中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (10): 1833-1837.doi: 10.3969/j.issn.1673-8225.2010.10.026

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

人胚胎滋养细胞和胎盘间充质干细胞的分离与纯化

沙文琼，王自能，王冬菊   

  1. 暨南大学附属第一医院妇产科，广东省广州市 510632
  • 出版日期:2010-03-05 发布日期:2010-03-05
  • 通讯作者: 王自能，博士，教授，暨南大学附属第一医院妇产科，广东省广州市 510632 twangzineng@ jnu.edu.cn
  • 作者简介:沙文琼，女，1980年生，河北省秦皇岛市人，回族，暨南大学附属第一医院在读博士，主要从事妊娠高血压疾病方面的研究。 swq1980@gmail.com

Isolation and purification of human cytotrophoblasts and placental mesenchymal stem cells

Sha Wen-qiong, Wang Zi-neng, Wang Dong-ju   

  1. Department of Gynaecology and Obstetrics, First Hospital of Jinan University, Guangzhou   510632, Guangdong Province, China
  • Online:2010-03-05 Published:2010-03-05
  • Contact: Wang Zi-neng, Doctor, Professor, Department of Gynaecology and Obstetrics, First Hospital of Jinan University, Guangzhou 510632, Guangdong Province, China twangzineng@jnu. edu.cn
  • About author:Sha Wen-qiong, Studying for doctorate, Department of Gynaecology and Obstetrics, First Hospital of Jinan University, Guangzhou 510632, Guangdong Province, China swq1980@gmail.com

PDF

650

被引次数

11

摘要：

背景：胎盘的细胞成分较复杂，其中所包含的滋养细胞在母胎免疫耐受过程中起重要作用，胎盘间充质干细胞具有多向分化潜能以及抑制淋巴细胞增殖的特性，常规分离方法通常难以获得大量且纯度较高的上述两种细胞。

目的：拟建立一次操作即可同时获得大量、较高纯度的滋养细胞和胎盘间充质干细胞的操作方案。

方法：人胎盘组织洗净剪碎，采用胰蛋白酶和DNAse Ⅰ共同消化，分3个阶段，每个阶段在恒温37 ℃下180 r/min消化2 min。重悬消化产物，200目筛网过滤后采用Percoll密度梯度分离液分离，分别收集滋养细胞层和胎盘间充质干细胞层。滋养细胞层以差速贴壁法去除成纤维细胞，胎盘间充质干细胞直接接种于75 cm2培养瓶中培养。观察胎盘组织消化情况，计数滋养细胞数量及其细胞角蛋白7的表达，观察胎盘间充质干细胞生长增殖、表型及成骨分化潜能。

结果与结论：经胰蛋白酶和DNAse Ⅰ消化后，胎盘组织仅剩少许残渣。差速贴壁后，滋养细胞数达(5.48±1.98)×108个，细胞角蛋白7阳性率为(90±4.36)%。胎盘间充质干细胞接种19~21 d达90%融合，细胞数为(1.96±0.24)×106个，强表达CD29，CD44和HLA-ABC，不表达CD34，CD45，CD14和HLA-DR，诱导后茜素红染色呈鲜艳橙红色，可向成骨细胞方向分化。提示采用胰蛋白酶和DNAseⅠ共同消化胎盘组织，并结合Percoll不连续密度梯度分离液分离细胞，可同时一次性获得大量的滋养细胞和较多的胎盘间充质干细胞，细胞纯度和活性均较好。

关键词: 胎盘组织, 滋养细胞, 胰蛋白酶, DNAseⅠ, Percoll, 纯度, 胎盘间充质干细胞

Abstract:

BACKGROUND: Cytotrophoblasts in placental cell components plays an important role in fetal immunological tolerance. Placental mesenchymal stem cells (pMSCs) have potential of multiple differentiation and inhibition of lymphocyte proliferation. However, conventional methods cannot acquire a large amount of purified human cytotrophoblasts or pMSCs.

OBJECTIVE: To establish a method to obtain large placenta tissue, and harvest plenty of cytotrophoblasts and pMSCs with high purity and activity.

METHODS: Human placenta tissues were dissected, minced, and dissociated in trypsin and DNAse I. The dissociation was performed in three stages of incubation at 180 r/min for 20 minutes at 37 ℃. The digesting suspension was filtered using a 200 mesh strainer before separated by Percoll gradients. The cytotrophoblast cells and pMSCs fractions were collected respectively. Fibroblasts of cytotrophoblast cells fraction were removed by differential adhesion. The pMSCs were seeds on 75-cm2 flask directly for culture. The dissociation of placenta tissue was observed. The number of harvesting cytotrophoblasts was quantified and Cytokeratin 7 expression was tested. The pMSCs primary culture time, cell passage, induced osteoblast differentiation were observed. The cell surface makers were also detected.

RESULTS AND CONCLUSION: After digesting in trypsin and DNAse I, there was only little residue left. (5.48±1.98)×108 cytotrophoblasts were obtained after differential adhesion. (90±4.36)% of these cells were positive for Cytokeratin 7. At 19-21 days after pMSCs reached approximately 90% confluency, the cell number was (1.96±0.24)×106. The subcultre cells could be passaged again in 4 or 5 days. Flow cytometric analysis of pMSCs showed that the cells expressed CD29, CD44 and HLA-ABC intensively and were negative for CD34, CD45, CD14 and HLA-DR. pMSCs differentiated into osteoblast-like cells after induction, which stained bright salmon pink by Alizarin Red. Dissociating the placenta tissue in trypsin and DNAse Ⅰ in combination with discontinuous Percoll gradient separation obtained a large number of cytotrophoblasts and pMSCs recovered from placenta tissue, with high purity and activity.

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