Pharmaceutical composition for promoting hematopoiesis and preparation method thereof
A composition and drug technology, applied in the direction of drug combination, pharmaceutical formula, medical preparations containing active ingredients, etc., can solve the problems of slow onset, slow relief, low cure rate, etc., to achieve rapid onset, promote hematopoiesis, High cure rate effect
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[0035] Another aspect of the present invention provides a preparation method of a hematopoietic pharmaceutical composition, which comprises: isolating and purifying hematopoietic stem cells and resuspending them in a pharmaceutically acceptable medium; dissolving soap alum in a pharmaceutically acceptable medium Medium; mix the above cell suspension with soap alum solution.
[0036] Another aspect of the present invention provides a preparation method of a hematopoietic pharmaceutical composition, which includes: isolating and purifying hematopoietic stem cells; introducing expression vectors carrying cytokines into the stem cells, and screening positive transfection cells; The cells are resuspended in a pharmaceutically acceptable medium; saponin is dissolved in the pharmaceutically acceptable medium; and the above cell suspension is mixed with the saponin solution.
[0037] In the present invention, the medium for resuspending cells and the medium for dissolving soap alum ma...
Embodiment 1
[0051] Separation and purification of umbilical cord blood stem cells to obtain CD34 + cell
[0052] The umbilical cord blood of normal full-term male neonates (provided by the Southwest Hospital Affiliated to the Third Military Medical University) was aseptically collected, anticoagulated with heparin, and the mononuclear cells were separated by Ficoll lymphocyte separation medium (density 1.077g / ml) to contain 10 Wash twice in IMDM with % fetal calf serum, resuspend in 300 μl of PBS solution containing 0.5% BSA and 2 mmol / L EDTA, and isolate CD34 from mononuclear cells by immunomagnetic bead sorting system (mini-MACS) + cell. The specific method is as follows: add 100 μl of blocking solution (3% goat serum) and anti-CD34 monoclonal antibody to the above cell suspension, mix well, and place at 4° C. for 15 minutes. After centrifugation and washing with 10 ml of the above-mentioned PBS buffer, 100 μl of goat anti-mouse IgG immunomagnetic beads were added and placed at 4°C fo...
Embodiment 2
[0056] Construction of IL-3, FL and co-expression of FL and IL-3 bicistronic eukaryotic vector
[0057] The eukaryotic expression vector pIRES2-EGFP was purchased from Clontech Company. Escherichia coli DH5α strain was preserved by our laboratory. The plasmid MXIL-3neo containing full-length mouse IL-3 cDNA (2.5kb) was donated by Professor Moroni of University of Basel, Switzerland; the plasmid pUMVC3-hFL containing full-length human FL cDNA (0.8kb) was purchased from Gene Therapy Center, University of Michigan, USA.
[0058] 1. Construction of pIRES2-EGFP-IL-3:
[0059] 1) Amplification of IL-3 target fragment: use primer design software Primer5.0 to design primers as follows:
[0060] Upstream: 5-GGAATTCCCTGTGGCTTCTTCA-3 (SEQ ID NO: 1) (containing EcoRI site);
[0061] Downstream: 5-TCCCCGCGGGGAAAACCCATTTGTTC-3 (SEQ ID NO: 2) (containing SacII site).
[0062]The PCR reaction was carried out using the MXIL-3neo plasmid as a template. The reaction conditions were: denatura...
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