Interleukin-3 cDNA cloning and transduction and its expression in umbilical cord blood CD34+ cells / 中国组织工程研究

ABSTRACT

BACKGROUND: One unit of umbilical cord blood does not have a sufficient number of peripheral blood stem cells to meet the requirements of transplantation in adults. One solution of this problem is their ex vivo expansion, which requires not only a longer time and higher culture conditions, but also easily leads to the differentiation of stern cells, thus affecting the effects of transplantation. OBJECTIVE: To transduce human interleukin-3(IL-3) gene into umbilical cord blood CD34" cells and to observe IL-3 expression. DESIGN, TIME AND SETTING: A cell-genomics in vitro experiment was performed in the Chengde Medical College in 2008. MATERIALS Human peripheral blood mononuclear cells (PBMC) of healthy adult were provided by Chengde Blood Center, and umbilical cord blood was provided by Chengde Maternal and Child Care Hospital. Written informed consent was obtained from each donor. METHODS: Peripheral blood mononuclear cells were isolated by Ficoll gradient centrifugation and umbilical blood CD34+ cells were isolated using immunomagnetic beads method. IL-3 mRNA was extracted. IL-3 cDNA was synthesized by RT-PCR, and IL-3 cDNA was subcloned into eukaryotic expressing vector pcDNA3. In the experimental group, pcDNA3/IL-3 vectors were transduced into umbilical cord blood CD34+ cells, while in the control group, transfection was not performed. MAIN OUTCOME MEASURES: Detection of IL-3 level in umbilical cord blood CD34" cells suspension using ELISA kits. RESULTS: Theoretically, the amplified IL-3 cDNA was 616 bp, and actually, after agarose gel electrophoresis, the PCR products exhibited a strip with expected size under ultraviolet ray. Extraction of IL-3mRNA was successful and reversely transcripted cDNA was complete. A 616-bp inserted fragment was observed by agarose gel electrophoresis after double digestion with BamH Ⅰ and Xba Ⅰ, and it was the same as IL-3 sequence. Within 1-7 days after transfection, IL-3 level in the umbilical cord CD34+ cells suspension was significantly higher in the experimental group than in the control group (t = 3.46, P < 0.05). CONCLUSION: IL-3 cDNA was successfully cloned, and eukaryotic expressing plasmid pcDNA3/IL-3 that could be effectively expressed within short term in umbilical cord CD34+ cells was successfully constructed.