CN104800891B - A kind of extracellular matrix biomaterial for strengthening In vitro culture mesenchymal stem cell biological anti-oxidation function, preparation method and applications - Google Patents
A kind of extracellular matrix biomaterial for strengthening In vitro culture mesenchymal stem cell biological anti-oxidation function, preparation method and applications Download PDFInfo
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- CN104800891B CN104800891B CN201510257496.5A CN201510257496A CN104800891B CN 104800891 B CN104800891 B CN 104800891B CN 201510257496 A CN201510257496 A CN 201510257496A CN 104800891 B CN104800891 B CN 104800891B
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Abstract
The present invention provides a kind of extracellular matrix biomaterial for strengthening In vitro culture mesenchymal stem cell biological anti-oxidation function, preparation method and applications, and the preparation process of the extracellular matrix biomaterial is as follows：Umbilical cord mesenchymal stem cells culture in complete medium, during density >=90%, adds complete culture solution culture 7 10 days, changed liquid once per 3 days, add de-cell liquid afterwards, be built into extracellular matrix biomaterial.The extracellular matrix biomaterial of the present invention can strengthen In vitro culture mesenchymal stem cell biological anti-oxidation function.
Description
Technical field
The invention belongs to biomedical sector, more particularly to a kind of enhancing In vitro culture mesenchymal stem cell biological antioxidation
The extracellular matrix biomaterial of function, preparation method and applications.
Background technology
Mescenchymal stem cell（Mesenchymal stem cells, MSCs）Be a kind of responsible tissue regeneration and repair into
Somatic stem cell, is originally found in bone marrow, due to its regeneration and immunoregulatory potential, causes extensive pass in various fields
Note.Human Umbilical Cord's mescenchymal stem cell, especially from Wharton's jelly（Two umbilical arterys and one are wrapped in umbilical cord
The mucoprotein sample connective tissue of bar umbilical vein）It is considered as preferable seed cell in cell therapy and regenerative medicine.Umbilical cord is one
Organize outside kind of embryo, therefore umbilical cord mesenchymal stem cells possess the double grading of embryonic stem cell and adult stem cell, its self
Update and multi-lineage potential is substantially better than mesenchymal stem cells MSCs.
There is increasing evidence that, active oxygen (ROS) cell proliferation, differentiation, death, the stable state of matrix environment,
Oxidoreduction signaling mechanism etc. is requisite, but the ROS of cell abnormal level can cause DNA, protein, lipid irreversible
The destiny of aging and death is moved towards in the infringement for turning, final cell.
Although applications of the MSCs in regenerative medicine obtains extensive concern, its expand in vitro with internal migration process
But inevitably it is exposed under sizable oxidative stress, causes ROS（Such as hydrogen peroxide, hydroxyl radical free radical, superoxides
Radical anion）It is exceeded.H in the MSCs of longterm culture in vitro2O2Level increases, and causes cell ageing and Proliferation Ability.Bone
In arthritis and rheumatoid arthritis patients body, chondrocyte or neutrophilic granulocyte and macrophage produce the ROS of abnormal level,
Affect the regeneration potential of MSCs.Research shows that the accumulation of ROS is caused due to contacting inflammatory cytokine, so as to suppress dry thin
The cell propagation of born of the same parents and multi-lineage potential.
ROS generate and eliminate between balance to cells survival, breed, break up most important.Superoxide dismutase
(SOD), including the isozyme containing copper, zinc (SOD1 and SOD2) and manganese (SOD2), MSCs is protected from superoxide anion
The H that catalysis is generated2O2Active oxygen infringement.The mesenchyme that oxidative stress can eliminate human synovial's origin that SOD2 causes is dry thin
The cartilage differentiation ability of born of the same parents declines and the expression of matrix metalloproteinase increases.Additionally, the H that SOD is produced2O2By catalase
（catalase）Neutralized, previous numerous studies show, improve the catalase activity in MSCs and be conducive to alleviating H2O2Induction
Apoptosis.
Extracellular matrix (ECM) component of MSC origins includes I types and type III collagen protein, fibronectin, layer adhesion egg
In vain, the microenvironment of vitro culture system can be served as, is promoted MSC amplifications, guided MSC to break up to specific direction.Cell free ECM
The outer microenvironment of internal stem cells not only can be simulated, the biological behaviour of somatomedin and hormone is can also adjust.This fortune
Cellular replication aging can be prevented with the cultural method of cell free ECM, strengthen the differentiation capability again of terminally differentiated cellses, such as cartilage
Cell and nucleus pulposus cell.It has recently been demonstrated that cell free ECM can improve mesenchymal stem cells MSCs opposing H2O2Induction
Oxidative stress and cell cycle arrest.However, impacts of the ECM to antioxidant system in umbilical cord mesenchymal stem cells but rarely has people
Know.
The content of the invention
The technical problem of solution：Expand in vitro and inevitably expose in internal migration process for existing MSCs
Under sizable oxidative stress, the shortcoming that ROS is exceeded, the present invention is caused to provide a kind of In vitro culture mesenchyme that strengthens and do thin
The extracellular matrix biomaterial of born of the same parents' biological antioxidant function, preparation method and applications, by umbilical cord mesenchymal stem cells point
Secrete substrate and prepare extracellular matrix biomaterial, the feature not only with multiple proteins composition, and mesenchyme can be used for and do
The amplification in vitro of cell, has the advantages that good biocompatibility, breeds efficiency high, significantly reduces intracellular reactive oxygen content.
Technical scheme：
The detection content of the present invention includes：（1）Impact of the extracellular matrix biomaterial to UC-MSCs multiplication capacities；（2）
Extracellular matrix biomaterial is to ROS in UC-MSCs and H2O2 The impact of expression；（3）Extracellular matrix biomaterial is to UC-
The impact of SOD-2 expression in MSCs；（4）Impact of the extracellular matrix biomaterial to Catalase activity in UC-MSCs.
Content described above is realized by following steps：
（A）Umbilical cord mesenchymal stem cells（Umbilical cord-derived mesenchymal stem cells,
UC-MSCs) culture is in complete medium（Complete medium component is α-MEM and hyclone, and their volume ratio is（4-
9）:1, and contain antibiotic, the content of antibiotic is 10 ~ 200 U/mL）, during density >=90%, addition contains ascorbic acid
(100-200uM) complete culture solution culture 7-10 days, changed liquid once per 3 days, added de-cell liquid afterwards（It is 7.4 by pH value
Phosphate buffered saline, containing 0.5 ~ 5% volume ratios of Triton X-100 and ammonia 10-40mM）, it is built into extracellular
Matrix biopolymers material；
（B）UC-MSCs is cultivated respectively in common orifice plate（TCPS plates）Extracellular matrix biomaterial is covered with bottom
Orifice plate（ECM plates）In, the DNA content in different time point detection orifice plates, diacetic acid fluorescein（FDA）When dyeing record is different
Between the cell quantity put and metamorphosis；
（C）ROS and H in UC-MSCs on detection TCPS plates and ECM plates2O2Content；
（D）On detection TCPS plates and ECM plates, in UC-MSCs, SOD-2 contents and Catalase are active.
The UC-MSCs takes from people source, pig source, Mus source or rabbit source.
The extracellular matrix biomaterial can be applicable to include the fields such as cellular transplantation therapy and regenerative medicine.
Content concrete analysis step described above is as follows：
（1）FDA dyeing, impact of the DNA quantitative detecting analysis extracellular matrix biomaterial to UC-MSCs multiplication capacities.
（2）DCF-DA fluorescent quantitations detect intracellular ROS level.
（3）H2O2Detection kit detects intracellular H2O2Level.
（4）Western bort detect intracellular SOD-2 contents.
（5）Catalase activity detection kits detect intracellular Catalase activity levels.
H2O2Be one of ROS main components, dual function is played in cell propagation.Low-level H2O2(<10 μM) thorn
Sharp fibroblastic growth, and cell is exposed to the H of high concentration2O2(>400 μM) then can rapid apoptosis.The solution of the present invention table
It is bright, the intracellular H of extracellular matrix biomaterial cultured cells2O2Level declines, and is sertoli cell survival in vitro and fast breeding
A major reason.Additionally, superoxide anion affects cell to breed in dose-dependent mode, low-level super oxygen
Thing anion stimulates cellular proliferation, and concentration increase then can cell growth inhibiting.
Strengthen ROS levels during oxidation resistance causes UC-MSCs to decline, have a wide range of applications in terms of clinical treatment.Bone
Property arthritis in the SOD-2 activity of chondrocyte impaired do not only result in its oxidative damage, moreover it is possible to accelerate the morbidity of osteoarthritis
Process.
MSCs is widely used in the research and development of cellular transplantation therapy and organizational project, but ROS exceeded under pathological conditions is produced
Raw inflammatory cytokine changes some characteristics of MSCs, affects which to survive.Therefore, during transplanting in vivo, prevent thin
Born of the same parents' oxidativestress damage and raising cell survival rate are particularly important.Previous research has shown that cell culture thin from synovial membrane
The de- cell ECM of born of the same parents or medullary cell significantly reduces ROS levels, so illustrates the potential of anti-oxidative defense in ECM regulating cells
Molecular mechanism will become highly significant.
Beneficial effect：A kind of cell of enhancing In vitro culture mesenchymal stem cell biological anti-oxidation function that the present invention is provided
Epimatrix biomaterial, preparation method and applications, MSCs cultivate ratio in the extracellular matrix biomaterial on TCPS plates
The expression of enzymes higher level of the intracellular antioxidant activity of culture, especially SOD-2 and Catalase；SOD-2 and Catalase
Increased activity causes ROS and H2O2Level is reduced, therefore extracellular matrix biomaterial can strengthen In vitro culture mesenchyme and do thin
Born of the same parents' biological antioxidant function.
Description of the drawings
Fig. 1 is the proliferative conditions figure that FDA dyes UC-MSCs on record different time points TCPS plate and ECM plates.
Fig. 2 is the proliferative conditions figure of UC-MSCs on DNA quantitative detecting analysis different time points TCPS plates and ECM plates.
Fig. 3 is ROS and H in UC-MSCs on TCPS plates and ECM plates2O2Changes of contents figure.
Fig. 4 is SOD-2 changes of contents figures in UC-MSCs on TCPS plates and ECM plates.
Fig. 5 is Catalase activity change figures in UC-MSCs on TCPS plates and ECM plates.
Specific embodiment
The present invention is further explained with reference to specific embodiment, but specific embodiment is not to the present invention
It is limited in any way.Unless stated otherwise, reagent involved in embodiment, method are reagent commonly used in the art and method.
Embodiment 1
Specific experiment step is as follows：
（1）People source UC-MSCs is in 37 DEG C, 5%CO2175cm is incubated in incubator2In culture bottle, changed once per three days
Liquid, the volume ratio of wherein culture medium each component is 89% α-MEM, 10% hyclone, 1% penicillin, streptomycin；
（2）When cell density >=90% with 0.25% trypsin-EDTA peptic cells, by 5000/cm2Density is inoculated in
Cultivate in the orifice plate of the plurality of specifications of pretreatment, until reaching 90% fusion, add 100 μM of ascorbic acid to continue culture 8 days, three
It changes liquid once, and wherein inoculation method is incubated 1 hour under 37 DEG C of environment for 0.2% gelatin of TCPS plates, then uses 1% glutaraldehyde
It is incubated at room temperature 30 minutes with 1M ethanolamine, the plate of plurality of specifications is divided into 12 orifice plates for FDA dyeing, DNA detection by quantitative, 6
Orifice plate is used for ROS, H2O2, Catalase assays, 75cm2Culture bottle is used for SOD-2 analysis of protein；
（3）In order to obtain free extracellular matrix biomaterial after 8 days, cultured cells adds de- cell mixture, in
It is incubated 5 minutes under 37 DEG C of environment, wherein de- cell mixture contains 0.5% by the phosphate buffered saline that pH value is 7.4
Triton X-100 and 20mM ammonia；
（4）Cell free extracellular matrix biomaterial is cleaned into 3 times with PBS, for the last time with dual anti-containing 1X
PBS simultaneously retains in the orifice plate, 4 DEG C be stored in it is standby in gnotobasiss.
（5）UC-MSCs is with 5000/cm2Density is seeded in TCPS plates and ECM plates respectively, in 37 DEG C, 5%CO2Train under environment
Support.
Detection project and method are as follows：
1st, extracellular matrix biomaterial is to UC-MSCs multiplication capacities, intracellular ROS, H2O2The impact of level.
A. impact of the extracellular matrix biomaterial to UC-MSCs multiplication capacities.
Cell viability is dyeed with FDA respectively at the 1st, 3,5,7 day using FDA dyeing assessments, and cell is by PBS
Once it is incubated 10 minutes in 37 DEG C of lucifuges with 5 μ g/ml FDA dye liquors afterwards, after PBS washed once, is inverted with Olympus IX51
Microscope is taken pictures, and wherein FDA acetone is made into 1000X storage concentration, is diluted with culture fluid during use；
Cell propagation is assessed by DNA quantitative detecting analysis, using Quant-iTTM PicoGreen®DsDNA analytical reagents
Box.Trypsin digestion cell was used at the 1st, 3,5,7 day respectively, DNA sample is collected in 1.5ml EP pipes, is removed after high speed centrifugation
Supernatant, collects 4 for per group and manages.Often pipe adds 200 μ L papains dissolving buffer (to be dissolved in final concentration of 125 in PBS
μ g/ml), 60 DEG C of water-baths 4 hours allow cell fully to crack.The lysate and reaction reagent of equal number is added to 96 orifice plates
In, room temperature lucifuge is incubated 5 minutes, with SynergyMx multi-function microplate readers under 485/520 nm (excitation wavelength/launch wavelength)
The fluorescence intensity of determination sample, 6 multiple holes of per group of setting.
As depicted in figs. 1 and 2, extracellular matrix biomaterial significantly improves the multiplication capacity of UC-MSCs to testing result.
FDA dyeing shows that the stem cell morphology cultivated on ECM presents spindle-type, and Each point in time cell quantity is significantly more than TCPS
Group（Scale bar = 100 μm）；On ECM plates, Each point in time DNA content is significantly more than TCPS plates.
B. extracellular matrix biomaterial is to ROS in UC-MSCs and H2O2The impact of changes of contents.
DCF-DA fluorescent quantitations detect intracellular ROS level.Often manage5Individual cell, at 10 μM 2 ',
37 C lucifuges water-bath 30 minutes in 7 ' dichlorofluorescein oxalic acid (DCF-DA).Using Cytomics FC500 flow cytometries
Instrument measures fluorescence intensity.
Intracellular H2O2Amplex is used in the detection of level®Red Hydrogen Peroxide detection kit.Culture exists
Supernatant is extracted after cell cell pyrolysis liquid cracking on TCPS plates and ECM plates, pyrolysis product is incubated after being mixed with reaction reagent
Educate 30 minutes.With SynergyMx multi-function microplate readers in the glimmering of 530/590 nm (excitation wavelength/launch wavelength) determination sample
Light intensity.
Testing result is as shown in figure 3, ROS and H in UC-MSCs on ECM plates2O2Content is substantially less than TCPS plates.
2nd, impact of the extracellular matrix biomaterial to SOD-2 contents and Catalase activity change in UC-MSCs.
A. impact of the extracellular matrix biomaterial to SOD-2 changes of contents in UC-MSCs
75cm21.5ml EP pipes are transferred to after the extracellular matrix biomaterial cell pancreatin digestion of culture bottle culture
In, supernatant is removed after high speed centrifugation, often pipe adds the cell pyrolysis liquid 100ul containing protease inhibitor to crack 30 points on ice
Clock, takes supernatant after high speed centrifugation, determine protein concentration using BCA protein quantification test kits (the green skies).The egg that equivalent is extracted
White degeneration and separation in 10% polyacrylamide gel, then by electrophoretic transfer to nitrocellulose filter, film is diluted in PBS
4 DEG C of night incubation in the SOD-2 for crossing, α-tubulin antibody, then film continuation incubation 1 in the horseradish peroxidase of dilution
Hour, chemical luminescence reagent kit post-exposure development is added, target protein is quantified using ImageJ softwares.
Testing result is as shown in figure 4, extracellular matrix biomaterial substantially increases SOD-2 contents in UC-MSCs.
B. impact of the extracellular matrix biomaterial to Catalase activity in UC-MSCs
Commercialization Catalase activity detection kits are used in the detection of intracellular Catalase activity levels.Culture is in TCPS
Albumen supernatant is extracted after cell cell pyrolysis liquid cracking on plate and ECM plates, total protein uses BCA protein quantification test kits
Determine, the protein sample of every group of equivalent mix with the chrominance response substrate solution in test kit after incubation at room temperature 15 minutes, as early as possible
With absorbance of the microplate reader determination sample at 520 nm.
Testing result is as shown in figure 5, extracellular matrix biomaterial significantly improves Catalase activated waters in UC-MSCs
It is flat.
To sum up experiment shows that extracellular matrix biomaterial can significantly improve the multiplication capacity of UC-MSCs, reduces UC-
ROS and H in MSCs2O2Content, increases SOD-2 contents in UC-MSCs, improves Catalase activity levels in UC-MSCs, effectively
The oxidative stress infringement of prevention stem cell, the prospect of being widely applied in the field such as cellular transplantation therapy and regenerative medicine.
Claims (4)
1. a kind of extracellular matrix biomaterial for strengthening In vitro culture mesenchymal stem cell biological anti-oxidation function, its feature exists
In being prepared from by following steps：Umbilical cord mesenchymal stem cells culture during density >=90%, has been added in complete medium
Full nutrient solution culture 7-10 days, changed liquid once per 3 days, adds de-cell liquid afterwards, is built into extracellular matrix biomaterial；
The complete medium component is α-MEM and hyclone, and their volume ratio is（4-9）:1, and contain antibiosis
Element, the content of antibiotic is 10 ~ 200 U/mL；
Contain ascorbic acid 100-200uM in the complete culture solution.
2. a kind of enhancing In vitro culture mesenchymal stem cell biological anti-oxidation function according to claim 1 is extracellular
Matrix biopolymers material, it is characterised in that：The de-cell liquid is contained by the phosphate buffered saline that pH value is 7.4
0.5 ~ 5% volume ratios of TritonX-100 and ammonia 10-40mM.
3. a kind of enhancing In vitro culture mesenchymal stem cell biological anti-oxidation function according to claim 1 is extracellular
Matrix biopolymers material, it is characterised in that：The umbilical cord mesenchymal stem cells take from people source, pig source, Mus source or rabbit source.
4. it is a kind of strengthen In vitro culture mesenchymal stem cell biological anti-oxidation function extracellular matrix biomaterial preparation side
Method, it is characterised in that preparation process is as follows：Umbilical cord mesenchymal stem cells culture in complete medium, during density >=90%, plus
Enter complete culture solution culture 7-10 days, liquid was changed once per 3 days, add de-cell liquid afterwards, be built into extracellular matrix biological
Material；
The complete medium component is α-MEM and hyclone, and their volume ratio is（4-9）:1, and contain antibiosis
Element, the content of antibiotic is 10 ~ 200 U/mL；Contain ascorbic acid 100-200uM in the complete culture solution.
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| CN107308497B (en) * | 2017-06-09 | 2019-11-26 | 浙江大学 | The building of nucleus pulposus cell source property activity microcarrier |
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| CN108546675A (en) * | 2018-05-17 | 2018-09-18 | 广东芙金干细胞再生医学有限公司 | Stem cell is promoted to be divided into the preparation method of the extracellular matrix of liver cell |
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