Chinese Journal of Tissue Engineering Research ›› 2014, Vol. 18 ›› Issue (6): 968-973.doi: 10.3969/j.issn.2095-4344.2014.06.024
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Zhang Ping-ping, Xiang Chuan
Revised:
2013-12-10
Online:
2014-02-05
Published:
2014-02-05
Contact:
Xiang Chuan, Associate professor, Master’s supervisor, Orthopedics Laboratory, Second Hospital of Shanxi Medical University, Taiyuan 030001, Shanxi Province, China
About author:
Zhang Ping-ping, Studying for master’s degree, Orthopedics Laboratory, Second Hospital of Shanxi Medical University, Taiyuan 030001, Shanxi Province, China
CLC Number:
Zhang Ping-ping, Xiang Chuan. Bone marrow mesenchymal stem cells for osteoarthritis: its possibility and future[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(6): 968-973.
2 检索结果 Results 初检得到文献79篇,其中英文62篇,中文17篇。首先阅读初检文献的标题和摘要进行初筛,研究目的与此文无关者排除英文11篇,中文3篇;内容重复性的研究英文8篇,中文7篇;最后保留其中50篇符合要求的中英文文献做进一步研究。其中文献[1-13]为骨关节炎机制的研究,文献[14-16]研究了软骨细胞的形态学特征,文献[17-20]研究了骨髓间充质干细胞的近期研究状况,文献[21-37]研究了骨髓间充质干细胞对软骨细胞的作用,文献[38-50]研究了骨髓间充质干细胞影响软骨细胞再生的可能机制。 3 骨关节炎中关节软骨损伤机制 Articular cartilage injury mechanism in osteoarthritis 3.1 骨关节炎时的软骨损伤 骨性关节炎最主要表现,是关节软骨的改变。正常关节软骨,含有大量的水合性软骨细胞外基质,它由少数特殊的软骨细胞群合成并加以保持。在成人,软骨细胞占整个软骨体积的2%以下。软骨基质主要由胶原和蛋白聚糖组成,蛋白聚糖主要以软骨聚集蛋白聚糖形式存在,它的分子巨大且与透明质酸聚合在一起[14]。关节软骨的Ⅱ、Ⅸ、Ⅺ型胶原链接形成一个纤维网,构成了纤维软骨基质的结构支架,纤维方向各异、排列不同的纤维网被蛋白聚糖聚集体的高浓缩溶液所包绕。蛋白聚糖,是一种蛋白核心与糖胺聚糖链相连的复合性巨型分子。关节软骨的糖胺聚糖,主要是硫酸软骨素和硫酸角质蛋白。这些糖胺聚糖在溶液中带负电荷,起到关节软骨组织水合和产生巨大膨胀压力的作用。在关节软骨组织内也有许多其他更小的蛋白聚糖分子,其中的一些分子可以直接与胶原纤维链接[14]。 骨关节炎所观察到的关节软骨丧失,开始为一种局灶性改变,例如老年骨关节炎患者,由于负荷产生的变化导致关节面改变,逐渐扩展累及一些特定的关节间隔。关节软骨退变,首先看到的是关节面的纤维化。裂缝大多与关节面平行,然后穿透损伤的关节软骨,最后抵达软骨下骨。早期可见关节的关节软骨细胞出现细胞克隆,不过已被证实这种现象的位置比较表浅。继而从表面开始,关节软骨厚度逐渐减少。骨关节炎发展到远期,还可见到骨、韧带和滑膜的变化。关节软骨除胶原减少外可见软骨聚集蛋白增加、软骨基质合成加快的软骨肥厚性表现。其后处于软骨基质循环加快,只消耗重要成分软骨基质的状态。最后可观察到关节软骨严重损坏,胶原网消失[15-16]。 3.2 骨髓间充质干细胞的研究现状 骨髓间充质干细胞是一种多能成体干细胞,具有多向分化及自我更新的潜能,还具有明显的可塑性,且获取简便,易于体外培养、扩增。研究表明,骨髓间充质干细胞可体外被诱导分化为成骨细胞、脂肪细胞、肌细胞、神经细胞、上皮细胞等,已经被认为是组织工程及再生医学领域中具有广泛运用前景的种子细胞。许多体内研究表明骨髓间充质干细胞可迁移到损伤处,参与损伤组织的修复。Sasaki等[17]学者通过体内实验研究发现骨髓间充质干细胞通过分化为各种皮肤细胞修复皮肤缺损;Beeres等[18]利用骨髓间充质干细胞成功修复心肌细胞传导阻滞;Giannoni等[19]发现骨髓间充质干细胞可以再生修复山羊长骨缺损;Aung等发现将骨关节炎患者的原代软骨细胞与骨髓间充质干细胞共培养,可以诱导骨髓间充质干细胞向软骨细胞分化,同时抑制了骨髓间充质干细胞的肥大。干细胞修复组织缺损方面的研究已经取得了丰硕的成果,并积累了充足的经验。现阶段有学者提出采用软骨细胞和骨髓间充质干细胞共培养[20],既利于成熟软骨细胞形成的微环境、分泌的细胞因子诱导骨髓间充质干细胞向软骨细胞分化,同时骨髓间充质干细胞分泌的细胞因子促进细胞增殖和合成基质,防治体外软骨细胞去分化。 3.3 骨髓间充质干细胞对退变软骨细胞作用及途径的研究进展 运用骨髓间充质干细胞治疗骨关节炎,就是通过采用软骨细胞和骨髓间充质干细胞共培养,来促进退变软骨细胞的恢复和新生软骨细胞的生成。共培养有两方面好处。一方面利于成熟软骨细胞形成的微环境、分泌的细胞因子诱导骨髓间充质干细胞向软骨细胞分化,另一方面骨髓间充质干细胞分泌的细胞因子又促进了细胞增殖和合成基质,以此来防治体外软骨细胞去分化。 在Rollin等[21]的文章中,阐述了“骨关节炎患者的骨髓间充质干细胞比正常人更多地表达转化生长因子β,转化生长因子β和转化生长因子的受体”其利用蛋白质组技术分析后,还发现骨关节炎患者比骨质疏松患者的骨髓间充质干细胞对趋化因子的反应更强,这表明骨关节炎患者的退变软骨可以拥有更多的趋化信号,以此来增加骨髓间充质干细胞的增殖分化,而这正是缺损组织的修复所需要的。 但是,也有不同观点。Murphy等[22]发现,来源于严重骨关节炎患者的骨髓间充质干细胞的成脂分化能力和成软骨分化能力,正常人明显下降。而在Dudics等[23]的研究中,发现骨关节炎和类风湿关节炎患者的骨髓间充质干细胞成软骨分化能力与正常人无明显差异。 有学者对骨髓间充质干细胞的体外向软骨细胞分化能力进行了研究。运用体外软骨诱导单层培养,诱导分化为软骨细胞。Zhen等[24]利用兔的骨髓间充质干细胞,将其与转化生长因子β1,地塞米松共培养,得到Ⅱ型胶原和x型胶原。Kasemkijwattana等[25]利用人体髌下脂肪垫的骨髓间充质干细胞与软骨诱导培养物共培养,2周后得到可以分泌蛋白聚糖、多功能蛋白聚糖和胶原Ⅱ、Ⅸ、Ⅹ、Ⅺ的软骨细胞。Hegewaid等[26]利用马的骨髓间充质干细胞,将其与转化生长因子β1,透明质酸、自体滑膜液共培养。成功诱导为软骨细胞。Hagmann等[27]的文献中也提到转化生长因子β3与胰岛素样生长因子1一起可以诱导骨髓间充质干细胞向软骨分化。Wan等[28]用人骨髓间充质干细胞经离心使细胞聚集,与地塞米松和转化生长因子β1共培养,同样使骨髓间充质干细胞分化成软骨细胞获得成功。Davatchi等[29]在上述体系中又添加了骨形成蛋白6或骨形成蛋白2,结果更加增强了对软骨细胞的诱导。这些实验提示我们,由骨髓间充质干细胞向软骨细胞分化是一个复杂的多因子参与的联合反应[30]。 关于动物的体内实验,文献中的实验均采用了兔作为实验对象。国外学者Madry等[31]将胰岛素样生长因子1和成纤维细胞性长因子2用质粒转染到NIH 3T3细胞中,再将转然后细胞植入兔股骨滑车凹陷骨软骨缺损处,3周后实验结果显示此基因加速了软骨的修复。Mason等[32]将骨形成蛋白7用病毒转染到兔骨髓间充质干细胞,再将转然后细胞植入到兔膝关节软骨缺损处,8周后,实验结果显示,有新的骨和软骨再生。Katayama等[33]将软骨形态发生蛋白1通过脂质转染到骨髓间充质干细胞,再将细胞移植到兔膝关节软骨缺损处,结果缺损处被透明软骨填充,同时深部区域有重建的软骨下骨。国内也有学者做了类似实验,霍建忠等[34]将转化生长因子β1用质粒转染骨髓间充质干细胞,再将转染后细胞植入兔关节软骨缺损处,12周后与正常软骨十分相近的透明软骨组织覆盖了缺损软骨。郭晓东等[35]把将转化生长因子β1用质粒转染至骨髓间充质干细胞再将转染后细胞植入兔关节软骨缺损处,同样得到缺损处生成新生透明软骨的结论。 关于人体在体实验,相关文献报道的比较少,Christopher[36]利用患有膝关节病变患者的髂嵴的骨髓间充质干细胞,采用经皮移植,经过24周的治疗后,可观察到患者关节运动情况明显改善,软骨和半月板显著生长,改进的目测类比VAS疼痛评分有所降低。Wakitani等[37]利用患者骨髓的骨髓间充质干细胞,在混合胶原凝胶后,植入到膝关节软骨缺损处,经过42周的治疗后,观察到软骨缺损处,有透明质酸软骨样组织生成,其组织学评分和关节镜检查也有明显改善。 3.4 骨髓间充质干细胞防治骨关节炎可能机制的研究进展 骨髓间充质干细胞是一种具有高度自我更新能力和多向分化潜能的多能干细胞,广泛存在于人体多种组织中,其具有多向分化、低免疫原性和免疫调节功能,在免疫调节、减轻炎症反应、损伤修复方面表现出广阔的应用前景,并在相关领域取得较大进展[38-39]。 现有实验数据的结果均提示骨髓间充质干细胞对软骨细胞的再生有促进作用,那么,其防治骨关节炎的可能机制是什么呢?相关研究提示骨关节炎的病变基础为骨相关性的,关节软骨改变是继发性的,其病理显示,骨关节炎会发生包括骨赘形成,软骨下骨硬化,部分患者最终进展为局灶软骨下骨坏死[40-41]。骨关节炎从多方面与骨缺损处的增殖有关,同时伴随骨量及骨矿物质密度的改变。骨结构的这种改变提示,骨髓中的软骨先导细胞对于正常软骨的维持有重要作用。再者,研究发现,由软骨细胞合成的聚集蛋白聚糖其与年龄因素负相关,其吸收入聚合体的速度随年龄增长而减慢[42]。这提示骨关节炎的发生与软骨细胞功能的改变也有关。而且,骨关节炎的发展常与严重的炎症并行。运用骨髓间充质干细胞防治骨关节炎,不仅能在结构上影响组织修复,同时可以进行有效的免疫修饰及抗炎[43]。 骨髓间充质干细胞的较强软骨分化能力,和其原材料的易获得性,使其广泛应用于软骨组织工程修复成为可能。骨髓间充质干细胞的多向分化潜能,已经在多重肌肉骨骼组织和受伤部位的嫁接中取得了良好的效果。其分泌的多种生物活性分子[44-45],用于激活内源性前体细胞,且对机体进行免疫调节,对局部缺损组织进行修复。形成了防治骨关节炎发展的完整链条。鉴于骨髓间充质干细胞的这些特性,使其在骨关节炎的治疗上成为非常有前景的研究方向。国内学者许键炜等[46]通过动物实验验证了骨髓间充质干细胞对关节炎的干预和治疗,实验收集了培养扩增SD大鼠骨髓间充质干细胞造模之后同体移植治疗,结果模型大鼠经骨髓间充质干细胞治疗后关节肿胀减轻,X射线片示骨质破坏明显修复。病理苏木精-伊红染色见关节滑膜病变减轻。干预组经干细胞治疗后,原发性炎症和继发病变均不明显,关节X射线片和病理检查均未见明显异常,与模型组大鼠相比,体质量、足爪体积以及踝关节周长差异无显著性意义。可见骨髓间充质干细胞治疗可有效减轻并逆转弗氏完全佐剂诱导的大鼠关节炎的进展。 国外一些专家也在尝试做了一些临床试验,Punwar等[47]利用一个运动员股骨髁的自体骨髓间充质干细胞治疗大关节软骨全层缺损。手术前4周骨髓穿刺抽取病人的髂骨细胞,扩大培养后去除红细胞,然后嵌入胶原凝胶,将其转移到关节软骨缺损处,最后用自体骨膜瓣内覆盖。7个月后手术,关节镜检查发现缺陷处覆盖着透明样软骨样组织。手术后1年,患者临床症状明显改善,患者已经回到了他以前的活动水平。Hamoud等[48]还对5例膝关节软骨缺损患者进行了试验,将骨髓间充质干细胞移植入富含血小板纤维蛋白胶(PR-FG)的支架上,对患者进行疗效跟踪,其结果是所有患者的症状和膝关节评分均有改善。"
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