Chinese Journal of Tissue Engineering Research ›› 2013, Vol. 17 ›› Issue (19): 3546-3550.doi: 10.3969/j.issn.2095-4344.2013.19.020
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Ju Ya-ping, Zhao Qing-hua
Received:
2012-06-25
Revised:
2012-07-27
Online:
2013-05-07
Published:
2013-05-07
Contact:
Zhao Qing-hua, M.D., Attending physician, Department of Orthopedics, Shanghai First People’s Hospital, Shanghai Jiao Tong University, Shanghai 200080, China
About author:
Ju Ya-ping, Department of Orthopedics, Shanghai First People’s Hospital, Shanghai Jiao Tong University, Shanghai 200080, China
juyaping1992@hotmail.com
CLC Number:
Ju Ya-ping, Zhao Qing-hua. Comparison of mesenchymal stem cells from three different perinatal tissues[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(19): 3546-3550.
2.1 将围产期组织用于细胞治疗的条件与背景 人类胚胎干细胞起源于内细胞团。内细胞团有很强的分化潜能,可以多向分化为三胚层的所有细胞。然而现今,有3个主要因素限制了胚胎干细胞的临床应用。①首先是伦理问题。②其次,在移植后残留的多能胚胎干细胞会致使畸胎瘤的形成。③在移植之后胚胎干细胞可能会引起免疫反应。 相比之下,胎儿娩出后的妊娠期组织具有实足年龄小,基因突变率低,获取途径不会对人体造成伤害等优点。目前国内情况下,围产期的组织都在胎儿出生时被遗弃,因此,可以在对胎儿和母亲没有任何风险的情况下随时获得细胞。因此,围产期组织以其充足的供应,便捷的来源,以及最小程度的道德与法律的纷争成为了间充质干细胞的良好来源。 2.2 间充质干细胞的生物学特性 多种组织中已经被证实含有间充质干细胞,包括脂肪组织,皮肤组织,胚胎组织,血液等[1]。间充质干细胞有体外扩增潜能,更为重要的是有多向分化潜能。它不仅可以分化成中胚层细胞(包括骨母细胞,脂肪细胞,软骨细胞),还可以分化为内胚层细胞(肝细胞样细胞)和外胚层细胞(神经细胞,神经胶质细胞)。这种能力可能与间充质组织的发育起源相关。间充质干细胞移植后主要分化为3种细胞类型;组织特异性细胞:可以修复受损伤组织,如移植后的间充质干细胞可以分化为心肌细胞;功能性相关细胞:此类细胞可能参与构成组织修复所需的特殊微环境[2];调整类细胞:此类细胞通过分泌具有营养或免疫调整功能的细胞活素类物质来达到促进组织修复和再生的目的。 在细胞疗法的整个领域中,间充质干细胞都是一个中心参与者。间充质干细胞表面表达CD29, CD44, CD71, CD90, CD106 ,CD120a 分子以及若干参与T淋巴细胞免疫的黏附分子如VCAM-1/CAM-1和LFA3;不表达CD14,CD34,CD45等造血细胞标志以及MHCⅡ类分子,FasL和B7-1、B7-2、CD40、CD40L。 间充质干细胞可产生大量的生长因子、细胞因子、趋化因子和蛋白酶,参与其免疫调节和迁移[3]。间充质干细胞具有趋化因子受体,可以迁移到特异性的趋化触发位点,因此间充质干细胞可以定位于多个器官,进行特异性组织分化[4]。此外,间充质干细胞可以通过细胞之间的相互作用或释放大量可溶性生长因子的方式来影响与它相邻的细胞,这些因子包括基质细胞衍生因子1,肝细胞生长因子,胰岛素样生长因子1,血管内皮生长因子,白细胞介素1,白细胞介素6 [5-6]。正由于这种局部细胞环境的构建能力,由间充质干细胞形成的基质层可以支撑并长时期保存克隆形成的细胞群。不仅如此,间充质干细胞也明显受到微环境中细胞因子的影响。间充质干细胞具有基本的免疫调节功能,它可以抑制NK细胞增殖以及其细胞毒作用,并通过释放前列腺素E2等可溶性分子抑制CD4+T细胞功能,从而导致B细胞增殖受阻,抗体功能受损。此外还可以抑制CD8+T细胞的毒性,并对Treg细胞的分化起促进作用。并能影响树突状细胞的功能和肿瘤细胞的生长。 2.3 来自脐血的间充质干细胞 脐血中间充质干细胞的数量非常少,不易分离。数据表明,每份脐血70-110 mL,其中有核细胞(0.7-3.2)×108个,间充质干细胞有3.5-896.0个,数量太少,不易分离[7]。干细胞分离主要方法有密度梯度离心法,贴壁筛选法,流式细胞仪分离法和免疫磁珠分离法。后两种方法对细胞活性影响交大,甚至使细胞完全失活。现多提倡密度梯度离心法和贴壁筛选法结合使用。对于培养基以及血清浓度,目前还没有统一方案,但多数实验发现,原代培养脐血间充质干细胞多在培养24-48 h后贴壁,1周左右呈现梭形,并呈克隆性生长。一般传至第3代即可获得纯度高且形态均一的间充质干细胞。 关于分化潜能,脐血间充质干细胞在体外比骨髓间充质干细胞分化为骨的潜能更大。而用胚胎细胞微团培养并在生长因子的诱导下也可使脐血间充质干细胞分化为软骨。脐血间质干细胞和骨髓间质干细胞都能分化为软骨,但是分化成软骨的种类并不相同[8]。而关于脐血间质干细胞分化为脂肪组织能力的高低也有不同的研究结果[9-11]。 2.4 来自脐带中的间充质干细胞 脐带中间充质干细胞的来源主要有3种:①来源于华通胶质(Wharton’s jelly)。②来源于脐血管周围。③来源于脐静脉血管内皮下。由于华通胶质中间充质干细胞易于分离,因此实验中多先分离出华通胶质,而后从中分离间充质干细胞。分离主要采取的方法有:植块法,酶消化法。在原代细胞克隆,细胞产率,传代时间以及扩增倍数上,植块法明显优于后者。因此多采用植块法从华通胶质中分离间充质干细胞。研究表明,足月分娩的脐带间充质干细胞比早产脐带间充质干细胞有更强的繁殖扩增能力。培养7 d后局部形成集落生长,14 d左右即可传代。传代后7 d左右可再次传代。随着传代,细胞呈现相对均一的成纤维细胞样,平行排列或漩涡生长。 脐带间充质干细胞表达的表面分子与脐血间充质干细胞相似。均表达CD29,CD44,CD90,CD105和SH3,低表达MHCⅠ类分子标记(HLA-ABC)等,不表达造血干细胞标记如CD34、CD45、CD14、内皮细胞标记如CD33、CD133及MHCⅡ分子标记(HLA-DR、-DA、-DP、-DQ)等。同时脐带间充质细胞高表达一些转录因子,这些转录因子多为人胚胎干细胞所表达,如Oct-4、Sox-2、Nanog等[12-14]。在人胚胎干细胞中,上述分子是细胞自我更新和多向分化的主要调控分子。再结合脐带间充质干细胞的强增殖能力与它同时具有体外成骨和成神经的能力等因素,可以认为脐带间充质干细胞是介于胚胎干细胞和成体干细胞之间的一种较为原始的干细胞。 2.5 来自胎盘中的间充质干细胞 储存有相当数量的干细胞和前体细胞。而不同区域细胞种类亦不尽相同。然而这些细胞中可能会混有母亲的细胞。需要一种能够检测出小于所占数量比例1%的母体细胞的敏感的方法来证明这些细胞是来自胎儿的。分娩过后,将羊膜剥离,并通过胰蛋白酶和胶原酶去除上皮层后就可以得到羊膜间充质干细胞。对于绒毛膜而言,因母体部分已经被移除,经中性蛋白酶和胶原酶消化滋养层后,可得到绒毛膜干细胞。 与其他来源的间充质干细胞相同的是,胎盘间充质干细胞强表达SH2,SH3,CD29,CD44,CD73,CD90,CD105,CD166和HLA-ABC,不表达CD34,CD45,CD14,CD80,CD86,CD40L和HLA-DR。并同骨髓间充质干细胞一样,羊膜间充质干细胞和绒毛膜间充质干细胞具有成纤维性,可以黏附在塑料上。它们可以形成典型的群落,表现出向中胚层谱系分化的潜能并表达间充质干细胞特征性的标记分子。此外,这些细胞也表达一些多潜能标记如SSEA-4,TRA-1-61,TRA-1-80以及干细胞标志Oct-4,Nanog,Sox-2以及具有免疫调节作用的HLA-G。羊膜间充质干细胞和绒毛膜间充质干细胞的分化潜能略有不同。羊膜间充质干细胞更倾向于分化为脂肪细胞,而绒毛膜间充质干细胞倾向于分化成骨细胞,肌肉细胞,纤维细胞[15]。"
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