人脐血间充质干细胞向甲状腺细胞的诱导分化

Differentiation of human umbilical cord blood mesenchymal stem cells into thyrocytes

目的探索人脐血间充质干细胞(UBMSCs)诱导分化为甲状腺细胞的可能性。方法采用羟乙基淀粉沉淀法和差速贴壁培养分离、纯化人UBMSCs,添加促甲状腺素(TSH)、胰岛素(insulin)、碘化钾(KI)等诱导因子体外共培养21 d,RT-PCR定量测定相关基因TSHR、PAX8、NIS、TPO、TG的表达;双色免疫荧光检测分化标记物PAX8和TTF-1、PAX8和TTF-2的共表达。结果分离、纯化的细胞不表达造血细胞相关的表面抗原如CD14(0.59%)、CD34(0.04%)、CD45(0.20%)等,不表达上皮细胞相关的表面抗原如CD106(0.68%)等,强表达与间充质干细胞相关的抗原如CD13(98.95%)、CD29(92.77%)、CD44(92.67%)等。原代UBMSCs多数呈梭形,平行排列,局部形成漩涡状排列,混有少量圆形细胞;第3代UBMSCs形态比较均一,呈长梭形,折光性好。诱导后UBMSCs边界清晰,部分呈类圆形或多角形贴壁生长,折光性好。诱导前人UBMSCs、诱导后细胞(包括向成骨细胞诱导和向甲状腺细胞诱导)均可见PAX8、NIS、TSHR、TPO、TG基因的表达,其中向甲状腺细胞诱导的目的细胞表达水平最高,诱导前人UBMSCs次之,向成骨细胞诱导的目的细胞表达水平最低。视野内可见诱导后细胞中有零星散在的PAX8和TTF-1、PAX8和TTF-2共表达细胞,DAPI染色核定位示PAX8和TTF-1、PAX8和TTF-2共表达于细胞核,作为对照的诱导前人UBMSCs中未见PAX8和TTF-1、PAX8和TTF-2共表达细胞。结论人UBMSCs在特定条件下可定向分化为甲状腺细胞。

Objective To explore the feasibility of directed differentiation of human umbilical cord blood mesenchymal stem cells（ UBMSCs） into thyrocyte-like cells in vitro. Methods Nucleated cells were isolated by sedimentation with hydroxyethyl starch and MSCs were obtained by adherent method. Then purified UCBMSCs were induced with growth factors-TSH,insulin and KI for 21 days. The induced cells were identified by reverse transcription-polymerase chain reaction（ RT-PCR） for TSHR,PAX8,NIS,TPO and Tg gene. In addition,the molecular markers of thyroid cells were identified by dual-fluorescent analysis under laser scanning confocal microscope. Results MSCs from cord blood did not express hematopoietic cells surface markers CD14（ 0. 59%）,CD34（ 0. 04%） or CD45（ 0. 20%）,nor express epithelial cells surface markers CD106（ 0. 68%）; but strongly expressed mesenchymal stem cells surface markers CD13（ 98. 95%）,CD29（ 92. 77%） and CD44（ 92. 67%）. The primary UBMSCs were mostly fusiform,arranged in parallel,partial spiral arranged,and mixed with a small number of round cells. The third generation of UBMSCs were homogeneous in shape,with long spindle shape and good refractive index. After induction,the boundary of UBMSCs was clear,and some of them were round or polygonal adherent wall growth,and with good refraction. The expression of PAX8,NIS,TPO,Tg and TSHR genes was the highest in UBMSCs-derived thyrocyte-like cells,followed by UBMSCs,and UBMSCs-derived osteoblasts. Dual-fluorescent analysis under laser scanning confocal microscope showed that co-expression of PAX8 and TTF-1,as well as PAX8 and TTF-2 were observed in several UBMSCs-derived thyrocyte-like cells. DAPI staining showed that PAX8 and TTF-1,PAX8 and TTF-2 were co-expressed in the nucleus. No PAX8 and TTF-1,or PAX8 and TTF-2 co-expressed cells were found in UBMSCs. Conclusion Human UBMSCs can differentiate into thyrocyte-like cells under given inductive conditions and related growth factors in vitro.