Chinese Journal of Tissue Engineering Research ›› 2023, Vol. 27 ›› Issue (6): 921-927.doi: 10.12307/2023.301
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Bai Siqi, Xiao Zhen, Liu Jing
Received:
2022-03-26
Accepted:
2022-06-02
Online:
2023-02-28
Published:
2022-08-12
Contact:
Xiao Zhen, MD, Associate professor, First Affiliated Hospital of Dalian Medical University, Dalian 116011, Liaoning Province, China
Liu Jing, MD, Professor, First Affiliated Hospital of Dalian Medical University, Dalian 116011, Liaoning Province, China
About author:
Bai Siqi, Master candidate, First Affiliated Hospital of Dalian Medical University, Dalian 116011, Liaoning Province, China
CLC Number:
Bai Siqi, Xiao Zhen, Liu Jing. Application potential of adipose-derived stem cells in female pelvic floor dysfunction diseases[J]. Chinese Journal of Tissue Engineering Research, 2023, 27(6): 921-927.
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2.1 脂肪源性干细胞的来源、培养及特点 脂肪组织中的基质血管细胞成分(stromal-vascular cell fraction,SVF)是多能干细胞的丰富来源,脂肪源性干细胞是指从脂肪组织中获取的基质血管细胞成分内具有多向分化潜能的细胞,这些干细胞被称为脂肪来源的干细胞或脂肪组织来源的基质细胞[10]。脂肪源性干细胞主要来源于脂肪组织,而脂肪组织遍布于身体各处,最常见的获取部位为皮下。由于白色脂肪中基质血管细胞成分的获取率较高[11],通常认为皮下白色脂肪组织是脂肪源性干细胞的主要来源。有研究发现,从200 mL经过处理的人类皮下脂肪组织中,可获取大约750万个干细胞[12],这提示脂肪源性干细胞的来源丰富,在人体中存储量巨大,使得分离和提取足够数量的干细胞难度显著降低,采集脂肪组织的外科过程本身相对收集其他来源干细胞对人体的影响也更小[13]。 细胞外基质是细胞生长的天然材料,其中盆底支持组织细胞外基质的主要成分是弹性蛋白、胶原蛋白、蛋白聚糖、层粘连蛋白和纤连蛋白[14]。在组织工程研究中,干细胞需要与支架材料结合形成细胞-材料复合体,在体外或体内适宜条件下培养,使干细胞在三维空间中生长、分化,最终形成所需的功能组织。相比细胞外基质,理想的支架材料应该具备良好的生物降解性、组织相容性和三维空间结构。水凝胶是一种很好的模拟天然细胞外基质的三维材料,用水凝胶搭载干细胞或细胞因子等用于盆底疾病的治疗也成为了近年来的研究热点[15]。聚丙烯酰胺作为目前最为常见的水凝胶材料,其化学性能稳定,无细胞毒性、无胚胎毒性、无致癌性,进入人体组织后不受催化剂影响,不易降解[16-17]。有研究显示,人脱细胞脂肪组织水凝胶能够携带脂肪源性干细胞到达相应部位,且脂肪源性干细胞包埋的人类脱细胞脂肪组织水凝胶在体温下即可形成,为脂肪源性干细胞提供了培养平台[18]。除此之外,以壳聚糖、β-甘油磷酸酯和胶原为原料制备的杂化水凝胶支架,能与有机物组织具有良好的组织相容性[17],其在低温态下为液状,体温下则糊化。而热敏可注射水凝胶能够将细胞、药物等生物活性物质包埋后注入生物体内发挥作用。例如:将脂源性干细胞包埋于凝胶内,再将凝胶注射到膀胱壁和尿道后,脂肪源性干细胞大量成活并形成具有收缩性的肌纤维[16-17];而且脂肪源性干细胞在体外培养过程能够长时间保持扩增能力,其增殖能力也不会因为传代次数的增加而有所减弱。由此可见,脂肪源性干细胞能够在体外培养,现有的培养材料和培养环境能够满足其增殖和分化。 在应用于临床之前,有必要对新鲜获取的脂肪源性干细胞进行体外扩增以确保在治疗期间给予临床上需要的细胞数量。因此,在早期传代时通过冷冻保存,可以延长新鲜分离细胞的储存时间。目前,冷冻保存被视为一种能够安全贮存脂肪源性干细胞的常规方法。尽管有人担心冷冻保存处理会对细胞特性造成影响。对此,DURANDT等[10]的研究显示,冷冻保存不会影响脂肪源性干细胞分化为脂肪细胞的能力,连续几轮的扩增并没有改变细胞表面标记的表达。KUMAR等 [19]评估了长期冷冻保存脂肪源性干细胞的特性及其在平均冷冻12年后的再生潜力,结果表明脂肪源性干细胞在长期冷冻保存后仍能保持分化能力,只是不同供者之间存在差异。而且一些冷冻保存溶液已经用于临床研究,且不会对细胞存活率产生较大影响,例如细胞因子系列、丝胶和麦芽糖、甲基纤维素、动物血清的替代品等[20]。 通过流式细胞技术可以鉴定出脂肪源性干细胞独特的表型。与其他间充质干细胞一样,脂肪源性干细胞表达 CD90、CD73、CD105、CD44,不表达 CD45 和 CD31,通过表面标记 CD36 阳性和 CD106 阴性能够将其与骨髓间充质干细胞进行鉴别[21]。脂肪源性干细胞属于成体干细胞的一种,研究表明其具有向中胚层细胞分化的巨大潜力,如脂肪细胞、成骨细胞、软骨细胞和心肌细胞[22]。体外条件下的研究显示,它们具有很高的可塑性和分化成外胚层来源细胞和内胚层来源细胞的能力[23]。因此,将脂肪源性干细胞分化为目标细胞,有望替换受损、患病和有缺陷的细胞和组织。而相比于同样具有多向分化能力的其他来源干细胞,脂肪源性干细胞具有储备量大、获取方便、获取过程中较少的痛苦性和培养条件的非苛刻性、可观的增殖分化性等特点,已成为干细胞领域一种可供选择的种子细胞来源。 2.2 脂肪源性干细胞治疗盆底功能障碍性疾病的可能机制 虽然盆底功能障碍性疾病的具体机制目前并不清楚,但是衰老和各种原因导致的盆底组织损伤是造成此病可能的主要危险因素。生理状态下,肌肉组织受损后主要由卫星细胞发挥修复作用,这些细胞被激活、增殖,并分化成肌纤维,从而重建受损的组织。而病理和衰老状态下,卫星细胞群发生功能障碍或衰竭,严重影响肌肉再生,从而影响其功能[24]。脂肪源性干细胞可能通过以下机制发挥促进组织修复作用,进而有助于盆底功能障碍性疾病的治疗。 2.2.1 通过旁分泌作用参与机体免疫调节 脂肪源性干细胞能够促进肌肉再生与其旁分泌能力参与机体免疫调节过程有关。研究表明,脂肪源性干细胞通过2种主要机制调节免疫系统,一种是直接通过细胞间通讯,另一种是间接通过分泌可溶性递质、生长因子和血管外小泡[25],其中脂肪源性干细胞的旁分泌活动是参与组织修复的主要机制。研究表明,脂肪源性干细胞能够在特定的区域进行特异性分化以确保细胞修复,而且还通过分泌多种类型的趋化因子、细胞因子和蛋白质生长因子等[23],从而能够减轻免疫反应,支持肌肉重建[24]。此外,脂肪源性干细胞还通过分泌白细胞介素2干预T细胞和B细胞的增殖、细胞毒作用和自然杀伤细胞的激活[26],并且支持T细胞活化、巨噬细胞相互作用、白细胞介素10分泌和Th1细胞增殖下调[27],降低了干细胞移植治疗后发生免疫排斥反应的风险。 2.2.2 分泌体的组织损伤修复作用 人脂肪来源干细胞中有一种名叫分泌体的结构,是细胞释放到胞外空间的囊泡[28],因此又称外泌体、胞外囊泡[29]。分泌体中富含积极参与再生过程的趋化因子、细胞因子和蛋白质生长因子[30]。外切体作为分泌体的一部分,被认为是一种多功能的、丰富的组织损伤修复工具,可抑制或激活信号通路,参与细胞外基质重塑、免疫调节、血管再生,以及细胞的迁移、增殖和分化过程[30],主要由蛋白质、mRNA、miRNA和细胞器共同构成[31]。人脂肪源性干细胞的分泌体具有强大的生物学功能和组织再生修复作用,它能够参与机体免疫和抗炎活动,影响细胞的生长、增殖和分化,发挥促进血管生成和细胞基质重建的作用[30]。MITCHELL等[32]研究表明,脂肪源性干细胞分泌体内的可溶性分子能够发挥促进肌肉生成、加速骨骼肌损伤的修复作用,并证实了脂肪源性干细胞分泌体能够刺激细胞向肌管分化,但对细胞的迁移没有影响。此外,分泌体可以避开干细胞治疗的相关不良反应,增加了干细胞治疗的安全性,降低了免疫原性[28]。由此可见,脂肪源性干细胞的分泌体在肌肉组织再生中具有一定作用。 正常盆底组织结构的维持主要与盆底结缔组织中的细胞外基质成分能否保持合成和降解的动态平衡有关,如果平衡破坏就可能产生以盆底结构改变为特征的女性盆底功能障碍性疾病[14]。有研究证实,脂肪干细胞来源分泌体通过增加压力性尿失禁患者阴道成纤维细胞胶原合成和减少胶原降解来增加Ⅰ型胶原含量,从而保持盆底组织细胞外基质的动态平衡[33]。这些关于干细胞分泌体作用的研究,将有助于扩大基于干细胞治疗组织缺陷相关盆底疾病的潜力,也为盆底功能障碍性疾病的治疗提供了合理解释的可能性。 2.3 脂肪源性干细胞治疗盆底功能障碍性疾病的临床前和临床试验 2.3.1 临床前试验 近年来,许多研究表明脂肪源性干细胞能在动物模型上改善盆底功能,其应用主要集中在尿失禁和便失禁方面。从病因学考虑,干细胞治疗失禁有很多优势,传统手术治疗失禁更多关注改善症状,较少根治具体的病因[34]。2010年LIN等[35]第1次探讨脂肪源性干细胞治疗压力性尿失禁的可行性,结果显示,经尿道和静脉注射脂肪源性干细胞的治疗组无论是膀胱计量学还是组织学分析,均显示疗效优于对照组。自此有关脂肪源性干细胞用于盆底功能障碍性疾病的研究逐渐开展,特别是针对失禁的研究备受关注。因此,针对脂肪源性干细胞对便失禁和压力性尿失禁的临床前试验做了整理,见表1和表2。 "
2.3.2 临床试验 一项自体脂肪来源间充质干细胞治疗尿失禁的两期临床试验中,招募了9名男性(8名治疗)和10名女性,行尿道镜下细胞腔内注射自体脂肪间充质干细胞用于尿失禁治疗,试验结果用于评价脂肪源性干细胞的可行性和安全性。在随访过程中,行尿道镜检查、尿动力学等客观检查和尿垫试验、生活质量、尿便失禁问卷等主观评价的检测,结果显示,在3/8的男性和5/10的女性中,客观改善率在50%,主观改善率达到了70%-80%,可见应用脂肪源性干细胞治疗尿失禁是一种安全可行的方法[44]。脂肪源性干细胞对于失禁的疗效,已经在很多动物实验中得到了验证,此外相关临床试验也在逐步开展,为了解其可行性并为临床应用提供进一步的理论支持,现将临床试验汇总至表3。 "
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