CN110368402A - Mescenchymal stem cell preparation and its preparation method and application - Google Patents
Mescenchymal stem cell preparation and its preparation method and application Download PDFInfo
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Abstract
The present invention provides a kind of mescenchymal stem cell preparation, includes following components: mescenchymal stem cell, human serum albumin, low molecular weight heparin, amino acid, vitamin C and compound electrolyte；The mescenchymal stem cell is the mescenchymal stem cell handled by inflammatory cytokine.The reparation of mescenchymal stem cell preparation alleviation of inflammation and damaged tissues suitable for rheumatoid arthritis disease, realizes the alleviation of patient symptom and the recovery of function.
Description
Technical field
The invention belongs to biomedicine fields, and in particular in treatment rheumatoid arthritis mescenchymal stem cell preparation and
Preparation method.
Background technique
Rheumatoid arthritis (RA) is a kind of unknown systemic disease chronic, based on inflammatory synovitis of cause of disease.Its
It is characterized in hand, the multi-joint of sufficient Minor articulus, symmetry, aggressive arthritis, is often accompanied by organ outside joint and is involved serum
Rheumatoid factor positive can cause joint deformity and function to be lost.RA women sends out well, and disease incidence is 2~3 times of male.It can
Any age is betided, high incidence age is 40~60 years old.The morbidity of RA may be related with heredity, infection, sex hormone etc..The joint RA
Scorching pathology mainly has synovial membrane stave cell hyperplasia, the new life of interstitial massive inflammatory cells infiltrated and capilary, pannus
Formation and cartilage and the destruction of bone tissue etc..The main purpose for the treatment of rheumatoid arthritis is to mitigate arthritis reaction, suppression
Pathological development processed and irreversible destruction of bone, the as far as possible function of Saving cortilage and muscle, be finally reached state of an illness complete incidence graph or
Reduce the target of disease activity.RA principle of reatment includes patient education, early treatment, drug combination, individualized treatment scheme
And functional training.Drug therapy mainly includes nonsteroidal anti-inflammatory agent, acts on antirheumatic drug, immunosuppressor, immune and life slowly
Object preparation and botanical medicine etc..
Mescenchymal stem cell is a kind of cell with self-renewing and Multidirectional Differentiation ability, and materials are easy, are from a wealth of sources.
The Various Tissues such as bone, cartilage, fat can be divided into, immunoloregulation function, for regenerative medicine and autoimmune disease
Disease has very big potentiality.Mescenchymal stem cell treatment osteoarthritis is a kind of new therapeutic modality, has there is a large amount of clinic
Preceding research confirms that mescenchymal stem cell, which treats rheumatoid arthritis, has good effect.
A kind of allosome interstitial blood vessel confluent monolayer cells (SVF) and allosome mesenchyma ancestral are provided in patent CN2015104069072
The purposes of cell (haMPCs), they be used to prepare prevention and/or treat the pharmaceutical composition of rheumatoid arthritis.But
It still needs further improvement for this method therapeutic effect, and cell extraction is complicated, is unfavorable for large-scale promotion use.
A kind of stem cell medicine for treating rheumatoid arthritis, including mesenchyma are disclosed in patent CN105833277A
Stem cell, human serum albumin and NSADs.The invention, can by using mescenchymal stem cell combination human serum albumin and NSADs
The therapeutic effect that rheumatoid arthritis is improved while mitigating patient pain, likewise, this method is in therapeutic effect
It needs to improve on a large scale, and wherein also uses human serum albumin, higher cost, three kinds of components work together, and technique is multiple
It is miscellaneous.
Above-mentioned patent to separate the mescenchymal stem cell obtained as main active, does not appoint mescenchymal stem cell
Where reason.But different tissue sources, Different Individual source, the mescenchymal stem cell quality difference of different batches are larger, cell
Function is uncertain.The mescenchymal stem cell that separation obtains is directly used in the treatment of rheumatoid inflammation, the effect is unsatisfactory.
Summary of the invention
That there are cell functions the purpose of the present invention is overcoming existing arthritis treatment preparation is uncertain, cannot give full play between
The problem of biology performance etc. of mesenchymal stem cells is unfavorable for arthritic recovery.For this purpose, treating class the present invention provides a kind of
The mescenchymal stem cell preparation of rheumatism inflammation, the cell preparation alleviation of inflammation and impaired group suitable for rheumatoid arthritis disease
The alleviation of patient symptom and the recovery of function are realized in the reparation knitted.
To achieve the purpose of the present invention, the present invention the following technical schemes are provided:
First aspect present invention provides a kind of mescenchymal stem cell preparation, and include following components: mescenchymal stem cell, people's blood are white
Albumen, low molecular weight heparin, amino acid, vitamin C and compound electrolyte；The mescenchymal stem cell is by inflammatory cell
The mescenchymal stem cell of factor treatment.
Preferably, the inflammatory cytokine handles the process of the mescenchymal stem cell are as follows: takes and is passaged between P4 generation
Mesenchymal stem cells are inoculated in the α-MEM culture solution containing 10% FBS and inflammatory cytokine, and in 37 DEG C, concentration is 5%
CO224-48h is cultivated in incubator, that is, obtains the mescenchymal stem cell by inflammatory cytokine processing.
Preferably, the inflammatory cytokine is selected from TNF-α, final concentration of 20-40 of the TNF-α in culture solution
ng/ml。
It is highly preferred that final concentration of 20 ng/ml, 25ng/ml, 30ng/ml, the 35ng/ of the TNF-α in culture solution
Ml or 40ng/ml.
Preferably, the concentration of the mescenchymal stem cell handled by inflammatory cytokine is 105~108A/mL.
It is highly preferred that the concentration of the mescenchymal stem cell handled by inflammatory cytokine is 105A/ml, 106A/
ml、107A/ml or 108A/ml.
Preferably, the mass volume ratio of human serum albumin is 5%, low molecular weight heparin in the mescenchymal stem cell preparation
Mass volume ratio is 0.5%, the mass volume ratio of amino acid is 10%, ascorbic mass volume ratio is 0.5%, surplus is
Compound electrolyte.
Preferably, the compound electrolyte includes following components: the sodium chloride of 5.26% mass volume ratio, 5.02% mass body
Accumulate potassium chloride, 0.30% mass body of the sodium gluconate of ratio, the sodium acetate of 3.68% mass volume ratio, 0.37% mass volume ratio
The magnesium chloride of product ratio, surplus is water.
Preferably, the mescenchymal stem cell is selected between mesenchymal stem cell, fat mesenchymal stem cell, synovial membrane and fills
One of matter stem cell, umbilical cord mesenchymal stem cells are a variety of.
Second aspect of the present invention provides a kind of preparation method of mescenchymal stem cell preparation, comprising the following steps:
(1) mescenchymal stem cell is separately cultured: after separating mesenchymal stem cell, being inoculated in the α-MEM culture containing 10% FBS
In base, in 37 DEG C, concentration is the CO of 5 %2It cultivates in incubator, when cell fusion degree is 80%-85%, is disappeared with 0.20% pancreatin
Change passage；
(2) it uses inflammatory cytokine inducing mesenchymal stem cell: taking the above-mentioned mescenchymal stem cell generation for being passaged to P4 generation, be inoculated with
In the α-MEM culture medium containing 10% FBS and inflammatory cytokine, in 37 DEG C, concentration is the CO of 5 %2It is cultivated in incubator
24-48h。
(3) preparation of mescenchymal stem cell preparation: human serum albumin stoste, low molecular weight heparin, amino acid, dimension are given birth to
Plain C is dissolved in compound electrolyte, 4 DEG C of pre- cold standbies, after the mescenchymal stem cell digestion after induction is counted, is resuspended in above-mentioned molten
In liquid, single cell suspension is made, obtains the mescenchymal stem cell preparation.
Preferably, the inflammatory cytokine is selected from TNF-α, final concentration of 20- of the TNF-α in culture solution
40ng/ml。
Preferably, the concentration of mescenchymal stem cell is 10 in the mescenchymal stem cell preparation5~108A/mL.
It is highly preferred that the concentration of the mescenchymal stem cell handled by inflammatory cytokine is 105A/ml, 106A/
ml、107A/ml or 108A/ml.
Preferably, the compound electrolyte includes following components: the sodium chloride of 5.26% mass volume ratio, 5.02% mass body
Accumulate potassium chloride, 0.30% mass body of the sodium gluconate of ratio, the sodium acetate of 3.68% mass volume ratio, 0.37% mass volume ratio
The magnesium chloride of product ratio, surplus is water.
Third aspect present invention provides above-mentioned mescenchymal stem cell preparation in preparing drugs for rheumatoid arthritis
Application.
Compared with the existing technology, the beneficial effects of the present invention are:
(1) TNF-α stem cell stimulates mesenchyma, can eliminate immune between mescenchymal stem cell separate sources, different batches
Regulatory function difference promotes mescenchymal stem cell immunoregulation capability and functional stabilization, improves entire articular cavity microenvironment, benefit
It is able to ascend the immunoregulation capability function of mescenchymal stem cell in the reparation of alleviation and the damage of inflammation, improves articular cavity inflammation
Microenvironment is conducive to alleviate inflammation and repairs damage.
(2) by the substances such as addition human serum albumin, amino acid, vitamin C and low molecular weight heparin be between fill it is dry
Cells with nutrient and viability environment promote the therapeutic effect of mescenchymal stem cell.Solution medium is compound electrolyte solution, can
To keep the osmotic pressure of cell, conducive to the survival of cell.
Detailed description of the invention
MICRO-CT result after the treatment of Fig. 1 arthritic mice；
MICRO-CT bone amount analyzes result after the treatment of Fig. 2 arthritic mice；
TNF-α inducing mesenchymal stem cell group joint HE result after the treatment of Fig. 3 A arthritic mice；
Mescenchymal stem cell group joint HE result after the treatment of Fig. 3 B arthritic mice；
Control group joint HE result after the treatment of Fig. 3 C arthritic mice；
TNF-α inducing mesenchymal stem cell group arthroncus degree after the treatment of Fig. 4 A arthritic mice；
Mescenchymal stem cell group arthroncus degree after the treatment of Fig. 4 B arthritic mice；
Control group arthroncus degree after the treatment of Fig. 4 C arthritic mice；
Fig. 5 arthritic mice treats posterior joint inflammation appraisal result.
Specific embodiment
Protection scope of the present invention is illustrated with reference to the accompanying drawings and examples, but is not construed as to this hair
The limitation of bright protection scope.
Embodiment 1
It include following components the present embodiment provides a kind of mescenchymal stem cell preparation: mescenchymal stem cell, human serum albumin, low
Molecular heparin, amino acid, vitamin C and compound electrolyte；The mescenchymal stem cell be by inflammatory cytokine at
The mescenchymal stem cell of reason.
In the present embodiment, the inflammatory cytokine handles the process of the mescenchymal stem cell are as follows: takes and is passaged to P4 generation
Mescenchymal stem cell, be inoculated in the α-MEM culture solution containing 10% FBS and inflammatory cytokine, in 37 DEG C, concentration 5%
CO224-48h is cultivated in incubator, that is, obtains the mescenchymal stem cell by inflammatory cytokine processing.
In the present embodiment, the inflammatory cytokine is selected from TNF-α, and the TNF-α is final concentration of in culture solution
20ng/ml。
In some embodiments, final concentration of 25ng/ml, 30ng/ml, 35ng/ml in culture solution of the TNF-α or
40ng/ml。
In the present embodiment, the concentration of the mescenchymal stem cell handled by inflammatory cytokine is 106A/mL.
In some embodiments, the concentration of the mescenchymal stem cell handled by inflammatory cytokine is 105A/
ml、107A/ml or 108A/ml.
In the present embodiment, the mass volume ratio of human serum albumin is 5%, low molecule liver in the mescenchymal stem cell preparation
The mass volume ratio of element is 0.5%, the mass volume ratio of amino acid is 10%, ascorbic mass volume ratio is 0.5%, remaining
Amount is compound electrolyte.
In the present embodiment, the compound electrolyte includes following components: the sodium chloride of 5.26% mass volume ratio, 5.02% matter
Measure the sodium gluconate of volume ratio, the sodium acetate of 3.68% mass volume ratio, the potassium chloride of 0.37% mass volume ratio, 0.30% matter
The magnesium chloride of volume ratio is measured, surplus is water.
In the present embodiment, the mescenchymal stem cell is selected from umbilical cord mesenchymal stem cells.
In some embodiments, the mescenchymal stem cell be selected from mesenchymal stem cell, fat mesenchymal stem cell,
One of Synovial Mesenchymal Stem Cells are a variety of.
Embodiment 2
The present embodiment provides a kind of preparation methods of mescenchymal stem cell preparation, comprising the following steps:
(1) mescenchymal stem cell is separately cultured: after separating mesenchymal stem cell, being inoculated in the α-MEM culture containing 10% FBS
In base, in 37 DEG C, concentration is the CO of 5 %2It cultivates in incubator, when cell fusion degree is 80%-85%, is disappeared with 0.20% pancreatin
Change passage；
(2) it uses inflammatory cytokine inducing mesenchymal stem cell: taking the above-mentioned mescenchymal stem cell generation for being passaged to P4 generation, be inoculated with
In the α-MEM culture medium containing 10% FBS and inflammatory cytokine, in 37 DEG C, concentration is the CO of 5 %2It is cultivated in incubator
24-48h。
(3) preparation of mescenchymal stem cell preparation: human serum albumin stoste, low molecular weight heparin, amino acid, dimension are given birth to
Plain C is dissolved in compound electrolyte, 4 DEG C of pre- cold standbies, after the mescenchymal stem cell digestion after induction is counted, is resuspended in above-mentioned molten
In liquid, single cell suspension is made, obtains the mescenchymal stem cell preparation.
In the present embodiment, the inflammatory cytokine is selected from TNF-α, and the TNF-α is final concentration of in culture solution
20ng/ml。
In some embodiments, final concentration of 25ng/ml, 30ng/ml, 35ng/ml in culture solution of the TNF-α or
40ng/ml。
In the present embodiment, the concentration of mescenchymal stem cell is 10 in the mescenchymal stem cell preparation6A/mL.
In some embodiments, the concentration of the mescenchymal stem cell handled by inflammatory cytokine is 105A/
ml、107A/ml or 108A/ml.
In the present embodiment, the compound electrolyte includes following components: the sodium chloride of 5.26% mass volume ratio, 5.02% matter
Measure the sodium gluconate of volume ratio, the sodium acetate of 3.68% mass volume ratio, the potassium chloride of 0.37% mass volume ratio, 0.30% matter
The magnesium chloride of volume ratio is measured, surplus is water.
In addition to mescenchymal stem cell, remaining ingredient need to be prepared in advance in mescenchymal stem cell preparation, 4 DEG C of pre- cold standbies,
Mesenchymal stem cells are finally resuspended in this solution, and single cell suspension is made, and the quantity of mescenchymal stem cell is 1 in every ml of formulation
×106.Mescenchymal stem cell is in 2-15 DEG C of environment temperature, and inner cell is maintained as single cell suspension state, cell within 48 hours
Vigor (Trypan Blue meter vigor) is maintained at 90% or more.
Compliance test result:
The safety and validity experiment that mescenchymal stem cell preparation treats rheumatoid arthritis mouse
Rheumatoid arthritis mouse is chosen in the experiment, is randomly divided into 3 groups, and every group 10, respectively between control group, TNF-α induction
Mesenchymal stem cells group and mescenchymal stem cell group.Control group gives 200ul physiological saline tail vein injection；It is filled between TNF-α induction
Matter stem cell group and mescenchymal stem cell group mouse give mescenchymal stem cell preparation 200ul(include mescenchymal stem cell 1.0 ×
106) tail vein injection.The 4th week posterior joint pathological section is treated, sees Fig. 1.
As the result is shown: mescenchymal stem cell intravenous injection does not cause acute toxicology reaction and rejection.With compare
Group is compared, and TNF-α inducing mesenchymal stem cell group and mescenchymal stem cell group have therapeutic effect, bone after arthritic mice treatment
Amount increases (see Fig. 1-2), and the occurring degree of arthritic mice mitigates (see Fig. 3 A-C), and the swelling degree of mouse claw is relieved
(see Fig. 4 A-C), arthritis score reduce (see figure 5), but between the therapeutic effect of TNF-α inducing mesenchymal stem cell group is better than
Mesenchymal stem cells group.
The content of present invention merely illustrates claimed some specific embodiments, one of them or more skill
Documented technical characteristic can be combined with arbitrary one or more technical solutions in art scheme, these are combined and obtain
Technical solution also in the application protection scope, the technical solution just as obtained from these are combined is disclosed in the present invention
It is specifically recorded in content the same.
Claims (12)
1. mescenchymal stem cell preparation, which is characterized in that include following components: mescenchymal stem cell, human serum albumin, low molecule
Heparin, amino acid, vitamin C and compound electrolyte；The mescenchymal stem cell is to handle by inflammatory cytokine
Mescenchymal stem cell.
2. mescenchymal stem cell preparation according to claim 1, which is characterized in that described in the inflammatory cytokine processing
The process of mescenchymal stem cell are as follows: take the mescenchymal stem cell for being passaged to P4 generation, be inoculated in containing 10% FBS and inflammatory cell because
In the α-MEM culture solution of son, in 37 DEG C, CO that concentration is 5%224-48h is cultivated in incubator, that is, obtain by inflammatory cell because
The mescenchymal stem cell of subprocessing.
3. mescenchymal stem cell preparation according to claim 2, which is characterized in that the inflammatory cytokine is selected from TNF-
α, final concentration of 20-40 ng/ml of the TNF-α in culture solution.
4. mescenchymal stem cell preparation according to claim 2, which is characterized in that described to be handled by inflammatory cytokine
Mescenchymal stem cell concentration be 105~108A/mL.
5. mescenchymal stem cell preparation according to claim 1, which is characterized in that people in the mescenchymal stem cell preparation
The mass volume ratio of blood albumin is 5%, the mass volume ratio of low molecular weight heparin is 0.5%, the mass volume ratio of amino acid
For 10%, ascorbic mass volume ratio be 0.5%, surplus is compound electrolyte.
6. mescenchymal stem cell preparation according to claim 1, which is characterized in that the compound electrolyte includes with the following group
Point: the sodium chloride of 5.26% mass volume ratio, the sodium gluconate of 5.02% mass volume ratio, 3.68% mass volume ratio acetic acid
Sodium, the potassium chloride of 0.37% mass volume ratio, 0.30% mass volume ratio magnesium chloride, surplus is water.
7. mescenchymal stem cell preparation according to claim 1, which is characterized in that the mescenchymal stem cell is selected from marrow
One of mescenchymal stem cell, fat mesenchymal stem cell, Synovial Mesenchymal Stem Cells, umbilical cord mesenchymal stem cells are more
Kind.
8. a kind of preparation method of mescenchymal stem cell preparation, which comprises the following steps:
(1) mescenchymal stem cell is separately cultured: after separating mesenchymal stem cell, being inoculated in the α-MEM culture containing 10% FBS
In base, in 37 DEG C, concentration is the CO of 5 %2It cultivates in incubator, when cell fusion degree is 80%-85%, is disappeared with 0.20% pancreatin
Change passage；
(2) it uses inflammatory cytokine inducing mesenchymal stem cell: taking the above-mentioned mescenchymal stem cell generation for being passaged to P4 generation, be inoculated with
In the α-MEM culture medium containing 10% FBS and inflammatory cytokine, in 37 DEG C, concentration is the CO of 5 %2It is cultivated in incubator
24-48h；
(3) preparation of mescenchymal stem cell preparation: by human serum albumin stoste, low molecular weight heparin, amino acid, vitamin C
It is dissolved in compound electrolyte, 4 DEG C of pre- cold standbies, after the mescenchymal stem cell digestion after induction is counted, is resuspended in above-mentioned solution
In, single cell suspension is made, obtains the mescenchymal stem cell preparation.
9. preparation method according to claim 8, which is characterized in that the inflammatory cytokine is selected from TNF-α, described
Final concentration of 20-40 ng/ml of the TNF-α in culture solution.
10. preparation method according to claim 8, which is characterized in that mesenchyma is dry in the mescenchymal stem cell preparation
The concentration of cell is 105~108A/mL.
11. preparation method according to claim 8, which is characterized in that the compound electrolyte includes following components:
The sodium chloride of 5.26% mass volume ratio, the sodium gluconate of 5.02% mass volume ratio, 3.68% mass volume ratio sodium acetate,
The magnesium chloride of the potassium chloride of 0.37% mass volume ratio, 0.30% mass volume ratio, surplus are water.
12. mescenchymal stem cell preparation described in any one of claims 1-6 is in preparing drugs for rheumatoid arthritis
Using.
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| CN111973632A (en) * | 2020-08-25 | 2020-11-24 | 陕西佰傲干细胞再生医学有限公司 | Stem cell preparation for treating diabetes and preparation method thereof |
| CN112190596A (en) * | 2020-09-14 | 2021-01-08 | 陕西佰傲干细胞再生医学有限公司 | Mesenchymal stem cell preparation for treating arthritis and preparation method thereof |
| CN112773817A (en) * | 2019-11-09 | 2021-05-11 | 佛山市善玺化妆品股份有限公司 | Compound synthetic protein preparation capable of replacing stem cells and preparation method thereof |
| CN113832099A (en) * | 2021-10-13 | 2021-12-24 | 浙江领蔚生物技术有限公司 | Mesenchymal stem cell preparation for preparing medicine for treating rheumatoid arthritis |
| CN114621919A (en) * | 2022-05-17 | 2022-06-14 | 天九再生医学(天津)科技有限公司 | Method for enhancing immunoregulation capability of mesenchymal stem cells |
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| CN114621919B (en) * | 2022-05-17 | 2022-12-06 | 天九再生医学(天津)科技有限公司 | Method for enhancing immunoregulation capability of mesenchymal stem cells |
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Application publication date: 20191025 |