人AB型血清培养体系体外培养扩增人脐血源基质细胞的试验研究

Empirical study on the amplification effect of coculturing human stromal cells derived from umbilical cord blood with human AB type serum

目的建立并优化AB型健康成人血清培养体系对人脐血源基质细胞的体外培养条件,观察基质细胞的生物学特性。方法人脐带血标本分离培养人脐血源基质细胞,经密度梯度分离后,在含有15%人AB型血清、bFGF(2,5～20.0μg/L)、10^(-6)mol/L氢化可的松的DMEM/F12培养液中进行原代培养和传代。倒置显微镜观察细胞生长情况,HE染色观察细胞形态特征,并采用常规HE染色和免疫细胞化学染色对培养细胞进行鉴定。结果成功获得人脐血源基质细胞并进行体外扩增培养和传代,培养细胞能形成纤维细胞样集落。优化的培养条件为:DMEM/F12培养基添加15%人AB型血清、10.0μg/LbFGF、10^(-6)mol/L氢化可的松。免疫细胞化学染色Vimentin+、CD34+、TdT-、CD45-、CK-,符合造血基质细胞特点。结论采用人AB型血清培养体系可成功培养扩增人脐血源基质细胞,为下一步临床应用研究打下坚实的基础。

Objective To evaluate the propagation effect of coculturing human stromal cells derived from umbilical cord blood with human AB type serum,and observe their biological behaviors. Methods Human stromal cells were isolated from the umbilical cord blood and inoculated into the DMEM/F12 media containing 15% human AB type serum,10.0 μg/L bFGF and 10 6 mol/L hydrocortisone. The growth status was investigated under the inverted microscope,and the cytomorphologi cal characteristics were observed by applying immunostaining assay. The cultured cells were identified with H-E staining and immunocytochemical staining assay. Results The human umbilical cord blood derived strom cells（hUCBDSCs） were isolated,and primary culture and serial subcuhivation were conducted successfully. The hUCBDSCs demonstrated positive immunostaining for vimentin, CD34, negative immunostaining for TdT,CD45 and CK; which was in accordance with the character of human stromal cells. Conclusion The hUCBDSCs can be successfully cultured and propagated with human AB type serum in vitro, which provides a foundation for further study on the clinical application of hUCBDSCs.