【作者】 尹健；

【导师】 郝希山； 张学慧；

【作者基本信息】 天津医科大学 ， 肿瘤学， 2003， 博士

【摘要】 目的：本研究的目的是寻找最佳细胞因子组合，以及利用RetroNectin提高逆转录病毒介导的多药耐药基因的转染效率，从而为临床治疗做准备。 实验材料和方法：外周血标本均来自2001年15月至2002年5月在天津医科大学附属肿瘤医院住院接受自体外周血干细胞移植的恶性肿瘤患者。标本共22份，男女患者各11人，平均年龄38.8岁。采用CE(卡铂+足叶乙甙)+G-CSF+GM-CSF方案动员后采集。利用磁性细胞分离器对造血干细胞进行分选后，在不同细胞因子组合下进行体外培养：①对照组，不加任何细胞因子；②SCF+IL-3+GM-CSF+TPO；③FL+GM-CSF+IL-4；④SCF+IL-3+FL+TPO；⑤SCF+IL-3+IL-6+FL+GM-CSF；⑥SCF+IL-3+FL+TPO+EPO。观察培养不同时间的细胞总数、CD34+细胞比例及集落形成能力。利用逆转录病毒作载体将多药耐药基因导入造血干细胞，在转染中予细胞因子支持，并利用RetroNectin以提高转染效率。通过Rhodamine 123排出法、RT－PCR、竞争性PCR、集落形成和抗性实验、细胞增殖实验(MTT)检测基因的转染效率及效果。 结果：22份外周血标本中CD34+细胞的比例为1.3±0.28％，分选后CD34+细胞的纯度为90.3±4.35％，回收率为62.39±5.21％。第6组细胞因子组合(SCF+IL-3+FL+TPO 天津医科大学博士学位论文十EPO)支持下，CD34+细胞的增殖能力最强，所占比例最高。在该组细胞因子组合支持下，利用RetroNectin可使逆转录病毒介导的基因转染效率超过20%，转染组的耐药性较未转染组增强。 结论:在SCF+IL一3+FL+TPO+EPO的支持下，利用RetroNectin可以使逆转录病毒介导的多药耐药基因的转染效率达到20%，基本满足了临床治疗的需要。更多还原

【Abstract】 Objective: Our research was designed to investigate the appropriate combination of hematopoietic growth factors (HGF) and increase MDRl transfer efficiency of retrovirus-mediated transduction into peripheral blood stem cells (PBSC).Materials and methods: From May 2001 to May 2002, we enrolled 22 patients with malignant tumors eligible for autologous peripheral blood stem cell transplantation in Tianjin cancer hospital. 11 patients were male, and the others were female. The median age was 38.8 years. PBSC were harvested after mobilization with CBP + Vp16 + G-CSF + GM-CSF. CD34+ cells were isolated by using a high-gradient magnetic cell sorting system (MACS), and expanded with different HGF combination in a liquid culture system. There were six groups: (1)control, without any HGF; (2)SCF + IL-3 + GM-CSF + TPO; (3)FL + GM-CSF + IL-4; (4)SCF + IL-3 + FL + TPO; (5)SCF + IL-3 + IL-6 + FL + GM-CSF; (6)SCF + IL-3 + FL + TPO + EPO. We characterized the expanded cells by total cells counting, flow cytometry and colony forming units. MDRl was transferred into PBSC by retrovirus in the presence of HGF and RetroNectin. We examined the transfection and expression of MDRl by Rhodamine 123 efflux assay, RT-PCR, competitivePCR, colony forming units culture after exposure to Taxol and MTT colorimetric assay.Results: The proportion of CD34+ cells in mobilized PB was l.3± 0.28%. Overall yield was 62.39±5.21% and overall purity was 90.3±4.35%. The proliferation and clonogenic potential of CD34+ cells were significantly higher in the presence of SCF + 1L-3 + FL + TPO + EPO, and the percentage of CD34+ cells in total cells were highest. MDR1 was tranducted into PBSC by retrovirus in the presence of HGF and RetroNectin. Gene tansfer efficiency was over 20%, and the resistance to Taxel and Vincristime was higher.Conclusion: The gene transfer efficiency was over 20% in the presence of SCF + IL-3 + FL + TPO + EPO and RetroNectin. The efficiency of this gene transfer system provides a foundation for ameliorating combination chemotherapy toxicity in clinical trial.更多还原