CN113166725A - 用于生成软骨组织的分离的中隔软骨外来体 - Google Patents
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Abstract
本发明涉及通过从中隔软骨分离的细胞释放到培养基中的外来体产生的制剂生成软骨。本发明的目的是,因为它诱导软骨形成并且还抑制炎症反应,所以生成可用于治疗软骨组织缺陷例如骨关节炎或关节病的软骨。
Description
技术领域
本发明涉及用于生成软骨并且通过从中隔软骨分离的细胞释放到培养基中的微泡产生的制剂。
背景技术
软骨在一些器官中是一种表现出骨功能的柔性、坚硬和白色的组织。在大多数原始脊椎动物和在发达脊椎动物中,胚胎的骨骼由软骨组成。在一个完全成熟的人体中,鼻、喉和耳中有软骨部位。它也用作覆盖彼此面对的形成关节的骨表面的垫层。关节软骨可以各种方式被损坏和腐蚀。这导致被称为骨关节炎或关节病的退行性关节疾病[1]。骨关节炎是一种非炎性、慢性和退行性疾病,其特征在于逐渐发生的软骨破坏、骨赘形成和软骨下硬化,特别是在承重关节中。在这种也被称为退行性关节炎、骨关节炎或肥大性关节炎的疾病中,关节软骨会逐渐损耗[2]。
与骨不同,软骨不需要为存活而与骨直接接触。当组织液到达软骨的纤维基质时,成软骨细胞被滋养,且与异质植入物不同,它们不必嵌入组织中。因此,它可容易地用在鼻嵴中和甚至在上皮下囊(subepithelial pocket)中。为此目的,可使用即用型软骨或者中隔软骨、甲软骨或肋软骨。由于其柔性结构,软骨可容易地成形并可用作鼻中的小缺陷和边缘不规则的支撑物和填充材料两者。所用的大多数软骨移植物为自体的。新鲜或储存的同源软骨和经辐照的异源软骨已被使用多年,但因为它们随时间推移被再吸收,所以它们的使用减少[3]。中隔软骨、无血管软骨和肋软骨被广泛用于头部和颈部区域的软组织缺陷中,和用来代替鼻再造过程。
有关组织工程的研究已尝试通过将合适的细胞接种到合适的再吸收生物材料支架中,以在体外和体内形成软骨。此外,用于头部-颈部区域内的软组织置换的人中隔软骨的组织工程具有在不久的将来提供临床益处的潜力[4]。
在软骨损耗中,组织的自愈能力非常有限。尽管发生有限的修复,但是所得到的组织为纤维软骨,其不具有与原始关节软骨相同的生物力学性质。因此,软骨组织工程的目标是所得到的人造软骨具有与正常的关节软骨相同的生物力学性质[5]。在进行的临床研究中,可见用于软骨修复的技术提供短期和中期结果。正在进行有关用于软骨修复的第二代组织工程解决方案的广泛研究。正在研究将允许关节镜下植入细胞并因此降低发病率的各种方法和新技术。当今可用的众多技术中没有一种能够一贯地再现正常的透明软骨,且最佳的长期治疗仍然为未知的[6]。生物力学测试已证明,组织工程化的软骨的生物力学性质与正常的中隔软骨的生物力学性质相容[4,7]。
当前针对软骨缺陷的治疗方法包括外科干预(例如,微骨折、镶嵌成型,包括高级和模拟生物材料支架的组织工程)、细胞移植(干细胞或软骨细胞植入物)、靶向疗法和疾病修饰疗法(消炎药)[8]。
目前技术水平下遇到的问题可列举如下:
编号为EP2551342的欧洲专利申请文件作为目前技术水平申请之一,公开了一种诱导人下鼻甲间充质基质细胞分化为软骨细胞、骨细胞、神经细胞或脂肪细胞的方法。所述发明的方法是一种用于分离和培养软骨细胞的方法。
编号为EP3145514的欧洲专利申请文件作为目前技术水平申请之一,公开了一种用于骨、软骨、牙齿和牙周组织再生的制剂。通过给予在所述发明中开发的制剂,刺激骨和/或软骨生长以治疗骨折和软骨损伤。在为开发该发明而进行的实验研究中,在DMEM培养培养基中,于培养皿中培养从牙髓分离的干细胞。
编号为EP1926507的欧洲专利申请文件作为目前技术水平申请之一,公开了一种用于修复软骨缺陷的植入物和一种用于制造所述植入物的方法。植入物包含包被有具有软骨形成潜力的自体细胞的天然软骨组织的植入物主体。通过从软骨活组织检查分离的软骨细胞开始的体外细胞增殖来产生这些细胞。
编号为US2017296590的美国专利申请文件作为目前技术水平申请之一,公开了一种用于诱导软骨细胞分化或软骨组织再生的组合物。所述发明的组合物包含从分化为软骨细胞的干细胞衍生的外来体。在所述发明中,脂肪干细胞被分化为软骨细胞并且外来体从软骨细胞分离。
发明内容
本发明的目的是出于美容和治疗目的,从分离的中隔软骨外来体诱导软骨形成。
本发明的另一个目的是,由于其抗炎性质而形成不会对身体和细胞生成免疫反应、炎症、毒性和刺激的软骨。
本发明的进一步目的是,因为它诱导软骨形成并且还抑制炎症反应,所以从分离的中隔软骨外来体得到用于治疗软骨组织缺陷例如骨关节炎或关节病的软骨组织。
具体实施方式
在附图中说明了为实现本发明的目的而开发的“用于生成软骨组织的分离的中隔软骨外来体”,其中;
图1. 是使用MTS测试评估以不同浓度向干细胞给予从中隔细胞得到的外来体24、48和72小时对细胞生存力的作用的图示。
图2. 是通过向干细胞给予不同浓度的从中隔细胞得到的外来体和软骨分化培养基,来评估其对CD44(a)和SOX9(b)基因表达水平的作用的图示。
图3. 是通过向干细胞给予不同浓度的从中隔细胞得到的外来体,来评估所述外来体对细胞的凋亡作用的图示。(a1-仅给予从中隔细胞得到的外来体(100%),a2-仅给予从中隔细胞得到的外来体(50%),a3-仅给予从中隔细胞得到的外来体(25%),a4-通过仅给予细胞培养基生长的细胞,b-图示a1、a2、a3、a4图3)。
图4. 光学显微镜下的中隔细胞外来体(a)、外来体/软骨分化培养基混合物(1:1)(b)、用软骨分化培养基处理10天的细胞的阿尔新蓝(Alcian Blue)染色的图像(c)和对照应用(d)。
图5. 显示了在本发明范围内,中隔细胞外来体对花粉(a)和螨(b)过敏原激活的白细胞的作用的图示(通过流式细胞术设备,使用作为表面标志物的抗体测量CD4 T辅助淋巴细胞、CD8 T细胞毒性淋巴细胞、CD19 B淋巴细胞和CD56自然杀伤细胞的图)。
图6. 显示了在本发明范围内,中隔细胞外来体对IL2 (a)和PHA (b)激活的白细胞的作用的图示(通过流式细胞术设备,使用作为表面标志物的抗体测量CD4 T辅助淋巴细胞、CD8 T细胞毒性淋巴细胞、CD19 B淋巴细胞和CD56自然杀伤细胞的图)。
本发明涉及开发通过用于生成软骨组织的微泡产生的制剂,所述微泡由从中隔软骨分离的细胞释放。在本发明的实施中,使用中隔软骨干细胞。观察到从软骨细胞得到的微泡对干细胞的软骨分化有影响。这些微泡的有效范围被确定为以体积计5-100%。微泡可利用dH2O、EtOH、细胞培养培养基、PBS、DMSO及其混合物的溶液溶解。这些外来体从软骨衍生的细胞中的分离为这些外来体提供形成软骨的能力,其同样并入了干细胞的炎症抑制性质。因此,已通过实验证明这些外来体增强软骨形成和抑制炎症两者的事实,并显示在图中。由于这些性质,这些外来体可用于治疗软骨损伤和免疫系统相关疾病。
本发明的制剂相对于目前技术水平的差异之一是使用从中隔软骨分离的细胞,并且在它被分离之处和使用特征不同的细胞类型的方面均产生显著差异。此外,在本发明的范围内,使用作为这些细胞的特定成分的外来体。这些外来体只是细胞释放到细胞外部的化学物质的一部分。在本发明的范围内,使用中隔细胞的外来体加强软骨组织的形成,并且即使它不是自体的,也不引起任何炎症。尽管它们不是自体的,但是这些具有干细胞的免疫抑制性质的干细胞衍生的外来体不引起炎症,并且还抑制已发生的炎症(图5和6)。在本发明的范围内,分离未暴露于任何化学物质的未分化的中隔细胞释放到培养基中的外来体。
分离的中隔软骨外来体诱导软骨组织的形成,以用于治疗软骨组织缺陷,例如骨关节炎、肋软骨关节炎症、Tietze综合征或关节病;而且由于它们的抗炎性质,它们能够形成对身体和细胞不产生免疫反应、炎症、毒性和刺激的软骨。在本发明的范围内,从这些分离的中隔软骨外来体形成软骨组织的方法包括以下步骤:
- 在37℃的温度下,使用5% CO2,在细胞培养箱中的包含10%外来体耗尽的胎牛血清(Invitrogen)和1% PSA (Biological Industries,Beit Haemek,以色列)的Dulbecco改良Eagle培养基(DMEM)中培养软骨细胞,
- 使用包含双相PEG-葡聚糖的外来体分离溶液,用于从在培养过的培养基中的细胞分离微泡,
- 为了除去废弃细胞,将从培养培养基收集的培养基以300 g离心10分钟,
- 为了除去可能的细胞成分,转移上清液至新的管中并以14000 g离心30分钟,
- 转移上清液至新的管中,随即加入1/1体积的PEG-葡聚糖溶液,以1000g离心10分钟,并随后收集残留在较低相中的外来体,
- 10天期间内每隔一天向中隔软骨外来体给予用于软骨分化的分化溶液,
- 得到作为分化结果的软骨组织。
实验研究
1. 毒性
在培养培养基中包含10%外来体耗尽的胎牛血清(Invitrogen)和1% PSA(Biological Industries, Beit Haemek, 以色列)的Dulbecco改良Eagle培养基(DMEM)中,将细胞以5000个细胞/孔接种在96孔培养平板(Corning Glasswork,Corning, NY)中后,在第1、2和3天测量细胞的生存力水平。通过使用3-(4,5-二甲基-噻唑-2-基)-5-(3-羧基-甲氧基-苯基)-2-(4-磺基-苯基)-2H-四唑(MTS)-方法(CellTiter96 AqueousOneSolution;Promega,Southampton,UK)测定细胞生存力。将10 μl MTS溶液加到100 μl培养基中的细胞上,并将其在37℃下黑暗中温育2小时。温育过程后,通过ELISA平板读数器(Biotek,Winooski,VT)装置在490 nm波长下进行吸光度测量,来观察细胞生存力。
2. 软骨分化
在培养培养基中包含10%外来体耗尽的胎牛血清(Invitrogen)和1% PSA(Biological Industries,Beit Haemek,以色列)的Dulbecco改良Eagle培养基(DMEM)中,将细胞以50,000个细胞/孔接种在6孔培养平板(Corning Glasswork,Corning,NY)中。次日起的10天内每隔一天给予中隔软骨外来体和文献中用于软骨分化的分化溶液。
比较了用于软骨分化的培养基和从中隔细胞得到的外来体对软骨分化的作用,并显示外来体为更有效的(图3、4和5)。
3.实时PCR
培养过的细胞可失去它们自身的性质并获得新的性质。这些性质可在形态学水平和基因表达水平两者上。应用实时PCR方法来观察在基因表达水平上的变化。从在Dulbecco改良Eagle培养基(DMEM)中以50,000个细胞/孔接种在6孔培养平板(Corning Glasswork,Corning,NY)中的细胞分离总RNA并合成cDNA。将合成的cDNA与Fermentas Maxima SYBRGreen混合产品中的引物混合,使得最终体积为20 µl,并使用BIO-RAD装置分析基因的表达水平。
从分离的中隔软骨外来体生成软骨组织的本发明的方法的优点可列举如下:
•诱导软骨形成
•具有炎症抑制性质
•不引起炎症
•不诱导细胞内毒性
•使用后可在细胞内代谢
•可用于治疗骨关节炎和关节病
•可用于治疗软骨组织缺陷
•可用于鼻再造
•具有出于美容和治疗目的诱导软骨形成的高潜力
•可用作组织工程中的有效试剂
•由于其抗炎性质,不诱导针对身体和细胞的免疫反应、炎症、毒性和刺激
•由于其免疫抑制活性,可用于自身免疫性疾病
•由于其能使软骨形成和抑制炎症反应的性质,可用于治疗类风湿关节炎。
参考文献
[1]. Deans, R. J., & Moseley, A. B. (2000). Mesenchymal stem cells:biology and potential clinical uses(间充质干细胞:生物和潜在的临床用途).Experimental hematology, 28(8), 875-884.
[2]. Doral, M. N., Dönmez, G., Atay, Ö. A., Bozkurt, M., Leblebicioğlu, G., Üzümcügil, A., & Aydoğ, T. 2007. “Dejeneratif eklem hastalıkları”,TOTBİD dergisi, 6, 56-65.
[3]. Dağlı, A. Ş., Özdem, C., Akalın, Y., Ensari, S. 1993.“Rinoplastide Biyomateryeller”, K.B.B. ve Baş Boyun Cerrahisi Dergisi, Cilt:l Sayı: 2.
[4]. Rotter, N., Bonassar, L. J., Tobias, G., Lebl, M., Roy, A. K.,Vacanti, C. A. 2002. “Age dependence of biochemical and biomechanicalproperties of tissue-engineered human septal cartilage”(“组织工程化的人中隔软骨的生物化学和生物力学性质的年龄依赖性”), Biomaterials, 23(15), 3087-3094.
[5]. Şenköylü, A., & Korkusuz, F. 2004. “Kıkırdak Onarımında Doku Mühendisliği Uygulamaları”, TOTBİD (Türk Ortopedi ve Travmatoloji Birliği Derne ği) Dergisi, 3, 3-4.
[6]. Smith, G. D., Knutsen, G., & Richardson, J. B. 2005. “A clinicalreview of cartilage repair techniques”(“软骨修复技术的临床综述”), Bone & Joint Journal, 87(4), 445-449.
[7]. Haisch, A., Duda, G. N., Schroeder, D., Gröger, A., Gebert, C.,Leder, K., & Sittinger, M. 2005. “The morphology and biomechanicalcharacteristics of subcutaneously implanted tissue-engineered human septalcartilage”(“皮下植入的组织工程化的人中隔软骨的形态学和生物力学特征”),European Archives of Oto-Rhino-Laryngology and Head & Neck, 262(12), 993-997.
[8]. Li, M. H., Xiao, R., Li, J. B., & Zhu, Q. 2017. “Regenerativeapproaches for cartilage repair in the treatment of osteoarthritis”(“骨关节炎治疗中用于软骨修复的再生方法”), Osteoarthritis and cartilage, 25(10), 1577-1587.
Claims (6)
1.分离的中隔软骨外来体,所述分离的中隔软骨外来体由于干细胞对软骨分化为有效的事实而用于诱导软骨组织形成。
2.权利要求1的分离的中隔软骨外来体,当使用5-100%时,所述分离的中隔软骨外来体为有效的。
3.权利要求1的分离的中隔软骨外来体,所述分离的中隔软骨外来体可利用选自dH2O、EtOH、细胞培养培养基、PBS、DMSO及其混合物的溶液溶解。
4.权利要求1的分离的中隔软骨外来体,所述分离的中隔软骨外来体用于治疗软骨组织缺陷,例如骨关节炎、肋软骨关节炎症、Tietze综合征或关节病。
5.从权利要求1的分离的中隔软骨外来体生成软骨组织的方法,所述方法包括以下步骤:
- 在细胞培养箱中的包含10%外来体耗尽的胎牛血清(Invitrogen)和1% PSA(Biological Industries,Beit Haemek,以色列)的Dulbecco改良Eagle培养基(DMEM)中培养软骨细胞,
- 使用包含双相PEG-葡聚糖的外来体分离溶液,用于从在培养过的培养基中的中隔软骨细胞分离微泡,
- 为了除去废弃细胞,将从培养培养基收集的培养基以300 g离心10分钟,
- 为了除去可能的细胞成分,转移上清液至新的管中并以14000 g离心30分钟,
- 转移上清液至新的管中,随即加入1/1体积的PEG-葡聚糖溶液,以1000 g离心10分钟,并随后收集残留在较低相中的外来体,
- 10天期间内每隔一天向中隔软骨外来体给予用于软骨分化的分化溶液,
- 得到作为分化结果的软骨组织。
6.从权利要求3的分离的中隔软骨外来体生成软骨组织的方法,其中在37℃的温度下,使用5% CO2在细胞培养箱中培养软骨细胞。
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1882602A (zh) * | 2003-10-10 | 2006-12-20 | 梅迪泰克研究有限公司 | 在疾病治疗中乙酰透明质酸合成的调节和降解 |
WO2016126122A2 (ko) * | 2015-02-04 | 2016-08-11 | 한양대학교 에리카산학협력단 | 연골세포로 분화되고 있는 줄기세포로부터 추출된 엑소좀을 포함하는 연골세포 분화 유도 또는 연골조직 재생용 조성물 |
CN107223153A (zh) * | 2015-02-04 | 2017-09-29 | 胞外体干细胞株式会社 | 包含源于正分化成软骨细胞之干细胞的外排体的用于软骨细胞分化诱导或软骨组织再生的组合物 |
KR20180092348A (ko) * | 2017-02-09 | 2018-08-20 | 주식회사 엑소코바이오 | 연골세포로 분화되고 있는 제대 및 제대혈 유래 줄기세포로부터 분리된 엑소좀을 포함하는 연골세포 분화 유도 또는 연골조직 재생용 조성물 |
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Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1882602A (zh) * | 2003-10-10 | 2006-12-20 | 梅迪泰克研究有限公司 | 在疾病治疗中乙酰透明质酸合成的调节和降解 |
WO2016126122A2 (ko) * | 2015-02-04 | 2016-08-11 | 한양대학교 에리카산학협력단 | 연골세포로 분화되고 있는 줄기세포로부터 추출된 엑소좀을 포함하는 연골세포 분화 유도 또는 연골조직 재생용 조성물 |
CN107223153A (zh) * | 2015-02-04 | 2017-09-29 | 胞外体干细胞株式会社 | 包含源于正分化成软骨细胞之干细胞的外排体的用于软骨细胞分化诱导或软骨组织再生的组合物 |
US20170296590A1 (en) * | 2015-02-04 | 2017-10-19 | Exostemtech Co., Ltd. | Composition for inducing chondrocyte differentiation and regenerating cartilage tissue |
KR20180092348A (ko) * | 2017-02-09 | 2018-08-20 | 주식회사 엑소코바이오 | 연골세포로 분화되고 있는 제대 및 제대혈 유래 줄기세포로부터 분리된 엑소좀을 포함하는 연골세포 분화 유도 또는 연골조직 재생용 조성물 |
Non-Patent Citations (2)
Title |
---|
YAHONG CHEN 等: "Exosomes derived from mature chondrocytes facilitate subcutaneous stable ectopic chondrogenesis of cartilage progenitor cells", 《STEM CELL RESEARCH & THERAPY》, 21 November 2018 (2018-11-21), pages 1 - 14 * |
周晓雯 等: "外泌体在骨性关节炎发病机制中的研究进展", 《中国骨科临床与基础研究杂志》, 15 December 2018 (2018-12-15), pages 361 - 366 * |
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