CN106929474A - A kind of M2 macrophages derivant - Google Patents
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Abstract
The invention provides a kind of derivant of M2 macrophages and preparation method thereof, the derivant of the M2 macrophages is a kind of mescenchymal stem cell conditioned medium of collagen/chitosan sponge bracket induction.The preparation method of the derivant of the M2 macrophages, its step includes：A) mescenchymal stem cell is seeded on collagen/chitosan sponge bracket；B) using mescenchymal stem cell collagen/chitosan sponge bracket described in serum free medium culture；C) mescenchymal stem cell and culture medium are collected；D) fresh serum-free media is added, and continues to cultivate the mescenchymal stem cell；And e) collect the conditioned medium of the mescenchymal stem cell.The M2 macrophages derivant that the present invention is provided can effectively facilitate polarization of the macrophage to M2.M2 macrophages derivant of the invention can be used to treat obesity.
Description
Technical field
The invention belongs to biomedicine field, more particularly to a kind of derivant of M2 macrophages and preparation method thereof, and
For the application of obesity medicine.
Background technology
Obesity is due to abnormal or excessive lipopexia.A large amount of evidences show, the pathology of fat and many disease
Physiology is related, such as insulin resistance, diabetes, angiocardiopathy and cancer etc..The World Health Organization reports recently, 2014,
Obesity has exceeded 600,000,000 in 18 years old and the adult of the above, and obesity rates are 13%.Global obesity rates are between 1980 and 2014
It is more than doubled.Therefore, fat problem is more and more paid attention to.
It is fat along with adipose tissue chronic low grade inflammation (Hotamisligil, G.S.,
2006.Inflammation and metabolic disorders.Nature 444,860e867.；Ferrante,A.W.,
et al.,2007.Obesity-induced inflammation:a metabolic dialogue in the language
of inflammation.J.Intern.Med.262,408e414.；Stienstra,R.,et al.,2012.The
inflammasome puts obesity in the danger zone.Cell.Metab.15,10e18.).More and more
Research shows that improving inflammation can improve insulin sensitivity, reduces the fat cell size of obesity mice and improves lipid generation
Thank.For example, the antagonist by giving IL-1 acceptors or IL-1b, the enhancing of high fat diet mouse islets element sensitiveness (Larsen,
C.M.,et al.,2007.Interleukin-1-receptor antagonist in type 2 diabetes
mellitus.N.Engl.J.Med.356,1517e1526.；Ridker,P.M.,et al.,2012.Effects of
interleukin-1beta inhibition with canakinumab on hemoglobin A1c,lipids,C-
reactive protein,interleukin-6,and fibrinogen:a phase IIb randomized,placebo-
controlled trial.Circulation 126,2739e2748.).Additionally, when CCR2-/- mouse can not be inflammation monokaryon
Cell from blood replenish tissue when, show the fat associated metabolic less than high fat diet mouse neurological susceptibility (Weisberg,
S.P.,et al.,2006.CCR2modulates inflammatory and metabolic effects of high-fat
feeding.J.Clin.Invest 116,115e124.).Therefore, the method for seeking to mitigate fat related inflammation is fat to solve
Hope is brought with the resistance of fat Related Insulin.
Macrophage is the key factor for influenceing inflammation and fat Related Insulin resistance, and it is huge that they can be divided into proinflammatory
Phagocyte M1 and anti-inflammatory type macrophage M2.Under obese state, proinflammatory macrophage M1 largely breeds, aggravation adipose tissue and its
The insulin resistance of his organ.Although the anti-inflammatory macrophage M2 in adipose tissue can suppress inflammation, in obesity, it
It is much lower relative to the ratio of macrophage M1.Generally speaking, in obesity, macrophage M1 replaces macrophage M2 simultaneously
The homeostasis controlled inflammation with macrophage, stabilization is in proinflammatory disease " set point " higher.These as shown by data correct fat
Inflammation/the immune homeostasis of tissue are necessary.Infusion M2 macrophages can adjust internal macrophage stable state, change so as to reach
It is apt to fat purpose.
Mescenchymal stem cell (MSC) is one of most important multipotent adult stem cells, with self-renewing and differentiation of self
Potential, and the various factors of secretion participate in the reparation and regeneration of the tissue and organ for damaging and are widely used in various diseases
Treatment.MSCs under specific inductive condition, can be divided into pancreas islet, nerve, blood vessel endothelium, bone, cartilage, flesh in vivo or in vitro
The Various Tissues cell such as meat, liver, cardiac muscle.Increasing research confirms cell of the mescenchymal stem cell (MSCs) by secretion
The factor can play immunological regulation and reduce the effect of inflammation.Research finds that mescenchymal stem cell can be by promoting M2 macrophages thin
Born of the same parents polarize to alleviate inflammatory reaction and improve insulin resistance, (Xie Z, Hao H, Tong C, et al.Human
umbilical cord‐derived mesenchymal stem cells elicit macrophages into an
anti‐inflammatory phenotype to alleviate insulin resistance in type 2
diabetic rats[J].2016,34(3):627-639.).The therapy mechanism of MSCs efficiency depends primarily on their side point
Activity is secreted, MSC can secrete substantial amounts of growth factor, and current research thinks that these bioactivators make mescenchymal stem cell
Function with immunological regulation, reduction inflammation, bioactie agent and cell factor in all of MSC secretion can be in bars
Collected in part culture medium, the evidence of accumulation shows that MSC conditioned mediums have the therapeutic effect similar to MSC.
There are some researches prove stem cell is inoculated into CCSS (collagen/chitosan sponge scaffolds) glue
After original-chitosan sponges bracket, the increased activity of stem cell, recovery dynatron effect also strengthens (Tong C, Hao H, Xia L, et therewith
al.Hypoxia pretreatment of bone marrow-derived mesenchymal stem cells seeded
in a collagen-chitosan sponge scaffold promotes skin wound healing in
diabetic rats with hindlimb ischemia.[J].Wound Repair&Regeneration,2015,24
(1):45.)。
Based on the studies above, the present invention is cultivated MSC with CCSS, the mescenchymal stem cell CMC model for being activated
Base, for promoting polarization of the macrophage to M2, for improving the resistance of fat and Related Insulin.
The content of the invention
The invention provides a kind of derivant of M2 macrophages and preparation method thereof, the derivant energy of the macrophage
Polarization of the macrophage to M2 is enough effectively facilitated, for improving the resistance of fat and Related Insulin.
For achieving the above object, the present invention is achieved using following technical proposals：
A kind of derivant of M2 macrophages, it is characterized by, it is a kind of mesenchyma of collagen/chitosan sponge bracket induction
Stem cell conditioned medium.After the collagen/chitosan sponge bracket is for collagen and chitosan compound freeze-drying, in N-
Shaken in (3- dimethylaminopropyls)-N'- ethyl carbodiimides (EDC) and N-hydroxy-succinamide (NHS) solution and obtained
's.Described mescenchymal stem cell be selected from umbilical cord mesenchymal stem cells, fat mesenchymal stem cell, mesenchymal stem cells MSCs or
Its combination.
Further, the collagen/chitosan sponge bracket is with 1 by collagen and shitosan:1 ratio mixing, then
Freezed at -80 DEG C 2 hours, then freezed 24 hours；After drying, mixture immersion is contained into N- (3- dimethylaminos third
Base) in-N'- ethyl carbodiimides (EDC) and 8mM N-hydroxy-succinamides (NHS), while slow shake 12 at room temperature
Hour obtains.
A kind of preparation method of the derivant of M2 macrophages, its step includes：A) mescenchymal stem cell is seeded in glue
On original/chitosan sponges bracket；B) using mescenchymal stem cell-collagen/chitosan sponge branch described in serum free medium culture
Frame；C) mescenchymal stem cell and culture medium are collected；D) fresh serum-free media is added, and continues to cultivate the mesenchyma
Stem cell；And e) collect the conditioned medium of the mescenchymal stem cell.
Further, mescenchymal stem cell is seeded in the step on collagen/chitosan sponge bracket in the step a)
Including：The mescenchymal stem cell single cell suspension is dripped into collagen/chitosan sponge bracket top surface and is incubated culture；So
Equivalent mescenchymal stem cell is seeded in afterwards training is incubated in the mescenchymal stem cell-collagen/chitosan sponge bracket construct
Support.
The step of step c) collects the mescenchymal stem cell and culture medium includes：Culture medium is removed, using pancreas egg
White enzyme-ETDA digests the mescenchymal stem cell, shrinks it and departs from collagen/chitosan sponge bracket；Removed described in adding
Culture medium, neutralizes trypsase-EDTA；And gently blow and beat the mescenchymal stem cell.
The mescenchymal stem cell is continued culture 12 ± 1 hours in the step d) in serum free medium.
The mescenchymal stem cell and culture medium is collected by centrifugation in the step e), to remove cell fragment；Then with one
Individual 7-kDa weight shutoffs bag collects the conditioned medium.
A kind of application of the derivant of M2 macrophages that the present invention is provided in obesity is treated.
Beneficial effects of the present invention：
The present invention cultivates MSC and obtains conditioned medium by CCSS, used as derivant, inducing macrophage polarization, hence it is evident that
The polarization efficiency for enhancing macrophage and the effect for improving fat and insulin resistance.The mesenchyma of activation prepared by the present invention
Stem cell conditioned medium, by simple pretreatment, this preparation system is easy to quality control, it is easy to industrialization, the bar of preparation
Part culture medium can be made freeze-dried powder, it is easy to preserve.
Brief description of the drawings：
Fig. 1：Total protein concentration in the MSC-CM of BCA protein detection reagent kits assessment purifying.
Fig. 2：RT-PCR detection Bone Marrow Macrophage from supernatant after 24h and 48h after different MSC co-incubations
The relative expression quantity of proinflammatory cytokine (IL-1, IL-6, TNF-α), anti-inflammatory cytokines (IL-10, TGF-β) and CD163.
Con represents control, and HG represents sugar high, and HG+CCSS-MSC-CM represents the condition training of the MSC of sugar high+collagen-chitosan support culture
Support base.
Fig. 3：Mouse chow intake, groin adipose tissue (INAT) weight after second infusion macrophage and attached
Testis adipose tissue (EAT) weight.A：Mouse chow intake；B：INAT weight；C：EAT weight.M0 is not by any place
The bone marrow macrophage of reason, un-M2 is the M2 macrophages of untreated MSC-CM inductions；CCSS-M2 is CCSS cultures
The M2 macrophages of MSC-CM inductions.
Fig. 4：Detection M0 treatment groups, un-M2 treatment groups and CCSS-M2 macrophages are dyeed by hematoxylin-eosin (HE)
Treatment group's group mouse INAT and EAT fat cell Volume Changes comparison diagram.
Fig. 5:A:The amount of the p-AKT in the EAT of western blot detections；B：Abdominal cavity sugar dosis tolerata (IPGTT) of mouse
Result of the test.
Fig. 6：Separate groups of mice fat metabolism situation.A：The triglyceride levels of blood；B：Low-density lipoprotein white level；C:
Blood high-density lipoprotein/ldl ratio.
Specific embodiment
Invention is described in further detail with specific embodiment below
The present invention is described with reference to specific embodiment, but it is clear that still various modifications may be made and conversion
And without prejudice to the spirit and scope of the present invention.Therefore, specification and drawings of the invention are it is believed that be illustrative rather than limit
Property processed.
The preparation of the mescenchymal stem cell of embodiment 1.
First, the preparation of umbilical cord mesenchymal stem cells
Operated in superclean bench, taken fresh mature healthy fetus umbilical cord, with containing 2 times of 100U/mL penicillin and
The normal saline flushing removal bloodstain of streptomysin, Wal Tong Shi glue is peeled off, and is cut into less than 1 cubic millimeter block, even spread,
10mL serum free mediums are slowly added to, 37 DEG C are put, is cultivated in the carbon dioxide that volume fraction is 5%, saturated humidity incubator.
After primitive cell culture 7 days, serum-free medium is changed, culture reaches 70% fusion to cell, removes free serum culture
Liquid, addition concentration is respectively 0.25% and 0.1% trypsin-EDTA sodium (EDTA) digestion 1min, umbilical cord MSC
Shrink and depart from bottle wall, now add the culture supernatant previously removed, neutralize trypsin-EDTA solutions, and use pipette
Gently blow and beat, by umbilical cord MSC suspensions immigration 50mL centrifuge tubes.1000rpm is centrifuged 8min, removes supernatant；Rejoin nothing
Serum complete medium is resuspended by MSC, and counts.1.5 are passed according to 1 be inoculated with 10cm culture dishes.During MSC cultures fusion 70% or so
With reference to above method Secondary Culture.
Select the MSC of third generation culture, in exponential phase, and cell fusion degree when being no more than 80% according to passage
It is required that digestion, neutralization, washing, collection cell, using cells frozen storing liquid (90% hyclone and 10% dimethyl sulfoxide (DMSO))
Resuspended MSC, fully mixes, and often pipe 1mL is sub-packed in and freezes the seal of tube.Cryopreservation tube is placed in the freezing storing box equipped with isopropanol, in-
80 DEG C of refrigerator overnights, next day moves into liquid nitrogen container and preserves.
The step of MSC recoveries is：MSC cryopreservation tubes are taken out, is immersed in 37 DEG C of warm water dissolve immediately；Take appropriate free serum culture
Base suspension cell is simultaneously centrifuged, abandoning supernatant；Cell is resuspended in serum free medium, and is inoculated in culture dish, in 37 DEG C,
Volume fraction be 5% carbon dioxide, saturated humidity incubator in cultivate.
2nd, the preparation of mesenchymal stem cells MSCs
Using conventional method in anterior superior spine point of puncture, 50mL bone marrow fluids are extracted with medullo-puncture needle, injection 50mL it is aseptic from
In heart pipe；Diluted with PBS liquid of the equivalent containing 20u/mL heparin, and it is the pouring of 1.073g/ml to add isometric proportion along tube wall
Bar cell separation liquid；2000rmp is centrifuged 30min, draws mononuclearcell layer；SPSS centrifuge washing 2 times, every time
1000rpm is centrifuged 5min, and abandoning supernatant takes precipitation；By marrow MSC obtained above, it is suspended with serum free medium, is counted
Cell, by 5 × 106In the blake bottle that individual/mL is inoculated in, the training of carbon dioxide, saturated humidity that 37 DEG C of volume fractions are 5% is put
Support culture in case.After culture 3 days, serum-free medium is changed, discard non-attached cell, later every 3 days 1 time replacing nutrient solution；7
After~9 days, marrow MSC is merged during up to 50%~60%, carries out first time passage.Serum-free medium is removed, using concentration point
Not Wei 0.25% and 0.1%-EDTA37 DEG C of trypsase digestion 1min, marrow MSC shunk and departs from bottle wall, and addition is previously removed
Culture supernatant, neutralize trypsase-EDTA, and gently blown and beaten using pipette, medulla mesenchyma cell suspension is moved into
In 50mL sterile centrifugation tubes.1000rpm is centrifuged 8min, abandoning supernatant；Rejoin Serum-free complete medium resuspended, count
Number, 1-1.5 inoculation 10cm culture dishes are passed according to 1.Marrow MSC is cultivated when merging 70% or so such as above-mentioned method Secondary Culture.
3rd, the preparation of fat mesenchymal stem cell
Belly white adipose tissue is conventionally separated, visible capilary and musculature, aseptic PBS washings is rejected
Three times, adipose tissue is shredded with sterile scissors then to less than 1 cubic millimeter of pasty state, add aseptic digestive juice and (contain
The serum-free DME culture mediums of 0.1% Collagenase I type and 0.05% trypsase), 45-50min is at the uniform velocity stirred in 37 DEG C of water-baths,
It is clean to tissue block digestion；200 eye mesh screens are collected by filtration filtrate, then use the low sugar containing 10% hyclone (FBS)
DMEM culture mediums equivalent is neutralized, 1500rpm centrifugation 10min, abandoning supernatant；With 1 × 106The cell density of individual/mL, with without blood
The clear resuspended fatty MSC of culture medium, and being inoculated in blake bottle, 37 DEG C, volume fraction be 5% carbon dioxide, saturated humidity culture
Cultivated in case, when fatty MSC is grown to when 80% fusion, MSC cells are digested using 0.25% trypsin solution, with 1：
3 inoculative proportion carries out Secondary Culture in being inoculated into new blake bottle.Fatty MSC is fused to and is centrifuged no more than 80%, 1000rpm
8min, abandoning supernatant rejoins the resuspended MSC of Serum-free complete medium, counts, and passing 1-1.5 inoculations 10cm according to 1 cultivates
Ware.Fatty MSC is cultivated when merging 70% or so such as above-mentioned method Secondary Culture.
The making of the collagen/chitosan sponge bracket of embodiment 2.
By collagen (5mg/mL) and shitosan (5mg/mL is in 0.5M acetic acid) with 1:1 ratio mixing, and stir,
Then freezed at -80 DEG C 2 hours, then freezed 24 hours；After drying, mixture immersion is contained into N- (3- dimethylaminos
Propyl group) in-N'- ethyl carbodiimides (EDC) and 8mM N-hydroxy-succinamides (NHS), while slow shake at room temperature
12 hours.With distilled water washing matrix until pH returns to physiological range (7.0-7.4), then freeze to obtain collagen-chitosan
Sugared sponge bracket (CCSS).CCSS Co60Sterilizing.
The preparation of the collagen/chitosan sponge bracket MSC conditioned mediums of embodiment 3.
MSC is inoculated on collagen/chitosan sponge bracket
CCSS is soaked with PBS and is placed in 6 orifice plates, the single cell suspension of 100 μ L third generation MSC nutrient solutions is added dropwise
To the top surface of CCSS, wherein MSC cell numbers are 1 × 106, and in 37 DEG C, 5%CO2It is middle to be incubated 4 hours, so that Premeabilisation of cells
To in support and adhere to.The MSC of equivalent is seeded in cell-CCSS constructs, and is incubated 4 hours.After after cell adherence, incite somebody to action
2mL serum free mediums are added in each hole, 5%CO at 37 DEG C2Middle culture 48 ± 2 hours.Using MSC in blank well as right
Shine into capable culture.
The preparation of collagen/chitosan sponge bracket MSC conditioned mediums
After MSC cultivates certain hour on CCSS, nutrient solution is removed, postvaccinal CCSS is rinsed tri- times with PBS, used
0.25% trypsase ETDA digestion 1min, MSC shrinks disengaging CCSS, now adds the supernatant previously removed, and neutralizes pancreas
Protease EDTA, and gently blown and beaten using pipette.MSC suspensions are collected with centrifuge tube, is fully washed with PBS 2 times, use 15mL
Plasma-free DMEM medium is resuspended by MSC, moves into T150 blake bottles, cultivates 12 ± 1 hours.MSC suspensions are moved into again
10min is centrifuged under 50mL centrifuge tubes, 3000rpm, supernatant is collected, is existed with a 7-kDa weight shutoff bag type filter
PEG20000, is dialysed at 4 DEG C, and culture medium is concentrated 20 times, is made conditioned medium, i.e. CCSS-MSC-CM.
By the lyophilized prepared dry powder of CCSS-MSC-CM
The supernatant that CCSS-MSC-CM freezes solidification at -80 DEG C is vacuumized, lyophilization, remove ice crystal prepared lyophilized
Powder, -20 DEG C of preservations.
The growth factor showed increased of the MSCs secretions of CCSS cultures
Growth factor is efficient protein, enzyme combination, and the egg of the MCS-CM of purifying is assessed by BCA protein detection reagent kits
White matter concentration, you can measure the change of growth factor concentration in MSC-CM.From the 5 × 10 of 80mL CCSS cultures7MSC supernatants
About 410 μ g albumen of middle separation.As shown in figure 1, it is about the cells of 95 μ g/ million with the total protein concentration in the MSC-CM of CCSS cultures,
And total protein concentration is about the cells of 55 μ g/ million in MSC-CM, horizontal total protein concentration is significantly higher than not in the MSC-CM of CCSS cultures
The MSC-CM for the treatment of, it was demonstrated that CCSS cultures MSC can promote the secretion of MSC growth factors.
The functional verification of the collagen/chitosan sponge bracket MSC conditioned mediums of embodiment 4.
(1) collagen/chitosan sponge bracket MSC-CM promotes the Bone Marrow Macrophage of inflammatory to M2 macrophages
Polarization
The preparation and induction of bone marrow macrophage
Crane one and put to death mouse, foot is put into the fur stripping of mouse hind leg, foot is cut off together with the fur taken off, after cutting
Leg, is put into the culture dish for filling aseptic PBS.After tweezers clamp thigh bone one end, muscle is rejected with scissors, leg is cut in joint
Bone.1640 culture mediums of serum-free are drawn with 2mL syringes, syringe needle are pierced into ossis, marrow to marrow rinsed repeatedly and is bleached,
Bone marrow irrigation liquid is added in 50mL centrifuge tubes, 1000rpm centrifugations 5min.The bone marrow cell for obtaining will be centrifuged with 10mL containing huge
Phagocyte colony stimulating factor (macrophage colony-stimulating factor) MCSF (50ng/mL) and containing serum
1640 culture mediums it is resuspended, be then dispensed into two 25cm2Tissue Culture Flask in, be placed in 37 DEG C and 5%CO2Gas concentration is fitted
Cultivated 3 days in the incubator of degree.Supernatant is abandoned, 1640 culture mediums of the 5mL containing MCSF (50ng/mL) and containing serum is changed and is continued to cultivate 4
My god.Then, in 6 orifice plates, with the glucose solution culture Bone Marrow Macrophage of 30mM, with phorbol 12-myristate
13- acetic acid esters (PMA, 160ng/mL) treatment induction differentiation.After 3 days, three removal non-adherent cells are rinsed with PBS.
MSC-CM inducing bone marrow macrophages
The patch after above-mentioned induction is broken up is processed with CCSS-MSC-CM (20 μ g/mL) and untreated MSC-CM (20 μ g/mL)
The Bone Marrow Macrophage of wall 48 hours, obtains the bone marrow macrophage of MSC-CMC treatment, is respectively designated as in the application
CCSS-M2 and un-M2.It is not to be named as M0 by the bone marrow macrophage of any treatment.In injecting and preparing, first with scraping
Knife takes out macrophage from culture dish, and is centrifuged 10 minutes with 1500rpm.Then, cell is diluted to 2.5 × 106/mL。
As a result：
Collagen/chitosan sponge bracket MSC-CM promotes the Bone Marrow Macrophage of inflammatory to the pole of M2 macrophages
Change
IL-1, IL-6 and TNF α are the proinflammatory cytokine that macrophage can be secreted, and IL-10, TGF-β can for macrophage
With the anti-inflammatory cytokines for producing, CD163 is the mark of M2 Macrophage Surfaces.This experiment is using RT-PCR to above-mentioned cell
The expression quantity of the factor is detected.As shown in Fig. 2 the treatment of glucose and PMA increased in macrophage proinflammatory cytokines because
The expression of son, extension over time, expression quantity further increases, and the treatment of MSC-CM inhibits the expression of proinflammatory factor,
And the expression of proinflammatory factor is maintained certain level；MSC-CM is processed relative to collagen/chitosan sponge bracket is provided without,
CCSS-MSC-CM is notable and significantly inhibits the expression of proinflammatory cytokine IL-1, IL-6 and TNF α.The treatment of glucose and PMA
The expression of anti-inflammatory cytokines in macrophage is reduced, extension over time, expression quantity is further reduced；CCSS-MSC-
CM remarkably promotes IL-10 and the TGF-β expression of Bone Marrow Macrophage, and remarkably promotes M2 Macrophage Surface marks
The expression of CD163, these as shown by data CCSS-MSC-CM promotes polarization of the macrophage to M2.Cultivated by CCSS
MSC, can secrete more growth factors, improve the efficiency that macrophage is induced into M2 macrophages.
(2) bone marrow macrophage of collagen/chitosan sponge bracket MSC-CM inductions improves lipid-metabolism
The induction and treatment of obesity mice
8 week old male C57BL/6N mouse are raised under 12 hours illumination/dark cycles in 22 DEG C of separate ventilation cages.It is suitable
After answering property is fed one week, it is divided into two groups, one group is 6 mouse, standard chow diet group (normal group), another group of 24 mouse,
Food rich in fat diet (HFD, 60% fat) group, feeds HFD mouse 12 weeks to meet obese model.
The mouse of HFD groups is separated into four groups, every group of 6 mouse：HFD and PBS infusions group (fat group), HFD is huge with M0
Phagocyte infusion group (M0 infusion groups, experiment contrast), the M2 macrophage infusion groups (CCSS- of HFD and CCSS-MSC-CM inductions
M2 infusions group) and the M2 macrophage infusions groups (un-M2 infusions group) that are induced with untreated MSC-CM of HFD.Then, will
In 0.2mL PBS 5 × 105The M2 macrophages of M0, CCSS-MSC-CM induction or the M2 macrophages of untreated MSC-CM inductions
Cell is infused into Mice Body by tail vein.After one week, second injection of same composition is carried out, and observed before execution
All mouse one week.In whole process, all mouse keep the diet before them, and record the food of complete cycle after second injection
Thing is absorbed.Before execution, anaesthetized to 1% yellow Jackets (60mg/kg) are injected in animal peritoneal.Mouse blood is taken, for surveying
The lipid-metabolism point of amount triglycerides (TG), HDL-C (HDLc) and LDL-C (LDLc)
Analysis；Record INAT (inguinal adipose tissue, groin adipose tissue) and EAT (epididymal adipose
Tissue, epididymal adipose) weight.Adipose tissue is fixed for pathological analysis in formalin.
As a result：
The infusion of M2 macrophages reduces the weight of INAT
As shown in Figure 3A, after macrophage infusion of therapeutic, the food intake of fat group mouse does not have substantially change.
And compared to the mouse of fat group, the weight and EAT weight of the INAT for reducing obesity mice of the infusion of macrophage.With M0
Treatment group compares with un-M2 treatment groups, and the weight for being transfused the INAT of the mouse of CCSS-M2 macrophages is decreased significantly (figure
3B), EAT weight reduces also more (Fig. 3 C).
The infusion of M2 macrophages reduces mouse adipocytes volume
Obesity causes the fat cell of mouse to become big, and the infusion of macrophage reduces mouse adipocytes volume.Such as
Shown in Fig. 4, hematoxylin-eosin (HE) dyeing finds that compared with M0 treatment groups and un-M2 treatment groups, CCSS-M2 macrophages are defeated
INAT and EAT (epididymal adipose tissue epididymal adiposes) fat cell volume of note group mouse has substantially
Reduce.
The infusion of M2 macrophages improves insulin resistance
P-AKT is the label for being widely used for assessing insulin sensitivity.By p- in western blot detections EAT
AKT, it is found that compared to M0 treatment groups and un-M2 treatment groups, the p-AKT of EAT dramatically increases (figure in CCSS-M2 infusion groups
5A).Additionally, insulin resistance has and substantially changes in the result display CCSS-M2 infusion groups of Fig. 5 B abdominal cavity carbohydrate tolerance tests (IPGTT)
It is kind.These results indicate that CCSS-M2 macrophages infusion improves the insulin resistance in obesity mice, in a way may be used
To stimulate the mitigation of body weight.
The infusion of M2 macrophages improves the metabolism of lipid
Found after lipid level to blood is measured, it is low close that obesity increased triglycerides (TG) and blood plasma in mouse
The concentration of degree lipoprotein (LDL-c).Compared with M0 treatment groups and un-M2 treatment groups, CCSS-M2 treatment groups TG and LDL-c has aobvious
Writing is reduced, and HDL-c (HDL)/LDL-c (low-density lipoprotein) is dramatically increased.These results of study show CCSS-
M2 macrophages infusion can improve lipid-metabolism.
The MSC cultivated by CCSS, can secrete more growth factors, and macrophage improves into that induced into M2 macrophages is thin
The efficiency of born of the same parents.M2 macrophages are a kind of immunocytes of anti-inflammatory, the cell by recovering the inflammation/macrophage stable state of EAT,
Further improve insulin resistance and lipid-metabolism.The M2 macrophages that the MSC-CM cultivated by CCSS is induced, therapeutic effect
It is more significantly, fat and fat Related Insulin resistance is treated by recovering macrophage homeostasis, be obese patient with
The treatment for having come newly is wished.
Claims (10)
1. a kind of derivant of M2 macrophages, it is characterized by, it is that a kind of mesenchyma of collagen/chitosan sponge bracket induction is done
Cell conditioned medium.
2. the derivant of M2 macrophages as claimed in claim 1, the collagen/chitosan sponge bracket is that collagen gathers with shell
After sugared mixture freeze-drying, in N- (3- dimethylaminopropyls)-N'- ethyl carbodiimides (EDC) and N- hydroxysuccinimidyl acyls
Shake what is obtained in imines (NHS) solution.
3. the derivant of M2 macrophages as claimed in claim 2, the collagen/chitosan sponge bracket is by collagen and shell
Glycan is with 1:1 ratio mixing, then freezes 2 hours at -80 DEG C, then freezes 24 hours；After drying, mixture is immersed
In containing N- (3- dimethylaminopropyls)-N'- ethyl carbodiimides (EDC) and 8mM N-hydroxy-succinamides (NHS), together
When at room temperature slowly shake 12 hours obtain.
4. mescenchymal stem cell as claimed in claim 1 is selected from umbilical cord mesenchymal stem cells, fat mesenchymal stem cell, marrow
Mescenchymal stem cell.
5. a kind of preparation method of the derivant of M2 macrophages, its step includes：A) by mescenchymal stem cell be seeded in collagen/
On chitosan sponges bracket；B) using mescenchymal stem cell-collagen/chitosan sponge bracket described in serum free medium culture；
C) mescenchymal stem cell and culture medium are collected；D) fresh serum-free media is added, and continues to cultivate the mesenchyma and done
Cell；And e) collect the conditioned medium of the mescenchymal stem cell.
6. the preparation method of the derivant of M2 macrophages as claimed in claim 5, dry thin by mesenchyma in the step a)
The step that born of the same parents are seeded on collagen/chitosan sponge bracket includes：The mescenchymal stem cell single cell suspension is dripped into glue
Original/chitosan sponges bracket top surface is incubated culture；Then equivalent mescenchymal stem cell is seeded in the mesenchyma dry thin
Culture is incubated in born of the same parents-collagen/chitosan sponge bracket construct.
7. the preparation method of the derivant of M2 macrophages as claimed in claim 5, the step c) collects the mesenchyma
The step of stem cell and culture medium, includes：Culture medium is removed, the mescenchymal stem cell is digested using trypsase-ETDA, made
It shrinks and departs from collagen/chitosan sponge bracket；The culture medium removed described in adding, neutralizes trypsase-EDTA；And gently blow
Beat the mescenchymal stem cell.
8. the preparation method of the derivant of M2 macrophages as claimed in claim 5, by the mesenchyma in the step d)
Stem cell continues culture 12 ± 1 hours in serum free medium.
9. the preparation method of the derivant of M2 macrophages as claimed in claim 5, is collected by centrifugation described in the step e)
Mescenchymal stem cell and culture medium, to remove cell fragment；Then the condition is collected with a 7-kDa weight shutoffs bag to train
Support base.
10. application of the derivant of any described M2 macrophages of claim 1-4 in obesity is treated.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110684732A (en) * | 2019-10-18 | 2020-01-14 | 广东唯泰生物科技有限公司 | Method for detecting differentiation of macrophages into M2 type macrophages |
| CN111727046A (en) * | 2018-07-17 | 2020-09-29 | 仿生技术支持有限公司 | Eicosanoid generation promoter |
| WO2020200295A1 (en) * | 2019-04-03 | 2020-10-08 | Nanjing Iaso Biotherapeutics Co., Ltd. | Ex-vivo cultured macrophage population and use thereof |
| CN113730439A (en) * | 2021-09-09 | 2021-12-03 | 陕西中鸿瑞康健康管理有限公司 | Stem cell factor freeze-dried powder capable of reducing triglyceride and preparation method and application thereof |
| CN114891728A (en) * | 2022-04-07 | 2022-08-12 | 广东医科大学附属医院 | Polyelectrolyte membrane, macrophage exosome and application of polyelectrolyte membrane and macrophage exosome in promotion of BMSCs differentiation |
Citations (31)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008049281A1 (en) * | 2006-10-27 | 2008-05-02 | Beijing Institute Of Transfusion Medicine | Construction method of hepatic tissue engineering construct and the hepatic tissue engineering construct |
| CN101379182A (en) * | 2005-12-14 | 2009-03-04 | 器官发生有限公司 | Skin care compositions and treatments |
| WO2011010966A1 (en) * | 2009-07-23 | 2011-01-27 | Agency For Science, Technology And Research (A*Star) | Pre-natal mesenchymal stem cells |
| WO2011106521A1 (en) * | 2010-02-25 | 2011-09-01 | Abt Holding Company | Modulation of macrophage activation |
| WO2011143411A1 (en) * | 2010-05-12 | 2011-11-17 | Abt Holding Company | Modulation of splenocytes in cell therapy for traumatic brain injury |
| AU2012258384A1 (en) * | 2001-12-07 | 2012-12-20 | Asterias Biotherapeutics, Inc. | Hematopoietic cells from human embryonic stem cells |
| EP2557153A2 (en) * | 2010-04-06 | 2013-02-13 | Gosudarstvennoye Obrazovatel'Noye Uchrezhdeniye Vysshyego Propyhsessional'Nogo Obrazovanija "Krasnojarskij Gosudarstvyennyj MyedItsinskij | Method for producing a neural matrix |
| CN103372233A (en) * | 2012-04-13 | 2013-10-30 | 杭州龙禧生物医药科技有限公司 | Preparation method and product of tissue-engineered cartilage graft |
| CN103420931A (en) * | 2013-05-02 | 2013-12-04 | 范国煌 | Pentacyclic compound capable of adjusting polarization of macrophages and resisting diabetes and application thereof |
| CN103687941A (en) * | 2011-01-12 | 2014-03-26 | 城户常雄 | Culture method to obtain and maintain a pure or enriched population of mammalian neural stem cells and/or neural progenitor cells that are prone to differentiate into oligodendrocyte-lineage cells in vitro |
| US20150258144A1 (en) * | 2013-03-15 | 2015-09-17 | Thomas A. Davis | Allograft tolerance induction |
| CN105154406A (en) * | 2015-09-18 | 2015-12-16 | 中国人民解放军第二军医大学 | Pluripotent stem cell as well as preparation method and application thereof |
| CN105316285A (en) * | 2015-11-06 | 2016-02-10 | 深圳爱生再生医学科技有限公司 | Method for culturing mesenchymal stem cells through manual simulation of bone marrow microenvironment |
| CN105362263A (en) * | 2015-12-05 | 2016-03-02 | 江苏省中国科学院植物研究所 | Application of compound Isomeranzin to treatment of inflammatory diseases |
| CN105481834A (en) * | 2016-01-12 | 2016-04-13 | 范国煌 | Tricyclic small-molecule inhibitor for macrophage movement inhibiting factor and application of tricyclic small-molecule inhibitor |
| CN105497005A (en) * | 2015-12-04 | 2016-04-20 | 江苏省中国科学院植物研究所 | Application of flavonoid compound in treatment of inflammatory diseases |
| CN106074602A (en) * | 2016-06-22 | 2016-11-09 | 深圳爱生再生医学科技有限公司 | Cell mixing preparation and preparation method and application |
| CN106244679A (en) * | 2016-07-27 | 2016-12-21 | 中国医学科学院基础医学研究所 | MiR 100 inhibitor purposes in reducing cancer metastasis |
| CN106318904A (en) * | 2015-06-15 | 2017-01-11 | 上海朗斯生物工程有限公司 | Use of mesenchymal stem cell conditioned culture medium for cosmetics field |
| CN106361771A (en) * | 2016-11-08 | 2017-02-01 | 北京恒峰铭成生物科技有限公司 | High-glucose activated mesenchymal stem cell injection and application thereof for diabetic drugs |
| CN106497886A (en) * | 2016-12-09 | 2017-03-15 | 北京恒峰铭成生物科技有限公司 | A kind of hair regeneration induction liquid and its preparation method and application |
| CN106492269A (en) * | 2016-12-09 | 2017-03-15 | 广东颜值科技有限公司 | A kind of temperature-sensitive hydrogel and preparation method thereof |
| CN107261213A (en) * | 2017-06-13 | 2017-10-20 | 北京恒峰铭成生物科技有限公司 | A kind of active hydrogel and preparation method thereof and the application in operation wound reparation |
| WO2018005780A1 (en) * | 2016-06-30 | 2018-01-04 | University Of Pittsburgh - Of The Commonwealth System Of Higher Education | Artificial cells and delivery devices for use in tissue engineering, and related methods |
| CN109394777A (en) * | 2018-12-12 | 2019-03-01 | 上海交通大学医学院附属第九人民医院 | Application of the anoectochilus roxburghii glycosides in osteoarthritis treatment drug |
| CN109762903A (en) * | 2019-01-31 | 2019-05-17 | 山东大学齐鲁医院 | Application of the miR-1246 and/or TERF2IP in diagnosis and treatment glioma |
| US10568911B2 (en) * | 2007-05-03 | 2020-02-25 | The Brigham And Women's Hospital, Inc. | Multipotent stem cells and uses thereof |
| RU2716159C2 (en) * | 2018-03-06 | 2020-03-06 | Андрей Александрович Темнов | Composition for preventive or therapeutic treatment of conditions associated with changing amount of hair |
| US10603407B2 (en) * | 2011-04-28 | 2020-03-31 | Warsaw Orthopedic, Inc. | Collagen matrix for cell therapy |
| US10624930B2 (en) * | 2014-06-10 | 2020-04-21 | Mesoblast International Sarl | Treatment of immune disorders |
| US10639355B2 (en) * | 2014-01-24 | 2020-05-05 | Quarrymen & Co. Inc. | Method of making conditioned medium from immortalized dental pulp stem cells |
-
2017
- 2017-03-31 CN CN201710209865.2A patent/CN106929474B/en active Active
Patent Citations (31)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2012258384A1 (en) * | 2001-12-07 | 2012-12-20 | Asterias Biotherapeutics, Inc. | Hematopoietic cells from human embryonic stem cells |
| CN101379182A (en) * | 2005-12-14 | 2009-03-04 | 器官发生有限公司 | Skin care compositions and treatments |
| WO2008049281A1 (en) * | 2006-10-27 | 2008-05-02 | Beijing Institute Of Transfusion Medicine | Construction method of hepatic tissue engineering construct and the hepatic tissue engineering construct |
| US10568911B2 (en) * | 2007-05-03 | 2020-02-25 | The Brigham And Women's Hospital, Inc. | Multipotent stem cells and uses thereof |
| WO2011010966A1 (en) * | 2009-07-23 | 2011-01-27 | Agency For Science, Technology And Research (A*Star) | Pre-natal mesenchymal stem cells |
| WO2011106521A1 (en) * | 2010-02-25 | 2011-09-01 | Abt Holding Company | Modulation of macrophage activation |
| EP2557153A2 (en) * | 2010-04-06 | 2013-02-13 | Gosudarstvennoye Obrazovatel'Noye Uchrezhdeniye Vysshyego Propyhsessional'Nogo Obrazovanija "Krasnojarskij Gosudarstvyennyj MyedItsinskij | Method for producing a neural matrix |
| WO2011143411A1 (en) * | 2010-05-12 | 2011-11-17 | Abt Holding Company | Modulation of splenocytes in cell therapy for traumatic brain injury |
| CN103687941A (en) * | 2011-01-12 | 2014-03-26 | 城户常雄 | Culture method to obtain and maintain a pure or enriched population of mammalian neural stem cells and/or neural progenitor cells that are prone to differentiate into oligodendrocyte-lineage cells in vitro |
| US10603407B2 (en) * | 2011-04-28 | 2020-03-31 | Warsaw Orthopedic, Inc. | Collagen matrix for cell therapy |
| CN103372233A (en) * | 2012-04-13 | 2013-10-30 | 杭州龙禧生物医药科技有限公司 | Preparation method and product of tissue-engineered cartilage graft |
| US20150258144A1 (en) * | 2013-03-15 | 2015-09-17 | Thomas A. Davis | Allograft tolerance induction |
| CN103420931A (en) * | 2013-05-02 | 2013-12-04 | 范国煌 | Pentacyclic compound capable of adjusting polarization of macrophages and resisting diabetes and application thereof |
| US10639355B2 (en) * | 2014-01-24 | 2020-05-05 | Quarrymen & Co. Inc. | Method of making conditioned medium from immortalized dental pulp stem cells |
| US10624930B2 (en) * | 2014-06-10 | 2020-04-21 | Mesoblast International Sarl | Treatment of immune disorders |
| CN106318904A (en) * | 2015-06-15 | 2017-01-11 | 上海朗斯生物工程有限公司 | Use of mesenchymal stem cell conditioned culture medium for cosmetics field |
| CN105154406A (en) * | 2015-09-18 | 2015-12-16 | 中国人民解放军第二军医大学 | Pluripotent stem cell as well as preparation method and application thereof |
| CN105316285A (en) * | 2015-11-06 | 2016-02-10 | 深圳爱生再生医学科技有限公司 | Method for culturing mesenchymal stem cells through manual simulation of bone marrow microenvironment |
| CN105497005A (en) * | 2015-12-04 | 2016-04-20 | 江苏省中国科学院植物研究所 | Application of flavonoid compound in treatment of inflammatory diseases |
| CN105362263A (en) * | 2015-12-05 | 2016-03-02 | 江苏省中国科学院植物研究所 | Application of compound Isomeranzin to treatment of inflammatory diseases |
| CN105481834A (en) * | 2016-01-12 | 2016-04-13 | 范国煌 | Tricyclic small-molecule inhibitor for macrophage movement inhibiting factor and application of tricyclic small-molecule inhibitor |
| CN106074602A (en) * | 2016-06-22 | 2016-11-09 | 深圳爱生再生医学科技有限公司 | Cell mixing preparation and preparation method and application |
| WO2018005780A1 (en) * | 2016-06-30 | 2018-01-04 | University Of Pittsburgh - Of The Commonwealth System Of Higher Education | Artificial cells and delivery devices for use in tissue engineering, and related methods |
| CN106244679A (en) * | 2016-07-27 | 2016-12-21 | 中国医学科学院基础医学研究所 | MiR 100 inhibitor purposes in reducing cancer metastasis |
| CN106361771A (en) * | 2016-11-08 | 2017-02-01 | 北京恒峰铭成生物科技有限公司 | High-glucose activated mesenchymal stem cell injection and application thereof for diabetic drugs |
| CN106492269A (en) * | 2016-12-09 | 2017-03-15 | 广东颜值科技有限公司 | A kind of temperature-sensitive hydrogel and preparation method thereof |
| CN106497886A (en) * | 2016-12-09 | 2017-03-15 | 北京恒峰铭成生物科技有限公司 | A kind of hair regeneration induction liquid and its preparation method and application |
| CN107261213A (en) * | 2017-06-13 | 2017-10-20 | 北京恒峰铭成生物科技有限公司 | A kind of active hydrogel and preparation method thereof and the application in operation wound reparation |
| RU2716159C2 (en) * | 2018-03-06 | 2020-03-06 | Андрей Александрович Темнов | Composition for preventive or therapeutic treatment of conditions associated with changing amount of hair |
| CN109394777A (en) * | 2018-12-12 | 2019-03-01 | 上海交通大学医学院附属第九人民医院 | Application of the anoectochilus roxburghii glycosides in osteoarthritis treatment drug |
| CN109762903A (en) * | 2019-01-31 | 2019-05-17 | 山东大学齐鲁医院 | Application of the miR-1246 and/or TERF2IP in diagnosis and treatment glioma |
Non-Patent Citations (7)
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111727046A (en) * | 2018-07-17 | 2020-09-29 | 仿生技术支持有限公司 | Eicosanoid generation promoter |
| CN111727046B (en) * | 2018-07-17 | 2024-03-22 | 仿生技术支持有限公司 | Use of mesenchymal stem cell secretion in preparation of eicosanoid production promoter |
| WO2020200295A1 (en) * | 2019-04-03 | 2020-10-08 | Nanjing Iaso Biotherapeutics Co., Ltd. | Ex-vivo cultured macrophage population and use thereof |
| CN110684732A (en) * | 2019-10-18 | 2020-01-14 | 广东唯泰生物科技有限公司 | Method for detecting differentiation of macrophages into M2 type macrophages |
| CN113730439A (en) * | 2021-09-09 | 2021-12-03 | 陕西中鸿瑞康健康管理有限公司 | Stem cell factor freeze-dried powder capable of reducing triglyceride and preparation method and application thereof |
| CN114891728A (en) * | 2022-04-07 | 2022-08-12 | 广东医科大学附属医院 | Polyelectrolyte membrane, macrophage exosome and application of polyelectrolyte membrane and macrophage exosome in promotion of BMSCs differentiation |
| CN114891728B (en) * | 2022-04-07 | 2023-01-03 | 广东医科大学附属医院 | Polyelectrolyte membrane, macrophage exosome and application of polyelectrolyte membrane and macrophage exosome in promotion of BMSCs differentiation |
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