原核表达重组人白介素-3及其对脐带血造血干细胞体外增殖的影响  

Prokaryotic expression of recombinant human interleukin 3 and its effects on extracorporeal proliferation of cord blood hematopoietic stem cells

作　　者：张彦西 戴卫[1] 蒋永平[1,2] 

机构地区：[1]中国医学科学院北京协和医学院苏州方舟生物医药研发中心,江苏苏州215126 [2]苏州方舟基因药业有限公司,江苏苏州215126

出　　处：《实用临床医药杂志》2013年第5期8-12,共5页Journal of Clinical Medicine in Practice

基　　金：中国高校医学期刊临床专项资金(11320030);国家"重大新药创制"科技重大专项(2011ZX09102-010-04);国家"重大新药创制"科技重大专项(2011ZX09401-027)

摘　　要：目的研究原核表达重组人白介素-3(rhIL-3)及其对脐带血造血干细胞体外增殖的影响。方法构建pET-43.1-rhIL-3表达载体,克隆至BL21菌株,并进行中试发酵生产,所得包涵体经透析复性后,采用阳离子交换柱纯化,纯化产物送N-端测序。Ficoll密度梯度离心结合免疫磁珠法富集人脐带血中CD34+细胞,以不同浓度rhIL-3及干细胞因子(SCF)、Fms样酪氨酸激酶受体-3配体(FL)和促血小板生成素(TPO)因子组合培养1周,测定细胞总数,计算CD34+细胞增殖倍数。结果在SCF和FL存在下,rhIL-3可明显促进造血干祖细胞增殖,但较无rhIL-3时CD34+细胞占细胞总数的比例有所下降。与SCF、FL共同作用时的最佳浓度在15 ng/mL左右,与SCF、FL、TPO共同作用时的最佳浓度在50 ng/mL左右。结论高效表达及纯化的rhIL-3与SCF、FL和TPO细胞因子组合对脐带血造血干细胞具有良好的增殖作用。Objective To study the prokaryotic expression of recombinant human interleukin 3 （rhIL-3） and its effects on extraeorporeal proliferation of CB-HSCs. Methods pET-43.1-rhIL-3 expression vector was established, cloned into BL21 strain and conducted pilot-scale fermentation. After dialysis and renaturation, the inclusion body was purified by cation exchange column, then the purified products were sent to N-terminal to sequencing. Ficoll density gradient centrifugation combined with immunomagnetic beads was adopted to collect CD34 ~ cells in cord blood. Different concentrations of rhIL-3, SCF, Fins-like receptor tyrosine kinase-3 ligand （FL） and TPO factors were combined to foster for 1 week. The total number of cells were detected, and the proliferation times of CD34 ＋ cells were calculated. Results Under the existence of SCF and FL, rhIL-3 can obviously enhance the proliferation of hematopoietic stem and progenitor cells, but without rhIL-3, the proportion of CD34 ＋ cells taking up the total number of cells decreased. At the time of combined action with SCF and FL. Conclusion The combination of rhIL-3 with high-effective expression and purification with SCF, FL and TPO cell factors has good proliferative functions on CB-HSC.

关 键 词：重组人白介素-3 脐带血造血干细胞 CD34+细胞 体外增殖 

分 类 号：R331.2[医药卫生—人体生理学]