An Experimental Study On The Co-transfection Of Human Marrow Stromal Cells With Three Main Hematopoietic Growth Factors

Objective:To construct three eukaryotic expression vectors TPO-pEGFP-N1, IL-6-pcDNA3.1(+)and IL-11-pcDNA3.1(-), co-transfect into human marrow stromal cells by liposometransfection, and then observe the expression of the three eukaryotic expression vectors inhuman marrow stromal cells.Methods:1. Three gene fragments of IPO, IL-6and IL-11were cloned respectively. Then thecopies of IPO, IL-6and IL-11gene was ligated to plasmid pEGFP-N1, pcDNA3.1(+) andpcDNA3.1(-) respectively by PCR amplification, to form eukaryotic expression vectorsTPO-pEGFP-N1, IL-6-pcDNA3.1(+) and IL-11-pcDNA3.1(-). By restriction mapping andsequence analysis, the correctness of cDNA open reading frame of both cloned genefragements and constructed gene fragments were examined.2. Human marrow stromal cells were isolated by adhesion screening. The highlyfissionable3rd generation cells were selected as gene-transfection target cells. By usingliposome transfection, human marrow cells were co-transfected with three eukaryoticexpression vectors TPO-pEGFP-N1,IL-6-pcDNA3.1(+) and IL-11-pcDNA3.1(-). Finally,the expression of the three genes on mRNA and protein level after transfecting the marrowstromal cells was examined by using various methods, such as RT-PCR, cellimmunohistochemistry, and Western blot.Results:1. The results of restriction mapping and sequence analysis confirmed the validity ofthe three eukaryotic expression vectors TPO-pEGFP-N1, IL-6-pcDNA3.1(+) andIL-11-pcDNA3.1(-).2. In vitro cultured human marrow stromal cells were morphologically heterogeneous.The primary cells depicted colonial growth pattern, while all the descendant generations depicted heterogeneous one.3. After co-transfection of human bone marrow stromal cells, expression of TPO incells was observed under fluorescence microscope, and25%of the cells emitted greenfluorescence. Expression of IL-6and IL-11was observed by cell immunohistochemistry. ByWestern blot analysis, TPO protein expression (in terms of TPO/GAPDH ratio) of controlgroup, empty vector transfected group, and multi gene co-transfected group were0.17±0.01,0.18±0.01, and0.62±0.01, respectively; IL-6protein expression (in terms of IL-6/GAPDHratio) of control group, empty vector transfected group, and multi gene co-transfected groupwere0.14±0.01,0.18±0.04, and0.34±0.06, respectively; IL-11protein expression (in termsof IL-11/GAPDH ratio) of control group, empty vector transfected group, and multi geneco-transfected group were0.08±0.23,0.19±0.14, and1.62±0.23, respectively. The proteinexpression of multi genes co-transfected group was significantly higher than that of controlgroup (p<0.01). The results showed that TPO, IL-6and IL-11factors had steady expressionin the marrow stromal cells that had been co-transfected with the three genes.Conclusion:1. Three eukaryotic expression vectors TPO-pEGFP-N1, IL-6-pcDNA3.1(+) andIL-11-pcDNA3.1(-) were successfully constructed.2. Human marrow stromal cells could be co-transfected with TPO, IL-6and IL-11genes. All three genes had effective expression in stromal cells, suggesting that they exerteda facilitating effect on platelet production.