猴骨髓间充质干细胞的分离及原代培养特性的研究

目的：探讨猴骨髓间充质干细胞(MSCs)低分化干细胞方面的特性。方法：以Percoll(质量密度1073g/L)非连续密度梯度离心分离出骨髓悬液中的骨髓间充质干细胞，用含100mL/L胎牛血清的DMEM培养液培养，观察细脑的形态、生长状况及其超微结构。结果：原代培养的骨横间充质干细胞增殖缓侵，细胞增殖率和形态分化不均一。如有一些细脑在4wk的培养时间内末见明显增殖，这些增殖不明显的细胞有的未见明显的伸展，呈圆形，有的伸展充分，成片状；在培养2wk后也可见一些明显增殖的细胞，分别形成克隆团状或条状的增生细胞群；培养过程中可见有神经元样及多核细胞出现。原代培养的骨髓间充质干细胞的超微结构也显示了低分化的干细胞特点，如细胞表面具有微绒毛，脑浆内内质网丰富、激离核糖体数量多、线粒体小，核仁大而不规则、可见核裂，超微结构还显示了一些中胚层来源于细瘤的结构特点，如大量的微丝。结论：经Percoll细胞分离介质分离出的骨髓间充质干细胞，在非诱导条件下原代培养过程中，从形态分化、增殖率等方面显示了其多向分化潜能；从超微结构等方面显示了其中胚层来源低分化细胞的特点。这些特点可作为骨髓间充质干细胞鉴定的佐证之一，说明Percoll非连续密度梯度离心法对骨髓间充质干细胞的分离具有可靠性。

Isolation of monkey marrow mesenchymal stem cells and biological features of the cells during primary culture

Objective:To investigate the low differentiation features of monkey marrow mesenchymal stem cells(MSCs).Methods:MSCs were isolated from marrow suspension by discontinuous gradient centrifugation on Percoll(density:1073g/L)and primarily cultured on the DMEM medium containing 100ml/L fetal bovne serum The growth and morphological character were evaluated.Results:With the heterogeneity of proliferation rate and morphological differentiation,the growth of primarily cultured MSCs was slow.For instance,no significant proliferation was observed in some MSCs during four-weeks culture,of which some were round and un-spread but some were lamellar and well-spread.However,there was significant proliferation in some other MSCs after two-weeks culture.These cells grew into cell polarity,which looked like a clone or a band.Neuron-like cells and multinucleated cells could be found during culture.The ultrastructure of primarily cultured MSCs,such as the microvilli,the abundant endoplasmic reticulum and free ribosome,small mitochondria,the irregular large cell nucleus,and the karyorrhexis,showed the structure characteristics of low differentiated stem cells.Such ultrastructure as microfilament also showed the structure characteristics of mesoderm stem cells.Conclusion:In the course of primarily non-conductive culture,the heterogeneity of proliferation rate and morphological differentiation showed the multi-differentiation potential of the Percoll-isolated MSCs,what's more,the ultrastructure showed the characteristics of the mesoderm stem cells.That is some of the supportng to identify the MSCs.It indicates that the discontinuous gradient centrifugation on Percoll should be a reliable way to isolate MSCs.