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目的:检测人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUCMSCs)在与大鼠胚胎中脑细胞共培养环境中酪氨酸羟化酶(tyrosine hydroxylase,TH)和多巴胺转运蛋白(dopamine transporter,DAT)表达的变化。方法:(1)植块法分离培养hUCMSCs,以细胞免疫化学法鉴定其表面特异性标记物的表达;(2)无菌条件下分离E15大鼠胚胎中脑组织,制成单细胞悬液及中脑组织提取液;(3)用大鼠胚胎中脑组织提取液做培养液,将5-溴脱氧尿嘧啶核苷(bromodeoxyuridine,BrdU)标记的hUCMSCs与大鼠胚胎中脑细胞共培养14 d,细胞免疫化学法检测hUCMSCs中TH和DAT的表达。结果:HUCMSCs高表达CD29、CD44、CD73、CD90和CD105,极低表达CD31和CD45。在与大鼠胚胎中脑细胞共培养14 d后,hUCMSCs中TH阳性表达率为(18.06±2.29)%,DAT阳性表达率为(11.14±2.10)%。结论:用植块法可成功分离并体外培养hUCMSCs,其免疫表型符合间充质干细胞(mesenchymal stem cells,MSCs)的特征。在以大鼠胚胎中脑组织提取液作为培养液,并与大鼠胚胎中脑细胞共培养的诱导条件下,hUCMSCs可部分向多巴胺能神经元分化。
OBJECTIVE: To detect the expression of tyrosine hydroxylase (TH) and dopamine transporter (hTERT) in human umbilical cord mesenchymal stem cells (hUCMSCs) co-cultured with rat embryonic mesencephalic cells , DAT) expression changes. Methods: (1) hUCMSCs were isolated and cultured by explant method, and their surface specific markers were identified by immunocytochemistry. (2) The embryonic midbrain tissues of E15 rats were isolated under aseptic conditions and made into single cell suspension (3) BrdU-labeled hUCMSCs were co-cultured with rat embryo mesencephalic cells for 14 days with medium from rat embryonic midbrain tissue culture Immunocytochemistry was used to detect the expression of TH and DAT in hUCMSCs. Results: HUCMSCs highly expressed CD29, CD44, CD73, CD90 and CD105, with very low expression of CD31 and CD45. After co-cultured with rat embryonic mesencephalic cells for 14 days, the positive expression rate of TH in hUCMSCs was (18.06 ± 2.29)%, and the positive expression rate of DAT was (11.14 ± 2.10)%. CONCLUSION: The hUCMSCs can be successfully isolated and cultured in vitro by immunophenotyping. The immunophenotypes are consistent with the characteristics of mesenchymal stem cells (MSCs). HUCMSCs can partially differentiate into dopaminergic neurons under the induction conditions of rat embryonic brain extracts as culture medium and co-culture with rat embryonic mesencephalic cells.