摘要
目的:初步研究VCAM-1修饰的hUCBSCs促进造血干/祖细胞体外扩增的作用。方法:在成功构建VCAM-1-pcDNA3.1(+)真核表达载体的基础上,转染原代培养的人脐血基质细胞,采用半定量RT-PCR法和免疫细胞化学技术检测转染前后hUCBSCs VCAM-1的表达情况;以VCAM-1修饰的人脐血基质细胞为滋养层,体外扩增脐血CD34+细胞,并对扩增后的脐血CD34+细胞进行CFU-GM、CFU-E、CFU-Mg半固体培养。结果:脂质体介导VCAM-1-pcDNA3.1(+)真核表达载体成功转染人脐血基质细胞;RT-PCR和免疫细胞化学从mRNA水平和蛋白质水平均证明转染后VCAM-1表达明显高于转染前(P<0.05);VCAM-1修饰的人脐血基质细胞能够有效支持脐血CD34+细胞的体外扩增(P<0.01),其中对CFU-Mg的扩增作用强于未转染组(S组,P<0.05)。结论:VCAM-1修饰的人脐血基质细胞能够有效地支持造血干/祖细胞的体外扩增,显示其在造血重建方面潜在的应用价值。
AIM ： To investigate the effect of vascular cell adhesion molecule - 1 （ VCAM - 1 ） modified human umbilical cord blood stromal cells （ hUCBSCs ） on the expansion of hematopoietic stem cells/progenitor cells in vitro. METHODS ： After the VCAM - 1 - pcDNA3.1 （ ＋ ） eukaryotic expression vector was constructed successfully, the recombinant vector was transfected into hUCBSCs via liposome mediation. The expression of the VCAM - 1 was detected by semiquantitative RT - PCR and immunocytochemistry. The expansion abilities of hematopoietic cell in vitro under different conditions were compared, including cytokine stimulation, hUCBSCs feeder layer and VCAM - 1 modified hUCBSCs feeder layer etc. The effect on hematopoietic reconstitution of different expanded hematopoietic cell groups was compared after CFU - GM, CFU - E and CFU - Mg semi - solid culture were proceed, respectively. RESULTS ： The positive clone and Neo^R gene detection proved that recombinant vector had been transfected into hUCBSCs. The results of semiquantitative RT - PCR and immunocytochemistry manifested that VCAM - 1 was high expressed not only at transcriptional level in the stable - transfected hUCBSCs （P 〈 0. 05 ）, but also on the membrane of hUCBSCs （ P 〈 0. 05 ）. VCAM - 1 modified hUCBSCs feeder layer was used for expansion of CD34 ^＋ cells. It expanded CFU - Mg more effectively than untransfected hUCBSCs did （P 〈0. 05）. CONCLUSION： VCAM- 1 modified human umbilical cord blood stromal cells could be useful for expansion of hematopoietic stem/progenitor cells in vitro. It shows that VCAM - 1 modified hUCBSCs has potential capability on supportting hematopoiesis.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2007年第10期1923-1926,共4页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.30070327)
第三军医大学科研基金资助项目(No.XG200347)
关键词
血管细胞黏附分子-1
胎血
间质细胞
转染
扩增
Vascular cell adhesion molecule - 1
Fetal blood
Stromal cells
Transfection
Expanding
