中培养,当细胞融合达到80-90%时,加入含1μM褪黑素的无血清DMEM培养基,24h后收集细胞上清液。细胞上清液经过300g在4℃环境下离心10min,16500g在4℃环境下离心10min去除细胞和细胞碎片,120000g在4℃环境下离心70min收集细胞外囊泡。
其中,上述采用的人间充质干细胞不局限于脂肪来源间充质干细胞,可以采用骨髓来源间充质干细胞、脐带间充质干细胞、胎盘间充质干细胞、尿源性间充质干细胞等;上述获得的细胞外囊泡包括微颗粒和外泌体。
2、制备注射用褪黑素预处理人间充质干细胞后的细胞外囊泡:
将褪黑素预处理人脂肪间充质干细胞后的细胞外囊泡在磷酸盐缓冲液中稀释1000倍,根据布朗运动和弥散系数在激光粒度分析仪(Zetasizer Pro,MalvernPanalytical Ltd,UK)中自动分析。图1为褪黑素预处理人脂肪间充质干细胞后的细胞外囊泡电镜图,标尺为100nm;图2为褪黑素预处理人脂肪间充质干细胞后的细胞外囊泡的粒径分布图。
其中,上述采用的磷酸盐缓冲液中含有0.27g/L的磷酸二氢钾、1.42g/L的磷酸氢二钠、8g/L的氯化钠和0.2g/L的氯化钾,pH值为7.2~7.4。上述获得的注射用褪黑素预处理人脂肪间充质干细胞后的细胞外囊泡中细胞外囊泡的浓度为5×1010个颗粒/毫升。
实施例2
治疗斯普拉格-道利大鼠神经损伤性勃起功能障碍:
(1)取实施例1制备所得的注射用褪黑素预处理人脂肪间充质干细胞后的细胞外囊泡。
(2)在8周龄斯普拉格-道利大鼠上构建神经损伤性勃起功能障碍模型,每只大鼠阴茎海绵体注射取100微升上述褪黑素预处理人脂肪间充质干细胞后的细胞外囊泡。
(3)治疗4周后评价大鼠勃起功能,测量阴茎海绵体内压(ICP)和平均动脉压(MAP),计算两者比值。
由图3可知,用1μM褪黑素预处理人脂肪间充质干细胞后来源的细胞外囊泡(MT-EVs)治疗神经损伤性勃起功能障碍大鼠后,代表勃起功能的ICP/MAP指标显著性改善(P<0.05)。正常大鼠(Sham)同一时间点阴茎海绵体内压/平均动脉压比值为0.803±0.04453(图3A),使用磷酸盐缓冲液(PBS)作为对照治疗4周后,大鼠阴茎海绵体内压/平均动脉压比值为0.2579±0.03048(图3B),利用未经褪黑素预处理的人脂肪间充质干细胞的细胞外囊泡(EVs)治疗4周后,大鼠阴茎海绵体内压/平均动脉压比值为0.4633±0.03512(图3C),利用褪黑素预处理人脂肪间充质干细胞后的细胞外囊泡治疗4周后,大鼠阴茎海绵体内压/平均动脉压比值为0.6043±0.03502(图3D)。经过统计学分析,利用褪黑素预处理人脂肪间充质干细胞后来源的细胞外囊泡能够显著促进大鼠勃起功能恢复(图3E)。
(4)治疗4周后评价海绵体内皮细胞eNOS表达水平。由图4可知,使用磷酸盐缓冲液(PBS)作为对照治疗4周后,海绵体内皮细胞eNOS表达水平较低(图4B)。采用MT-EVs治疗后(图4D),eNOS表达水平显著恢复,且比EVs治疗后(图4C)表达水平更高。经过统计学分析,MT-EVs能够更好的促进内皮功能修复(图4E)。
(5)治疗4周后评价阴茎背神经nNOS表达水平。由图5可知,使用磷酸盐缓冲液(PBS)作为对照治疗4周后,阴茎背神经nNOS表达水平较低(图5B)。采用MT-EVs治疗后(图5D),nNOS表达水平显著恢复,且比EVs治疗后(图5C)表达水平更高。经过统计学分析,MT-EVs具有更好的修复神经损伤的效果(图5E)。
上述实施例仅为本发明的优选实施例,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。
Claims (6)
1.一种细胞外囊泡在制备治疗神经损伤性勃起功能障碍的药物中的应用,其特征在于,所述的细胞外囊泡来源于褪黑素预处理离体的人间充质干细胞后提取得到的细胞外囊泡。
2.如权利要求1所述的应用,其特征在于,所述褪黑素的浓度为1μM。
3.如权利要求1所述的应用,其特征在于,所述的人间充质干细胞包括脂肪来源间充质干细胞、骨髓来源间充质干细胞、脐带间充质干细胞、胎盘间充质干细胞和尿源性间充质干细胞中的至少一种。
4.如权利要求1所述的应用,其特征在于,所述的药物包括注射剂。
5.如权利要求4所述的应用,其特征在于,所述注射剂为细胞外囊泡溶于溶剂后制得,所述溶剂包括磷酸盐缓冲液,所述磷酸盐缓冲液的pH值为7.2~7.4。
6.如权利要求4所述的应用,其特征在于,所述的注射剂中细胞外囊泡的浓度为5×1010个颗粒/毫升。
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115006543A (zh) * | 2022-03-01 | 2022-09-06 | 中国人民解放军总医院第二医学中心 | 负载褪黑素细胞外囊泡及制备方法 |
CN115850480A (zh) * | 2022-09-23 | 2023-03-28 | 北京诺赛国际医学研究院 | 一种干细胞外泌体的制备方法以及在治疗男性勃起功能障碍中的应用 |
WO2023179615A1 (en) * | 2022-03-21 | 2023-09-28 | U-Neuron Biomedical Inc. | Treatment of erectile dysfunction using mesenchymal stem cells of amniotic fluid |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
IE20050639A1 (en) * | 2004-09-27 | 2006-09-06 | Sigmoid Biotechnologies Ltd | Minicapsule formulations |
CN101879184A (zh) * | 2009-05-07 | 2010-11-10 | 浙江医药股份有限公司新昌制药厂 | 用于提高生殖能力的药物组合物及其制备方法和应用 |
US20110236903A1 (en) * | 2008-12-04 | 2011-09-29 | Mcclelland Michael | Materials and methods for determining diagnosis and prognosis of prostate cancer |
CN105441385A (zh) * | 2007-02-23 | 2016-03-30 | 奥卡塔治疗公司 | 重编程分化细胞和从重编程的细胞产生动物和胚胎干细胞的高效方法 |
CN108392493A (zh) * | 2018-04-10 | 2018-08-14 | 上海交通大学医学院附属仁济医院 | 一种用于治疗勃起功能障碍的含细胞外囊泡的可注射水凝胶及其制备方法 |
CN108498452A (zh) * | 2018-04-10 | 2018-09-07 | 上海交通大学医学院附属仁济医院 | 一种用于治疗勃起功能障碍的含干细胞可注射水凝胶及其制备方法 |
CN111484969A (zh) * | 2020-05-07 | 2020-08-04 | 内蒙古农业大学 | 毛囊干细胞来源外泌体在促进毛囊干细胞增殖以及向毛囊细胞分化中的用途 |
CN111971063A (zh) * | 2018-04-06 | 2020-11-20 | 4阵营疗法公司 | 通过靶向调节基因信号传导网络治疗疾病 |
-
2021
- 2021-10-20 CN CN202111220183.4A patent/CN113925878B/zh active Active
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
IE20050639A1 (en) * | 2004-09-27 | 2006-09-06 | Sigmoid Biotechnologies Ltd | Minicapsule formulations |
CN105441385A (zh) * | 2007-02-23 | 2016-03-30 | 奥卡塔治疗公司 | 重编程分化细胞和从重编程的细胞产生动物和胚胎干细胞的高效方法 |
US20110236903A1 (en) * | 2008-12-04 | 2011-09-29 | Mcclelland Michael | Materials and methods for determining diagnosis and prognosis of prostate cancer |
CN101879184A (zh) * | 2009-05-07 | 2010-11-10 | 浙江医药股份有限公司新昌制药厂 | 用于提高生殖能力的药物组合物及其制备方法和应用 |
CN111971063A (zh) * | 2018-04-06 | 2020-11-20 | 4阵营疗法公司 | 通过靶向调节基因信号传导网络治疗疾病 |
CN108392493A (zh) * | 2018-04-10 | 2018-08-14 | 上海交通大学医学院附属仁济医院 | 一种用于治疗勃起功能障碍的含细胞外囊泡的可注射水凝胶及其制备方法 |
CN108498452A (zh) * | 2018-04-10 | 2018-09-07 | 上海交通大学医学院附属仁济医院 | 一种用于治疗勃起功能障碍的含干细胞可注射水凝胶及其制备方法 |
CN111484969A (zh) * | 2020-05-07 | 2020-08-04 | 内蒙古农业大学 | 毛囊干细胞来源外泌体在促进毛囊干细胞增殖以及向毛囊细胞分化中的用途 |
Non-Patent Citations (8)
Title |
---|
CHEN Z等: "Melatonin-Primed Mesenchymal Stem Cells-Derived Small Extracellular Vesicles Alleviated Neurogenic Erectile Dysfunction by Reversing Phenotypic Modulation", 《ADV HEALTHC MATE》, 27 January 2023 (2023-01-27) * |
XUE-FENG QIU等: "Mobilisation of endothelial progenitor cells: one of the possible mechanisms involved in the chronic administration of melatonin preventing erectile dysfunction in diabetic rats", 《ASIAN J ANDROL 》, 27 February 2012 (2012-02-27), pages 481 - 486 * |
乐岭: "氧化应激在糖尿病大鼠勃起功能障碍中的作用", 《CNKI》, 15 July 2011 (2011-07-15), pages 1 - 87 * |
刘威: "预处理的外泌体对骨质疏松骨整合及糖尿病创面修复作用的研究", 《万方》, pages 6 - 7 * |
张蒙夏等: "褪黑素对大鼠骨髓基质干细胞诱导分化及抗凋亡作用的研究", 《美国中华临床医学杂志》, pages 51 - 53 * |
惠宇等: "褪黑素对糖尿病性勃起功能障碍大鼠的神经保护作用及可能的机制研究", 《现代泌尿外科杂志》, 28 June 2016 (2016-06-28), pages 301 - 306 * |
申艳超等: "褪黑素对雄性动物生殖机能的研究进展", 《河南农业科学》, pages 1 - 7 * |
陈钰萌: "褪黑素对内蒙古绒山羊毛囊干细胞外泌体分泌的影响", 《CNKI》, pages 1 - 75 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115006543A (zh) * | 2022-03-01 | 2022-09-06 | 中国人民解放军总医院第二医学中心 | 负载褪黑素细胞外囊泡及制备方法 |
CN115006543B (zh) * | 2022-03-01 | 2023-08-29 | 中国人民解放军总医院第二医学中心 | 负载褪黑素细胞外囊泡及制备方法 |
WO2023179615A1 (en) * | 2022-03-21 | 2023-09-28 | U-Neuron Biomedical Inc. | Treatment of erectile dysfunction using mesenchymal stem cells of amniotic fluid |
CN115850480A (zh) * | 2022-09-23 | 2023-03-28 | 北京诺赛国际医学研究院 | 一种干细胞外泌体的制备方法以及在治疗男性勃起功能障碍中的应用 |
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