脐血造血干/祖细胞与脂肪干细胞共培养的作用距离研究

Effective Action Distance of Co-Culture between Umbilical Cord Blood-Derived Hematopoietic Stem/Progenitor Cells and Human Adipose Derived Stem Cells

设计了一种细胞间距可调的trans well共培养方法，以研究脐带血来源的造血干／祖细胞（HS／PCs）和人脂肪干细胞（human-adipose derived stem cells，h-ADSCs）体外共培养时细胞间作用距离对造血干细胞扩增能力和脂肪干细胞在共培养后干细胞特性的影响。采用不同规格的砂纸打磨孔板的上壁面，经高精度游标卡尺测量，使两种细胞之间的有效接触间距为10--450μm不等。然后以h-ADSCs为基质细胞，分别考察HS／PCs和h-ADSCs在不同的作用距离下的共培养情况。其间每24小时对细胞进行计数，观察细胞形态。培养7天后对MNCs表面抗原CD34＋、CFU-GM集落扩增倍数进行了检测；同时也对h．ADSCs表面抗原（CD13、CD29、CD34、CD44、CIM5、CD73、CD105、CD166、HLA-DR）及其多向分化潜能（成骨、成软骨、成脂肪）进行分析以鉴定其干细胞性能。结果表明，通过transwell共培养，细胞之间在作用距离为350gm时HS／PCs的扩增效果最好，其中造血MNCs扩增了15．1±0．2倍，CD34＋扩增了5．0±0．1倍：扩增的h-ADSCs表达CD29、CD44、CD166，而不表达CD34、CD45，且在适当诱导条件下可以向成骨细胞、软骨细胞、脂肪细胞分化。

In order to investigate the effects of in vitro co-culture distance between umbilical cord blood-derived hematopoietic stem/progenitor cells （HS/PCs） and human adipose derived stem cells （h-ADSCs） on the expansion of HS/PCs and the stem cell properties of h-ADSCs, a novel transwell co-culture protocol in which the distance between the two culture chambers could be adjusted was designed. Sand papers with different specifications were utilized to adjust the cellular action distance between two kinds of cells mentioned above in the range of 10 to 450 μm which was measured by a high-precision vernier caliper. Then the HS/PCs were cultured in the modified transwell supported by ADSCs for 7 days. The total cell number was counted by using a hemacytometer and the cell morphology was observed by an inverted microscope every day. After 7 days of co-culture, the expansion fold of surface antigen CD34~ and CUF-GM of the hematopoietic mononuclear cells （MNCs） were analyzed. Meanwhile, the surface markers （CD 13, CD29, CD34, CD44, CD45, CD73, CD105, CD166 and HLA-DR） and the multi-differentiation potential （adipogenic, osteogenic and chondrogenic） of ADSCs were also assayed to identify the sternness of expanded stromal cells. The results show that there exists an optimal communication distance （around 350 ~tm） between these two stem cells during their co-culture. At this distance, the expansion folds of the MNCs and CD34~ cells achieve 15.14-0.2 and 5.0 4-0.1 fold, respectively. It was found that the expanded ADSCs still show positive expression of CD29, CD44, CD166 and negative expression of CD34, CD45, and could also differentiate into osteoblasts, adipocytes and chondrocytes after respective inductions.