人脐带和胎盘来源的干细胞分离鉴定及其组份分析

Isolation and Identification of Human Umbilical Cord and Placentaderived Stem Cells and Their Component Analysis

目的:脐带血(UCB)内含有丰富的造血干细胞,能够有效地用于造血干细胞移植,挽救了许多患病儿童的生命,但由于从UCB采集的有核细胞(NC)和造血干细胞(CD34+)数量有限,在成年病人造血干细胞移植治疗中效果不理想。为此,本研究分析和对比UCB和残留在胎盘内的干细胞(UPB)的数量和质量。方法:采集UCB的同时,无菌收集来自于同一UCB的胎盘。采用0.9%生理盐水在脐带动、静脉血管灌注和抽取,收集灌注后的UPB;采用脐带组织贴壁培养、扩增和收集脐带间充质干细胞(UM SC)以及胎盘组织间充质干细胞(PM SC);按照浓缩、冰冻和保存UCB的常规方法保存来自于各组份的细胞,应用干/祖细胞集落培养和流式细胞术方法分析各细胞组份。结果:在62例的UPB和UCB的样本中MNC和CD34+总数分别为(17.45±16.86)×108、(6.9±4.61)×108和(2.97±2.25)×106,(1.91±1.7)×106;UPB和UCB的早期造血前体干细胞(early precursor of hematopoietic stem cells)(CD33+CD4-)分别为(6.2±13.5)×105和(0.2±0.8)×105;;此外,在UPB中有比UCB含量更高的间充质干细胞(M SC),分别占NC的百分比为25.21±18.69和0.05±0.10(%)。UCB、UPB、PM SC和UM SC这些不同部位来源的M SC具有相同的细胞形态和免疫表型。结论:胎盘内含有丰富的造血干细胞和间充质干细胞,从数量和分化能力方面都优于UCB,因而胎盘可以为造血干细胞研究和细胞治疗提供另一干细胞来源。

Objective:Clinical transplantation evidence has been indicated that umbilical cord blood(UCB) can be useful in the hematopoietic reconstitution in the children,but can not be well in the adult hematopoietic transplantation because of the low count.This study was to evaluate a new method for collecting stem cells from human placenta and umbilical cord,and to comparatively analyze the similarity and difference of quality and quantity of the cells.Methods:The UCB was collected,in same time the placental tissue was sterily collected;the umbilical placenta was collected by perfusing the blood(UPB) cord arterial and venous vascules with 0.9%saline;the mesenchymal stem cells(MSC)from same source of umbilical cord and placental tissues were isolated and cultured.The cell colony assay and flow cytometry were performed to determine the proliferation capacities and cell markers of UMSC and PMSC.Results:The total nuclear cells(NC) and hematopoietic stem cells(CD34^+) from UPB and UCB were(17.45 ± 16.86) × 10~8,(6.9 ±4.61) ×10~8 and(2.97 ±2.25) ×10~6,(1.91 ±1.7) × 10~6,respectively.Furthermore,the UPB contained more early precursor of hematopoietic stem cells(CD33^+ CD34^-)(6.2 ± 13.5) ×10~5,(0.2 ± 0.8) × 10~5;and high proportion of MSC to NC(25.21 ± 18.69,0.05 ± 0.10)%,respectively in 62 samples.There were no difference of the MSC level in UPB and UCB,as well as in the morphology and cell markers.Conclusion:UPB has rich hematopoietic stem cells and mesenchymal stem cells.Placenta may offer another source for hematopoietic stem cells in research of hematopoietic stem cells and regeneration medicine.