CN1978656A - Induced differentiation of hemopoietic stem/progenitor cell into erythroid progenitor cells - Google Patents

Abstract

The invention involves biomedical field, concretely speaking, it involves the method and application of inducing hematopoietic stem / progenitor cells to erythroid differentiation of hematopoietic progenitor cells in vitro. This invention combines the genetic engineering and stem cell engineering, using the multipotency of stem cells and the bone marrow stromal cells of WNT3a in protein secretion activity as trophoblast cells, hematopoietic stem / progenitor cells to erythroid differentiation of hematopoietic progenitor cells is induced. this in vitro inducing system not only provides corroboration for the 'adult stem cell plasticity' theory, but also provides rich source of the cells as clinical preparation for diseases such as anemia, leukemia and other blood diseases, cancer chemotherapy and radiotherapy in the treatment of hematopoietic support, immune system diseases and infectious diseases such as liver and kidney dysfunction cell disease.

Description

A kind of method and application of inducing hematopoietic stem to the CFU-E differentiation

Technical field

The present invention relates to biomedical sector, specifically relate to use of the foundation of the external evoked hematopoietic stem of marrow stromal cell of transfection WNT3a gene to the CFU-E differentiation method.

Background technology

Anaemia and leukemic treatment all need the satisfactory red corpuscle of capacity clinically.Be the required cell concentration that obtains medical treatment, many scientists attempt the red corpuscle that obtains by vitro culture, mainly are by adding various cytokines etc.But currently used method amplifying candidate stem cell limited amount, and the red corpuscle purity that obtains is not high.

Reya confirmation WNT signal path is brought into play crucial effects in the process of hematopoietic stem self.WNT3a is a very important gene in the WNT path.Experiment confirm under certain condition, WNT3a albumen can promote that hemopoietic stem cell breaks up to CFU-E.Willert finds that the proteic activity of WNT3a is inseparable with the fat modification structure of himself.The WNT3a albumen that purifying obtains is very easy to remove palmitoylation under ambient conditions, thus loss of activity.

In our research with the WNT3a gene transfection to marrow stromal cell, marrow stromal cell excretory WNT3a albumen is in native state thereby has activity under this condition, so it can realize the purpose that it promotes CFU-E differentiation.Can use this culture system hematopoietic stem is induced differentiation, induce CFU-E after the differentiation to can be preparation and treat the cell preparation of the diseases such as hematopoiesis supportive treatment, disease of immune system infectious diseases and hepatic renal dysfunction of hematologic diseases such as anaemia that a variety of causes causes, leukemia, tumor chemoradiotherapy clinically abundant cell source is provided.

Summary of the invention

Technical problem to be solved by this invention is to set up the method that a kind of external evoked hemopoietic stem cell is divided into CFU-E.For solving the problems of the technologies described above, the technical solution used in the present invention is:

1.WNT3a the gene transfection marrow stromal cell, and transgenic cell is identified from molecule and cell levels.

2. external enlarged culturing is changeed the marrow stromal cell of WNT3a gene, treats that cell grows to acceptance when converging more than 90% ⁶⁰The Co gammairradiation is set up the trophocyte.

3. collection hematopoietic stem.

4. hematopoietic stem and the trophocyte who obtains cultivated altogether, induce differentiation to red system rent cell external.

5. the CFU-E after the differentiation is detected, comprise that colony forms the mensuration of ability and cell surface marker etc.

Description of drawings:

Fig. 1 is the pcr amplification electrophoretogram of goal gene Wnt3a

Fig. 2 carries out the electrophoretogram that enzyme is cut evaluation for Pac I to pAd-WNT3a recombinant virus plasmid

Fig. 3 is the expression photo of GFP in transfected 293 cells

Fig. 4 is for changeing Wnt3a marrow stromal cell Fluirescence observation result

Fig. 5 respectively organizes the CD117/CD71 expression level detected result of cell surface

Fig. 6 respectively organizes the red assembly of cell and falls to cultivating statistics

Fig. 7 pMSCV-Wnt3a recombinant retroviral vector carries out the electrophoretogram that enzyme is cut evaluation

Fig. 8 carries out the result that virus titer is measured for pMSCV-Wnt3a infects the NIH3T3 cell

(A): 10 ^-2Doubly dilution; (B): 10 ^-4Doubly dilution; (C): 10 ^-6Doubly dilution.

Fig. 9 detects the expression of Wnt3amRNA in PT67 cell and the NIH3T3 cell for RT-PCR.

1: people's placenta lysate (positive control);

The 2:PT-Wnt3a cell;

The 3:PT67 cell;

4: the NIH3T3 cell that infects pMSCV-Wnt3a;

The 5:NIH3T3 cell.

Specific embodiments:

Embodiment 1, adenovirus vector construct

1, the acquisition of WNT3a gene

For touching plate, also introduce XbaI respectively with mouse placenta cDNA according to the sequence of WNT3a-cDNA coding region, Hpa I and BamHI restriction enzyme site synthetic primer, the condition of primer sequence and polymerase chain reaction thereof (PCR) is as follows:

P1 5′-

ATGGCTCCTCTCGGATACCT-3′

P2 5′-

CTACTTGCAGGTGTGCACGT-3′

20 μ l reaction systems comprise: 1ul cDNA (100ng/ul), primer1 and 2 each 0.5 μ l (20 μ M), 1.5 μ l dNTP (2.5mM), 0.25 μ l La Taq archaeal dna polymerase (TakaRa company, 5u/ μ l), 2 μ l, 5 * GC Buffer I, 14.75 μ l aqua sterilisas.Reaction conditions is: 94 ℃ of sex change 3min:94 ℃ 45s, 56 ℃ of 45s, 72 ℃ of 2min, 28 circulations; 72 ℃ of 7min.Electrophoresis result as shown in Figure 1, among the figure l, swimming lane 1 be a goal gene, swimming lane 2 is a dna molecular amount mark (DL2000), the arrow indication be goal gene WNT3a electrophoretic band, big or small at 1058bp.Reaction product is cloned into pGEM-T eassy carrier (Promega company, the U.S.), obtains cloned plasmids T-WNT3a.

2, homologous recombination produces recombinant adenovirus plasmid (AdEasy Vector System, Quantum Biotechnologies) in the bacterium

Extract the T-WNT3a plasmid with restriction enzyme XbaI digestion, reclaim the purpose fragment and be inserted on the XbaI site of pAdTrack carrier, obtain recombinant plasmid pAdTrack-WNT3a.It is carried out forward and reverse order-checking and analysis revealed, and its open reading frame is correct, frameshit does not take place change.Show that the target gene sequences that we clone is correct, recombinant plasmid pAdTrack-WNT3a successfully constructs.

Get 1ug pAdTrack-WNT3a " pAdTrack with pAdEasy-1 all from AdEasy Vector System (QuantumBiotechnologies) " pAdTrack and WNT3a respectively through XbaI but enzyme be connected after cutting obtain pAdTrack-WNT3a) with after the Pme I linearizing, carry out 1% agarose gel electrophoresis, gel reclaims the purpose fragment of 10000bp, use the calf intestinal alkaline phosphatase dephosphorylation, ethanol sedimentation is standby.The method that provides by AdEasy Vector System specification sheets, earlier adenovirus skeleton plasmid pAdEasy-1 is changed in the BJ5183 bacterium, the BJ5183 bacterium that picking contains pAdEasy-1 prepares efficient competence bacteria, change the linearizing pAdTrack-WNT3a of 1ug over to contain pAdEasy-1 BJ5183 bacterium again, in the LB of 50mg/L kantlex culture plate, cultivate 16-20h for 37 ℃, the picking clone, the extracting plasmid, carry out 0.7% agarose gel electrophoresis, the result as shown in Figure 2, the size that shows plasmid is 43000bp, obtain the recombinant virus plasmid, called after pAd-WNT3a.By transforming pAd-WNT3a is changed in the DH5 α bacterium again.Among Fig. 2, swimming lane 1 is a recombinant plasmid 1, and swimming lane 2 is a recombinant plasmid 2, and swimming lane 3 is a dna molecular amount mark (DL15000), the arrow indication be the plasmid electrophoretic band.

3, to the evaluation of goal gene among the recombinant virus plasmid pAd-WNT3a

With Pac I pAd-WNT3a recombinant virus plasmid is carried out enzyme respectively and cut evaluation, qualification result shows to obtain two bands that size is respectively 39000bp and 4500b as shown in Figure 3, according to the success of recombinating as can be known of adenovirus characteristic.Among Fig. 3, swimming lane 1 is a recombinant plasmid, and swimming lane 2 is a dna molecular amount mark (DL15000), the arrow indication be the plasmid electrophoretic band.

4, recombinant viral vector is at the packing of 293 cells, the purifying and the titer determination of virus

The middle extraction plasmid DNA 10ug of the DH5 α bacterium that contains recombinant virus plasmid pAd-WNT3a from step 1 cuts with the PacI enzyme and to make it linearization.Carry out rotaring redyeing 293 cell according to producer's explanation with lipofect AMINE (GIBCO company).Observe with fluorescent microscope behind the 7-10d, the result observes the expression of GFP (Fig. 4) in most 293 cells.Collect cell then, 4 cracking cells of multigelation under-70 ℃/37 ℃ conditions, the supernatant that centrifuging and taking contains virus infects 293 cells once more with amplification recombinant virus, collecting cell behind the 3d, multigelation 3 times.Carry out viral purifying with CsCl density gradient ultracentrifuge method, the viral stock solution after concentrating is done the dilution of different ratios.Getting 400 μ l diluents adds in 293 Tissue Culture Flasks, hatched 3 hours in 37 ℃, renew bright substratum and continue to cultivate 18-24h, count the GFP positive cell number down in fluorescent microscope, calculate virus titer as follows: virus titer=GFP cell positive number * viral supernatant extension rate/0.4ml (pfu/ml).The result shows that virus titer is 3.16 * 10 ⁷Pfu/ml.

The marrow stromal cell of embodiment 2, commentaries on classics WNT3a gene is to the effect of hematopoietic stem

One, the separation of hematopoietic stem and purity check

(1) Cord blood mononuclearcell (mononuclear cell, separation MNC)

Material and reagent

Cord blood is taken from the umbilical cord of healthy full-term pregnancy natural labor fetus.

PBS: every 1L deionized water dissolving 8.0g NaCl, 0.2g KCl, 1.44g Na ₂HPO ₄, 0.24g KH ₂PO ₄, regulate pH7.4 with HCl or NaOH.

0.5% methylcellulose gum: the 1g methylcellulose gum adds 200mL physiological saline, puts into 4 ℃ of refrigerators behind the autoclaving while hot and makes its dissolving, with before shaking up.

1, the fresh Cord blood of anticoagulant heparin by 4: 1 volume ratio and 0.5% methylcellulose gum mixing, leaves standstill 30min under the room temperature again by 1: 1 and PBS mixing, treat that the red corpuscle natural subsidence is to boundary when clearly demarcated, the sucking-off supernatant is partly put in the 50mL centrifuge tube, room temperature 1, the centrifugal 5min of 000rpm;

2, abandon supernatant, add 10mL PBS suspension cell, the same centrifuge washing;

3, abandon supernatant, with 5mL PBS re-suspended cell;

4, in the 10mL centrifuge tube, add the 5mL human leukocyte parting liquid [Ficoll-Hypaque liquid, (1.077 ± 0.0002) g/L] of pre-balance, slowly add the 5mL cell suspension along tube wall then, room temperature, the centrifugal 25min of 1000rpm to room temperature.Be divided into PBS as can be seen from top to bottom, totally four layers of MNCs, Ficoll-Hypaque liquid, red corpuscle;

5, collect interface MNC cellular layer, room temperature, 1, the centrifugal 5min of 000rpm abandons supernatant;

6, use 1mL CD ₃₄ ⁺(PBS+0.5%BSA+2mmol/L EDTA, pH7.2) suspension cell move in the 1.5mLEp pipe counting to the cellular segregation damping fluid.

(2) derived from bone marrow mononuclearcell (mononuclear cell, separation MNC)

Material and reagent

Derived from bone marrow is in transplanting section of 307 hospitals

PBS: every 1L deionized water dissolving 8.0g NaCl, 0.2g KCl, 1.44g Na ₂HPO ₄, 0.24g KH ₂PO ₄, regulate pH7.4 with HCl or NaOH.

Percoll parting liquid (Amersham company)

1, marrow is transferred in the 50ml centrifuge tube, add the DMEM nutrient solution contain 10% serum (with the marrow volume ratio be 3: 1);

2, the centrifugal 5-10min of 1500rpm abandons supernatant;

3, PBS suspension cell is crossed 57% (1.073) Percoll;

The preparation of 57%Percoll (10ml):

1) 10 * PBS 0.6ml+Percoll stoste 5.4ml

2) discard the 300ul aforesaid liquid and add 1 * PBS 4.3ml, be mixed with 10ml experiment Percoll;

4,10ml experiment is added to respectively in 2 10ml centrifuge tubes with Percoll, respectively with 1: 1 ratio and the centrifugal 20min of cell 1500rpm;

5, get cell and wash one time with PBS, the centrifugal 5min of 1500rpm moves in the 1.5mL Ep pipe counting.

(3) separation of C D from MNC ₃₄ ⁺Cell (adopting the miniMACS separation system)

1, per 10 ⁸Individual mononuclearcell (MNC) is suspended in the CD of 4 ℃ of pre-temperature of 300 μ L ₃₄ ⁺The cellular segregation damping fluid adds the non-specific blocking antibody FcR of 100 μ L encapsulant, mixing.And then add 100 μ L magnetic bead link coupled CD ₃₄Monoclonal antibody (encapsulant, monoclonal antibody, separator column magnetic field are all available from Miltenyi Biotec), mixing is hatched 30min for 6-12 ℃;

2, add 500 μ lCD ₃₄ ⁺The cellular segregation damping fluid, 4 ℃, the centrifugal 3min of 2000rpm abandons supernatant;

3, use the CD of 1mL degasification ₃₄ ⁺Cellular segregation damping fluid re-suspended cell, the preparation single cell suspension;

4, the MACS separator column is fixed in the MACS magnetic field, with the 2mL CD of degasification ₃₄ ⁺Cellular segregation damping fluid flushing separator column;

5, with the slow adherent adding separator column of single cell suspension, avoid producing bubble, treat that it flows out the CD of back with 500 μ L degasification naturally ₃₄ ⁺The cellular segregation damping fluid washs uncombined cell, totally 4 times;

6, separator column is shifted out magnetic field, use 1mL CD ₃₄ ⁺Cellular segregation damping fluid pressurization wash-out, the collection component is CD ₃₄ ⁺Cell, counting.

(4) purity check

The cell of immunomagnetic beads mark passes through the magnetic field purifying twice, detects CD through flow cytometer ₃₄ ⁺The purity of cell can reach 97.74% (Fig. 5).

Two, the trophoblastic foundation of stroma cell

1, former generation the mouse stroma cell separation and cultivation

4 ages in week, male BABL/c mouse was 2, and disconnected neck is put to death, and femur and shin bone are got in 70% alcohol immersion sterilization 20-30 minute, remove the soft tissue and the epiphysis end that adhere on the bone.Isolated femur and shin bone are moved into another aseptic plate, take out 5mlPBS with needle tubing marrow is gone out, filter the centrifugal 4min of 1500rpm with 200 mesh filter screens.Sedimentary cell is resuspended in the a-MEM substratum, and the counting back is with 2 * 10 ⁶Individual/bottle is inoculated in the plastic culture bottle.Inoculate back 7 days and change liquid first.Afterwards, changed liquid once in 3-4 days, went down to posterity in 7-10 days.It is standby for the back to pass 2-3.

2, WNT3a gene transfection marrow stromal cell

Discard the nutrient solution in the culturing bottle, add the a-MEM substratum of 1-2ml.Add the packaged venom of 100ul to it then, placed 3 hours in the CO2gas incubator, add the 2ml fresh medium again, hatch 72h, can observe under the fluorescent microscope.

External enlarged culturing is changeed the marrow stromal cell of WNT3a gene, 5.0 * 10 ⁵/ ml inoculates 24 orifice plates, treats that cell grows to acceptance when converging more than 90% ⁶⁰The trophocyte is set up in Co gamma-rays 11Gy irradiation.

3, the carrying out of cultivating altogether

With CD ₃₄ ⁺Cell adds the early stage hematopoietic cell long-term cultivation base Myelocult that contains 50ng/ml rhMGF (rhSCF), 10ng/ml rhIL-3 (recombination human interleukin-3) and 20ng/ml rhFL (recombinant human Flt3 aglucon) ^TMAmong the H5110, be inoculated in 24 orifice plates, cell density is 1.0 * 10 ⁴/ ml, every hole 1ml culture system.Trophocyte in the test group orifice plate is for changeing the marrow stromal cell of WNT3a gene.Control group is not for changeing the marrow stromal cell of WNT3a gene.Every group three hole.24 orifice plates place 37 degrees centigrade, 5%CO ₂Cultivated for 3 weeks in the incubator, half amount is changed liquid, 3 weeks of cultured continuously weekly.

Stream measuring CD117 CD71

Cultivate after 12 days, collect the CD34+ cell (8.0 * 10 that each group is cultivated altogether ⁵/ ml), detecting by the expression level of streaming technology to the CD117/CD71 on co-cultured cell surface, result (Fig. 5) shows the CD117 that changes WNT3A trophoderm group cell, the CD71 expression level all is higher than common trophoderm group respectively.

Red assembly falls to cultivating

In 12 days process of Short-term Culture,, from each hole, got 2000+8000CD in 12 days respectively the 3rd, 7 ₃₄ ⁺Cell inoculation has added the methylcellulose gum premix substratum MethoCult of 5U/mL EPO in 0.5mL ^TMAmong the SF H4236, add, be cultured to the various colonies of counting formation in 16 days by every hole 0.5mL in 24 orifice plates.Blood counting chamber is adopted in cell, colony count cell counting, gets weighted mean value 3 times.Colony count is in the following direct viewing of inverted microscope (Nikon), is a colony greater than the calculation of 50 cells, gets the mean value of 3 parallel holes.Colony density is per 10 ⁴Individual plastidogenetic colony number.Result (as Fig. 6) shows with control group and compares, and changes the ability that the WNT3a marrow stromal cell has the red assembly of tangible promotion to fall to forming as trophoderm.

The structure of embodiment 3, recombinant retrovirus plasmid pMSCV-WNT3a.

For touching plate, also introduce XbaI respectively with mouse placenta cDNA according to the sequence of WNT3a-cDNA coding region, Hpa I and BamHI restriction enzyme site synthetic primer, the condition of primer sequence and polymerase chain reaction thereof (PCR) is as follows:

P1 5′- ATGGCTCCTCTCGGATACCT-3′

P2 5′-

CTACTTGCAGGTGTGCACGT-3′

20 μ l reaction systems comprise: 1ul cDNA (100ng/ul), primer1 and 2 each 0.5 μ l (20 μ M), 1.5 μ l dNTP (2.5mM), 0.25 μ l La Taq archaeal dna polymerase (TakaRa company, 5u/ μ l), 2 μ l, 5 * GC Buffer I, 14.75 μ l aqua sterilisas.Reaction conditions is: 94 ℃ of sex change 3min; 94 ℃ of 45s, 56 ℃ of 45s, 72 ℃ of ο 2min, 28 circulations; 72 ℃ of 7min.It behind the electrophoresis goal gene WNT3a (see figure 1).Reaction product is cloned into pGEM-T eassy carrier (Promega company, the U.S.), obtains cloned plasmids T-WNT3a.

Plasmid digests with restriction enzyme Hpa I and BamH I, reclaiming the purpose fragment is inserted on the Hpa I and Bgl II site of pMSCVneo carrier, the retroviral vector pMSCV-WNT3a of goal gene is carried in acquisition, recombinant plasmid is respectively behind Hpa I, BamH I double digestion, the TAE agarose gel electrophoresis obtains 5.15 respectively, the band of 1kb, meets with expected results; It is carried out forward and reverse order-checking and analysis revealed, and its open reading frame is correct, frameshit does not take place change.Show that the target gene sequences that we clone is correct, recombinant retroviral vector pMSCV-WNT3a successfully constructs (see figure 7).

Foundation and screening that embodiment 4, retrovirus produce cell strain.

The PT67 cell is cultured to 95% with the DMEM/F12 nutrient solution (complete culture solution) that contains 10% foetal calf serum and converges.PMSCV-WNT3a 5 μ g are added 250 μ l serum-free mediums, get LF2000 10 μ l and add the above-mentioned nutrient solution of 250 μ l, both mixing room temperatures are placed 20min.The PT67 cell is washed 2 times with nutrient solution, slowly splashed into DNA-LF2000 mixed solution with the dilution of 1ml nutrient solution.After cultivating 6h, add the nutrient solution that 1ml contains 20% foetal calf serum, change complete culture solution after continuing to cultivate 24h.Went down to posterity by 1: 15 behind the cell transfecting 48h, add and contain 600 μ g/ml G418 screening 14d, form until drug-resistant colonies.Select the cell colony of big health, use the complete culture solution enlarged culturing, contain packing cell supernatant, mensuration virus titer and the freeze-stored cell of virus with preparation.

The mensuration of embodiment 5, recombinant retrovirus pMSCV-WNT3a virus titer and the foundation of PT-WNT3a cell strain.

The NIH3T3 cell routine that growth conditions is good is cultivated 24h to 60% and is converged, and the viral liquid phase of preparation should be done 10 ^-2, 10 ^-4, 10 ^-6Doubly dilution, add polybrene to final concentration be 8 μ g/ml.Remove cell culture fluid, the viral liquid of drawing the 1ml dilution respectively adds in the culture dish, the conventional 5h that cultivates adds the conventional 24h of cultivation of substratum that 2ml contains foetal calf serum, changes the fresh substratum that contains 500 μ g/mlG418, every 3d changes liquid once, after cultivation 14d waited to grow macroscopic colony, the nutrient solution that inclines was to dye with the female Sa of a Ji after the pure formaldehyde fixed, automatically count with clone's calculating instrument, multiply by again behind the extension rate virus titer (cfu).We pick out virus titer＞10 altogether ⁶6 of the packing cells of cfu filter out and have the highest titre (8.8 * 10 ⁷Cfu/ml) packing cell (see figure 8) is set up the packaging cell line PT-WNT3a that high titre is expressed WNT3a after the enlarged culturing.

The detection of wild-type virus in embodiment 6, the recombinant retrovirus liquid.

Above virus titer is measured gained NIH3T3 polyclone with complete culture solution enlarged culturing 48h, collect the gained cell conditioned medium and infect the NIH3T3 cell once more, add 500 μ g/ml G418 screening, cell is all dead behind the 5d.Prompting PT67 cell does not produce detectable helper virus, with the retrovirus of its preparation can infected person and other mammalian cells can be extensive use of and safer.

The expression of embodiment 7, recombinant retrovirus and evaluation.

According to the sequences Design synthetic primer of WNT3a-cDNA coding region, and carry out reverse transcription PCR (RT-PCR) reaction.

Primer sequence P1:5 '-ATGGCTCCTCTCGGATACCT-3 ',

P2：5′-CTACTTGCAGGTGTGCACGT-3′，

Extracting with TRIzol reagent, PT67 cell and the NIH3T3 cell total rna of Buddhist monk's untransfected pMSCV-WNT3a carry out RT-PCR and agarose gel electrophoresis, observe size and be the amplified band of 1058bp, the control cells of untransfected pMSCV-WNT3a is not then seen positive band, and confirmation WNT3a gene is transcribed (the results are shown in Figure 9) in the NIH3T3 cell of PT-WNT3a cell strain and infection pMSCV-WNT3a.

Sequence table

<160>2

<210>1

<211>1059

<212>DNA

＜213〉mouse (Mus musculus)

<400>1

atggctcctc?tcggatacct?cttagtgctc?tgcagcctga?agcaggctct?gggcagctac 60

ccgatctggt?ggtccttggc?tgtgggaccc?cagtactcct?ctctgagcac?tcagcccatt 120

ctctgtgcca?gcatcccagg?cctggtaccg?aagcagctgc?gcttctgcag?gaactacgtg 180

gagatcatgc?ccagcgtggc?tgagggtgtc?aaagcgggca?tccaggagtg?ccagcaccag 240

ttccgaggcc?ggcgttggaa?ctgcaccacc?gtcagcaaca?gcctggccat?ctttggccct 300

gttctggaca?aagccacccg?ggagtcagcc?tttgtccatg?ccatcgcctc?cgctggagta 360

gctttcgcag?tgacacgctc?ctgtgcagag?ggatcagctg?ctatctgtgg?gtgcagcagc 420

cgcctccagg?gctccccagg?cgagggctgg?aagtggggcg?gctgtagtga?ggacattgaa 480

tttggaggaa?tggtctctcg?ggagtttgcc?gatgccaggg?agaaccggcc?ggatgcccgc 540

tctgccatga?accgtcacaa?caatgaggct?gggcgccagg?ccatcgccag?tcacatgcac 600

ctcaagtgca?aatgccacgg?gctatctggc?agctgtgaag?tgaagacctg?ctggtggtcg 660

cagccggact?tccgcaccat?cggggatttc?ctcaaggaca?agtatgacag?tgcctcggag 720

atggtggtag?agaaacaccg?agagtctcgt?ggctgggtgg?agaccctgag?gccacgttac 780

acgtacttca?aggtgccgac?agaacgcgac?ctggtctact?acgaggcctc?acccaacttc 840

tgcgaaccta?accccgaaac?cggctccttc?gggacgcgtg?accgcacctg?caatgtgagc 900

tcgcatggca?tagatgggtg?cgacctgttg?tgctgcgggc?gcgggcataa?cgcgcgcact 960

gagcgacgga?gggagaaatg?ccactgtgtt?ttccattggt?gctgctacgt?cagctgccag 1020

gagtgcacac?gtgtctatga?cgtgcacacc?tgcaagtag 1059

<210>2

<211>352

<212>PRT

＜213〉mouse (Mus musculus)

<400>2

Met?Ala?Pro?Leu?Gly?Tyr?Leu?Leu?Val?Leu?Cys?Ser?Leu?Lys?Gln?Ala

1 5 10 15

Leu?Gly?Ser?Tyr?Pro?Ile?Trp?Trp?Ser?Leu?Ala?Val?Gly?Pro?Gln?Tyr

20 25 30

Ser?Ser?Leu?Ser?Thr?Gln?Pro?Ile?Leu?Cys?Ala?Ser?Ile?Pro?Gly?Leu

35 40 45

Val?Pro?Lys?Gln?Leu?Arg?Phe?Cys?Arg?Asn?Tyr?Val?Glu?Ile?Met?Pro

50 55 60

Ser?Val?Ala?Glu?Gly?Val?Lys?Ala?Gly?Ile?Gln?Glu?Cys?Gln?His?Gln

65 70 75 80

Phe?Arg?Gly?Arg?Arg?Trp?Asn?Cys?Thr?Thr?Val?Ser?Asn?Ser?Leu?Ala

85 90 95

Ile?Phe?Gly?Pro?Val?Leu?Asp?Lys?Ala?Thr?Arg?Glu?Ser?Ala?Phe?Val

100 105 110

His?Ala?Ile?Ala?Ser?Ala?Gly?Val?Ala?Phe?Ala?Val?Thr?Arg?Ser?Cys

115 120 125

Ala?Glu?Gly?Ser?Ala?Ala?Ile?Cys?Gly?Cys?Ser?Ser?Arg?Leu?Gln?Gly

130 135 140

Ser?Pro?Gly?Glu?Gly?Trp?Lys?Trp?Gly?Gly?Cys?Ser?Glu?Asp?Ile?Glu

145 150 155 160

Phe?Gly?Gly?Met?Val?Ser?Arg?Glu?Phe?Ala?Asp?Ala?Arg?Glu?Asn?Arg

165 170 175

Pro?Asp?Ala?Arg?Ser?Ala?Met?Asn?Arg?His?Asn?Asn?Glu?Ala?Gly?Arg

180 185 190

Gln?Ala?Ile?Ala?Ser?His?Met?His?Leu?Lys?Cys?Lys?Cys?His?Gly?Leu

195 200 205

Ser?Gly?Ser?Cys?Glu?Val?Lys?Thr?Cys?Trp?Trp?Ser?Gln?Pro?Asp?Phe

210 215 220

Arg?Thr?Ile?Gly?Asp?Phe?Leu?Lys?Asp?Lys?Tyr?Asp?Ser?Ala?Ser?Glu

225 230 235 240

Met?Val?Val?Glu?Lys?His?Arg?Glu?Ser?Arg?Gly?Trp?Val?Glu?Thr?Leu

245 250 255

Arg?Pro?Arg?Tyr?Thr?Tyr?Phe?Lys?Val?Pro?Thr?Glu?Arg?Asp?Leu?Val

260 265 270

Tyr?Tyr?Glu?Ala?Ser?Pro?Asn?Phe?Cys?Glu?Pro?Asn?Pro?Glu?Thr?Gly

275 280 285

Ser?Phe?Gly?Thr?Arg?Asp?Arg?Thr?Cys?Asn?Val?Ser?Ser?His?Gly?Ile

290 295 300

Asp?Gly?Cys?Asp?Leu?Leu?Cys?Cys?Gly?Arg?Gly?His?Asn?Ala?Arg?Thr

305 310 315 320

Glu?Arg?Arg?Arg?Glu?Lys?Cys?His?Cys?Val?Phe?His?Trp?Cys?Cys?Tyr

325 330 335

Val?Ser?Cys?Gln?Glu?Cys?Thr?Arg?Val?Tyr?Asp?Val?His?Thr?Cys?Lys

340 345 350

Claims (9)

1, a kind of method of inducing hematopoietic stem to the hemopoietic progenitor cell of red blood cell line differentiation, it is characterized in that setting up with transfection WNT3a marrow stromal cell is trophoblastic hematopoietic stem vitro culture system, is divided into CFU-E by the external evoked hematopoietic stem of the active WNT3a albumen of stroma cell excretory.

2, method according to claim 1 is characterized in that described hematopoietic stem can derive from tissues such as human or animal's peripheral blood, Cord blood, marrow.

3, method according to claim 1 is characterized in that described WNT3a gene is one of following nucleotide sequences:

1) dna sequence dna of sequence 1 in the sequence table;

2) with sequence table in the dna sequence dna that limits of sequence 1 have 90% above homology, and the identical function protein DNA sequence of encoding.

4, method according to claim 1, it is characterized in that described marrow stromal cell is the marrow stromal cell of transfection WNT3a gene, this marrow stromal cell people that can originate also can derive from other all animals, comprise build the stroma cell that is and former generation marrow stromal cell.

5, method according to claim 1 is characterized in that the method for described WNT3a transfection marrow stromal cell, is to utilize the method for electric commentaries on classics, viral vector infection or liposome transfection to set up the marrow stromal cell that changes the WNT3a gene.

6, method according to claim 1 is characterized in that the marrow stromal cell to change the WNT3a gene is a trophoderm, by being total to best cultivation and it is carried out amplification in vitro with hematopoietic stem.

7, the marrow stromal cell trophoderm of the described commentaries on classics of claim 4 WNT3a gene is characterized in that adopting the marrow stromal cell digestion of the commentaries on classics WNT3a through cultivating to collect and usefulness ⁶⁰The Co gammairradiation is inoculated and be can be used as trophoderm after adherent.

8, the described virus vector transfection marrow stromal cell that utilizes of claim 5, used virus vector comprises adenovirus, retrovirus, slow virus and adeno-associated virus etc.

9, hematopoietic stem is to the purposes of hemopoietic progenitor cell of red blood cell line differentiation back CFU-E, it is characterized in that inducing erythroid cells after the differentiation to can be preparation and treats the cell preparation of hemopoietic system damages such as hematologic disease such as anaemia that a variety of causes causes, leukemia and chemicotherapy clinically abundant cell source is provided.

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