Conditioned medium from cultured mesenchymal stem cells: a potential for repairing multiple diseases and injuries

doi:10.3969/j.issn.2095-4344.1797

Abstract

Abstract:

BACKGROUND: Mesenchymal stem cells have provided new ideas and new methods for the development of regenerative medicine and the treatment of a variety of diseases. However, due to the low transplantation rate and survival rate of stem cells in the implanted bed, the mechanism of action of stem cells is inclined to involve the paracrine effect of soluble molecules released by stem cells during wound healing. The conditioned medium contains a variety of active substances secreted by stem cells. It is characterized by being easy to obtain, easy to use and safe, and may be used as a new technology for the treatment of a variety of human diseases.
OBJECTIVE: To review the?composition, function, acquisition and application of conditioned medium from mesenchymal stem cells, attempting to better apply it in the treatment of diseases.
METHODS: The keywords were “mesenchymal stem cells AND conditioned medium” in English and Chinese, respectively. Relevant articles published were retrieved by the first author in the PubMed, CNKI, and WanFang. According to the inclusion and exclusion criteria, finally 46 articles were retained.
RESULTS AND CONCLUSION: Conditioned medium obtained from mesenchymal stem cells contains growth factors, cytokines, inflammatory factors, extracellular matrix proteins, and tissue remodeling enzymes that are secreted by mesenchymal stem cells during the growth. The conditioned medium has the functions of immunomodulation, promotes angiogenesis, maintains stem cell activity, supports the growth of cells, has chemoattraction properties, and promotes cell differentiation into stem cells. The culture medium from cultured mesenchymal stem cells can be filtrated or concentrated to be an active liquid containing no cell components but higher concentrations of growth factors and cytokines, which is convenient and safe to use. This medium has been used for the basic research, a variety of animal disease models and some human clinical studies, and has achieved good results. With the in-depth research the stem cell conditioned medium will be continuously developed and exert their advantages to better benefit humans.

Key words: mesenchymal stem cells, conditioned medium, paracrine, cytokines, growth factors, regenerative medicine, National Natural Science Foundation of China

CLC Number:

Qiu Jiling, Wang Xiaotong, Zhou Haowen, Yang Pishan, Song Aimei. Conditioned medium from cultured mesenchymal stem cells: a potential for repairing multiple diseases and injuries [J]. Chinese Journal of Tissue Engineering Research, 2019, 23(29): 4743-4748.

Figures/Tables 1

2.1 间充质干细胞条件培养液的成分间充质干细胞在生长过程中会将一些活性物质分泌到培养环境中，这些活性物质会影响其他细胞的生长和/或功能，在创伤修复和内环境稳态的各个方面发挥重要作用，这称为间充质干细胞的旁分泌功能[6]。相关研究多在维持干细胞干性的培养过程中收集条件培养液，而对于干细胞接受诱导培养后定向分化过程中收集的条件培养液少见报道。因此，该综述所指的条件培养液，除非特别说明，均指的是维持间充质干细胞干性及生长的基础培养液。Engel等[7]研究表明干细胞的旁分泌物由分泌蛋白(如生长因子、血管生成因子、激素、细胞因子、细胞外基质蛋白、细胞外基质蛋白酶和激素)和遗传物质(如双链DNA、mRNA、microRNA和lncRNA)组成，通过细胞外囊泡或外泌体释放。Pawitan等[8]获取的干细胞条件培养液中通常含有多种活性物质，条件培养液的主要成分有：①生长因子，包括血管内皮生长因子、血小板衍生生长因子、胰岛素样生长因子、肝细胞生长因子、碱性成纤维细胞生长因子等；②炎性因子，包括转化生长因子β和一些白细胞介素，例如白细胞介素6、白细胞介素10、白细胞介素27、白细胞介素17E、白细胞介素13、白细胞介素12p70、白细胞介素8、白细胞介素9、白细胞介素1β等；③细胞外基质蛋白和组织重塑酶等，包括血管生成素、集落刺激因子、单核细胞趋化蛋白1，金属蛋白酶组织抑制剂1、丝氨酸蛋白酶抑制剂E1、尿激酶型纤溶酶原激活物等。虽然同是间充质干细胞，但由于组织来源不同，可能会受到特异组织来源的影响而导致某些特异因子的表达不同，包括种类和数量。例如Inukai等[9]采用ELISA法检测人骨髓间充质干细胞条件培养液(常氧培养48 h)中胰岛素样生长因子1、血管内皮生长因子、转化生长因子β1、肝细胞生长因子水平分别为(1 515.6±211.8) ng/L，(465.8±108.8) ng/L，(339.8±14.4) ng/L和(20.3±7.9) ng/L，而未检测到碱性成纤维细胞生长因子、血小板衍生生长因子BB、骨形态发生蛋白2和基质细胞衍生因子1。然而，Kim等[10]在常氧下培养48 h采用ELISA法检测到人脐血间充质干细胞中表皮生长因子、血管内皮生长因子、粒细胞集落刺激因子、粒细胞-巨噬细胞集落刺激因子水平分别为(3 286±419) ng/L，(2 463±151) ng/L，(3 615±173) ng/L，(3 623±345) ng/L，碱性成纤维细胞生长因子、角化细胞生长因子、转化生长因子β1、血小板衍生生长因子水平与人脐血内皮细胞条件培养液中含量相近。即使来自同一种类干细胞，条件培养液所含成分还会随培养条件的不同而发生变化。Song等[11]研究将低氧和常氧条件下得到的间充质干细胞条件培养液进行比较，检测到66种蛋白质表达差异，其中2种原肌球蛋白亚型在低氧条件培养液中显著增高，进一步分析表明这些蛋白质大多与心脏调节有关。Overath等[12]研究发现脂肪干细胞经过低氧处理(0.5%)，条件培养液中有68种蛋白表达增加了2倍以上，从而显著提升了脂肪干细胞条件培养液对顺铂诱导大鼠急性肺损伤的治疗效果。Jiang等[13]研究表明，与常氧培养获得的骨髓间充质干细胞条件培养液比较，经过低氧预处理的骨髓间充质干细胞条件培养液中有14种蛋白明显增加，低氧预处理骨髓间充质干细胞条件培养液在减少大鼠缺血性脑卒中后脑组织损伤和改善神经系统恢复方面起着至关重要的作用。因此，研究者可根据需要调整干细胞的培养环境，引导干细胞分泌目标因子，从而更好地将干细胞条件培养液用于疾病的治疗。 2.2 间充质干细胞条件培养液的作用骨髓间充质干细胞是较早培养和研究较多的间充质干细胞，骨髓间充质干细胞参与修复组织损伤过程中可能至少有2种不同的作用方式：旁分泌机制以及分化替代机制[14]。干细胞的旁分泌作用包括趋化性能、免疫调节功能、细胞生长支持功能、抗细胞凋亡及血管生成功能等，是通过干细胞分泌的生长因子等实现的。干细胞条件培养液还具有促进其他细胞向干细胞方向转化以及使其保持干细胞活性等功能。 2.2.1 趋化性能多项体外研究表明干细胞条件培养液能影响内皮细胞、角化细胞、成纤维细胞的增殖和/或迁移[15-16]。Chen等[17]研究表明大鼠骨髓间充质干细胞条件培养液可以促进肌腱细胞的增殖和迁移。Ando等[18]研究表明间充质干细胞条件培养液中所包含的因子可以募集小鼠骨髓基质细胞和内皮细胞/内皮祖细胞，起到抑制炎症和细胞凋亡，促进成骨细胞分化、血管生成和细胞增殖的作用，并认为单核细胞趋化因子1/3和白细胞介素3/6是募集小鼠骨髓基质细胞和内皮细胞/内皮祖细胞的关键因素。 2.2.2 免疫调节功能 Nagata等[19]研究表明，牙周膜干细胞条件培养液能降低牙周组织中肿瘤坏死因子α mRNA水平，抑制单核巨噬细胞原始细胞群体中肿瘤坏死因子α mRNA水平，同时降低白细胞介素1水平，提高白细胞介素10水平，通过抑制炎症反应、调节免疫，从而起到促进牙周再生的作用。白海等[20]研究表明骨髓间充质干细胞条件培养液可能通过影响T淋巴细胞分泌γ-干扰素和白细胞介素4进而抑制异体外周血T淋巴细胞增殖。 2.2.3 血管生成、细胞生长支持刘新宾等[21]研究表明间充质干细胞条件培养液中含有多种与血管生成、组织修复、细胞存活和抗炎作用相关联的生物活性物质，例如肿瘤坏死因子α、碱性成纤维细胞生长因子2、转化生长因子β、白细胞介素1、白细胞介素6等细胞因子，在胶原合成和血管生成、促进心肌细胞肥大、代偿心功能以及心肌重塑作用中起到了重要作用。 2.2.4 保持干细胞活性或促进其他细胞转化为干细胞 Qi等[22]研究表明脐带间充质干细胞条件培养液能使高糖诱导的髓核间充质干细胞Ⅱ型胶原蛋白和蛋白多聚糖表达升高，从而减轻细胞外基质降解，缓解高糖诱导的干细胞凋亡。Hu等[23]研究表明，骨髓间充质干细胞条件培养液可以诱导外周血单核细胞获得间充质干细胞的特征，为临床实践中获得间充质干细胞提供了新的途径。 2.3 间充质干细胞条件培养液的制取现有众多研究表明，即使是对于同一种间充质干细胞来源的条件培养液，不同研究所采取的制取标准等也有所不同，例如干细胞的数量、代数、培养基种类、培养条件以及条件培养液的进一步加工，尚未达成一致。不同的研究使用的基础培养基种类也不尽相同，还可能采用完全培养基或无血清培养基，制取条件培养液所选用的培养时间从6 h到5 d不等，其中24 h或48 h比较常见，Ando等[18]将间充质干细胞采用完全培养基培养至融合度70%-80%时，更换无血清培养基，48 h后收集上清液用于大鼠实验，结果显示间充质干细胞条件培养液对牵张成骨有促进作用。不同研究所选取的干细胞培养条件也可能不同。例如一些研究在制取干细胞条件培养液时对细胞进行常氧(氧体积分数为21%)培养，一些研究则选取不同的低氧条件(氧体积分数为0.5%，1%，1.5%，2%等)。Jiang等[24]研究表明低氧使骨髓间充质干细胞条件培养液中血管内皮生长因子、肝细胞生长因子、血小板衍生生长因子等表达增加，从而促进骨髓间充质干细胞的增殖和干性维持，并且增强其促血管生成特性。根据研究目的不同，研究者会对获得的条件培养液进行进一步加工，例如浓缩得到不同浓度的干细胞条件培养液。一般来说，体外研究使用的是非浓缩的干细胞条件培养液，体内研究常使用干细胞条件培养液的浓缩液。Nagata等[19]使用离心机以及切断界点为10 kD的超滤离心管获得不同浓缩倍数的条件培养液，植入大鼠牙周骨缺损，结果显示低浓度条件培养液没有明显作用，高浓度条件培养液组可观察到明显的牙周组织再生。 2.4 间充质干细胞条件培养液在组织再生中的应用移植间充质干细胞条件培养液已经在一些体外研究、多种动物创伤及疾病的组织再生模型中取得较好效果，例如心肌梗死、脑损伤、脑缺血、脊髓损伤、骨缺损、肾纤维化、结肠炎、关节炎、牙周缺损等。人体相关临床应用虽较少，但也已有报道。 2.4.1 体外实验研究 Lee等[25]研究显示间充质干细胞条件培养液可以诱导人胚胎干细胞和多能干细胞的成骨及成软骨方向分化。Cortes-Dericks等[26]研究表明人肺源性间充质干细胞条件培养液对体外恶性胸膜间皮瘤细胞系有抗癌作用。Hendudari等[27]发现联合骨髓间充质干细胞条件培养液与激光有利于人皮肤成纤维细胞在高糖培养基中的生长，为研究皮肤成纤维细胞特性提供了实验方法。 2.4.2 动物体内研究 He等[28]研究表明低氧诱导下脂肪间充质干细胞条件培养液可以促进心肌细胞增殖和迁移，有利于大鼠心肌梗死的恢复；Faezi等[29]研究表明间充质干细胞条件培养液可以促进大鼠局灶脑缺血功能性恢复；Szekiova等[30]研究表明大鼠脂肪间充质干细胞条件培养液在体外模拟局部炎症的脊髓损伤模型中可以对神经元起到保护作用；Liu等[31]研究发现人脐带间充质干细胞条件培养液可以减少组织炎症反应，通过TLR-4/NF-κB信号通路减弱大鼠肾纤维化；Pouya等[32]研究发现将间充质干细胞条件培养液注射应用于小鼠急性结肠炎具有免疫调节功能，可降低炎症反应；Kay等[33]研究表明间充质干细胞条件培养液能改善小鼠炎症性关节炎的严重程度。Nagata等[19]将牙周膜干细胞条件培养液植入大鼠牙周组织缺损，获得了较好的牙周组织再生，并且缺损的再生效果与条件培养液的浓度显著相关，进一步研究表明牙周膜干细胞条件培养液是通过抑制牙周创区的炎症因子，主要是肿瘤坏死因子α，从而促进牙周组织再生。 2.4.3 人体临床应用 Zhou等[34]研究表明脂肪间充质干细胞条件培养液用于二氧化碳激光表面处理后的人体皮肤创面，具有良好的促进修复愈合效果；Prakoeswa等[35]研究比较局部单独应用人羊膜间充质干细胞条件培养基以及和维生素C、E联合对麻风病慢性足底溃疡愈合的影响，结果显示与维生素E联合治疗效果最佳；Fukuoka等[36]研究表明脂肪间充质干细胞条件培养液局部注射于22例秃头症患者进行随访研究，结果显示实验组头发再生的数量明显高于对照组，因此局部注射条件培养液有望成为毛发再生的一种新技术。综上所述，间充质干细胞条件培养液已在多种疾病中取得了显著效果，具体详见表1[26-27，31-46]。"

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