论文部分内容阅读
目的:分离纯化及体外定向诱导人卵黄囊间质干细胞(hYS -MSC)分化为成骨细胞及神经细胞。方法:卵黄囊细胞经贴壁培养、传代纯化得到hYS -MSC ,检测其表面抗原表达、对其进行核型分析、细胞周期检测并测定AKP活性;采用地塞米松、β-甘油磷酸钠、维生素C作成骨诱导剂、β-巯基乙醇或复方丹参注射液作为神经诱导剂诱导hYS -MSC向成骨细胞及神经细胞定向分化。组织化学方法作成骨检测;免疫组化方法检测NSE、NF及GFAP在经神经诱导hYS -MSC中的表达。结果:hYS -MSC易于纯化,在培养过程中保持正常核型,具有较大增殖能力。hYS -MSCCD2 9、CD4 4、CD16 6及CD10 5表达阳性,CD34、CD4 5和CD86为阴性;AKP弱阳性。hYS -MSC经成骨诱导AKP强阳性,诱导两周后形成钙盐沉积形成的矿化区。hYS -MSC经神经诱导可见NSE、NF或GFAP阳性细胞,符合神经元及胶质细胞的生物学特征。结论:hYS -MSC在体外培养过程中具有较大增殖能力并保持正常核型,与成体MSC表型一致,在体外可以诱导分化为成骨细胞、神经元及胶质细胞。
OBJECTIVE: To isolate, purify and differentiate human yolk sac mesenchymal stem cells (hYS-MSCs) into osteoblasts and neural cells in vitro. Methods: The yolk sac cells were cultured in adherent culture. The hYS-MSCs were subcultured and purified to detect the expression of surface antigens. Karyotype analysis, cell cycle detection and AKP activity assay were carried out. Dexamethasone, β-glycerophosphate, C as osteoinductive agents, β-mercaptoethanol or compound Salvia miltiorrhiza injection as a neural induction agent to induce hYS-MSCs to differentiate into osteoblasts and neurons. Histochemistry was used to detect bone formation. Immunohistochemistry was used to detect the expression of NSE, NF and GFAP in neural-induced hYS-MSCs. Results: hYS-MSC was easy to purify and maintained normal karyotype during culture. It had a large proliferative capacity. hYS -MSCCD2 9, CD4 4, CD16 6 and CD10 5 were positive, while CD34, CD4 5 and CD86 were negative; AKP was weakly positive. hYS-MSCs were strongly positive for osteoinductive AKP and formed a mineralized zone formed by calcium salt deposition two weeks later. NSE, NF or GFAP-positive cells were induced by hYS-MSCs, which was in line with the biological characteristics of neurons and glial cells. CONCLUSION: hYS-MSCs have a large proliferative capacity and maintain normal karyotype during in vitro culture, which is consistent with the phenotype of adult MSCs. In vitro, hYS-MSCs can differentiate into osteoblasts, neurons and glial cells.