论文部分内容阅读
目的:建立转hLIF基因腺病毒载体的饲养层细胞,观察对CD34+造血干/祖细胞的扩增作用,并研究移植辐射损伤模型SCID小鼠的效果。方法:建立转hLIF基因腺病毒载体的饲养层细胞,并用RT-PCR法鉴定目的基因;采用免疫磁珠法分离脐带血CD34+造血干/祖细胞,流式细胞术检测纯度;将CD34+造血干/祖细胞与饲养层细胞共培养,流式细胞术检测各组增殖效果,建立辐射损伤模型SCID小鼠,将扩增后的CD34+造血干/祖细胞经CFDASE荧光标记后移植入SCID小鼠体内,通过RT-PCR和观察荧光标记细胞来检测小鼠内的人源细胞。结果:建立的转基因饲养层细胞均有绿色荧光,RT-PCR法证实有目的基因表达,免疫磁珠法分离的CD34+造血干/祖细胞纯度可达(95.6±2.58)%,与饲养层细胞共培养后CD34+造血干/祖细胞可扩增13.2倍,表面粘附分子CXCR4和CD54表达量仍较高,移植入SCID小鼠四周后,仍可见带有荧光标记的人源细胞,RT-PCR证明人源基因Alu的存在。结论:建立的转hLIF基因腺病毒载体饲养层细胞可以有效地扩增CD34+造血干/祖细胞,延缓其分化,并且有较高的移植效率和造血活性。
OBJECTIVE: To establish feeder cells of hLIF gene adenovirus vector to observe the expansion of CD34 + hematopoietic stem / progenitor cells and to study the effect of transplanted radiation-damaged SCID mice. Methods: The feeder cells of hLIF gene adenovirus vector was established and the target gene was identified by RT-PCR. Cord blood CD34 + hematopoietic stem / progenitor cells were isolated by immunomagnetic beads method. The purity of CD34 + hematopoietic stem / The progenitor cells and feeder cells were co-cultured. The proliferation of each group was detected by flow cytometry. The radiation-damaged SCID mice were established. The expanded CD34 + hematopoietic stem / progenitor cells were labeled with CFDASE and then transplanted into SCID mice. Human-derived cells in mice were detected by RT-PCR and observation of fluorescently labeled cells. Results: The green fluorescent cells were established in all the transgenic feeder layers. The target gene expression was confirmed by RT-PCR. The purity of CD34 + hematopoietic stem / progenitor cells isolated by immunomagnetic beads method was (95.6 ± 2.58)%, After culturing, the CD34 + hematopoietic stem / progenitor cells expanded 13.2 times and the expression of surface adhesion molecules CXCR4 and CD54 were still high. Four weeks after transplantation into SCID mice, fluorescent labeled human cells could still be seen. RT-PCR proved The presence of human gene Alu. CONCLUSION: The established feeder cells of hLIF gene adenovirus vector can effectively expand CD34 + hematopoietic stem / progenitor cells, delay their differentiation, and have high transplantation efficiency and hematopoietic activity.