Chinese Journal of Tissue Engineering Research ›› 2013, Vol. 17 ›› Issue (32): 5877-5882.doi: 10.3969/j.issn.2095-4344.2013.32.020
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Chen Ming-xing, Ouyang Gui-fang
Received:
2012-10-27
Revised:
2012-12-18
Online:
2013-08-06
Published:
2013-08-06
Contact:
Ouyang Gui-fang, Master’s supervisor, Chief physician, Medical School of Ningbo University, Ningbo 315000, Zhejiang Province, China
ouyangguifang@medmail.com.cn
About author:
Chen Ming-xing★, Master, Medical School of Ningbo University, Ningbo 315000, Zhejiang Province, China
cmxcmx2008@yahoo.com.cn
Supported by:
the Natural Science Foundation of Ningbo City, No. 2010C50019*
CLC Number:
Chen Ming-xing, Ouyang Gui-fang. Differentiation potential of umbilical cord blood-derived mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(32): 5877-5882.
2.1 脐血来源间充质干细胞的分离培养及其生物学特性 脐血的采集:选择健康产妇,胎儿娩出后,在距胎儿5-7 cm的脐带处进行结扎,剪断脐带,穿刺脐静脉,用含抗凝保护液的采血袋抽取50 mL左右的脐血,采集后对脐血进行生物学检测、细菌污染检查、血型及HLA分型检测、血清学病毒检查以及遗传病检测。 4 ℃冰箱保存,4 h内处理标本。 脐血间充质干细胞的分离:脐血间充质干细胞的研究开展较晚,直到1999年,Erices等从脐血中分离培养出间充质干细胞后,相继有学者也分别从脐血中成功分离出间充质干细胞并对之进行鉴定。但培养成功率较低,可能与间充质干细胞在脐血中的出现频率低相关。 细胞形态呈均一的长梭形,具有贴壁生长的特点;适当诱导培养条件下,具有谱系内多向分化的特点和跨系分化的潜能;表达CD13、CD29、CD44、CD54、CD58、CD90、CD95、CD105、CD166等免疫表型,不表达CD14、CD34、CD40、CD45、CD50、CD68、CD80、CD86、CD117和CD152等免疫表型,强阳性表达抗原标记SH-2、SH-3、SH-4(人间充质干细胞特异性抗体),同时表达HLA-ABC等抗原和胶原Ⅰ,不表达HLA-DR抗原、共刺激分子B7-1(CD80)、B7-2(CD86)、CD40/ CD40L以及胶原Ⅱ、Ⅲ[9-10];细胞多处于G0/G1期,具有良好的增殖分化能力,简便易行的分离、纯化程序,而且能分泌一些造血细胞因子,具有促进造血的作用[11];作为一种原始的免疫缺陷细胞,具有良好的免疫耐受性[9],在体外培养既不能刺激也不能抑制淋巴细胞反应[12],同时具有非常好的免疫节作用,能预防移植物抗宿主病,其在造血干细胞移植中显示出了其优越性[13-14]。 2.2 脐血来源充质干细胞的分化潜能 2.2.1 脐血来源间充质干细胞的成骨分化潜能 成骨分化潜能:脐血来源间充质干细胞在含有体积分数为10%的胎牛血清+10 mmol/L β-甘油磷酸+ 10-7 mol/L地塞米松和0.2 mmol/L维生素的成骨诱导培养基培养下能发生成骨分化并表达碱性磷酸酶、Ⅰ型胶原、骨黏连蛋白、Cbfa-1和骨钙蛋白,同时高度表达成骨标记骨桥蛋白。尽管低浓度的细胞外基质层黏蛋白、纤连蛋白、胶原促进细胞贴壁和细胞生长,但是这些条件并不是成骨祖细胞生长的充分条件。 骨桥蛋白常常用来证实成骨发生的状态,它在骨中高表达;然而它同样出现在其他非骨组织中[15]。在一系列基因表达分析骨髓间充质干细胞中钙粘连蛋白mRNA水平非常高,非诱导骨髓间充质干细胞和脐血来源间充质干细胞能显著地表达糖蛋白。基质钙沉淀是成骨分化过程中的最新阶段,钙粘连蛋白称为终端分化的一个标记物[16]。Plant和Tobias等研究了成骨细胞分化,在20-24 d诱导分化后观察到了骨钙蛋白和骨黏连蛋白中等水平的表达结果。虽然碱性磷酸酶好像是一种成骨发生很好的标记,但是却存在着许多争议,Chang等[17]认为碱性磷酸酶在骨诱导下能显著增加。目前有关研究证实诱导间充质干细胞分化为成成骨系的基因是非常复杂的[18],仍然需要做进一步研究。 脐血来源间充质干细胞成骨分化时的影响因素:目前脐血间充质干细胞的培养主要建立在骨髓源间充质干细胞培养的经验上。而关于诱导分化条件的信息还是很匮乏。在脐血间充质干细胞的分化过程中,培养基的条件对其有重大影响。Hildebrandt等[19]用3种不同成分的培养基(alpha MEM+体积分数为15%胎牛血清,DMEM+体积分数为15%胎牛血清,MSCGM+10%SingleQuot),同时用两种不同浓度的地塞米松对脐血间充质干细胞培养。结果发现MSCGM+10%SingQuot能促进成骨分化;细胞外基质钙化沉淀只出现在含有10-8 mol/L地塞米松+骨形态发生蛋白2的培养基里;地塞米松浓度为10-7 mol/L能产生更高浓度的细胞外基质和在加有骨形态发生蛋白2时脐血源间充质干细胞独立表达Ⅰ型胶原产物。根据以上实验数据表明地塞米松是成骨分化诱导因子,而骨形态发生蛋白2有支持脐血间充质干细胞的作用,MSCGM+10%SingQuot+10-7 mol/L地塞米松是诱导成骨分化最好的培养条件。有人研究表明成骨诱导培养基只有加了维生素D或者维生素D+成纤维细胞生长因子9之后骨钙蛋白标记物才会明显增多[20]。此外通过碱性磷酸酶活性染色证实了瘦蛋白能加强成骨分化的能力[21],且CD105对下调脐血间充质干细胞成骨分化有非常密切的关系。有证据表明磷酸甘油酸酯变位酶、热休克蛋白27等在脐血间充质干细胞成骨分化中是上调的[22],在体外成骨分化过程中,白细胞介素6、白细胞介素9、白细胞介素10、白细胞介素12、成纤维生长因子的含量是比较多的,随着进一步分化这些因子会减少。尤其在成骨分化过程中白细胞介素6水平的高低对成骨分化是有影响的,据报道白细胞介素6能促进骨吸收和破骨形成[23-24]。Mekala等[25]研究发现维生素C能促进脐血间充质干细胞的成骨分化,维生素的浓度与脐血间充质干细胞的增殖和分化起着至关重要作用。Liu 等[26]证实了部分脱矿物质骨基质能促进脐血间充质干细胞的成骨分化。 2.2.2 脐血来源间充质干细胞的脂肪分化潜能 脂肪分化潜能:Goodwin等[27]和Lee等[28]发现脐血间充质干细胞在脂肪诱导培养基条件下可以分化成脂肪细胞,见图3。并证明脐血中确实含有能分化成脂肪细胞的间充质干细胞。这些脂肪细胞表达过氧化物酶增殖激活受体Gamma 2(PPAR Gamma 2)、低密度脂蛋白和脂肪酸结合蛋白[1],同时能表达脂肪酸相关蛋白4(FABP-4)[29]。然而Kern等[30]研究当中未见脐血间充质干细胞发生脂肪分化,而其成骨分化、软骨分化与报道一致。一直以来研究者报道脐血间充质干细胞的脂肪分化结果不一致,其脂肪分化需要进一步研究。"
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