CN100572528C - A kind of method of amplification in vitro hematopoietic stem - Google Patents
A kind of method of amplification in vitro hematopoietic stem Download PDFInfo
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Abstract
The present invention relates to a kind of method of amplification in vitro hematopoietic stem.The method of amplification in vitro hematopoietic stem provided by the present invention is to cultivate hematopoietic stem with the feeder cell that import the Wnt3a gene as trophoderm.The present invention as trophoderm, experimental results show that this trophoderm secretion provides the Wnt3a albumen of activated native state with the feeder cell that import the Wnt3a gene, express Wnt3a.Compare with the Wnt3a albumen that direct interpolation purifying obtains, not only expanding effect is stable but also application is convenient, with low cost, can realize external a large amount of amplifying candidate stem cell, thereby help solving the problem that hematopoietic stem cell transplantation clinically faces the donor critical shortage.
Description
Technical field
The present invention relates to a kind of method of amplification in vitro hematopoietic stem.
Background technology
Hematopoietic stem cell transplantation has become the treatment means of various pernicious, non-pernicious neoplastic hematologic disorder diseases and heavy dose of cancer chemotherapy patient marrow-reconstitution clinically.Be the required cell concentration that obtains medical treatment, many scientists attempt the means expanding hemopoietic ancestral cells by exsomatize (ex vivo), comprise and use feeder cell and various cytokine couplings etc.But currently used method amplifying candidate stem cell limited amount, and hemopoietic stem cell self ability drop and differentiation is arranged in the process of amplification in vitro, this still can not fundamentally solve the problem that hematopoietic stem cell transplantation faces the donor critical shortage.
Hematopoietic stem has the characteristic of self and reconstitute hematopoiesis.Reya confirmation WNT signal path is brought into play crucial effects in the process of hematopoietic stem self.The albumen of Wnt coded by said gene is a class secretor type glycoprotein, is made up of 350 to 400 amino acid, is positioned at proteic carboxyl terminal comprising 23 or 24 conservative halfcystines and about 50%.Improving its expression level not only can expanding hemopoietic ancestral cells quantity (＞100 times), alleviate its differentiation; And do not influence the function of its reconstitute hematopoiesis.Willert finds that the characteristic of Wnt3a albumen amplification ancestral cells is inseparable with the fat modification structure of himself.The Wnt3a albumen that purifying obtains is very easy to go palmitoylation under ambient conditions, purifying obtains albumen and just loses its activity thereby the ancestral cells that can not be used for increasing (Wnt proteins are lipid-modified and can act as stem cell growthfactors.Nature.2003 May 22 like this; 423 (6938): 448-52.Epub 2003 Apr 27).
At present, in the research of expanding hemopoietic ancestral cells, adopt marrow stromal cell (former generation mouse stroma cell and the ATCC stromal cell lines of including, MS-5, TC-1 etc.) as trophoderm, by cells contacting effect and collaborative other cytokines of its excretory cytokine, thereby hematopoietic stem is produced amplification effect.
Disclosure of the Invention
The method of amplification in vitro hematopoietic stem provided by the present invention is to cultivate hematopoietic stem with the feeder cell that import the Wnt3a gene as trophoderm.
Described Wnt3a gene is the proteinic nucleotide sequence that coding has the amino acid residue sequence of sequence 2 in the sequence table.
Described Wnt3a gene can have one of following nucleotide sequence:
1) SEQ ID № in the sequence table: 1 dna sequence dna;
2) with sequence table in SEQ ID №: 1 dna sequence dna that limits has 90% above homology, and code sequence tabulate in the dna sequence dna of amino acid residue sequence of sequence 2;
3) nucleotide sequence of the dna sequence dna hybridization that under the rigorous condition of height, can limit with the sequence 1 in the sequence table.
Wherein, the rigorous condition of described height for hybridization back with contain 0.1 * SSPE (or 0.1 * SSC), the solution of 0.1%SDS washes film under 65 ℃.
SEQ ID № in the sequence table: 1 by 1059 based compositions, and its encoding sequence is from 5 ' end the 1st to the 1059th bit base.
Described feeder cell can be marrow stromal cell.
Described marrow stromal cell can derive from the people, also can derive from other all vertebratess.Comprise former generation marrow stromal cell and built the marrow stromal cell that is, as MS-5, TC-1.
Can described Wnt3a gene be imported feeder cell by liposome or virus vector.
But described virus vector comprises the carrier of transfecting animal cells such as adenovirus carrier, retroviral vector and adeno-associated virus.
In the aforesaid method, can described Wnt3a gene be imported feeder cell by recombinant adenoviral vector pAd-Wnt3a.
In the aforesaid method, can described Wnt3a gene be imported feeder cell by recombinant retroviral vector pMSCV-Wnt3a.
In order to obtain better effect, import the feeder cell warp of Wnt3a gene
60Co gamma-rays 18Gy irradiation is as trophoderm.
In order to obtain stable expanding effect, the feeder cell of described importing Wnt3a gene are for expressing the cell of Wnt3a.
Description of drawings
Fig. 1 is the pcr amplification electrophoretogram of Wnt3a
Fig. 2 is the electrophoretogram of recombinant virus plasmid pAd-Wnt3a
Fig. 3 carries out the electrophoretogram that enzyme is cut evaluation for Pac I to pAd-Wnt3a recombinant virus plasmid
Fig. 4 is the expression photo of GFP in transfected 293 cells
Fig. 5 detects the proteic expression map of Wnt3a for the Western-blot experiment
Fig. 6 carries out the electrophoretogram that enzyme is cut evaluation to the pMSCV-Wnt3a recombinant retroviral vector for Hpa I and BamH I
Fig. 7 carries out the result that virus titer is measured for pMSCV-Wnt3a infects the NIH3T3 cell
Fig. 8 detects the expression of Wnt3a mRNA in PT67 cell and the NIH3T3 cell for RT-PCR
Fig. 9 detects the expression map of Wnt3a for RT-PCR
Figure 10 detects CD34 for flow cytometer
+The purity curve of cell
Figure 11 forms experiment for the marrow stromal cell of expressing Wnt3a as trophoblastic CFC colony
Figure 12 forms experiment for the marrow stromal cell of expressing Wnt3a as trophoblastic HPP-CFC colony
The best mode that carries out an invention
One, the structure of recombinant adenoviral vector
1, homologous recombination produces recombinant adenovirus plasmid in the bacterium
(1) 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 BamH I 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), each 0.5 μ l (20 μ M) of P1 and P2,1.5 μ l dNTP (2.5mM), 0.25 μ l La Taq archaeal dna polymerase (TakaRa company, 5u/ μ l), 2 μ l, 5 * GC damping fluid I (TakaRa company), 14.75 μ l aqua sterilisas.Reaction conditions is: 94 ℃ of sex change 3min; 94 ℃ of 45s, 56 ℃ of 45s, 72 ℃ of 2min, 28 circulations; 72 ℃ of 7min.Electrophoresis result as shown in Figure 1, among Fig. 1, swimming lane 1 is a goal gene, swimming lane M is DNA marker (DL2000), the arrow indication be goal gene Wnt3a electrophoretic band, size is 1059bp.Amplified fragments reclaims rear clone to pGEM-T easy carrier (Promega company, the U.S.), obtain cloned plasmids T-Wnt3a, carry out gene sequencing, the result shows the nucleotide sequence that the Wnt3a gene that obtains of amplification has SEQ ID NO:1 in the sequence table (1059 bases), its encoding sequence is from 5 ' end the 1st to the 1059th bit base, the amino acid residue sequence of sequence 2 in the code sequence tabulation.
Extracting the T-Wnt3a plasmid digests with restriction enzyme XbaI, recovery Wnt3a gene fragment is inserted into pAdTrack and (on the XbaI site of pAdTrack from AdEasy Vector System (QuantumBiotechnologies) carrier, obtains 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.The target gene sequences that shows the clone is correct, and recombinant plasmid pAdTrack-Wnt3a successfully constructs.
After getting 1ug pAdTrack-Wnt3a usefulness Pme I linearizing, carry out 1% agarose gel electrophoresis, gel reclaims the purpose fragment of 10000bp, uses the calf intestinal alkaline phosphatase dephosphorylation, and 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.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 recombinant plasmid 2 (recombinant plasmid 1, recombinant plasmid 2 are respectively from the difference of pAd-Wnt3a clone), and swimming lane M is DNA marker (DL15000), the arrow indication be the plasmid electrophoretic band.
2, 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 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 be pAd-Wnt3a through Pac I restriction enzyme digestion and electrophoresis result, swimming lane M is DNA marker (DL15000), the arrow indication cut the electrophoretic band that obtains for pAd-Wnt3a recombinant virus plasmid through Pac I enzyme.
3, recombinant viral vector is at the packing of 293 cells, the purifying and the titer determination of virus
Extract plasmid DNA 10ug in the DH5 α bacterium that contains recombinant virus plasmid pAd-Wnt3a from step 1, cut with Pac I enzyme and make it linearization.Carry out rotaring redyeing 293 cell according to producer's explanation with lipofectAMINE (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/L.
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 70% ethanol disinfection 20-30 minute, get femur and shin bone, remove the soft tissue and the epiphysis end that adhere on the bone.Isolated femur and footpath bone are moved into another aseptic plate, take out 5ml PBS with needle tubing marrow is gone out, filter the centrifugal 4min of 1500rpm with 200 mesh filter screens.Sedimentary cell is resuspended in the 10ml glass centrifuge tube 2 * 10
6Individual cell inoculation is 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 3-4.
2, set up marrow stromal cell efficient, stably express Wnt3a
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, fluorescent microscope can be observed the expression of green fluorescent protein down.
External enlarged culturing is changeed the marrow stromal cell of Wnt3a, 5.0 * 10
5/ ml inoculates 24 orifice plates, treats that cell grows to acceptance when converging more than 90%
60Lysing cell after 48 hours is cultivated in Co gamma-rays 18Gy irradiation, with anti-Wnt3a (available from R﹠amp; D company) being one anti-, is two anti-with the rabbit anti-mouse igg of horseradish peroxidase-labeled (available from middle mountain company), carries out the Western-blot experiment, and the result has detected the proteic expression of Wnt3a as shown in Figure 5.Proof obtains changeing the marrow stromal cell of Wnt3a, thereby sets up the trophocyte.Swimming lane 1 is the marrow stromal cell of untransfected Wnt3a among Fig. 5, and swimming lane 2 is the marrow stromal cell of transfection Wnt3a.
One, the structure of recombinant retrovirus plasmid pMSCV-Wnt3a and evaluation thereof
1, the structure of recombinant retrovirus plasmid pMSCV-Wnt3a
The T-Wnt3a plasmid that extracts among the embodiment 1 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 (available from Clontech company), 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.The target gene sequences that shows the clone is correct, recombinant retroviral vector pMSCV-Wnt3a successfully constructs (Fig. 6), swimming lane 1 is the electrophoresis result of pMSCV-Wnt3a through Hpa I, BamH I double digestion among Fig. 6, swimming lane M is DNA marker (DL2000), the arrow indication be goal gene Wnt3a band, size is 1059bp.
2, retrovirus produces the foundation and the screening of cell strain
PT67 cell (available from Clontech company) 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 Lipofectamine
TM2000 (available from Invitrogen) 10 μ l add the above-mentioned nutrient solution of 250 μ l, and both mixing room temperatures are placed 20min.The PT67 cell is washed 2 times with nutrient solution, slowly splashed into DNA-Lipofectamine with the dilution of 1ml nutrient solution
TM2000 mixed solutions.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, collect supernatant, with the membrane filtration of 0.45um, packing is frozen.
3, the foundation of the mensuration of recombinant retrovirus pMSCV-Wnt3a virus titer and 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 adds 1, and poly-Methobromide (polybrene) to the final concentration of 5-dimethyl-1,5 phenodiazine 11 methylene radical is 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/ml G418, every 3d changes liquid once, after cultivation 14d grew 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).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 (Fig. 7) is set up the packaging cell line PT-Wnt3a that high titre is expressed Wnt3a after the enlarged culturing.Among Fig. 7, A:10
-2Doubly dilution; B:10
-4Doubly dilution; C:10
-6Doubly dilution.
4, the detection of wild-type virus in the recombinant retrovirus liquid
Virus titer in the above step is measured gained NIH3T3 polyclone complete culture solution enlarged culturing 48h, and centrifugal back is collected supernatant and is infected the NIH3T3 cell once more, adds 500 μ g/ml G418 screening, and 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.
5, the expression of 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 P3:5 '-ATGGCTCCTCTCGGATACCT-3 ', P4:5 '-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 the amplified band of 1059bp, the control cells of untransfected pMSCV-Wnt3a (NIH3T3 cell) is not then seen positive band, and confirmation Wnt3a gene is transcribed (Fig. 8) in the NIH3T3 cell of PT-Wnt3a cell strain and infection pMSCV-Wnt3a.Among Fig. 8, swimming lane 1 is people's placenta lysate (positive control), and swimming lane 2 is the PT-Wnt3a cell, and swimming lane 3 is the PT67 cell, and swimming lane 4 is for infecting the NIH3T3 cell of pMSCV-Wnt3a, and swimming lane 5 is the NIH3T3 cell.
Two, trophoblastic foundation
Before the virus infection 20h, inoculation 1 * 10
5Individual separation and Culture former generation mouse bone marrow in 6 orifice plates, routine is cultured to 40% and converges, filter the reorganization reversion rate virus supernatant liquor infecting mouse marrow stromal cell of collecting, pMSCV-Wnt3a repeated infection 3 times (each 24h at interval) with nylon leaching film respectively through 0.45um.The substratum of changing the fresh 700ug/ml of containing G418 screens, every 3d changes liquid once, single resistance clone enlarged culturing behind the cultivation 14d, extract the resistance clone cell total rna with TRIzol reagent, with P3 and P4 is that primer carries out RT-PCR detection and agarose gel electrophoresis according to ordinary method, and the result obtains the purpose band of 1059bp size as shown in Figure 9, the stably express strain of proof commentaries on classics Wnt3a is set up thus, can be used as the trophocyte.Among Fig. 9, swimming lane 1 is the RT-PCR amplification of resistance clone, and swimming lane M is DNA marker (DL2000).
Embodiment 3, with by the marrow stromal cell of the expression Wnt3a of recombinant adenoviral vector transfection as the trophoderm expanding hemopoietic do/ancestral is thin
One, the acquisition of hematopoietic stem
(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, 0.24gKH
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 is pressed 1: 1 volume ratio and PBS mixing, by 4: 1 volume ratio and 0.5% methylcellulose gum mixing, leaves standstill 30min under the room temperature again, 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 the 10mLPBS 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, 1, the centrifugal 25min of 000rpm 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 1.5mL Ep pipe counting to the cellular segregation damping fluid.
(2) separation of C D from MNC
34 +Cell (adopting the miniMACS separation system)
1, per 10
8Individual Cord Blood Mononuclear Cell (MNC) is suspended in 4 ℃ of CD that preset 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 (FcR encapsulant, magnetic bead link coupled CD
34Monoclonal antibody is all available from Miltenyi Biotec company), mixing is hatched 30min for 6-12 ℃;
2, add 500 μ lCD
34 +The cellular segregation damping fluid, 4 ℃, 2, the centrifugal 3min of 000rpm 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 (MACS separator column and magnetic field are all available from Miltenyi Biotec company), 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.
(3) purity check
Twice in the cell of immunomagnetic beads mark is divided into two groups by the magnetic field purifying, and one group is test group, detects the CD34 that separation obtains with the anti-CD34 antibody (available from Miltenyi Biotec company) of PE mark
+Cell purity, another group be for control group, the anti-mouse IgG (available from middle mountain) that adopts the PE mark with separate the CD34 that obtains
+Cell is hatched.Detect CD34 through flow cytometer
+The purity of cell can reach 97.74% (Figure 10).
Separate the hemopoietic stem cell surface that obtains and all express CD34,
Wherein, test group is
So the anti-CD34 antibody (available from Miltenyi Biotec company) with the PE mark in the experiment detects the CD34 that separation obtains
+The anti-mouse IgG (available from middle mountain) that cell purity, control group adopt the PE mark with separate the CD34 that obtains
+Cell is hatched.
Two, expanding hemopoietic ancestral cells
The trophoderm amplification expanding hemopoietic ancestral cells of using embodiment 1 to set up in accordance with the following methods:
1, the carrying out of cultivating altogether
With CD
34 +Cell adds and contains 50ng/ml rhMGF (rhSCF, available from peprotech company), 10ng/ml rhIL-3 (recombination human interleukin-3, available from peprotech company) and the early stage hematopoietic cell long-term cultivation base Myelocult of 20ng/ml rhFL (recombinant human Flt3 aglucon is available from peprotech company)
TMAmong the H5110 (available from Stem cell company), 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 the trophocyte that embodiment 1 sets up.Control group is not for changeing the marrow stromal cell of Wnt3a.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.
2, mixing colony cultivates
From step 1, get 2000-8000CD respectively in each hole in 3 weeks of cultured continuously
34 +Cell inoculation contains 5ug/L GM-CSF in 0.5mL, and (grain is a macrophage colony stimulating factor, available from peprotech company), 5ug/L IL-3,50ug/L SCF (stem cell factor, available from peprotech company), 2U/mL EPO (erythropoietin is available from peprotech company), 5 * 10
-5The methylcellulose gum premix substratum MethoCult of mol/L 2-ME (the β mercaptoethanol is available from peprotech company), 10%FBS, 10%HS (horse serum is available from peprotech company)
TMAmong the SF (available from Stemcell company), 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 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.The result shows the CD that the trophocyte that sets up with embodiment 1 cultivates as trophoderm (experimental group) as shown in figure 11
34 +Plastidogenetic colony number is compared CFC (colony forming cell) with control group increase 1.8-3.2 doubly, and among Figure 11, Image to left is contrast, and Image to right is an experimental group.
3, the colony of high proliferative potential colony forming cell (HPP-CFC) is cultivated and counting
From step 1, get 2000-8000CD respectively in each hole in 3 weeks of cultured continuously
34 +Cell inoculation is in 0.5mL methylcellulose gum premix substratum MethoCult
TM(Stem cell company among the SF, H4434), add by every hole 0.5mL in 24 orifice plates, every hole drips the IMDM that 0.2mL contains 20% cord blood plasma when being cultured to 14d, continue to be cultured to 28d, the cell colony of counting diameter＞0.5mm (cell count＞50,000) at inverted microscope (Nikon) down is a HPP-CFC, gets the mean value of 3 parallel holes.HPP-CFC content is promptly through converting per 10
4Individual plastidogenetic HPP-CFC colony number.The result shows the CD that the trophocyte that sets up with embodiment 1 cultivates as trophoderm (experimental group) as shown in figure 12
34 +The HPP-CFC colony density of cell is 2.2-4.3 times of control group.Each figure is respectively the observed difform colony of microscopically among Figure 12.
Commercial Application
The present invention with the feeder cells that import the Wnt3a gene, express Wnt3a as trophoderm, experiment Prove that the secretion of this trophoderm provides the Wnt3a albumen of activated native state. With direct interpolation purifying The Wnt3a albumen that obtains is compared, and not only expanding effect is stable but also application is convenient, with low cost, Can realize external a large amount of amplifying candidate stem cell, candidate stem cell moves thereby be conducive to solve clinically Plant the problem that faces the donor critical shortage. The foundation of the method is expected to be clinically bone-marrow transplantation, blood The researchs such as the cell therapy of systemic disease treatment, hematopoietic stem/progenitor and gene therapy provide enough Desirable target cell, and be that research hematopoietic regulation and the self mechanism of inquiring into stem cell are offered help, Also amplification and the clinical practice for other stem cell provides new theory and method; While the method Foundation will have in the regenerative medicine on basis take genetic engineering, cell engineering and even organizational project etc. Splendid application prospect, and will have a tremendous social and economic benefits.
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 (14)
1, a kind of method of amplification in vitro hematopoietic stem is to cultivate hematopoietic stem with the feeder cell that import the Wnt3a gene as trophoderm.
2, method according to claim 1 is characterized in that: described Wnt3a gene is the proteinic nucleotide sequence of the amino acid residue sequence of sequence 2 in the code sequence tabulation.
3, method according to claim 2 is characterized in that: described Wnt3a gene is SEQ ID № in the sequence table: the dna sequence dna of 1 expression.
4, method according to claim 1 is characterized in that: described feeder cell are marrow stromal cell.
5, according to the described method of claim 4, it is characterized in that: described marrow stromal cell comprises former generation marrow stromal cell or built the marrow stromal cell that is.
6, according to the described method of arbitrary claim among the claim 1-5, it is characterized in that: described Wnt3a gene imports feeder cell by liposome or virus vector.
7, method according to claim 6 is characterized in that: described virus vector comprises adenovirus carrier, retroviral vector or adeno-associated virus.
8, method according to claim 7 is characterized in that: in the described method, by recombinant adenoviral vector pAd-Wnt3a described Wnt3a gene is imported feeder cell.
9, method according to claim 7 is characterized in that: in the described method, by recombinant retroviral vector pMSCV-Wnt3a described Wnt3a gene is imported feeder cell.
10, according to the described method of arbitrary claim among the claim 1-5, it is characterized in that: the feeder cell of described importing Wnt3a gene are for expressing the cell of Wnt3a.
11, method according to claim 6 is characterized in that: the feeder cell of described importing Wnt3a gene are for expressing the cell of Wnt3a.
12, method according to claim 7 is characterized in that: the feeder cell of described importing Wnt3a gene are for expressing the cell of Wnt3a.
13, method according to claim 8 is characterized in that: the feeder cell of described importing Wnt3a gene are for expressing the cell of Wnt3a.
14, method according to claim 9 is characterized in that: the feeder cell of described importing Wnt3a gene are for expressing the cell of Wnt3a.
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| PCT/CN2004/001109 WO2006034602A1 (en) | 2004-09-28 | 2004-09-28 | Methods for porliferation of hematopoietic stem/progenitor cell in vitro |
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| CN100572528C true CN100572528C (en) | 2009-12-23 |
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| CN114958744A (en) * | 2022-07-12 | 2022-08-30 | 上海派思维新生物医药科技有限公司 | In-vitro stem cell expanded culture method and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1122368A (en) * | 1994-11-04 | 1996-05-15 | 上海贝特生物技术有限公司 | Cloning method of hemotopoietic stem cell, ancestor cell and megacaryocyte |
| CN1280187A (en) * | 1999-07-13 | 2001-01-17 | 中国人民解放军第二军医大学 | Method of extracorporeal cloning hemopoietic stem cell |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5851984A (en) * | 1996-08-16 | 1998-12-22 | Genentech, Inc. | Method of enhancing proliferation or differentiation of hematopoietic stem cells using Wnt polypeptides |
| US6159462A (en) * | 1996-08-16 | 2000-12-12 | Genentech, Inc. | Uses of Wnt polypeptides |
| WO1999057248A1 (en) * | 1998-04-30 | 1999-11-11 | President And Fellows Of Harvard College | Induction of neuronal regeneration |
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2004
- 2004-09-28 CN CNB2004800439973A patent/CN100572528C/en active Active
- 2004-09-28 WO PCT/CN2004/001109 patent/WO2006034602A1/en active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1122368A (en) * | 1994-11-04 | 1996-05-15 | 上海贝特生物技术有限公司 | Cloning method of hemotopoietic stem cell, ancestor cell and megacaryocyte |
| CN1280187A (en) * | 1999-07-13 | 2001-01-17 | 中国人民解放军第二军医大学 | Method of extracorporeal cloning hemopoietic stem cell |
Non-Patent Citations (1)
| Title |
|---|
| Wnt proteins are lipid-modified and can act as stem cellgrowth factors. Willert K等.Nature,Vol.423 No.6938. 2003 * |
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| WO2006034602A1 (en) | 2006-04-06 |
| CN101018855A (en) | 2007-08-15 |
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