Vascular tissue engineering by cord blood stem cell

在获得知情同意书后，在正常足月分娩期间获得人脐带血。通过Ficoll-Hypaque密度梯度离心分离单核细胞，并通过磁珠分离法纯化CD 34 ^+细胞。回收的细胞中95%以上为CD 34 ^+细胞，将纯化的CD 34 ^+细胞在无血清悬浮培养中与各种细胞因子组合孵育，并在第14天和第21天使用一组单克隆抗体通过流式细胞术进行分析。使用IL 6、SCF、TPO、IL 3、EPO、FLT 3和G-CSF的组合来测定在每个时间点在悬浮培养物中产生的各种祖细胞。本实验将端粒酶全长cDNA（hTERT）质粒和模拟载体转染到具有白血病干细胞特征的K562细胞中，检测巨核细胞中CD 41 ^+和/或CD 42 ^+细胞的百分率，为端粒酶基因治疗提供实验依据。TPO对巨核细胞的分化具有生长优势，而SCF对巨核细胞的分化具有抑制作用。在含有TPO作为单一生长因子的培养物中，CD 41 ^+细胞在第14天达到最大扩增，当在培养基中加入巨大戟二萜醇时，CD 41 ^+细胞的总百分比稳步增加。hTERT在k562细胞中的异位表达在无血清的情况下显示出存活优势。转导的细胞保留表型特征、分化能力和对TPA的信号转导应答。这些数据表明，异位表达hTERT的正常造血干细胞可能会赋予生存优势，而不改变先天的生物学特性。

Human umbilical cord blood was obtained during normal full-term deliveries after obtaining informed consent. Mononuclear cells were separated by Ficoll-Hypaque density gradient centrifugation and CD34^+ cells were purified by a magnetic bead separation method. More than 95% of recovered cell was CD34^+.Purified CD34^+ cells were incubated in serum-free suspension culture in the presence of various combination of cytokines and analyzed by flowcytometry using a panel of monoclonal antibodies at day 14 and 21. A combination of IL6, SCF, TPO, IL3, EPO, FLT3 and G-CSF was used for the determination of various progenitors generated in suspension culture at each time points. In some experiments, the plasmid containing the full-length cDNA of telomerase(hTERT) and mock vector were transfected to K562 cells with the characteristics of leukemic stem cells in order to provide data on a possible approach to telomerase gene therapy.The megakaryocytic fraction was determined as the percentage of CD41^+ and/or CD42^+ cells. TPO always resulted in growth advantage for megakaryocytic differentiation, whereas SCF suppressed the differentiation. In cultures containing TPO as a single growth factor, maximal expansion of CD41^+ cells was achieved at day 14 and total percentage of CD41^+ cells steadily increased when ingenol was added in culture medium. Ectopic expression of hTERT in k562 cells showed a survival advantage in the absence of serum. Transduced cells retained phenotypic characteristics, differentiation ability, and the signal transduction response to TPA. These data suggest that ectopic expression of hTERT by normal hematopoietic stem cells may confer a survival advantage without changing innate biological characteristics.