Effect of feeder cells transduced with Ad-hLIF on the expansion of CD34+ cells in indirect co-culture
10.3760/cma.j.issn.0254-5101.2009.03.012
- VernacularTitle:Ad-hLIF转基因饲养层细胞对CD34+造血干/祖细胞增殖和分化的影响
- Author:
Yingying JING
;
Jicheng YANG
;
Weihua SHENG
;
Zhiqing HU
;
Xin YU
;
Yunbo SHAN
;
Tielian LIU
;
Yali HAN
;
Wanrong BAO
;
Ri ZHANG
;
Nankang ZHU
;
Jingcheng MIAO
- Publication Type:Journal Article
- Keywords:
hLIF;
Recombinant adenovirus vector;
Hematopoietic stem/progenitor cell;
SCID mice
- From:
Chinese Journal of Microbiology and Immunology
2009;29(3):234-238
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish Ad-hLIF transgenic feeder cells for the expansion of umbilical cord blood CD34+ HSPC in vitro and study the SCID mice model of hematopoietic stem/progenitor cell (HSPC) transplantation. Methods The expression of objective gene in Ad-hLIF transgenic feeder cells was detected by RT-PCR and ELISA. The purity of umbilical cord blood CD34+ HSPC separated by magnetic-activated cell sorting(MACS) was detected by the flow cytometry. After expanded with various combinant of cytokines and transgenic feeder layer cells for 28 d, the quantity of mono-nuclear cell (MNC) and CD34+ cells rate was detected in different time. MNC after expansion stained by CFDA SE was injected to the sublethally irradiated SCID mice. Humanize gene Alu was detected by RT-PCR and fluorescence microscope. Results The green fluorescence was observed in the transgenic cells infected with 50MOI( multiplicity of infection) Ad-hLIF, and the objective gene was confirmed by RT-PCR and ELISA. The purity of umbilical cord blood CD34+ HSPC separated by MACS could reach 95.60% ±2.58%, Ad-hLIF transgenic feeder cells and various cytokines system increased MNC by 356.95±0.87 fold, and maximal expansion of CD34+ cells was observed during 0-14 d of culture, then down-expansion gradually. Four weeks after transplanted in SCID mice, fluorescently-labeled humanize cells still can be observed. The existence of the humanized gene Alu was confirmed by RT-PCR. Conclusion Ad-hLIF transgenic feeder cells can effectively proliferate umbilical cord blood CD34+ HSPC in vitro and delay it differentiate, what's more, it has high transplant efficacy and haematogenesis activity.
