人羊膜间充质干细胞体外传代培养对染色体核型的影响

The effects of human amniotic mesenchymal stem cells in vitro culture on chromosome karyotype

目的:分离培养人羊膜间充质干细胞(hAMSCs)并进行体外长期传代培养,通过对连续传代的细胞形态、膜表面标志、增殖、分化特性以及染色体核型进行研究,以观察体外培养对该细胞的影响,为其储备和临床应用提供实验依据。方法:无菌条件下从新鲜人胎盘组织分离羊膜,采用组织贴壁法分离培养hAMSCs,并对原代细胞～P11代细胞利用流式细胞技术检测其膜表面标志物;取P3～P4代细胞向神经细胞和成骨细胞诱导,免疫组化染色、PCR检测基因表达情况;采用染色体G显带法制备染色体核型。结果:hAMSCs形态似长梭型,呈流水状生长,细胞活性高,冻存复苏后仍能保持正常形态,并可在体外稳定扩增,细胞生长曲线呈"S"型;细胞高表达CD90、CD73、CD44、CD105;细胞经诱导后可以向神经细胞和成骨细胞分化,神经元特异性烯醇化酶和成骨细胞矿化结节检测为阳性,PCR检测巢蛋白和骨桥蛋白基因表达为阳性;P3～P10代的hAMSCs染色体核型均为正常二倍体核型。结论:hAMSCs在体外经数代培养后能保持间充质干细胞的外显和遗传特性,并且未见染色体核型异常改变。

Objective：After isolated and established human amniotic mesenchymal stem cells（hAMSCs）,and cultured for long-term in vitro,then to study in vitro subculture influence on hAMSCs by observing morphology of hAMSCs,the membrane surface marks,proliferation and differentiation,and its chromosome karyotype.And to provide the experimental evidences for reserving and clinical application of hAMSCs.Methods：The hAMSCs were isolated from human placenta tissues through tissue attachment culture methods.The immunophenotypes were detected by flow cytometry.The cells were induced to differentiate into nerves and osteoblasts by different culture system.The osteopontin and nestin genes were measured by PCR and Karyotype analysis was carried out with chromosome Giemsa banding method.Results：hAMSCs were dominated as long spindle cells and had grown in water shaped with high cell activity.After cryopreservation,hAMSCs could be recovered normal morphology,proliferated stably and showed typical＇S＇curve of propagation.Positive expression of CD90,CD73,CD44 and CD105in hAMSCs were all high.After induction,hAMSCs could be differentiated into the nerve cell and osteoblast.The cells had positive of alizarin red staining and neuron specific enolase.Nestin and osteopontin genes were also positive in these cells.The chromosome karyotype of hAMSCs kept normal dipoid karyotype after P3 to P10in vitro culture.Conclusion：After several generations in vitro subculture,hAMSCs can keep explicit features,genetic characteristics of mesenchymal stem cells and has no any change in chromosome karyotype.