CN108251363A - A kind of polysaccharide composition for promoting human umbilical cord mesenchymal stem cells proliferation and its application - Google Patents
A kind of polysaccharide composition for promoting human umbilical cord mesenchymal stem cells proliferation and its application Download PDFInfo
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- CN108251363A CN108251363A CN201711496917.5A CN201711496917A CN108251363A CN 108251363 A CN108251363 A CN 108251363A CN 201711496917 A CN201711496917 A CN 201711496917A CN 108251363 A CN108251363 A CN 108251363A
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- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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Abstract
The invention discloses a kind of ophiopogonpolysaccharide and soluble polysaccharide composition and preparation method thereof, and above-mentioned ophiopogonpolysaccharide and soluble polysaccharide composition are disclosed in the application for promoting human umbilical cord mesenchymal stem cells in-vitro multiplication.Pharmaceutical composition of the present invention can make mescenchymal stem cell keep greater activity, and growth rate increases substantially, and prevents its differentiation.The umbilical cord mesenchymal stem cells prepared using the present invention are of great significance to scientific research and clinical practice.
Description
Technical field
The invention belongs to biomedicine technical fields, are related to preparation and the purposes of a kind of polysaccharide composition, and in particular to wheat
Winter polysaccharide and purposes of the soluble polysaccharide composition in terms of human umbilical cord mesenchymal stem cells proliferation is promoted.
Background technology
Human umbilical cord mesenchymal stem cells（Mesenchymal Stem Cells, MSCs）Refer to be present in neonatal umbilical cord group
A kind of early stage cell for having the characteristics that the cell of myeloid-lymphoid stem cell, being mesoderm development in knitting, can be to mesoderm and ectoderm
The tissue differentiation in source, it is similar to bone marrow derived MSCs with multi-lineage potential, form and Biological characteristics（Br J
Haematology,2000,109:235-242）.Umbilical cord mesenchymal stem cells（MSCs）With higher differentiation potential, certain
Experiment condition under can be divided into osteoblast, cartilage cell, adipocyte, nerve cell, liver cell, Skeletal Muscle Cell etc.
(Blood,2004,103:1669-1675).MSCs is isolated from people's umbilical cord, cell content, amplification ability are better than marrow
MSCs, immunogenicity is lower than bone marrow MSCs, and has many advantages, such as convenient material drawing, no ethics dispute, can be used as it is a kind of newly
Cell derived is substituted, for the cell transplantation and gene therapy of each systemic disease.
Radix Ophiopogonis is the dried root of Liliaceae Ophiopogon plant Radix Ophiopogonis, has the effect of nourishing Yin and promoting production of body fluid, moistening lung clears away heart-fire（In
State pharmacopeia .2015:155-156）.Radix Ophiopogonis is mainly containing steroid saponin, homoisoflavone, polysaccharide isoreactivity ingredient.Containing big in Radix Ophiopogonis
The polysaccharide constituents of amount, and be made of monosaccharide and oligosaccharides compound.Document report, ophiopogonpolysaccharide through hydrolysis after mainly by
Fructose forms, and the ratio of fructose and glucose is 35: 1（Chinese herbal medicine, 2005,36 (5):1488）.Modern pharmacological studies have shown that
Ophiopogonpolysaccharide has extensive pharmacological action, including blood sugar reducing function, resists myocardial ischemia with myocardial infarction effect, to human body siberian crabapple
Effect, antioxidation, antitumor action, exocrine gland protective effect of system etc..Ophiopogonpolysaccharide content is higher, extraction process phase
To simple.
Rhizoma Atractylodis Macrocephalae is the dry rhizome of feverfew Rhizoma Atractylodis Macrocephalae, and warm-natured, sweet-bitter flavor has the effects that invigorating the spleen, QI invigorating.Soluble polysaccharide
(PAM) be Rhizoma Atractylodis Macrocephalae main active, the heteroglycan being mainly made of glucose, xylose, arabinose, galactolipin, tool
Have adjust immune, anti-oxidant and anti-aging, it is antitumor, hypoglycemic the effects that（Chinese medicine, 2006:107–109）.
Human umbilical cord mesenchymal stem cells are widely used to the clinical trial of various basic research and disease treatment at present, but
Culture scheme used at present is different, and culture effect is simultaneously unstable, often occurs that stem cell growth speed is slow, morphologic change causes
The problems such as character changes, Cell viability reduces.Human umbilical cord mesenchymal stem cells can be promoted to increase the object of the present invention is to provide a kind of
The polysaccharide composition grown, for obtaining reliable and stable human umbilical cord mesenchymal stem cells.
Invention content
Inventor has found in an experiment, and ophiopogonpolysaccharide and soluble polysaccharide composition are under appropriate proportioning and concentration to people's umbilical cord
Mescenchymal stem cell, which has, promotes increment effect, promotes the proliferative capacity of the umbilical cord mesenchymal stem cells of in vitro culture, and enhancing is thin
Cytoactive.Polysaccharide composition provided by the invention can be used as human umbilical cord mesenchymal stem cells enhancer of proliferation and be used to prepare
The research of the drug or human umbilical cord mesenchymal stem cells proliferation mechanism of human umbilical cord mesenchymal stem cells proliferation is promoted to provide greatly
Measure stem cell.
The present invention is to provide the preparation method of a kind of ophiopogonpolysaccharide and soluble polysaccharide composition, the preparation method technique first
Simply, extraction efficiency is high.The technical solution is prepared using ophiopogonpolysaccharide and soluble polysaccharide as raw material after water extraction and alcohol treatment
It obtains.
In above-mentioned preparation method：The weight of Radix Ophiopogonis of the present invention and Rhizoma Atractylodis Macrocephalae is 300-100: 100；Good choosing
It is 300:100.During water of the present invention extraction, the dosage of water is 5-20 times of raw material gross weight during extraction every time, extraction 2-3 times, often
Secondary extraction time is 1-2h.After water of the present invention extraction, filtrate is concentrated after filtering, gained concentrate use volumn concentration for
The ethanol water of 60-95% carries out alcohol precipitation processing, and it is ophiopogonpolysaccharide and soluble polysaccharide composition to obtain sediment.It is wherein dense
The preferred freezing vacuum that contracts concentrates.
Polysaccharide composition of the present invention includes ophiopogonpolysaccharide and soluble polysaccharide.It is more that Radix Ophiopogonis is extracted according to preceding method
Sugar and soluble polysaccharide, preferred portfolio ratio are 3: 1.
Second purpose of the invention is to provide above-mentioned ophiopogonpolysaccharide and soluble polysaccharide composition as between promotion people's umbilical cord
Application in mesenchymal stem cell proliferation.Ophiopogonpolysaccharide and soluble polysaccharide are added in basal medium according to 1% ratio.Base
Basal culture medium includes DMEM/F12,1640, α-MEM etc..
Stem cells hyperplasia situation is detected using mtt assay test cell activity, cell activity uses following equation：
The relevant experimental data of the present invention proves that being combined using polysaccharide of the present invention can be to the increasing of human umbilical cord mesenchymal stem cells
It grows culture and plays facilitation.By micro- sem observation, it can protect mescenchymal stem cell using pharmaceutical composition of the present invention
Greater activity is held, growth rate increases substantially, and prevents its differentiation.The present invention grinds the science of umbilical cord mesenchymal stem cells
Study carefully and be of great significance with clinical practice.
Beneficial effects of the present invention：
1st, the present invention adds in ophiopogonpolysaccharide and soluble polysaccharide culture human umbilical cord mesenchymal stem cells in basal medium, this is dry thin
The activity and form of born of the same parents can be kept for a long time, effectively maintain the differentiation capability of stem cell itself, ensure that the quality of stem cell.
2nd, the stem cell of medium culture using the present invention, competence for added value are significantly higher than the dry of base culture base
Cell is effectively increased the human umbilical cord mesenchymal stem cells speed of growth.
3rd, significant effect of the invention, the culture of extremely suitable human umbilical cord mesenchymal stem cells, suitable for promoting.
Description of the drawings
3 umbilical cord mesenchymal stem cells proliferative conditions of Fig. 1 embodiments.
4 umbilical cord mesenchymal stem cells proliferative conditions of Fig. 2 embodiments.
5 umbilical cord mesenchymal stem cells proliferative conditions of Fig. 3 embodiments.
Specific embodiment
With reference to specific embodiment and Figure of description, it is more particularly described present disclosure.It should be appreciated that these
Embodiment is merely to illustrate the present invention, and the invention is not limited in the following examples.The reagent and instrument that the present invention uses
It is all common commercially available product, can be all bought in market.If without specializing, the method that embodiment uses is technology generally in the art；
If the experimental method without special noted provisos, usual routinely condition, such as《Molecular Cloning:A Laboratory guide》（Fourth edition）（Green
M.R, Sambrook J. write；He Fu is elementary to be translated, Science Press, and 2017）Described in condition or built according to manufacturer
The condition of view.
Embodiment 1
Human umbilical cord mesenchymal stem cells detach and purifying:By people's umbilical cord PBS buffer solution under aseptic condition（Containing penicillin, strepto-
Element）It rinses 3 times, umbilical cord outer membrane and blood vessel is rejected, by 1 after shredding：3 volume ratio is with containing pancreatin (w/v 0.05%) and IV
Low sugar DMEM (Gibco) mixed liquor of Collagenase Type (1.5mg/mL) mixes, and 1h is digested at 37 DEG C, and the filtering of 200 mesh filter screens does not disappear
The tissue block of change is added under hyaluronic acid the enzymic digestion 10min, 100-200r/min of final concentration of 25ug/mL and is centrifuged
10min abandons supernatant, cleans precipitation 1 time with the low sugar DMEM culture mediums containing 10%FBS, is centrifuged under 100-200r/min
10min, it is primary umbilical cord mesenchymal stem cells that removal supernatant, which obtains precipitation,；The primary umbilical cord mesenchymal stem cells of gained are connect
Kind cultivates in 37 DEG C, 5%CO2, saturated humidity incubator in culture medium, a subculture was changed every 3-4 days.Treat that cell melts
It is right be 70-80% when, with the trypsin solution had digestive transfer culture containing EDTA.MSC was cultivated to P3 generations, was photographed to record carefully with microscope
Born of the same parents' form, and for subsequent experimental.
Embodiment 2
Ophiopogonpolysaccharide and soluble polysaccharide composition and preparation method thereof:Take dry Radix Ophiopogonis medicinal material 300g and Rhizoma Atractylodis Macrocephalae 100g powder
Broken mixing adds in 4000ml pure water, boils 2h；2000ml pure water is added in again, boils 1h；Filtering removal impurity, filtrate
Be concentrated in vacuo, concentrate concentration of volume percent be 70% ethanol water alcohol precipitation, centrifugation obtain sediment be Radix Ophiopogonis it is more
Sugar and soluble polysaccharide composition.
Embodiment 3
The facilitation that ophiopogonpolysaccharide is proliferated umbilical cord mesenchymal stem cells with soluble polysaccharide composition:By ophiopogonpolysaccharide and Rhizoma Atractylodis Macrocephalae
(embodiment 2 obtains or buy to filter after ophiopogonpolysaccharide and soluble polysaccharide monomer are mixed according to 3: 1 ratio goes out polysaccharide composition
Bacterium）, it is dissolved in DMEM/F12 culture mediums in 1% ratio.With the medium culture umbilical cord mesenchymal stem cells (mescenchymal stem cell
It is obtained from umbilical cord by 1 method of embodiment).Experiment packet is：Medium controls (are not added with polysaccharide composition)；Culture medium+more
Sugar composite.Culture adds in serum-free DMEM/F12 culture mediums and washes after twice the final concentration processing for using MTT with 5mg/mL after four days,
And supernatant is carefully drawn after being incubated about 4 hours in 37 DEG C, the DMSO of 100 μ L is added to enter dissolving purple in 24 orifice plates and is precipitated, and
At room temperature in being shaken on shaking table 10 minutes, crystal is allowed fully to dissolve；After purple precipitation is completely dissolved in full-automatic microplate reader
OD490 values are detected, experiment is repeated 3 times detection cell activity.
The height of Fig. 1 block diagrams represents chemiluminescence intensity, directly proportional to cell number.As a result ophiopogonpolysaccharide and Rhizoma Atractylodis Macrocephalae are shown
Polysaccharide（3:1）Composition is added in DMEM/F12 culture mediums and is cultivated, and umbilical cord mesenchymal stem cells activity has 25.78% increase,
Confirm that the ophiopogonpolysaccharide and soluble polysaccharide composition have the function of umbilical cord mesenchymal stem cells to remarkably promote proliferation.
Embodiment 4
The facilitation that ophiopogonpolysaccharide is proliferated umbilical cord mesenchymal stem cells with soluble polysaccharide composition:By ophiopogonpolysaccharide and Rhizoma Atractylodis Macrocephalae
(embodiment 2 obtains or buy to filter after ophiopogonpolysaccharide and soluble polysaccharide monomer are mixed according to 2: 1 ratio goes out polysaccharide composition
Bacterium）, it is dissolved in DMEM/F12 culture mediums in 1% ratio.With the medium culture umbilical cord mesenchymal stem cells (mescenchymal stem cell
It is obtained from umbilical cord by 1 method of embodiment).Experiment packet is：Medium controls (are not added with polysaccharide composition)；Culture medium+more
Sugar composite.Culture adds in serum-free DMEM/F12 culture mediums and washes after twice the final concentration processing for using MTT with 5mg/mL after four days,
And supernatant is carefully drawn after being incubated about 4 hours in 37 DEG C, the DMSO of 100 μ L is added to enter dissolving purple in 24 orifice plates and is precipitated, and
At room temperature in being shaken on shaking table 10 minutes, crystal is allowed fully to dissolve；After purple precipitation is completely dissolved in full-automatic microplate reader
OD490 values are detected, experiment is repeated 3 times detection cell activity.
The height of Fig. 2 block diagrams represents chemiluminescence intensity, directly proportional to cell number.As a result ophiopogonpolysaccharide and Rhizoma Atractylodis Macrocephalae are shown
Polysaccharide（2:1）Composition is added in DMEM/F12 culture mediums and is cultivated, and umbilical cord mesenchymal stem cells activity has 16.51% increase,
Confirm that the ophiopogonpolysaccharide has the function of umbilical cord mesenchymal stem cells with soluble polysaccharide composition to promote proliferation.
Embodiment 5
The facilitation that ophiopogonpolysaccharide is proliferated umbilical cord mesenchymal stem cells with soluble polysaccharide composition:By ophiopogonpolysaccharide and Rhizoma Atractylodis Macrocephalae
(embodiment 2 obtains or buy to filter after ophiopogonpolysaccharide and soluble polysaccharide monomer are mixed according to 3: 1 ratio goes out polysaccharide composition
Bacterium）, it is dissolved in α-MEM culture mediums in 1% ratio.With the medium culture umbilical cord mesenchymal stem cells, (mescenchymal stem cell is pressed
1 method of embodiment is obtained from umbilical cord).Experiment packet is：Medium controls (are not added with polysaccharide composition)；Culture medium+polysaccharide
Composition.Culture adds in serum-free DMEM/F12 culture mediums and washes after twice with MTT with the final concentration processing of 5mg/mL after four days, and
Supernatant is carefully drawn after being incubated about 4 hours in 37 DEG C, the DMSO of 100 μ L is added to enter dissolving purple precipitation in 24 orifice plates, and in room
In being shaken on shaking table 10 minutes under temperature, crystal is allowed fully to dissolve；It is examined in full-automatic microplate reader after purple precipitation is completely dissolved
OD490 values are surveyed, experiment is repeated 3 times detection cell activity.
The height of Fig. 3 block diagrams represents chemiluminescence intensity, directly proportional to cell number.As a result ophiopogonpolysaccharide and Rhizoma Atractylodis Macrocephalae are shown
Polysaccharide（3:1）Composition is added in α-MEM culture mediums and is cultivated, and umbilical cord mesenchymal stem cells activity has 21.39% increase, card
The real ophiopogonpolysaccharide has the function of umbilical cord mesenchymal stem cells to remarkably promote proliferation with soluble polysaccharide composition.
Claims (7)
1. a kind of have the polysaccharide composition for promoting mescenchymal stem cell proliferation function, it is characterised in that：The polysaccharide composition
It is made of ophiopogonpolysaccharide and soluble polysaccharide.
2. ophiopogonpolysaccharide according to claim 1 and soluble polysaccharide composition are to dry Radix Ophiopogonis with Rhizoma Atractylodis Macrocephalae as raw material, through water
It carries and being prepared with after alcohol treatment.
3. the ophiopogonpolysaccharide prepared according to claim 2 and soluble polysaccharide composition, it is characterised in that：The Radix Ophiopogonis with it is white
The weight of art is 300-100:100；When water extracts, the dosage of water is 5-20 times of raw material gross weight during extraction every time,
Extraction 2-3 times, each extraction time are 1-2h；After water extraction, filtrate is concentrated after filtering, gained concentrate uses volume basis
The ethanol water that content is 60~95% carries out alcohol precipitation processing, and it is ophiopogonpolysaccharide and soluble polysaccharide composition to obtain sediment.
A kind of 4. external method for promoting umbilical cord mesenchymal stem cells proliferation, it is characterised in that：It is 2-3 in the ratio that is added to:1
Umbilical cord mesenchymal stem cells are cultivated in the basal medium of the polysaccharide composition of ophiopogonpolysaccharide and soluble polysaccharide.
5. basal medium according to claim 4, it is characterised in that：Including DMEM/F12 basal mediums, 1640 bases
Culture medium, α-MEM basal mediums.
6. the polysaccharide composition of ophiopogonpolysaccharide and soluble polysaccharide according to claim 4, it is characterised in that：Concentration ratio is base
The 1% of basal culture medium.
7. the high activity umbilical cord mesenchymal stem cells prepared according to claim 4 are in disease treatment stem cell drugs are prepared
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111057679A (en) * | 2019-12-30 | 2020-04-24 | 贵州汉氏联合生物技术有限公司 | Mesenchymal stem cell guiding agent and guiding method thereof |
| CN113754790A (en) * | 2021-10-15 | 2021-12-07 | 山东元裕生物科技有限公司 | Biological agent for promoting cell growth |
| CN117106712A (en) * | 2023-10-25 | 2023-11-24 | 山东博森医学工程技术有限公司 | Stem cell exosome for skin regeneration, application and product thereof |
-
2017
- 2017-12-31 CN CN201711496917.5A patent/CN108251363A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111057679A (en) * | 2019-12-30 | 2020-04-24 | 贵州汉氏联合生物技术有限公司 | Mesenchymal stem cell guiding agent and guiding method thereof |
| CN113754790A (en) * | 2021-10-15 | 2021-12-07 | 山东元裕生物科技有限公司 | Biological agent for promoting cell growth |
| CN113754790B (en) * | 2021-10-15 | 2022-03-18 | 山东元裕生物科技有限公司 | Biological agent for promoting cell growth |
| CN115010821A (en) * | 2021-10-15 | 2022-09-06 | 山东元裕生物科技有限公司 | Application of polysaccharide in promoting growth of umbilical cord mesenchymal stem cells |
| CN115010821B (en) * | 2021-10-15 | 2023-10-20 | 北京恒生佳合细胞科技有限公司 | Application of polysaccharide in promoting growth of umbilical cord mesenchymal stem cells |
| CN117106712A (en) * | 2023-10-25 | 2023-11-24 | 山东博森医学工程技术有限公司 | Stem cell exosome for skin regeneration, application and product thereof |
| CN117106712B (en) * | 2023-10-25 | 2024-01-05 | 山东博森医学工程技术有限公司 | Stem cell exosome for skin regeneration, application and product thereof |
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