CN106754712A - The method that induction of cord blood mononuclear cell is divided into myeloid cell - Google Patents
The method that induction of cord blood mononuclear cell is divided into myeloid cell Download PDFInfo
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Abstract
The invention discloses method, kit and system that a kind of induction of cord blood mononuclear cell is divided into myeloid cell.The method that the induction of cord blood mononuclear cell is divided into myeloid cell includes：Amplification cultivation is carried out to the human umbilical cord blood mononuclear cell using the first culture medium, so as to the candidate stem cell after being expanded；And Fiber differentiation is carried out to the candidate stem cell after the amplification using the second culture medium, to obtain the myeloid cell, first culture medium is spemspan culture mediums, and second culture medium is the X VIVO containing the first additiveTM15 culture mediums, and first additive includes recombined human thrombopoietin, rhMGF and recombinant human granulocyte colony stimulating factor.The method of the present invention is obtained in that a large amount of myeloid cells, and induction differentiation efficiency is higher.
Description
Technical field
The present invention relates to bioengineering field.In particular it relates to induction of cord blood mononuclear cell is divided into grain
It is the method for cell.More particularly it relates to induction of cord blood mononuclear cell is divided into the method for myeloid cell, reagent
Box and system.
Background technology
Myeloid cell is the important component of blood non-specific immune systems, particularly neutrophil leucocyte, in non-spy
Important physiological function is performed in specific immunological response, when inflammation occurs, the neutrophil leucocyte with chemotaxis reaches scorching
Disease position, swallows microbial pathogens, because neutrophil leucocyte is contained within a large amount of lysosomal enzymes, will can swallow into intracellular thin
Bacterium and fragment of tissue are decomposed, killed.The reasons such as chemicals, ionising radiation (chemicotherapy), severe infections can cause bone marrow injury
Reduced with myeloid cell.Myeloid cell lack often result in it is dizzy, weak, in addition it may also occur that severe infections symptom, infection site
It is common with lung, urinary tract, skin etc., easily there is septicopyemia or septicemia, the death rate is up to 25%.Neutrophilic granulocytopenia is
The syndrome produced because peripheral blood neutrophil quantity is reduced, is most common complication in oncotherapy, is being received
Incidence is 10%-20% in the entity tumor patient of chemotherapy, and the death rate reaches 9.8%.Candidate stem cell induction differentiation grain is thin
Born of the same parents' approach new for clinical practice is provided.
However, at present the amplification of myeloid cell and induction system also in the presence of induction, differentiation efficiency is low, cell quantity is few etc. asks
Topic, still requires study.
The content of the invention
It is contemplated that at least solving one of technical problem present in prior art to a certain extent.Therefore, this hair
It is bright to propose method, kit and system that a kind of induction of cord blood mononuclear cell is divided into myeloid cell.Using the method,
Kit and system are obtained in that a large amount of myeloid cells, and induction differentiation efficiency is higher.
Therefore, in one aspect of the invention, the present invention proposes a kind of induction of cord blood mononuclear cell and is divided into grain
It is the method for cell.Embodiments in accordance with the present invention, methods described includes：Using the first culture medium to the single core of the Cord blood
Cell carries out amplification cultivation, so as to the candidate stem cell after being expanded；And after utilizing the second culture medium to the amplification
Candidate stem cell carries out Fiber differentiation, and to obtain the myeloid cell, first culture medium is spemspan culture mediums, institute
It is the X-VIVO containing the first additive to state the second culture mediumTM15 culture mediums, and first additive includes that recombinant human is small
Plate generation element, rhMGF and recombinant human granulocyte colony stimulating factor.
It is surprisingly found by the inventors that, human umbilical cord blood mononuclear cell is carried out into amplification cultivation in advance, to obtain substantial amounts of hematopoiesis
Stem cell, then Fiber differentiation is carried out to it, to obtain substantial amounts of myeloid cell.Obtained using the induction pattern of above-mentioned two benches culture
Myeloid cell amount apparently higher than human umbilical cord blood mononuclear cell directly is carried out into Fiber differentiation.Further, inventor's discovery,
First medium component and the second medium component significantly affect induction differentiation efficiency, if the first medium component and the second culture
Based component is unsuitable for human umbilical cord blood mononuclear cell amplification for candidate stem cell and its is divided into myeloid cell, can cause myeloid cell
Induction differentiation efficiency is relatively low.If for example, the first medium component is uncomfortable, may easily cause that human umbilical cord blood mononuclear cell amplification is
The quantity of candidate stem cell is reduced, so as to cause myeloid cell yield relatively low；If the second medium component is uncomfortable, may easily make
Candidate stem cell directed differentiation is obtained for the quantity of myeloid cell is reduced, myeloid cell yield is also resulted in relatively low.Therefore, inventor
Optimal first culture medium and the second medium component are obtained by further investigation, as a result, myeloid cell induction differentiation efficiency
It is higher, so as to obtain a large amount of myeloid cells.
Embodiments in accordance with the present invention, the method that above-mentioned induction of cord blood mononuclear cell is divided into myeloid cell can be with
Further there is following additional technical feature：
Embodiments in accordance with the present invention, the spemspan culture mediums contain the second additive, the second additive bag
Include：Recombined human thrombopoietin, rhMGF, recombinant human interleukin -3 and combining human interleukins
Element -6.Thus, method according to embodiments of the present invention is obtained in that a large amount of candidate stem cells, and induction differentiation efficiency is higher.
Embodiments in accordance with the present invention, second additive further includes following at least one：FMS sample tyrosine
The reinforcing agent of kinases 3, aryl hydrocarbon receptor inhibitor and aryl hydrocarbon receptor antagonist.According to a preferred embodiment of the invention, it is described
Second additive further includes the reinforcing agent of aryl hydrocarbon receptor inhibitor and aryl hydrocarbon receptor antagonist.Thus, according to this hair
The method of bright embodiment is obtained in that a large amount of candidate stem cells, and induction differentiation efficiency is higher.
Embodiments in accordance with the present invention, in second culture medium, the concentration of the recombined human thrombopoietin is
30~100ng/mL, preferably 50ng/mL；The concentration of the rhMGF is 30~100ng/mL, preferably 50ng/
mL；The concentration of the granulocyte colony stimulating factor is 30~100ng/mL, preferably 50ng/mL.Thus, implemented according to the present invention
The method of example is obtained in that a large amount of myeloid cells, and induction differentiation efficiency is higher.
Embodiments in accordance with the present invention, in first culture medium, the concentration of the recombined human thrombopoietin is 5
~20ng/mL, preferably 10ng/mL；The concentration of the rhMGF is 30~100ng/mL, preferably 50ng/mL；Institute
The concentration for stating recombinant human interleukin -3 is 10~50ng/mL, preferably 20ng/mL；The recombinant human interleukin-6
Concentration is 10~50ng/mL, preferably 20ng/mL；The concentration of the aryl hydrocarbon receptor inhibitor is 0.3~0.8 μM, preferably 0.5 μ
M；The concentration of the reinforcing agent of the aryl hydrocarbon receptor antagonist is 0.3~0.8 μM, preferably 0.5 μM.Thus, according to of the invention real
The method for applying example is obtained in that a large amount of candidate stem cells, and induction differentiation efficiency is higher.
Embodiments in accordance with the present invention, the time of the amplification cultivation is 5~10 days, preferably 7 days.Thus, according to this hair
The method of bright embodiment is obtained in that a large amount of candidate stem cells, and induction differentiation efficiency is higher.
Embodiments in accordance with the present invention, the time of the Fiber differentiation is 14~25 days, preferably 21 days.Thus, according to this
The method of inventive embodiments is obtained in that a large amount of myeloid cells, and induction differentiation efficiency is higher.
In another aspect of this invention, the present invention proposes one kind and is divided into grain system for induction of cord blood mononuclear cell
The kit of cell.Embodiments in accordance with the present invention, the kit includes：First culture medium, first culture medium is used for
Amplification cultivation is carried out to the human umbilical cord blood mononuclear cell, so as to the candidate stem cell after being expanded；And second culture medium,
Second culture medium is used to carry out Fiber differentiation to the candidate stem cell after the amplification, to obtain the myeloid cell,
Wherein, first culture medium and the second culture medium are that induction of cord blood mononuclear cell as previously described is divided into grain system
The method definition of cell.Thus, kit according to embodiments of the present invention is obtained in that a large amount of myeloid cells, induction differentiation effect
Rate is higher.
In another aspect of this invention, the present invention proposes one kind and is divided into grain system for induction of cord blood mononuclear cell
The system of cell.Embodiments in accordance with the present invention, the system includes：First culture apparatus, first culture apparatus is used for
Amplification cultivation is carried out to the human umbilical cord blood mononuclear cell using the first culture medium, so as to the candidate stem cell after being expanded；
And second culture apparatus, second culture apparatus is used to enter the candidate stem cell after the amplification using the second culture medium
Row Fiber differentiation, to obtain the myeloid cell, wherein, first culture medium and the second culture medium are as described previously
Induction of cord blood mononuclear cell be divided into myeloid cell method definition.Thus, system according to embodiments of the present invention
A large amount of myeloid cells are obtained in that, induction differentiation efficiency is higher.
Additional aspect of the invention and advantage will be set forth in part in the description, and will partly become from the following description
Obtain substantially, or recognized by practice of the invention.
Brief description of the drawings
Of the invention above-mentioned and/or additional aspect and advantage will become from description of the accompanying drawings below to embodiment is combined
Substantially and be readily appreciated that, wherein：
Fig. 1 shows that induction of cord blood mononuclear cell according to an embodiment of the invention is divided into the side of myeloid cell
The schematic flow sheet of method；
Fig. 2 shows external evoked myeloid cell aspect graph (1000 ×) according to an embodiment of the invention；
Fig. 3 shows external evoked myeloid cell CD18 expression (400 ×) according to an embodiment of the invention；And
Fig. 4 shows the microscope figure of external evoked myeloid cell phagocytic function according to an embodiment of the invention
(1000×)。
Specific embodiment
Embodiments of the invention are described below in detail.The embodiments described below is exemplary, is only used for explaining this hair
It is bright, and be not considered as limiting the invention.
It should be noted that term " first ", " second " are only used for describing purpose, and it is not intended that indicating or implying phase
To importance or the implicit quantity for indicating indicated technical characteristic.Thus, " first " is defined, the feature of " second " can be with
Express or implicitly include one or more this feature.Further, in the description of the invention, unless otherwise saying
Bright, " multiple " is meant that two or more.
The present invention proposes a kind of induction of cord blood mononuclear cell and is divided into the method for myeloid cell, kit and is
System, will be described in greater detail respectively below.
The method that induction of cord blood mononuclear cell is divided into myeloid cell
In one aspect of the invention, the present invention proposes a kind of induction of cord blood mononuclear cell and is divided into myeloid cell
Method.Embodiments in accordance with the present invention, referring to Fig. 1, the method includes amplification cultivation S100 and Fiber differentiation S200.By
This, method according to embodiments of the present invention is obtained in that a large amount of myeloid cells, and induction differentiation efficiency is higher.It is described more fully below
The method that induction of cord blood mononuclear cell is divided into myeloid cell.
Embodiments in accordance with the present invention, the method that induction of cord blood mononuclear cell is divided into myeloid cell includes：
S100 amplification cultivations
In this step, amplification cultivation is carried out to human umbilical cord blood mononuclear cell using the first culture medium, to obtain hematopoiesis
Stem cell.
S200 Fiber differentiations
In this step, Fiber differentiation is carried out to the candidate stem cell after amplification using the second culture medium, to obtain grain
It is cell.
It is surprisingly found by the inventors that, human umbilical cord blood mononuclear cell is carried out into amplification cultivation in advance, to obtain substantial amounts of hematopoiesis
Stem cell, especially CD34+Candidate stem cell, then Fiber differentiation is carried out to it, to obtain substantial amounts of myeloid cell.Using above-mentioned
The myeloid cell amount that the induction pattern of two benches culture is obtained by human umbilical cord blood mononuclear cell apparently higher than directly carrying out induction training
Support.Further, inventor has found that the first medium component and the second medium component significantly affect induction differentiation efficiency, if
First medium component and the second medium component are unsuitable for human umbilical cord blood mononuclear cell amplification and its are divided into myeloid cell, meeting
So that induction differentiation efficiency is relatively low.If for example, the first medium component is uncomfortable, may easily cause that human umbilical cord blood mononuclear cell expands
The quantity for increasing to candidate stem cell is reduced, so as to cause myeloid cell yield relatively low；If the second medium component is uncomfortable, Ke Nengrong
Easily cause that candidate stem cell directed differentiation is reduced for the quantity of myeloid cell, also result in myeloid cell yield relatively low.
Therefore, inventor obtains optimal first culture medium and the second medium component, specifically, first by further investigation
Culture medium is spemspan culture mediums, and the second culture medium is the X-VIVO containing the first additiveTM15 culture mediums, and the first addition
Thing includes restructuring recombined human thrombopoietin (rhTPO), rhMGF (rhSCF) and recombinant humangranulocyte collection
G-CSF (rhG-CSF).
Inventor has found, using X-VIVOTMMyeloid cell quantity obtained by the induction differentiation of 15 culture mediums is significantly more than it
His culture medium, such as SCGM culture mediums.Further, inventor's discovery, X-VIVOTMCell factor (the is added in 15 culture mediums
One additive) so that candidate stem cell is further divided into myeloid cell, and the species of cell factor significantly affects induction differentiation
Efficiency.And then, inventor by many experiments find, restructuring recombined human thrombopoietin, rhMGF and
Recombinant human granulocyte colony stimulating factor plays synergy, can remarkably promote human umbilical cord blood mononuclear cell and be induced to differentiate into grain
It is cell.
Embodiments in accordance with the present invention, spemspan culture mediums contain the second additive, and the second additive includes：Restructuring weight
Group human thrombopoietin, rhMGF, recombinant human interleukin -3 and recombinant human interleukin-6.Hair
A person of good sense by many experiments find, recombined human thrombopoietin, rhMGF, recombinant human interleukin -3 with
And recombinant human interleukin-6 is used as growth factor, can remarkably promote human umbilical cord blood mononuclear cell amplification, acquisition it is a large amount of
And activity candidate stem cell higher, especially CD34+Candidate stem cell, consequently facilitating subsequently induction differentiation obtains substantial amounts of grain
It is cell.
Embodiments in accordance with the present invention, the second additive further includes following at least one：FMS sample EGFR-TKs
The reinforcing agent (UM729) of 3 (FLt3), aryl hydrocarbon receptor inhibitor (StemRegenin 1, SR1) and aryl hydrocarbon receptor antagonist.
Inventor has found, by adding at least one expansion that can further improve human umbilical cord blood mononuclear cell of FLt3, SR1 and UM729
Increasing Efficiency, the especially a large amount of and activity of acquisition candidate stem cell higher, CD34+Candidate stem cell, consequently facilitating subsequently luring
Lead differentiation and obtain substantial amounts of myeloid cell, it has further been found that, when simultaneously containing SR1 and UM729, best results.
Embodiments in accordance with the present invention, in the first culture medium, the concentration of recombined human thrombopoietin is 5~20ng/
ML, preferably 10ng/mL；The concentration of rhMGF is 30~100ng/mL, preferably 50ng/mL；Recombinant human leucocyte
The concentration of interleukin -3 is 10~50ng/mL, preferably 20ng/mL；The concentration of recombinant human interleukin-6 is 10~50ng/mL,
It is preferred that 20ng/mL；The concentration of SR1 is 0.3~0.8 μM, preferably 0.5 μM；The concentration of UM729 is 0.3~0.8 μM, preferably 0.5 μ
M。
Inventor has found that the amplification that the concentration of cell factor significantly affects human umbilical cord blood mononuclear cell in the first culture medium is
Candidate stem cell, and then influence the induction differentiation efficiency of myeloid cell.And then, inventor obtains above-mentioned optimal by many experiments
Concentration, thereby, it is possible to effectively improve the amplification efficiency of human umbilical cord blood mononuclear cell, the hematopoiesis higher of a large amount of and activity of acquisition
Stem cell, especially CD34+Candidate stem cell, consequently facilitating subsequently induction differentiation obtains substantial amounts of myeloid cell.However, other
Concentration effect is not good.
Embodiments in accordance with the present invention, in the second culture medium, the concentration of recombined human thrombopoietin for 30~
100ng/mL, preferably 50ng/mL；The concentration of rhMGF is 30~100ng/mL, preferably 50ng/mL；Granulocyte
The concentration of colony stimulating factor is 30~100ng/mL, preferably 50ng/mL.Inventor's discovery, cell factor in the second culture medium
Concentration significantly affect induction differentiation efficiency.And then, inventor obtains above-mentioned optimal concentration by many experiments, thereby, it is possible to
A large amount of myeloid cells are obtained, induction differentiation efficiency is higher.However, other concentration effects are not good.
Embodiments in accordance with the present invention, the time of amplification cultivation is 5~10 days.Thus, the Hematopoietic Stem obtained by cultivating is thin
Born of the same parents are in exponential phase of growth, and the activity of cell is high.According to a preferred embodiment of the invention, the time of amplification cultivation is 7 days.By
This, candidate stem cell form after the amplification in exponential phase of growth is good, cytoactive is higher, is conducive to induction differentiation to obtain grain
It is cell.
Embodiments in accordance with the present invention, the time of Fiber differentiation is 14~25 days.Thus, the grain system obtained by induction differentiation
Cell number is more.According to a preferred embodiment of the invention, the time of Fiber differentiation is 21 days.Thus, induction differentiation efficiency is optimal.
The kit of myeloid cell is divided into for induction of cord blood mononuclear cell
In another aspect of this invention, the present invention proposes one kind and is divided into grain system for induction of cord blood mononuclear cell
The kit of cell.Embodiments in accordance with the present invention, the kit includes：First culture medium, the first culture medium is used for umbilical cord
Blood mononuclear cell carries out amplification cultivation, so as to the candidate stem cell after being expanded；And second culture medium, the second culture medium
For carrying out Fiber differentiation to the candidate stem cell after amplification, to obtain myeloid cell, wherein, the first culture medium and second is trained
Foster base is the method definition that induction of cord blood mononuclear cell as previously described is divided into myeloid cell.
The kit that myeloid cell is divided into for induction of cord blood mononuclear cell according to embodiments of the present invention, first
Culture medium is spemspan culture mediums, and first culture medium carries out amplification cultivation to human umbilical cord blood mononuclear cell, so as to be made
Hemocytoblast, the second culture medium is the X-VIVO of factor-containingTM15 culture mediums, second culture medium is to the Hematopoietic Stem after amplification
Cell carries out Fiber differentiation, promotes candidate stem cell to be induced to differentiate into myeloid cell.Using the induced module of above-mentioned two benches culture
The myeloid cell amount that formula is obtained by human umbilical cord blood mononuclear cell apparently higher than directly carrying out Fiber differentiation.
It will be appreciated to those of skill in the art that above for the feature described by the first culture medium and the second culture medium
And advantage, the kit that myeloid cell is divided into for induction of cord blood mononuclear cell is equally applicable to, no longer go to live in the household of one's in-laws on getting married herein
State.
The system of myeloid cell is divided into for induction of cord blood mononuclear cell
In still another aspect of the invention, the present invention proposes one kind and is divided into grain system for induction of cord blood mononuclear cell
The system of cell.Embodiments in accordance with the present invention, the system includes：First culture apparatus, the first culture apparatus is used to utilize the
One culture medium carries out amplification cultivation to human umbilical cord blood mononuclear cell, so as to the candidate stem cell after being expanded；And second training
Device is supported, the second culture apparatus is used to carry out Fiber differentiation to the candidate stem cell after amplification using the second culture medium, so as to
To myeloid cell, wherein, the first culture medium and the second culture medium are induction of cord blood mononuclear cell differentiation as described
For myeloid cell method define.
The kit that myeloid cell is divided into for induction of cord blood mononuclear cell according to embodiments of the present invention, first
Culture medium is spemspan culture mediums, and first culture medium carries out amplification cultivation to human umbilical cord blood mononuclear cell, so as to be made
Hemocytoblast, the second culture medium is the X-VIVO of factor-containingTM15 culture mediums, second culture medium is to the Hematopoietic Stem after amplification
Cell carries out Fiber differentiation, promotes candidate stem cell to be induced to differentiate into myeloid cell.Using the induced module of above-mentioned two benches culture
The myeloid cell amount that formula is obtained by human umbilical cord blood mononuclear cell apparently higher than directly carrying out Fiber differentiation.
It will be appreciated to those of skill in the art that above for the feature described by the first culture medium and the second culture medium
And advantage, this is equally applicable to for the system that induction of cord blood mononuclear cell is divided into myeloid cell, will not be repeated here.
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that following
Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Unreceipted particular technique or bar in embodiment
Part, carried out according to the technology or condition described by document in the art or according to product description.Agents useful for same or instrument
The unreceipted production firm person of device, be can by city available from conventional products.
Conventional method
1 data
1.1 physical data
The Cord blood of healthy pregnant women, obtains the informed consent of volunteer.
1.2 instruments and reagent
It is public that 6% HES and human lymphocyte separating liquid are purchased from Tianjin Hao oceans biological products science and technology Limited Liability
Department；X-VIVOTM15 culture mediums are purchased from Lonza companies；SCGM serum free mediums are purchased from CellGenix companies of Germany；Growth because
Sub- rhSCF, rhFlt-3, rhIL-6, rhIL-3 and rhG-CSF are purchased from peprotech companies；Hyclone (FBS) is purchased from the U.S.
GIBCO companies.RhTPO is purchased from Shenyang Sansheng Pharmaceutical Co., Ltd.；Anti-human CD15-PE monoclonal antibodies, anti-human CD35-
APC monoclonal antibodies and anti-human CD11b-Percp monoclonal antibodies are purchased from eBioscience companies；Mouse anti human CD-18 Dan Ke
Grand antibodies Antibodies are purchased from Abcam companies；Goat anti-mouse IgG-FITC antibody is purchased from ZSGB-BIO companies of Zhong Shan Golden Bridge；Rui Shi-
Ji's nurse Sa A liquid and B liquid are purchased from BaSo Zhuhai Bei Suo bio tech ltd；Obtain pavilion prepared Chinese ink has purchased from Beijing bright Heng Tong industry and trade of profit
Limit company；0.9% physiological saline is purchased from Shijiazhuang Siyao Co., Ltd.Vi-CELLXR cell viabilities analyzer is purchased from German shellfish
Gram Man；5910 type refrigerated centrifuges are purchased from Japanese Kubo field company；CKS31 inverted microscopes are purchased from OLYMPUS companies；
TXD3 cell smears centrifuge is purchased from Xiang Yi centrifuges company；Inverted fluorescence microscope and microscope is just being put purchased from Nikon companies；
Flow cytometer is purchased from BD companies.
2 human umbilical cord blood mononuclear cells are obtained
Cord blood uses 6% 15~30min of HES room temperature sedimented red cell.It is careful to draw supernatant, physiological saline
Re-suspended cell.It is slowly added in the centrifuge tube containing lymphocyte separation medium, room temperature density gradient centrifugation 20min, collects middle white
Film layer, brine 2 times, cell count.Inoculating cell, is placed in 37 DEG C, 5%CO2Induced amplification is carried out in incubator, is passed
Generation, induction takes cell and is detected after 14~21 days, obtains human umbilical cord blood mononuclear cell.
3 myeloid cell morphological observations
Rejection tablet：Take 2 × 105Individual/piece cell 1200rpm centrifugations 3min.The drop of addition 2 Rui Shi-Ji nurse Sa A liquid dyeing 1~
3min, adds 2 to drip left and right Rui Shi-Ji nurse Sa B liquid dyeing 10min, and residual dye is cleaned in ultra-pure water.Micro- sem observation grain system is thin
Born of the same parents dye.
4 Immunofluorescence test myeloid cell CD18 are expressed
Take 2 × 107In EP pipes, PBS is washed once individual cell, after the fixed 1h of 4% 4 DEG C of paraformaldehyde, abandons supernatant.Add
In PBS of the 300 μ l containing 0.1%Triton, supernatant is abandoned in 4 DEG C of 15~30min for the treatment of, centrifugation.PBS is washed, plus 1%BSA is mixed,
Add 1:The mouse anti human CD18 antibody of 300 dilutions, 4 DEG C of incubation 1h.PBS is washed, and abandons supernatant.PBS re-suspended cells, add 1:
Supernatant is abandoned in the anti-mouse secondary antibody of the FITC marks of 200 dilutions, 4 DEG C of incubation 1h, PBS washings, centrifugation.PBS re-suspended cells,
1000rpm is centrifuged 5min rejection tablets, fluorescence microscope.
5 flow cytomery myeloid cell surface markers
Often pipe takes 2 × 105Individual cell, physiology salt is washed 2 times, adds 100 μ l physiological saline resuspended, with different fluorescence labelings
IgG antibody is control group, and experimental group 1-3 is to be separately added into CD15-PE antibody, CD35-APC antibody, CD11b-percp antibody,
Experimental group 4 repeats three to add above-mentioned three kinds of antibody, each sample simultaneously.4 DEG C of incubation 30min.Physiology salt is washed 2 times, plus
Enter 200 μ l resuspended, screen filtration, upper machine testing.
6 granulocyte phagocytic functions are detected
Physiological saline presses 1:1000 dilution prepared Chinese ink, take 1 × 106Individual cell is suspended from 100 μ l physiological saline, adds 100 μ l
The prepared Chinese ink for having diluted, 37 DEG C of incubation 3h, physiology salt is washed 2~3 times, 1000rpm centrifugation 5min rejection tablets.Switzerland's Giemsa staining,
The phagocytosis situation of prepared Chinese ink in 100 cells of micro- sem observation.
The selection of the human umbilical cord blood mononuclear cell amplification culture medium of embodiment 1
Using rhSCF containing 50ng/ml, 10ng/ml rhTPO, 20ng/ml IL-3,10ng/ml IL-6 stemspan
Culture medium, add respectively 0.5 μM of SR1 and 0.5 μM of UM729 share, 50ng/ml Flt3,0.5 μM of SR1,0.5 μM of UM729,
Amplification cultivation 7 days, compares cell survival rate and CD34+Cell number.Result as shown in table 1, adds 0.5 μM in above-mentioned culture medium
SR1 and 0.5 μM of UM729 shares CD34 positive cells, best results in amplification human umbilical cord blood mononuclear cell.Therefore can select
50ng/ml rhSCF, 10ng/ml rhTPO, 20ng/ml IL-3,10ng/ml IL-6,0.5 μM of SR1 and 0.5 μM of UM729
Stemspan culture mediums as CD34+The amplification culture medium of candidate stem cell.
Influence of the different culture formulas of table 1 to human umbilical cord blood mononuclear cell amplification
| Testing index | FLt3 | SR1 | UM729 | SR1+UM729 |
| Survival rate (%) | 88.17±1.08 | 90.62±1.01 | 94.55±1.08 | 94.74±0.36 |
| CD34+Cell number | 3.41×106 | 4.09×106 | 3.33×106 | 4.97×106 |
The selection of the myeloid cell inducing culture of embodiment 2
1st, the influence that hyclone (FBS) and rhTPO are expanded and broken up to myeloid cell
Using the X-VIVO containing rhSCF, rhG-CSF cell factorTM15 culture mediums, TPO is added according to the mode shown in table 2
And FBS, wherein "+" represents addition；"-" is represented without the influence to myeloid cell induced amplification is observed in induction 14 days.Knot
Fruit shows, myeloid cell amplification is substantially inhibited after addition hyclone and is expressed.Obtained by addition rhTPO, myeloid cell
Cell number, survival rate and CD15+、CD15+CD11b+Expression percentage is more preferably.Therefore, selection containing rhSCF, rhG-CSF,
The X-VIVO of rhTPOTM15 culture mediums are used as the culture medium for inducing myeloid cell.
The influence that the FBS of table 2 and rhTPO is induced myeloid cell
| Title | Cell number (× 107) | Survival rate (%) | CD15+(%) | CD15+CD11b+(%) |
| rhTPO+/FBS- | 2.53±0.02 | 94.92±1.59 | 96.05±0.40 | 52.60±3.54 |
| rhTPO+/FBS+ | 1.03±0.03 | 89.63±2.27 | 88.22±0.38 | 44.13±4.40 |
| rhTPO-/FBS+ | 0.74±0.01 | 86.08±3.49 | 84.53±0.21 | 47.85±4.88 |
2、X-VIVOTMThe influence that 15 culture mediums and SCGM culture mediums are expanded and broken up to myeloid cell
Using containing 50ng/ml rhTPO, 50ng/ml rhSCF, 50ng/ml rhG-CSF X-VIVOTM15 culture mediums
With SCGM culture mediums, cell number and marker molecule are detected in amplification cultivation human umbilical cord blood mononuclear cell 14 days in 24 orifice plates
The expression of CD15, CD11b and CD35, as a result as shown in table 3.Myeloid cell is in X-VIVOTMCell number, surface mark in 15 culture mediums
Will CD15+、CD15+CD11b+Expression is higher than SCGM culture mediums；Two kinds of ripe myeloid cell phenotype CD35 of culture medium induction+
CD15+Expression without significant difference (p ＞ 0.05), therefore, first-selected X-VIVOTM15 culture mediums are cultivated as myeloid cell basis
Base.Continued to induce Fiber differentiation 21 days with the cultivating system, flow cytometer detection cell phenotype CD15+Be expressed as (91.68 ±
0.32) %, CD15+CD11b+It is expressed as (47.50 ± 2.55) %, CD35+CD15+(78.65 ± 1.63) %, with induction 14 days
Compare, ripe granulocyte CD35+CD15+Increase.
The different culture media culture of table 3 induces myeloid cell
| Title | Cell number (× 107) | CD15+(%) | CD15+CD11b+(%) | CD35+CD15+(%) |
| X-VIVOTM15 | 1.46±0.03 | 97.73±0.37 | 53.15±2.33 | 49.35±1.06 |
| SCGM | 0.86±0.01 | 84.35±1.91 | 51.35±4.74 | 49.90±8.06 |
The selection of the induction pattern of embodiment 3
Direct induction pattern：Using 50ng/ml rhTPO, 50ng/ml rhSCF, 50ng/ml rhG-CSF X-
VIVOTM15 culture mediums, induction of cord blood mononuclear cell 14 days；
First expand the two benches induction pattern for inducing afterwards：Using containing 50ng/ml rhSCF, 10ng/ml rhTPO,
The stemspan culture medium single cores of amplification cultivation Cord blood of 20ng/mlIL-3,10ng/ml IL-6,50ng/ml Flt-3 are thin
Born of the same parents 7 days, by cell after resulting amplification according to 4 × 106Individual/mL is inoculated in and contains 50ng/ml rhTPO, 50ng/ml
The X-VIVO of rhSCF, 50ng/mlrhG-CSFTM15 culture mediums are induced 14 days.
Result is as shown in table 4.FCM analysis show that two benches induction pattern, myeloid cell differentiation efficiency is lagged behind
Direct induction pattern.But, cells expanded, hence it is evident that higher than direct induction pattern.
The induction culturing model comparision of table 4
| CD15+(%) | CD15+CD11b+(%) | CD35+CD15+(%) | |
| Direct induction pattern | 97.73±0.37 | 53.15±2.33 | 49.35±1.06 |
| Two benches induction pattern | 69.6±1.06 | 34.11±1.27 | 26.83±1.17 |
After being expanded, induced using two benches induction pattern, amplification times are 132 times.After induction 21 days, amplification times
It is 2590.5 times.Rui Shi-Ji's nurse Sa dyes the granulocyte of visible different phase, including segmented granulocyte.As shown in Fig. 2 into
Ripe segmented granulocyte, karyon is finer and close, and in banding is blunt curved or lobulated, nucleocytoplasmic ratio is smaller, and kytoplasm basophilla disappears, coloring
It is light red.
CD18 is the lysosomal protein in granulocyte, and Immunofluorescence test, human umbilical cord blood mononuclear cell is obtained through external evoked
The myeloid cell CD18 antibody tests for obtaining are the positive, and fluoresced green is specific as shown in Figure 3.
Fig. 4 is the microscope figure of prepared Chinese ink phagocytosis experiment, the granulocyte to swallow prepared Chinese ink that arrow is pointed to, prepared Chinese ink phagocytosis experiment
Show, the myeloid cell of amplification in vitro induction has a function of certain phagocytosis prepared Chinese ink, phagocytosis efficiency for (51.43 ±
0.05) %.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means to combine specific features, structure, material or spy that the embodiment or example are described
Point is contained at least one embodiment of the invention or example.In this manual, to the schematic representation of above-mentioned term not
Identical embodiment or example must be directed to.And, the specific features of description, structure, material or feature can be with office
Combined in an appropriate manner in one or more embodiments or example.Additionally, in the case of not conflicting, the skill of this area
Art personnel can be tied the feature of the different embodiments or example described in this specification and different embodiments or example
Close and combine.
Although embodiments of the invention have been shown and described above, it is to be understood that above-described embodiment is example
Property, it is impossible to limitation of the present invention is interpreted as, one of ordinary skill in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, changes, replacing and modification.
Claims (9)
1. a kind of method that induction of cord blood mononuclear cell is divided into myeloid cell, it is characterised in that including：
Amplification cultivation is carried out to the human umbilical cord blood mononuclear cell using the first culture medium, so that the Hematopoietic Stem after being expanded is thin
Born of the same parents；And
Fiber differentiation is carried out to the candidate stem cell after the amplification using the second culture medium, to obtain the myeloid cell,
First culture medium is spemspan culture mediums,
Second culture medium is the X-VIVO containing the first additiveTM15 culture mediums, and first additive includes restructuring
Human thrombopoietin, rhMGF and recombinant human granulocyte colony stimulating factor.
2. method according to claim 1, it is characterised in that the spemspan culture mediums contain the second additive, institute
Stating the second additive includes：Recombined human thrombopoietin, rhMGF, recombinant human interleukin -3 and weight
Group human interleukin-6.
3. method according to claim 2, it is characterised in that second additive further includes following at least one
Kind：The reinforcing agent of FMS samples EGFR-TK 3, aryl hydrocarbon receptor inhibitor and aryl hydrocarbon receptor antagonist,
Preferably, second additive further includes the enhancing of aryl hydrocarbon receptor inhibitor and aryl hydrocarbon receptor antagonist
Agent.
4. method according to claim 1, it is characterised in that in second culture medium,
The concentration of the recombined human thrombopoietin is 30~100ng/mL, preferably 50ng/mL；
The concentration of the rhMGF is 30~100ng/mL, preferably 50ng/mL；
The concentration of the granulocyte colony stimulating factor is 30~100ng/mL, preferably 50ng/mL.
5. method according to claim 3, it is characterised in that in first culture medium,
The concentration of the recombined human thrombopoietin is 5~20ng/mL, preferably 10ng/mL；
The concentration of the rhMGF is 30~100ng/mL, preferably 50ng/mL；
The concentration of the recombinant human interleukin -3 is 10~50ng/mL, preferably 20ng/mL；
The concentration of the recombinant human interleukin-6 is 10~50ng/mL, preferably 20ng/mL；
The concentration of the aryl hydrocarbon receptor inhibitor is 0.3~0.8 μM, preferably 0.5 μM；
The concentration of the reinforcing agent of the aryl hydrocarbon receptor antagonist is 0.3~0.8 μM, preferably 0.5 μM.
6. method according to claim 1, it is characterised in that the time of the amplification cultivation is 5~10 days, preferably 7 days.
7. method according to claim 1, it is characterised in that the time of the Fiber differentiation is 14~25 days, preferably 21
My god.
8. a kind of kit that myeloid cell is divided into for induction of cord blood mononuclear cell, it is characterised in that including：
First culture medium, first culture medium is used to carry out amplification cultivation to the human umbilical cord blood mononuclear cell, to obtain
Candidate stem cell after amplification；And
Second culture medium, second culture medium is used to carry out Fiber differentiation to the candidate stem cell after the amplification, so as to
To the myeloid cell,
Wherein, first culture medium and the second culture medium are single induction of cord blood as described in any one of Claims 1 to 5
Monocyte differentiation is defined for the method for myeloid cell.
9. a kind of system that myeloid cell is divided into for induction of cord blood mononuclear cell, it is characterised in that including：
First culture apparatus, first culture apparatus is used to carry out the human umbilical cord blood mononuclear cell using the first culture medium
Amplification cultivation, so as to the candidate stem cell after being expanded；And
Second culture apparatus, second culture apparatus is used to enter the candidate stem cell after the amplification using the second culture medium
Row Fiber differentiation, to obtain the myeloid cell,
Wherein, first culture medium and the second culture medium are single induction of cord blood as described in any one of Claims 1 to 5
Monocyte differentiation is defined for the method for myeloid cell.
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