CN109439613A - MICE FETAL LIVER stroma cell construction method and its application in hematopoiesis auxiliary - Google Patents
MICE FETAL LIVER stroma cell construction method and its application in hematopoiesis auxiliary Download PDFInfo
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Abstract
The invention discloses a kind of MICE FETAL LIVER stroma cell construction method and its applications in hematopoiesis auxiliary.It is obtained by Fetal liver stromal cell and in vitro culture, the separation of marrow LK cell and the operating procedure of cell marrow intraluminal grafting is completed.Fetal liver stromal cell prepared by the present invention can promote the recovery of PLT and RBC early stage hematopoietic reconstitution, promote the proliferation and rapid amplifying of HSC, and it is significant to promote hematopoietic reconstitution efficiency to the hematopoieticmicroenviron-ment for improving host in HSC transplanting for this.
Description
Technical field
The invention belongs to hematopoietic cell constructing technology fields, and in particular to MICE FETAL LIVER stroma cell construction method and its
Application in hematopoiesis auxiliary.
Background technique
Stroma cell is isolated from cavy BM by Friedenstein A etc. earliest, different from hematopoietic cell, they
Be it is a kind of can on plastic material vessel adherent growth fibroblast-like cells, later this kind of cell be found can to skeletonization,
Cartilage and Adipocyte Differentiation, therefore stroma cell is just slowly referred to as mescenchymal stem cell (mesenchymal stem
Cell, MSC), but since the identification to mouse MSC lacks unified standard, the MSC difference that difference researcher establishes both at home and abroad is very
Greatly, these differences are mainly reflected in surface marker expression.It is generally acknowledged that MSC does not express blood system mark CD45, but also there is research
Person is separated to CD45+Mouse BM MSC.CD34 is also regarded as in MSC not expressing as a kind of mark of hematopoietic cell,
But MSC CD34 positive rate Peister et al. isolated from B1/6 mouse BM is more than 75%.CD44 is hyaluronic acid
Receptor generally believes the high expression in MSC, but still the two class stroma cells for having researcher to cultivate do not express CD44.
Sca-1 is the mark of mouse HSC, and there is also disputes for the expression rate in MSC, and difference research difference is larger, Post S et al.
Obtained BM MSC Sca-1 positive rate is up to 90%, and there was only 32%, Saeed et al. separation in the research of Zhang Rongyao et al.
Obtained BM MSC, Sca-1 positive rate is then between 60%-90%.It should be pointed out that MSC high expresses integrin
CD29, does not express lymphocyte markers CD11b, is at present still generally acknowledged viewpoint.The MSC of expression different surfaces mark usually has
Different forms, Hui great Yang et al. cultivate the BM for obtaining subcircular, fibroblast shape and spindle polygon three types
MSC, wherein subcircular cell CD90 expression rate is 16%, and the cell of other two type is then 90% or so；Yuan Chunhui et al. points
It is 97% from obtained epithelium shape FL MSC CD105 positive rate, is only 14% into fiber shape cell.
Candidate stem cell (hematopoietic stem cell, HSC) is used as the most commonly used adult of current clinical application
Stem cell be used to treat all kinds of acute and chronic leukemias, lymthoma, alpastic anemia, thalassemia, severe and combine and exempts from
The Hemic and immune systems disease such as epidemic disease defect, but due to deriving from peripheral blood (peripheral blood, PB), marrow
The HSC scarcity of resources of (bone marrow, BM) and Cord blood, HSC transplanting can not be carried out in time by resulting in a large amount of patients, therefore
How to obtain a large amount of HSC in vitro becomes the research hotspot of related fields.Mammal tire liver (fetal liver, FL)
Hematopoiesis period is the period of HSC maturation and rapid amplifying, in addition to blood system is extracellular in FL, there are also it is all kinds of can adherent growth in vitro
Non- blood system cell, they are collectively referred to as stroma cell, and the hematopoieticmicroenviron-ment being made of FL stroma cell plays the amplification of HSC
Important booster action.Research both domestic and external is concentrated on using FL stroma cell as feeder cells at present, induces other classes
The cell differentiation of type is HSC and massive amplification, but in current research, rarely has scientific research personnel to explore FL stroma cell in vivo
The hematopoiesis booster action of performance: its in vivo the hematopoiesis booster action under environment whether with difference in vitro, can promote outer
Source HSC still needs to be explored in receptor intracorporal the problems such as returning tabernacle, proliferation and differentiation.
Summary of the invention
The purpose of the present invention is to provide a kind of MICE FETAL LIVER stroma cell construction method and its answering in hematopoiesis auxiliary
With.
A kind of MICE FETAL LIVER stroma cell construction method carries out in accordance with the following steps:
(1) Fetal liver stromal cell acquisition and in vitro culture
Cervical dislocation puts to death pregnant mouse, and it is cell suspension that tire liver is taken out under aseptic condition and is blown and beaten, and crosses 25-35 μm of strainer preparation
Single cell suspension separates tire liver mononuclearcell with lymphocyte separation medium, according to 0.5*106-2*106/cm2Density inoculation
On culture solution in culture dish, 37 DEG C are placed in, 5%CO2It is cultivated in concentration incubator, changes liquid for the first time after being inoculated with 20-28h, this
Liquid is changed within every 2-3 days afterwards, when cell convergence degree reaches 78-82% according to 1:2 secondary culture；
(2) marrow LK cell separates
Female mice is put to death through cervical dislocation, shin bone and femur are taken out under aseptic condition, goes out bone marrow cell, crosses 25-35 μm
Strainer prepares single cell suspension, then with the isolated bone marrow mononuclear cells of lymphocyte separation medium, hereafter according to magnetic bead sorting
Kit operating process isolates Lin-c-Kit+Cell, cell count be placed on 4 DEG C it is spare；
(3) cell marrow intraluminal grafting
Skin at knee joint is cut off after taking 1% Nembutal sodium solution anesthetized mice, with 0.5-2mL syringe from opening
It is pierced into shin bone ossis, extracts syringe needle, then draw cell suspension with 20-30 μ L sample injector and be inserted into ossis from pin hole just
And cell is injected, injection of bone marrow LK cell and Fetal liver stromal cell are cultivated 6-12 weeks.
Culture solution in the culture dish is DMEM in high glucose, 15% fetal calf serum (v/v), 1% blueness/streptomysin (v/v) and
0.1mM beta -mercaptoethanol.
Preferably, the injection volume of step (3) the marrow LK cell is 1.7*104It is a, the injection volume of Fetal liver stromal cell
For 2.3*105It is a.
Preferably, step (1) and step (2) the aperture of filter screen size are 30 μm.
Preferably, step (3) syringe is 1mL syringe, and sample injector is 25 μ L sample injectors.
Application of the MICE FETAL LIVER stroma cell of above-mentioned preparation in hematopoiesis auxiliary.
Beneficial effects of the present invention: Fetal liver stromal cell prepared by the present invention can promote early stage hematopoietic reconstitution PLT and
The recovery of RBC promotes the proliferation and rapid amplifying of HSC, this promotes hematopoiesis to the hematopoieticmicroenviron-ment for improving host in HSC transplanting
It is significant to rebuild efficiency.
Detailed description of the invention
Fig. 1 is that stroma cell form and surface marker are expressed；
In figure, A:FL stroma cell；B:BM stroma cell；Bar:100 μm.
Fig. 2 is receptor survival rate and every blood index；
In figure, A: survival rate；B:PB items physiochemical indice；C: the last fortnight PLT, RBC and HGB content after transplanting；p<0.05
Indicate statistically significant.
Fig. 3 is by peripheral body and marrow external source blood system's cell chimerism rate and dyeing (800X)；
In figure, A: receptor chimera rate；B: Rui Shi-Giemsa staining.
Specific embodiment
The present invention will be further described in the following with reference to the drawings and specific embodiments.
The experimental animal and main agents that following embodiments use are as follows:
FL stroma cell derives from C57BL/6CD45.2/CD45.2The tire of female mice and male mouse post-coitum pregnancy 13.5 days (E13.5)
Mouse, BM stroma cell source and cell transplantation Recipient mice are C57BL/6CD45.2/CD45.2Female mice；BM LK cell origin in
C57BL/6CD45.1/CD45.2Female mice.Mouse is purchased in Shanghai Ling Chang Biotechnology Co., Ltd [production permit: SCXK (Shanghai)
2013-0018] and Shanghai south model organism Science and Technology Co., Ltd. [production permit: SCXK (Shanghai) 2017-0010].Lymph
Cell separating liquid (Stemcell)；Lin-c-Kit+Cell magnetic bead sorting kit (Miltenyi)；CD44,CD29,CD45.1,
CD45.2 and Sca-1 antibody is purchased in BD Phamingen company.
1 stroma cell of embodiment obtains and in vitro culture, surface marker detection of expression
Cervical dislocation puts to death pregnant mouse, and it is cell suspension that FL is taken out under aseptic condition and is blown and beaten, cross 30 μm of strainers prepare it is slender
Born of the same parents' suspension separates FL mononuclearcell with lymphocyte separation medium, according to 0.5*106-2*106/cm2Density be inoculated in culture
On ware, culture solution is+15% fetal calf serum of DMEM in high glucose (v/v)+1% blueness/streptomysin (v/v)+0.1mM beta -mercaptoethanol, is set
In 37 DEG C, 5%CO2It is cultivated in concentration incubator.Liquid is changed for the first time after inoculation for 24 hours, changes liquid within hereafter every 2-3 days, when cell converges journey
According to 1:2 secondary culture when degree is up to 80% or so.BM stroma cell is derived from mouse tibia and femur, in addition to culture solution is low sugar
Outside DMEM, remaining training method is the same as FL stroma cell.
FL the and BM stroma cell in culture 1-4 generation is taken then to use phosphoric acid buffer with 0.25% trypsin solution vitellophag
Liquid (phosphate buffered saline, PBS) is washed once, is added 4 DEG C of CD44, CD29 and Sca-1 antibody and is protected from light incubation
25min, PBS are washed to be analyzed with flow cytometer (Beckman, CytoFlex) afterwards twice.
After primary cell inoculation for 24 hours, it can be seen that adherent stroma cell occurs under the microscope, the base of originally culture
Cell plastid form is mostly subcircular, short shuttle shape and fibroblast shape, separately has some irregular cells for having protrusion, afterwards
Incubation in can be to short spindle cell transition (Fig. 1).
Embodiment 2FL stroma cell marrow intraluminal grafting
BM LK cell is separated two C57BL/6CD45.1/CD45.2Female mice is put to death through cervical dislocation, is taken out under aseptic condition
Shin bone and femur go out BM cell, cross 30 μm of strainers and prepare single cell suspension, then is mono- with the isolated BM of lymphocyte separation medium
Hereafter a nucleus isolates Lin according to magnetic bead sorting kit operating process-c-Kit+Cell, cell count are placed on 4 DEG C
It is spare.
The E13.5FL stroma cell and P4MEF in the 4th generation (P4) of culture are selected in FL stroma cell and the preparation of MEF cell suspension,
Pancreatin digestion after be centrifuged, with PBS be resuspended cell after count, be placed in 4 DEG C it is spare.
Cell marrow intraluminal grafting: by 20 C57BL/6CD45.2/CD45.2(the previous day receives 9.0Gy to mouse60CO γ is penetrated
Beta radiation) it is divided into 4 groups, every group 5, it is respectively designated as " PBS " group, " LK " group, " LK+FL " group, " LK+MEF " group, 1% penta bars
Than cutting off skin at knee joint after appropriate sodium solution anesthetized mice, it is pierced into shin bone ossis from opening with 1mL syringe, extracts needle
Head, then be inserted into ossis from pin hole just with 25 μ L sample injectors absorption cell suspension and inject cell.PBS group injects 20 μ L
PBS；LK group injects 1.7*104BM LK cell；LK+FL group injects 1.7*104LK cell adds 2.3*105FL stroma cell；LK+
MEF group injects 1.7*104LK cell adds 2.3*105MEF。
It is taken a blood sample by tail point within the 1st, 2,3,4,6,8 week after cell transplantation and obtains Recipient mice PB, and pass through blood point
Analyzer (Sysmex, XT-2000i) detects and records every blood routine data.
Through eyeball sacrificed by exsanguination Recipient mice, acquisition PB and BM mononuclearcell is added within the 9th week after cell transplantation
CD45.1 and CD45.2 antibody, 4 DEG C are protected from light incubation 25min, and PBS is washed carries out Flow cytometry afterwards twice.Receptor chimera rate
Calculation method are as follows: CD45.1+CD45.2+Cell concentration/(CD CD45.1+CD45.2+Cell concentration+CD45.1-CD45.2+Cell concentration) *
100%.
9th week production receptor PB and breastbone BM smear after cell transplantation.PB smear is dyed with Rui Shi-Ji's nurse Sa, the dyeing of A liquid
After 1min plus B liquid contaminates 3min；BM smear: A liquid adds B liquid to contaminate 7min after dyeing 3min.It is rinsed after dyeing with tap water, it is dry
It is placed on optical microphotograph under the microscope.
Statistical analysis is carried out using 7 software of GraphPad Prism.Experimental data withIt indicates, P < 0.05 is indicated
Difference has statistical significance.
In order to inquire into the hematopoiesis booster action of FL stroma cell in vivo, inventor is by E13.5P4FL stroma cell and BM
Receive 9.0Gy on the day before being transplanted to after LK mixing with cells60COThe tibial bone of the Recipient mice of gamma-rays (lethal dose) radiation
In pulp cavity.PBS group receptor all death in 8-10 days after the transfer as the result is shown, LK and LK+MEF group difference the 10th day after the transfer
The phenomenon of receptor death had occurred with the 8th day, and LK+FL group receptor survival rate in two months is 100% (A in Fig. 2), table
Bright FL stroma cell may play important hematopoiesis booster action during hematopoietic reconstitution.
In order to further verify the specific effect of FL stroma cell performance, each group receptor after inventor has recorded before transplantation
The content (B in Fig. 2) of blood platelet (PLT), red blood cell (RBC), hemoglobin (HGB) and granulophilocyte (RET) in PB,
In the 0th week to radiate previous day data.Recipient mice via radiation after in PB PLT quantity drastically reduce, it is equal in the 1st week each group numerical value
It is the 1/10 of insufficient normal level, and the content of RBC and HGB is after the transfer in one month all on the 75% of normal value
Lower fluctuation, is hereafter gradually recovered, and one and a half months or so restores normal after the transfer.Although first and last three transplantation group PLT,
The recovery situation of RBC and HGB is almost the same, but the last fortnight after the transfer, and LK+FL group PLT content is above remaining two groups；?
The 2nd week after transplanting, RBC and HGB content is also significantly greater than LK group (C in Fig. 2), this shows that FL stroma cell may be in hematopoiesis
The generation of PLT and RBC of the early promotion of reconstruction.The particularity of the 1st week to the 2nd week this period can pass through RET after transplanting
Variation illustrated, RET is immature RBC, and the content back in PB has answered the generating state of RBC in BM, the 1st after transplanting
All each group receptor RET quantity all significantly reduces, and is 10% or so of normal level, shows the BM RBC generative capacity of receptor itself
It is badly damaged；But the 2nd week after the transfer, each group RET quantity steeply rises, and reaches 3 times of normal value or so.RET is being transplanted
The abnormal increase in 1 to 2 weeks shows that foreign hematopoietic cells have begun rapid Proliferation, Differentiation at this time afterwards, but not yet generates a large amount of mature
Haemocyte, and immature all kinds of haemocytes have been merely creating, corresponding biological function cannot be effectively played, therefore receptor is small
Mouse is easy to die of Haematopoietic failure in this stage.Inventor's research shows that hematopoietic reconstitution early stage (1-2 after transplanting
Week), all kinds of haemocytes of host itself are exhausted substantially, and foreign hematopoietic cells not yet generate a large amount of mature blood cells, therefore by
Body is easy to die of Haematopoietic failure, and FL stroma cell has played important hematopoiesis booster action in this stage --- it promotes
The fast quick-recovery of PLT and RBC, so as to avoid the death of receptor.
External source blood system cell chimerism rate is respectively 78.8%, 80.8% in LK, LK+FL and LK+MEF group PB after transplanting 9 weeks
With 70.0%；It is respectively 89.9%, 90.0% and 88.5% (A in Fig. 3) in BM.This is the result shows that 9.0Gy60CO γ is penetrated
Beta radiation almost destroys the hematopoiesis function of receptor itself, and BM LK cell can expand and break up in host's ossis
As the haemocyte of all kinds of maturations, there is outstanding hematopoietic reconstitution ability, while also indicating that ossis may be one efficient
HSC transplanted sites.
Blood film, which is shown in each group PB, has a large amount of typical biconcave discoid RBC, and has leaflet core
The haemocytes such as neutrophil leucocyte, the lymphocyte of single core and monocyte；Cell in BM is all karyocyte, various types of cells
The universal larger but number of nucleus it is different, with the presence of the cell (B in Fig. 3) of a large amount of multicores.In three transplantation group PB and BM
Cell type and ratio and Control group show that foreign hematopoietic cells have rebuild the blood of health in host without significant difference
Liquid system do not cause blood class disease, it was demonstrated that the reliability and safety of marrow intraluminal grafting HSC and stroma cell.
In the present invention, inventor's tentative confirmation FL stroma cell can promote PLT's and RBC early stage hematopoietic reconstitution
Restore.Although using BM or umbilical cord to be more easier as cell origin ratio FL for materials and perspective of ethics, from promotion HSC
Proliferation and rapid amplifying angle for, FL stroma cell has very big advantage, thus in subsequent research, inventor
It explores further in vivo really with the FL stroma cell monoid of hematopoiesis booster action, and finds out crucial cell factor and base
Cause, it is significant to promote hematopoietic reconstitution efficiency to the hematopoieticmicroenviron-ment for improving host in HSC transplanting for this.
Claims (6)
1. a kind of MICE FETAL LIVER stroma cell construction method, which is characterized in that carry out in accordance with the following steps:
(1) Fetal liver stromal cell acquisition and in vitro culture
Cervical dislocation puts to death pregnant mouse, and it is cell suspension that tire liver is taken out under aseptic condition and is blown and beaten, cross 25-35 μm of strainer prepare it is slender
Born of the same parents' suspension separates tire liver mononuclearcell with lymphocyte separation medium, according to 0.5*106-2*106/cm2Density be inoculated in training
It supports on the culture solution in ware, is placed in 37 DEG C, 5%CO2It is cultivated in concentration incubator, changes liquid for the first time after being inoculated with 20-28h, hereafter often
Liquid is changed within 2-3 days, when cell convergence degree reaches 78-82% according to 1:2 secondary culture.
(2) marrow LK cell separates
Female mice is put to death through cervical dislocation, shin bone and femur are taken out under aseptic condition, goes out bone marrow cell, crosses 25-35 μm of strainer
Single cell suspension is prepared, then with the isolated bone marrow mononuclear cells of lymphocyte separation medium, hereafter according to magnetic bead sorting reagent
Box operating process isolates Lin-c-Kit+Cell, cell count be placed on 4 DEG C it is spare；
(3) cell marrow intraluminal grafting
Skin at knee joint is cut off after taking 1% Nembutal sodium solution anesthetized mice, is pierced into 0.5-2mL syringe from opening
Shin bone ossis extracts syringe needle, then is inserted into ossis from pin hole just with 20-30 μ L sample injector absorption cell suspension and infuses
Enter cell, injection of bone marrow LK cell and Fetal liver stromal cell, cultivates 6-12 weeks.
2. MICE FETAL LIVER stroma cell construction method according to claim 1, which is characterized in that the culture in the culture dish
Liquid is DMEM in high glucose, 15% fetal calf serum (v/v), 1% blueness/streptomysin (v/v) and 0.1mM beta -mercaptoethanol.
3. MICE FETAL LIVER stroma cell construction method according to claim 1, which is characterized in that step (3) the marrow LK
The injection volume of cell is 1.7*104A, the injection volume of Fetal liver stromal cell is 2.3*105It is a.
4. -3 MICE FETAL LIVER stroma cell construction method according to claim 1, which is characterized in that step (1) and step (2)
The aperture of filter screen size is 30 μm.
5. MICE FETAL LIVER stroma cell construction method according to claim 1, which is characterized in that step (3) described syringe
For 1mL syringe, sample injector is 25 μ L sample injectors.
6. the application of MICE FETAL LIVER stroma cell prepared by claim 1 in hematopoiesis auxiliary.
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