CN101530427B - Pharmaceutical composition for promoting hematopoiesis and preparation method thereof - Google Patents
Pharmaceutical composition for promoting hematopoiesis and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a pharmaceutical composition for promoting hematopoiesis, which comprises hemopoietic stem cells and melanterite, wherein the preferred hemopoietic stem cells are cells which are imported with an expression vector with cell factor genes. The invention also relates to a method for preparing the pharmaceutical composition and application of the pharmaceutical composition in preparing a medicament for treating the damage state of hematopoietic function, wherein the hemopoietic stem cells and the melanterite have the function of synergistically promoting the hematopoiesis.
Description
Technical field
The present invention relates to a kind of pharmaceutical composition of hematopoiesis and method for preparing this pharmaceutical composition of promoting.The invention still further relates to this pharmaceutical composition and be used for the treatment of purposes in the medicament of the impaired state of hemopoietic function in preparation.
Background technology
Hematopoiesis is human life activity's integral part.The hemopoietic tissue of adult animals mainly is arranged in marrow.Under physiological condition, hematopoiesis is a hemopoietic stem cell (HSC) through propagation, differentiation, until the dynamic process that forms various mature blood cells.Multiple clinically common disease in the blood system, for example aplastic anemia, multiple myeloma, myelodysplastic syndrome etc., and the bone marrow injury that causes of radiation and chemotherapy all can cause hemopoietic function of bone marrow to be suppressed or depleted, brings serious harm to body health.
Hematopoietic stem cell transplantation (HSCT) can promote hematopoietic reconstitution, recover the hemopoietic function of marrow to a certain extent, become critical treatment means such as diseases in the blood system such as leukemia, multiple myeloma, aregeneratory type anaemia and some genetic anemias.In recent years, HSCT also is used for the supportive treatment after cancer radiations such as mammary cancer, neuroblastoma, ovarian cancer or the chemotherapy gradually.Known some other ionization radiation injury also can be treated by hematopoietic stem cell transplantation such as the bone marrow depression that acute radiation sickness causes.The hemopoietic stem cell that the inventor is previous studies show that cytokine gene to the zoografting transfection of suffering from acute radiation sickness can promote marrow hemopoiesis to rebuild people such as [, " Chinese radiological medicine and protection magazine " the 28th volume, 14-17 page or leaf] Zhang Yong effectively.
But because the influence of problems such as cell amplification, transfection efficiency, the interior transplanting vigor of body, simple hematopoietic stem cell transplantation is also unsatisfactory to the result of treatment of marrow hemopoiesis obstacle.
Chinese medicine preparation is because its higher security also is used to treat some diseases in the blood system.Wherein use more traditional Chinese medicine ingredients to be melanterite.The main component of melanterite is a ferrous sulfate, also contains the trace element that copper, arsenic, cobalt etc. stimulate marrow hemopoiesis.The melanterite compound ball that with the melanterite is main ingredient is usually used in some hematopoietic disorders type disease of assisting therapy, hemopoietic function to the radiation injury animal [people such as Xiao Yang that also has some improvement, " new Chinese medicine and clinical pharmacology ", the 15th volume, the 387-389 page or leaf], alleviate the shortcoming slow, that onset is slow, curative ratio is low but exist.
Summary of the invention
The object of the present invention is to provide a kind of pharmaceutical composition, said composition comprises hemopoietic stem cell and melanterite and pharmaceutically acceptable medium, the effect that have remarkable promotion hematopoietic reconstitution, recovers hemopoietic function of bone marrow.
Another object of the present invention is to provide a kind of preparation of drug combination method.
A further object of the present invention is to provide the purposes of a kind of pharmaceutical composition in the medicament of the impaired state of preparation treatment hemopoietic function.
In embodiments of the invention, described hemopoietic stem cell is CD34
+Hemopoietic stem cell.In the preferred embodiment of the invention, this CD34
+Stem cell is CD34
+Cord blood stem cell.
In another embodiment of the present invention, described hemopoietic stem cell can be the hemopoietic stem cell that has imported expression vector, and wherein said expression vector carries at least a cytokine gene.
In a preferred embodiment of the invention, the cytokine that described expression vector carries be selected from the Flt3 part (Flt3 ligand, FL), interleukin-3 (IL-3) or its combination.More preferably, described expression vector is pIRES2-EGFP-IL-3, pIRES2-EGFP-FL or its combination or pIRES2-EGFP-FL-IL-3.Most preferably, described carrier is pIRES2-EGFP-FL-IL-3.
In one embodiment of the invention, the pharmaceutically acceptable medium in the pharmaceutical composition is selected from damping fluid, physiological saline, cell culture medium, balanced salt solution or its combination.
In embodiments of the invention, preparation of drug combination method of the present invention comprises: the separation and purification hemopoietic stem cell also is resuspended in it in pharmaceutically acceptable medium; Melanterite is dissolved in the pharmaceutically acceptable medium; The resuspended liquid of above-mentioned cell is mixed with melanterite solution.
In another embodiment of the present invention, preparation of drug combination method of the present invention comprises: the separation and purification hemopoietic stem cell; To carry the cytokine expression carrier and import described stem cell, screening transfection positive cell; Described transfection positive cell is resuspended in the pharmaceutically acceptable medium; Melanterite is dissolved in the pharmaceutically acceptable medium; Then the resuspended liquid of above-mentioned cell is mixed with melanterite solution.
In pharmaceutical composition provided by the invention, hemopoietic stem cell and melanterite have the collaborative effect that promotes hematopoiesis, make this pharmaceutical composition have rapid-action, efficient height, advantage that curative ratio is high in hematopoietic reconstitution.
In embodiments of the invention, described pharmaceutical composition is with simple hematopoietic stem cell transplantation or give melanterite separately and compare, significantly improve the individual survival rate and the quantity of peripheral blood cells, shown the advantage of composition of the present invention in the impaired state of treatment hemopoietic function.Especially, pIRES2-EGFP-FL-IL-3 of the present invention is than using pIRES2-EGFP-IL-3, pIRES2-EGFP-FL or its combination to demonstrate better effect separately.
Brief Description Of Drawings
Fig. 1 shows the PCR product electrophorogram of IL-3.The 1st swimming lane is a molecular weight marker, and the 2nd, 3 swimming lanes are the PCR product of IL-3.
Fig. 2 shows that the enzyme of recombinant chou pIRES2-EGFP-IL-3 cuts the result.The 1st swimming lane is a molecular weight marker; The recombinant chou pIRES2-EGFP-IL-3 of the 2nd swimming lane for cutting without enzyme; The 3rd swimming lane is the recombinant chou pIRES2-EGFP-IL-3 through the HindIII single endonuclease digestion; The 4th swimming lane is the recombinant chou pIRES2-EGFP-IL-3 through the BglII+Xmal double digestion.
Fig. 3 shows that the enzyme of recombinant chou pIRES2-EGFP-FL cuts the result.The 1st swimming lane is molecular weight marker (DL2000); The 2nd swimming lane is through the pUMVC3-hFL of SalI and BglII double digestion (3.9kb+0.8kb); The 3rd swimming lane is the recombinant chou pIRES2-EGFP-FL (6.0kb) that cuts without enzyme; The 4th swimming lane is through the recombinant chou pIRES2-EGFP-FL of NheI+XhoI double digestion (5.25kb+0.75kb).
Fig. 4 shows that the enzyme of recombinant chou pIRES2-EGFP-FL-IL-3 cuts the result.The 1st swimming lane is molecular weight marker (DL2000); The 2nd swimming lane is recombinant chou pIRES2-EGFP-IL-3 (7.5kb); The 3rd swimming lane is recombinant chou pIRES2-EGFP-FL (6.0kb); The 4th swimming lane is IL-3 fragment (2.5kb); The 5th swimming lane is recombinant chou pIRES2-EGFP-FL-IL-3 (8.2kb); The 6th swimming lane is through the pIRES2-EGFP-FL-IL-3 of XmalI+XhoI double digestion (6.0kb+2.2kb); The 7th swimming lane is through the pIRES2-EGFP-FL-IL-3 of NheI+KpnI double digestion (4.7kb+3.5kb).
Fig. 5 detects IL-3, FL respectively at the CD34 that has imported pIRES2-EGFP-IL-3, pIRES2-EGFP-FL carrier by sxemiquantitative RT-PCR
+Intracellular expression.A, the 1st swimming lane are molecular weight marker, and the 2nd swimming lane is a non-transfected cells, and the 3rd swimming lane is the PCR product that replaces template to obtain with PBS, the 4th, the 5 swimming lanes CD34 of IL-3 that has been transfections
+Cell; B, the 1st swimming lane are molecular weight marker, and the 2nd swimming lane is a non-transfected cells, the 3-5 swimming lane the has been transfection CD34 of FL
+Cell.
Fig. 6 is presented at the CD34 that has imported pIRES2-EGFP-IL-3, pIRES2-EGFP-FL or pIRES2-EGFP-FL-IL-3 carrier
+In the cell, the protein expression of FL and IL-3.A is at the CD34 that imports pIRES2-EGFP-IL-3 and two kinds of carriers of pIRES2-EGFP-FL
+In the cell, the protein expression of IL-3, the 1st swimming lane is contrast, the 2nd swimming lane is two carrier transfectional cells; B is at the CD34 that imports pIRES2-EGFP-IL-3 and two kinds of carriers of pIRES2-EGFP-FL
+In the cell, the protein expression of FL, the 1st swimming lane is contrast, the 2nd swimming lane is two carrier transfectional cells; C is at the CD34 that imports the pIRES2-EGFP-FL-IL-3 carrier
+In the cell, the protein expression of IL-3, the 1st swimming lane is contrast, the 2nd swimming lane is the co-expression carrier transfectional cell; D is at the CD34 that imports the pIRES2-EGFP-FL-IL-3 carrier
+In the cell, the protein expression of FL, the 1st swimming lane is contrast, the 2nd swimming lane is the co-expression carrier transfectional cell.
Embodiment
An aspect of of the present present invention provides the pharmaceutical composition that promotes hematopoiesis, and this pharmaceutical composition comprises hemopoietic stem cell and melanterite and pharmaceutically acceptable medium.
" hemopoietic stem cell " of the present invention can be marrow hemopoietic stem cells, peripheral hematopoietic stem cells, cord blood stem cell, preferred cord blood stem cell.The antigenicity of cord blood stem cell immunocyte a little less than, few and light with relative marrow of generation and the peripheral blood of transplanting relevant graft versus host disease (GVH disease), and gather and preserve easily, donor is not had any injury.Therefore, Cord blood is considered to ideal hemopoietic stem cell source.
In one embodiment of the invention, described hemopoietic stem cell is CD34
+Hemopoietic stem cell.
In the preferred embodiment of the invention, this CD34
+Stem cell is CD34
+Cord blood stem cell.
" CD34 " is meant the I type transmembrane protein of high glycosylation, and its selectivity is distributed in hemopoietic stem cell surface, and along with the continuous differentiation and maturation of hematopoietic cell, its expression also reduces until disappearance gradually.The investigator is usually CD34
+Cell is as the source cell that carries out HSC biological characteristic research and clinical application.
In another embodiment of the present invention, hemopoietic stem cell can be the hemopoietic stem cell that has imported expression vector, and at least a cytokine gene is carried in wherein said expression.
" expression vector " of the present invention is reorganization or the synthetic nucleic acid construct that produces, and it has the specific nucleic acid element that the specific nucleic acid of a series of permissions is transcribed in host cell.Expression vector of the present invention can be the plasmid vector such as pIRES2-EGFP, pcDNA3.1, pCI-neo, pDC516, pVAC, pcDNA4.0, pGEM-T, pDC315, or such as the virus vector of adenovirus, adeno-associated virus, retrovirus, semliki forest virus (sFv) carrier.Virus vector Yin Qiyi is incorporated in the target cell karyomit(e), causes poor stability.Therefore, in a preferred embodiment of the invention, described expression vector is a plasmid vector, preferred pIRES2-EGFP carrier, because the latter comprises: 1. enhanced green fluorescence protein (EGFP) is easy to merge and be convenient to goal gene detect; 2. contain SV40 ori reproduction element, might divide, follow endochylema and entail daughter cell, can guarantee the goal gene stable delivery to a certain extent with host cell; 3. contain IRES, can strengthen the expression of goal gene translation skill.
" cytokine " of the present invention be meant existence to hemopoietic stem cell, transfer and die, breed and break up the protein families that plays the important regulating and controlling effect.Cytokine normally by at the special acceptor of its target cell surface bonding with the activation signal path of transduceing.The example of described cytokine comprises, but be not limited to, such as FL, IL-1, IL-3, IL-6, IL-11, IL-12, granulocyte colony-stimulating factor (G-CSF), leukaemia inhibitory factor (LIF) and STEM CELL FACTOR (SCF) etc. act on the cytokine of G0 phase, IL-3 for example, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 etc. act on the cytokine of the HSC of differentiation state, and erythropoietin (EPO) for example, thrombopoietin (TPO), macrophage colony stimulating factor (M-CSF) and IL-5 etc. act on the cytokine in differentiation later stage.Wherein FL finds recently, can stimulate the cytokine of early stage hematopoiesis, and it can regulate the propagation and the differentiation of primary hematopoietic stem, can with various kinds of cell factor synergy, hemopoietic function improvement.IL-3 claims the polyphyly G CFS again, and it is mainly produced by activated T lymphocyte, and hematopoietic cell is had the stimulation division of wide spectrum and the effect of differentiation.IL-3 is in the presence of FL or SCF, and the karyocyte sum that can effectively increase is the key regulatory factor that strengthens hematopoiesis and colony forming cell.
In a preferred embodiment of the invention, the described expression vector cytokine of carrying is selected from IL-3, FL or its combination.
" pharmaceutically receivable medium " of the present invention is meant the not carrier of the biological activity validity of interferon activity composition.Medium of the present invention includes but not limited to cell culture medium, damping fluid, physiological saline and balanced salt solution.The example of cell culture medium comprises BME, MEM, DMEM, IMEM, HAM F12, PRMI1640, M199 etc.The example of damping fluid comprises phosphoric acid salt, acetate, Citrate trianion, borate and carbonate.Described medium also can further comprise one or more compositions of pharmaceutically acceptable vehicle, adjuvant, emulsifying agent, stablizer, sanitas etc.
In embodiments of the invention, described medium is an isotonic buffer solution, is preferably isotonic phosphate buffer liquid (PBS).
Another aspect of the present invention provides the preparation of drug combination method of short hematopoiesis, and it comprises: the separation and purification hemopoietic stem cell also is resuspended in it in pharmaceutically acceptable medium; Melanterite is dissolved in the pharmaceutically acceptable medium; The resuspended liquid of above-mentioned cell is mixed with melanterite solution.
Another aspect of the present invention provides the preparation of drug combination method of short hematopoiesis, and it comprises: the separation and purification hemopoietic stem cell; To carry the cytokine expression carrier and import described stem cell, screening transfection positive cell; Described transfection positive cell is resuspended in the pharmaceutically acceptable medium; Melanterite is dissolved in the pharmaceutically acceptable medium; The resuspended liquid of above-mentioned cell is mixed with melanterite solution.
In the present invention, the medium of re-suspended cell can be identical or different with the medium of dissolving melanterite.In a preferred embodiment of the invention, the medium of re-suspended cell is identical with the medium of dissolving melanterite, and is isotonic phosphate buffer liquid.
In embodiments of the invention, the resuspended concentration of hemopoietic stem cell is 5 * 10
4/ ml to 1 * 10
7/ ml is preferably 5 * 10
5/ ml to 5 * 10
6/ ml most preferably is 2.5 * 10
6/ ml.
In embodiments of the invention, the concentration of ordinary dissolution of melanterite is 0.5mg/ml to 50mg/ml, is preferably 2mg/ml to 10mg/ml, most preferably is 5mg/ml.
In embodiments of the invention, the resuspended liquid of cell is 1: 2 to 3: 1 with the mixed volume ratio of melanterite solution, and preferred volume ratio is 1: 1 to 2: 1, most preferably is 2: 1.
In embodiments of the invention, described expression vector can be plasmid vector or virus vector, plasmid vector preferably, and most preferably be the pIRES2-EGFP plasmid vector.Described " transfection positive cell " is meant the cell that has successfully imported expression vector.Described expression vector carries the gene of the accelerated haematogenous cell factor, the express cell factor in stem cell.
In another embodiment of the present invention, the stem cell of the expression vector importing separation and purification of cytokine gene, preferred CD34 will be carried
+Stem cell, most preferably CD34
+Cord blood stem cell.
Another aspect of the present invention provides the purposes of pharmaceutical composition in the medicament of the impaired state of preparation treatment hemopoietic function of short hematopoiesis.
Hemopoietic stem cell of the present invention and melanterite " working in coordination with " effect is meant that the pharmaceutical composition that comprises hemopoietic stem cell and melanterite obviously is better than the effect of simple hematopoietic stem cell transplantation or independent melanterite to the promoter action of hematopoietic reconstitution.
" the impaired state of hemopoietic function " of the present invention is meant that hemopoietic function of bone marrow is suppressed, and causes the generation quantity of one or more hemocytes to be reduced to the state that endangers body health.The impaired state of hemopoietic function of the present invention includes but not limited to bone marrow injury that aplastic anemia, leukopenia, thrombocytopenia, multiple myeloma, myelodysplastic syndrome, radiation and chemotherapy cause and the marrow hemopoiesis obstacle that causes such as other ionization radiation injuries of acute radiation sickness." acute radiation sickness " is meant that whole body is subjected to the systemic disease that the above irradiation of 1 gray(Gy) (Gy) back takes place in the short period of time, mainly show as hematopoietic disorder.
In one embodiment of the invention, in several minutes, give the irradiation of Reconstruction in Sever Combined Immunodeciency (SCID) mouse 2.0-8.0Gy, duplicate the animal model of acute radiation injury; The marrow pathological examination shows that marrow hematopoiesis function failure degree and radiation dose are proportionate.In the preferred embodiment of the invention, give SCID mouse 8.0Gy the irradiation of (dose rate is 1.07Gy/min), so that observe the effect of pharmaceutical composition of the present invention better.
In embodiments of the invention, giving individual method with pharmaceutical composition can be intravenous injection or intravenous drip, preferred intravenous injection.
" individuality " of the present invention is meant Mammals, includes but not limited to primate, ox, horse, pig, sheep, goat, dog, cat and such as the rodent of rat and mouse.
Above-mentioned disclosure has been described the present invention generally, by the further example the present invention of the following examples.Describe these embodiment and only be explanation the present invention, rather than limit the scope of the invention.Although used special term and value herein, these terms and value are understood that exemplary equally, not delimit the scope of the invention.
The isolation and purification cord blood stem cell obtains CD34
+Cell
The aseptic normal full-term normal delivery male neonate Cord blood (providing) of taking by attached southwestern hospital of Third Military Medical University, use anticoagulant heparin, separate mononuclearcell through Ficoll lymphocyte separation medium (density 1.077g/ml), wash 2 times with the IMDM that contains 10% foetal calf serum, be resuspended in the PBS solution that 300 μ l contain 0.5%BSA and 2mmol/L EDTA, (mini-MACS) isolates CD34 from mononuclearcell with magnetic activated cell (sorting)
+Cell.Concrete grammar is as follows: add 100 μ l confining liquid (3% sheep blood serum) and anti-CD34 monoclonal antibodies in the resuspended liquid of above-mentioned cell respectively, mixing is placed 15min for 4 ℃.Behind the above-mentioned PBS damping fluid of 10ml centrifuge washing, add 100 μ l sheep anti-mouse igg immunomagnetic beadses, place 15min for 4 ℃.Cell is resuspended in the 1ml PBS damping fluid with after the aforesaid method washing, is added drop-wise to the separator column that places magnetic field, cell suspension is flowed out naturally.Wash post 4 times with described damping fluid, each 0.5ml.Separator column is shifted out magnetic field, add the 1ml damping fluid, use the separator column nook closing member that cell is released.The gained cell is mainly CD34
+Cell.
Get 1 * 10 before the screening and after the screening respectively
5The anti-CD34 monoclonal antibody of individual mononuclearcell and phycoerythrin (PE) mark is hatched 30min under 4 ℃.With cell with after the damping fluid washing, with the fixing 30min of 2% Paraformaldehyde 96 of 300 μ l.Discard Paraformaldehyde 96, after cell is washed with damping fluid, measure CD34 with the method for routine immunization groupization again
+Cell percentage.
The result: in the mononuclearcell before the screening, CD34
+Cell only is 0.62%-9.05%, and average out to 3.24% ± 0.46% (x ± sx).In the mononuclearcell after the screening, the CD34+ cell is 46.42%-95.61%, and average out to 79.35% ± 5.24% (x ± sx).CD34 before and after the screening purifying
+Cell concn has on average increased by 24.49 times.
The structure of IL-3, FL and coexpression FL and IL-3 bicistronic mRNA eukaryotic vector
Carrier for expression of eukaryon pIRES2-EGFP is available from Clontech company product.E.colistraindh5 is preserved by this laboratory.Containing total length mouse IL-3 cDNA (2.5kb) plasmid MXIL-3neo is so kind as to give by professor Moroni of Univ Basel Switzerland; Contain total length people FL cDNA (0.8kb) plasmid pUMVC3-hFL available from U.S. Michigan university gene therapy center.
1.pIRES2-EGFP-IL-3 structure:
1) the segmental amplification of IL-3 purpose: utilize primer-design software Primer5.0 design primer as follows:
Upstream: 5-GGAATTCCCTGTGGCTTCTTCA-3 (SEQ ID NO:1) (containing the EcoRI site);
Downstream: 5-TCCCCGCGGGGAAAACCCATTTGTTC-3 (SEQ ID NO:2) (containing the SacII site).
With the MXIL-3neo plasmid is that template is carried out the PCR reaction, and reaction conditions is: 94 ℃ of sex change 5min (1 circulation); 94 ℃ of sex change 30sec, 56 ℃ of annealing 1min, 72 ℃ are extended 3min (30 circulations); Last 72 ℃ are extended 10min.The result as shown in Figure 1, amplification obtains the IL-3 fragment (Fig. 1) of 2501bp.
2) with IL-3 fragment directed cloning to the pIRES2-EGFP carrier: utilize EcoRI, SacII double digestion PCR product IL-3 fragment and pIRES2-EGFP respectively, reclaiming the back connection spends the night, transformed into escherichia coli DH5 α, several single bacterium colonies of picking extract plasmid DNA on that resistant panel from blocking.Identify with HindIII single endonuclease digestion and BglII+Xmal double digestion recombinant chou respectively, and carry out the dna sequencing confirmation.Positive recombinant chou called after pIRES2-EGFP-IL-3.
Recombinant chou pIRES2-EGFP-IL-3 enzyme is cut the result as shown in Figure 2, the HindIII single endonuclease digestion: pIRES2-EGFP 623, there is the HindIII site at 892bp place and the segmental 801bp of IL-3 place, therefore three fragments of 0.8kb, 1.7kb and 5.0kb appear in positive recombinant chou after this enzyme is cut.The BglII+Xmal double digestion: pIRES2-EGFP 621, the 657bp place has BglII, Xmal site respectively, therefore 2.2kb and two fragments of 5.3kb appear in positive recombinant chou behind this double digestion.Determined dna sequence shows that the nucleotide sequence of IL-3 gene and GenebankK03233's is in full accord.
2.pIRES2-EGFP-FL construction of eukaryotic expression vector:
1) the segmental amplification of FL purpose: utilize Primer5.0 design primer as follows:
Upstream: 5-ATTGTGGCTTACCCTTGGTCTTCA-3 (SEQ ID NO:3);
Downstream: 5-GTTGCGGGGAACTCCCCATTTGTTC-3 (SEQ ID NO:4).
With the PUMVC3-hFL plasmid is that template is carried out the PCR reaction, and condition is: 94 ℃ of sex change 5min (1 circulation); 94 ℃ of sex change 30sec, 56 ℃ of annealing 1min, 72 ℃ are extended 3min (30 circulations); Last 72 ℃ are extended 10min.
2) FL bicistronic mRNA construction of eukaryotic expression vector: SalI and BglII double digestion pUMVC3-hFL obtain total length people FL sequence (0.8kb), and the floating rear clone in two ends is to the BglII site of pIRES2-EGFP carrier.After NheI and XhoI enzyme are cut evaluation, with the recombinant chou called after pIRES2-EGFP-FL of forward connection.
Enzyme is cut the result as shown in Figure 3, and pUMVC3-hFL obtains the 3.9kb+0.8kb fragment after the SalI+BglII enzyme is cut, and recombinant chou pIRES2-EGFP-FL obtains the 5.25kb+0.75kb fragment after the NheI+XhoI enzyme is cut.Determined dna sequence shows that the nucleotide sequence of FL gene and GenebankK03233's is in full accord.
3.pIRES2-EGFP-FL-IL-3 co-expression carrier makes up:
The pIRES2-EGFP-IL-3 that utilizes EcoRI and SacII site double digestion to successfully construct reclaims the corresponding site of IL-3 purpose fragment rear clone to pIRES2-EGFP-FL.Use XhoI and XmalI double digestion, NheI and KpnI double digestion to identify respectively, with positive recombinant chou called after pIRES2-EGFP-FL-IL-3.
Enzyme is cut the result as shown in Figure 4, and after the XmalI+XhoI enzyme was cut, 6.0kb and two fragments of 2.2kb appearred in this recombinant chou; After the NheI+KpnI enzyme was cut, two fragments of 4.7kb and 3.5kb appearred.Dna sequencing shows that the result is correct, mistakes such as no frameshit.
Gene transfection
Get the cord blood CD 34 that is in exponential phase of growth through separation and purification
+Cell carries out transfection when it merges to 60-80%.5 μ l plasmid vector DNA (53 μ g/ml) are added mixing in the 20 μ l transfection damping fluids (pH 7.4 for 20mmol/L HEPES, 150mmol/L NaCl), obtain A liquid; 15 μ l DOTAP liposomes are added mixing in the above-mentioned transfection damping fluid of 35 μ l, obtain B liquid; Under the room temperature A liquid and B liquid are mixed gently, 1, the centrifugal 30sec of 200rpm leaves standstill 15min, and it is slowly allocated among the IMDM that contains 10% foetal calf serum.Nutrient solution suction in the culturing bottle is abandoned, wash cell 1 time with 0.01mol/L PBS, the nutrient solution that will contain rotaring redyeing system then adds in the culturing bottle, in 37 ℃, 5%CO
2Incubation 24h in the incubator changes liquid afterwards.G418 is added about January in the culturing bottle by 800 μ g/ml concentration, and first quarter moon changes 1 not good liquor, and it is constant to keep G418 concentration.Cell amplification after the screening is standby.
The evaluation of transfection plasmid
1. detect the green fluorescence of EGFP
After the acid of common lid slide bubble, cleaning, dry, put into diameter 6cm culture dish, warp
60The conventional illumination-based disinfection of Co gamma-radiation.With 0.25% trysinization transfection the cell of cytokine gene, then with cell with about 1 * 10
5The concentration of/ml is inoculated in the culture dish of above-mentioned illumination-based disinfection.Discard nutrient solution behind the 24h, the PBS washed cell of usefulness 0.01mol/L 2 times.Take out cover glass then, place on the slide glass, under the blue laser of 488nm excites, with the green fluorescence of fluorescence microscope EGFP.
The result shows that the cell space of visible cell all sends green fluorescence under the fluorescent microscope, shows that plasmid successfully imports CD34
+In the cell.
2. sxemiquantitative RT-PCR detects the expression of FL and IL-3
1. utilize the single stage method total RNA extraction reagent box of Invitrogen company, extract the total RNA among the embodiment 3 through transfection screening and expanded cells.
2. primer design is with synthetic: it is as follows to use Primer5.0 software design primer,
IL-3: upstream (P1): 5-TGTTTCCACTCCGTCCAT-3 (SEQ ID NO:5);
Downstream (P2): 5-CCAAAGCAGGGCTCTTAC-3 (SEQ ID NO:6).(Tm53 ℃; 30 circulations; 585bp);
FL：P1：5-CTGGAGCCCAACAACCTATC-3(SEQ?ID?NO：7)；
P2：5-TCTGGACGAAGCGAAGACA-3(SEQ?ID?NO：8)。(57 ℃ of Tm; 27 circulations; 353bp).
3. RT-PCR: adopt two-step approach to carry out reverse transcription amplification.Reverse transcription: 42 ℃ 30 minutes, 99 ℃ 5 minutes, 5 ℃ 5 minutes, 1 circulation.PCR:94 ℃ of pre-sex change 4 minutes; 94 ℃ of sex change are 30 seconds then, 56 ℃ of annealing 45 seconds, and 72 ℃ were extended totally 40 circulations 1 minute; Last 72 ℃ are extended 10 minutes polishing ends.
The result is contrast with normal non-transfected cells as shown in Figure 5, CD34 after the transfection
+IL-3 of cell (Fig. 5 A) and FL express (Fig. 5 B) and all obviously raise.
3.Western blot detects the protein expression situation of FL and IL-3
Extract among the embodiment 3 and screen the also total protein of expanded cells, measure protein content with the Lowry method through transfection.Get equal protein and carry out the SDS-PAGE electrophoresis, change film, albumen is gone on the pvdf membrane.With film TBST (TBS, 0.05%Tween20) Xi Shi confining liquid (5% skim-milk) room temperature sealing is after 1 hour, resists 4 ℃ of overnight incubation of (Sigma) diluent with the FL one of 1: 800 IL-3, one anti-(Sigma) diluent (being diluted among the TBST) or 1: 500.One anti-hatch end after, wash film 3 times with TBST, each 10 minutes.Use two anti-(the anti-people of rabbit) diluents (being diluted among the TBST) of 1: 300 correspondence to hatch 1h more respectively in 37 ℃.Wash film 3 times with TBST then, each 10 minutes.Then film is hatched 1h in 37 ℃ in 1: 500 horseradish enzyme streptavidin three anti-diluents of TBST dilution.At last with DAB colour developing, the pvdf membrane of the clear band of colour developing placed on the Gel Doc2000 gel scanning system analyze, carry out statistical study with the ratio of scan sample value/control scan value.
The result imports the CD34 of pIRES2-EGFP-IL-3 and two kinds of carriers of pIRES2-EGFP-FL as shown in Figure 6 simultaneously
+In the cell, the proteic expression of IL-3 is higher than control group (Fig. 6 A), and the proteic expression of FL is higher than control group (Fig. 6 B); Import the CD34 of pIRES2-EGFP-FL-IL-3 co-expression carrier
+In the cell, the IL-3 of expression and FL albumen also all be higher than control group (Fig. 6 C, 6D).
Comprise CD34
+The preparation of drug combination of cord blood stem cell
Melanterite is available from its pharmacy company limited of army of Shaanxi Hao; CD34
+Cord blood stem cell is obtained by embodiment 1.Preparation process is as follows:
(1) will be from 5 * 10 of embodiment 1
5Individual CD34
+Cord blood stem cell is resuspended in the 200 μ l isotonic phosphate buffer liquid (0.03mol/L, pH 7.4), and obtaining concentration is 2.5 * 10
6The stem cell suspension of/ml.
(2) 500 μ g melanterite are dissolved in the above-mentioned phosphate buffered saline buffer of 100 μ l, obtaining concentration is the melanterite solution of 5mg/ml.
(3) above-mentioned stem cell suspension is mixed with melanterite solution, promptly obtain medicine composition injection.
Comprise the CD34 that has imported expression vector
+The preparation of drug combination of cord blood stem cell
Melanterite is available from its pharmacy company limited of army of Shaanxi Hao; Imported the CD34 of expression vector
+Cord blood stem cell is from embodiment 3.
1. comprise the CD34 that has imported pIRES2-EGFP-IL-3 and two kinds of carriers of pIRES2-EGFP-FL simultaneously
+The preparation of drug combination of cord blood stem cell
As the preparation method of embodiment 5, with 5 * 10
5The individual CD34 that has imported pIRES2-EGFP-IL-3 and pIRES2-EGFP-FL expression vector
+Cord blood stem cell is resuspended in the 200 μ l isotonic phosphate buffer liquid (0.03mol/L, pH 7.4), and obtaining concentration is 2.5 * 10
6The transfection of/ml the cell suspension of IL-3 gene; 500 μ g melanterite are dissolved in the above-mentioned phosphate buffered saline buffer of 100 μ l, and obtaining concentration is the melanterite solution of 5mg/ml; With above-mentioned two kinds of liquid mixing, obtain 300 μ l medicine composition injections.
2. comprise the CD34 that has imported the pIRES2-EGFP-FL-IL-3 co-expression carrier
+The preparation of drug combination of cord blood stem cell
Basic identical with top preparation method, difference is CD34
+What cord blood stem cell imported is the pIRES2-EGFP-FL-IL-3 co-expression carrier.
Observation is from the curative effect of the pharmaceutical composition of the present invention of embodiment 5
Experimental subjects: 48 4-6 age in week, the SCID female mice of body weight 18-20g (the Third Military Medical University animal provides).
Experimental technique: mouse is raised in aseptic laminar flow cabinet.The feed warp
60The Co irradiation, tap water and bedding and padding are all through the autoclave sterilization aseptically process, and administration began to drink the water that adds amphotericin B (80mg/L) and Ciprofloxacin (80mg/L) after the autoclave sterilization acidifying in preceding 3 days.All operations to mouse all carries out under aseptic laminar flow condition.Mouse places aseptic gas-pervious plastics casing, and the aseptic disinfecting towel of outer cover is used
60A general irradiation of Co gamma-rays 2.0-8.0Gy (dose rate is 1.07Gy/min) duplicates the animal model of radiation injury.After radiation, pass through the tail vein injection administration in 4 hours, after this keep microbiotic and raise clothes.
The experiment grouping: 48 mouse through irradiation are divided into blank group, CD34 at random
+Cord blood stem cell group (CD34 group), melanterite group, pharmaceutical composition group, every group of 12 animals.Wherein every mouse of CD34 group gives 300 μ l cell suspensions, contains 5 * 10
5Individual from the CD34 that implements 1
+Cord blood stem cell; Every mouse of melanterite group gives 300 μ l melanterite solution (containing melanterite 500 μ g); Every mouse of pharmaceutical composition group gives 300 μ l pharmaceutical composition from embodiment 5, promptly comprises melanterite and CD34
+The composition of cord blood stem cell.
Observation index: observe mouse existence situation; The 2nd week after the administration, 4 weeks and 6 all tail veins are got blood and are carried out routine blood test and detect.
Statistical procedures: all data represent that with x ± s SPSS 10.0 softwares carry out statistical procedures, mouse survival rate x
2Check, remainder data is checked with t, and there is statistical significance P＜0.05 for difference.
Experimental result:
It is as shown in table 1 that each organizes the mouse survival rate, and blank group mouse is depleted and all dead at 2 all internal cause hemopoietic functions; CD34
+The survival rate of group and melanterite group prolongs in time and reduces; The survival rate of pharmaceutical composition group is significantly higher than each group same period.
The result is as shown in table 2 for the peripheral blood conventional sense, and blank group does not have related data because of whole death; When the 2nd week and the 4th week, the quantity of leucocyte of pharmaceutical composition group, erythrocyte number and platelet counts are significantly higher than CD34 group and melanterite group (p＜0.05), and significant statistical significance is arranged; The 6th when week all the other groups except that the blank group each index between do not have obviously difference, this may be can survive because of every group all more satisfactory to the hematopoiesis recovery extent of the mouse in 6 weeks.
These data show, pharmaceutical composition of the present invention and CD34
+Cord blood stem cell is compared with melanterite, and the impaired state of hemopoietic function that radiation injury is caused has the obvious treatment effect.
Table 1 is respectively organized mouse survival quantity and survival rate
Annotate: compare with control group,
aP＜0.01; Compare with the CD34 group,
bP＜0.05;
dP＜0.01; Compare with the melanterite group,
cP＜0.01
Table 2 is respectively organized mouse peripheral blood conventional sense result (x ± s)
Annotate: the pharmaceutical composition group
AbExpression is for each index in the 2nd week and the 4th week, and the pharmaceutical composition group is compared with the CD34 group,
aP＜0.05; Compare with the melanterite group,
bP＜0.01.
Embodiment 8
Observation is from the curative effect of the pharmaceutical composition of the present invention of embodiment 6
Experimental subjects: 80 4-6 age in week, the SCID female mice of body weight 18-20g (the Third Military Medical University animal provides).
Experimental technique: identical with embodiment 7.
The experiment grouping: 80 mouse through irradiation are divided into the CD34 that imports two carriers at random
+Cord blood stem cell group (A group), import the pIRES2-EGFP-FL-IL-3 co-expression carrier stem cell group (B group), melanterite group (C group), comprise melanterite and imported the stem cell of two carriers pharmaceutical composition group (D group), comprise melanterite and imported the pharmaceutical composition group (E group) of the stem cell of co-expression carrier, every group of 16 animals.Because the mouse without treatment is all dead in two weeks, so do not establish the blank group.Every mouse of 5 groups all gives 300 μ l injection liquids by the tail vein, and wherein the mouse of A group gives cell suspension, and it contains 5 * 10
5The individual CD34 that has imported pIRES2-EGFP-IL-3 and two kinds of carriers of pIRES2-EGFP-FL
+Cord blood stem cell; B group mouse also gives cell suspension, and it contains 5 * 10
5The individual CD34 that imports the pIRES2-EGFP-FL-IL-3 expression vector
+Cord blood stem cell; C group mouse gives melanterite solution (containing melanterite 500 μ g); The D group gives pharmaceutical composition, the CD34 that it comprises melanterite and has imported pIRES2-EGFP-IL-3 and two kinds of carriers of pIRES2-EGFP-FL
+Cord blood stem cell; E group mouse also gives pharmaceutical composition, and said composition comprises melanterite and imported the CD34 of pIRES2-EGFP-FL-IL-3 co-expression carrier
+Cord blood stem cell.
Observation index: identical with embodiment 7.
Statistical procedures: identical with embodiment 7.
Experimental result:
It is as shown in table 3 that each organizes the mouse survival rate, the survival rate of E group all be significantly higher than A, B the 2nd, 4 and 6 weeks, C organizes (p＜0.05); The survival rate of D group all is higher than A, B, C group at different times, but is lower than the survival rate of the E group of the same period.
The result is referring to table 4 for the peripheral blood conventional sense, and when the 2nd week and the 4th week, quantity of leucocyte, erythrocyte number and the platelet counts of E group all is higher than A, B, C and D group (p＜0.05), and significant statistical significance is arranged; Each index in the D group is higher than A, B and C group, but all is lower than the E group of the same period.
This result shows, pharmaceutical composition of the present invention with transfection separately the CD34 of cytokine gene
+Cord blood stem cell is compared, and can significantly improve the survival rate of radiation injury animal, and hematopoietic reconstitution is had obvious facilitation.This result also shows simultaneously, and melanterite is more more outstanding than the synergy of melanterite and pIRES2-EGFP-IL-3 and pIRES2-EGFP-FL plasmid vector with the synergy of pIRES2-EGFP-FL-IL-3 co-expression carrier.
Table 3 is respectively organized mouse survival quantity and survival rate
Table 4 is respectively organized mouse peripheral blood conventional sense result (x ± s)
Be illustrated with certain form although be appreciated that the present invention, the present invention is not limited to the shown and content described in this specification sheets.It should be apparent to those skilled in the art that under the prerequisite that does not depart from scope of the present invention and also can make various variations.These change all in the scope of protection of present invention.
Sequence table
＜110〉Third Military Medical University southwest hematology of hospital
Zhang Yong
Du Hongwei
Guo Chaohua
Dong Shiwu
＜120〉pharmaceutical composition of short hematopoiesis and preparation method thereof
<130>08C11137CN
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<220>
＜223〉be used for the primer sequence of PCR
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ctggagccca?acaacctatc 20
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<220>
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Claims (6)
1. pharmaceutical composition, it is by CD34
+Hemopoietic stem cell and melanterite and isotonic phosphate buffer liquid are formed.
2. pharmaceutical composition, it is by the CD34 that has imported expression vector
+Hemopoietic stem cell, melanterite and isotonic phosphate buffer liquid are formed, and wherein said expression vector carries the cytokine gene that is selected from Flt3 part, interleukin-3 or its combination.
3. pharmaceutical composition according to claim 2, wherein said expression vector is for expressing the pIRES2-EGFP-FL-IL-3 of Flt3 part, interleukin-3 simultaneously.
4. the purposes of each described pharmaceutical composition in the medicament of the impaired state of preparation treatment hemopoietic function among the claim 1-3.
5. purposes according to claim 4, melanterite in the wherein said pharmaceutical composition and hemopoietic stem cell have the collaborative effect that promotes hematopoiesis.
6. purposes according to claim 5, the impaired state of wherein said hemopoietic function are selected from the hematopoietic disorders that aplastic anemia, leukopenia, thrombocytopenia, myelodysplastic syndrome, multiple myeloma and ionization radiation injury, chemotherapy cause.
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Non-Patent Citations (3)
| Title |
|---|
| Marie-Catherine Giarratana1 et al.Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells.《NATURE BIOTECHNOLOGY》.2005,第23卷(第1期),69-74. * |
| 吴鹏飞等.再生障碍性贫血临床治疗进展.《实用诊断与治疗杂志》.2007,第21卷(第12期),928-931. * |
| 张勇等.pIRES2-FL-IL-3转染协同促进脐血干细胞增殖的研究.《第三军医大学学报》.2007,第29卷(第24期),2330-2332. * |
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