CN106511381A - Composition and oral spray which contain stem cell secreted factors, and preparation method thereof - Google Patents
Composition and oral spray which contain stem cell secreted factors, and preparation method thereof Download PDFInfo
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- CN106511381A CN106511381A CN201610962034.8A CN201610962034A CN106511381A CN 106511381 A CN106511381 A CN 106511381A CN 201610962034 A CN201610962034 A CN 201610962034A CN 106511381 A CN106511381 A CN 106511381A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- 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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
- A61K9/006—Oral mucosa, e.g. mucoadhesive forms, sublingual droplets; Buccal patches or films; Buccal sprays
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Abstract
The invention relates to a method used for preparing a composition containing stem cell secreted factors. The method comprises following steps: mesenchymal stem cells are separated from embryonal connective tissues; proliferation of the mesenchymal stem cells is carried out so as to obtain a cultured product containing the stem cell secreted factors; a solution containing the stem cell secreted factors is obtained via removing the mesenchymal stem cells in the cultured product; and the solution is purified, and is subjected to vacuum freeze drying so as to obtain the composition containing the stem cell secreted factors. The active ingredients contained by the composition and the oral spray are absorbed through oral cavities and lungs, so that human immunity is improved, and it is beneficial for body health.
Description
Technical field
The present invention relates to biomedical sector, especially, be related to a kind of compositionss comprising stem cell secretion factor and its
Preparation method, and the mouth sprays comprising stem cell secretion factor prepared with said composition.
Background technology
Stem cell (stem cells, SC) is that a class has the pluripotency of the of self-replication capacity (self-renewing) thin
Born of the same parents, under certain condition, it can be divided into several functions cell.Stem cell is a kind of inabundant differentiation, still jejune thin
Born of the same parents, with the potential function for regenerating various histoorgans and human body, medical circle is referred to as " general-purpose cell ".Stem cell can be in machine
Indefinite duration division in body life process, and in suitable condition or after giving appropriate condition, can break up produce it is various different
Cell type.
Stem cell can secrete many bioactive molecules, such as mescenchymal stem cell at least secretory immune regulatory factor (HGF,
LIF), chemotactic factor (RANTES, SDF-1 α, fractalkine, MIP-1 α, MCP-1, MCP-2), the nutrition of support CFU-GM
The factor (IL-6, FGF-2, PDGF-AA, PDGF-BB, EGF), the revascularization factor (VEGF165, FGF-2, PDGF-AA, PDGF-
BB, EGF), suppress the cicatrix factor (HGF, FGF-2), anti-apoptosis factor (VEGF165, FGF-2, HGF), wound healing related because
Sub (IL-6, IL-8, TGF-β 1, MCP-1, VEGF, GM-CSF), TIMP-1), each collagen type (I, II, III, IV Collagen Type VI
Albumen) and various lysozyme etc..
The sublingual mucosal epithelium of human mouth not by keratinization, most beneficial for the systemic Absorption of biologically effective molecule；Next to that
Gums and hard palate, in general, permeability of the human oral mucosa to biomolecule：Hypoglossis mucous membrane>Buccal mucosa>Gingiva.
The respiratory system of human body is mainly thin by nose, pharynx, larynx, trachea-bronchial epithelial cell, bronchioless, terminal bronchiole, breathing
Bronchus, alveolar duct and alveolar sac composition.From trachea to alveolar, the caliber of air flue is less and less, but quantity is greatly increased.Exhale
Inhale road surface and cover pseudostratified ciliated columnar epithelium, between ciliated cell, accompany goblet cell, the visible cilium of cylindrical cell free surface.
Biologically effective molecule is alveolar in the main absorption site of pulmonary.About several hundred million alveolars of adult normal, summary table
Area is about 200m2, it is roughly the same with the microvilluss gross area of mucous membrane of small intestine.Alveolar is hemispherical vesicle, by simple squamous
Epithelial cell is constituted, only 0.1-0.5 μm of thickness, and the thin layer connective tissue between adjacent alveolar is alveolar septum.Have in alveolar septum rich
Rich blood capillary and collagen fiber and elastic fiber.Only about 1 μm of thickness between alveolar wall and capillary wall, is gas exchange
With the good place of biologically effective molecule absorption.As alveolar sorbent surface product is big, capillary network is abundant, transhipment is apart from pole
It is little, therefore pulmonary administration absorbs rapid.In addition, the metabolism for pulmonary absorption is with respect to gastrointestinal absorption, to biologically effective molecule
Effect is little, and the first pass effect of liver can be avoided to affect.
The content of the invention
For solving above technical problem, the invention provides a kind of side for preparing the compositionss comprising stem cell secretion factor
Method, which comprises the following steps：
S1：Mescenchymal stem cell is isolated from embryonal connective tissue；
S2：Mescenchymal stem cell described in amplification cultivation, obtains the culture comprising stem cell secretion factor；
S3：Mescenchymal stem cell is removed from the culture obtains the solution comprising stem cell secretion factor；
S4：Solution described in purification, and carry out vacuum lyophilization and obtain the compositionss comprising stem cell secretion factor.
Further, the embryonal connective tissue is from umbilical cord.
Further, umbilical cord adventitia is removed, obtains huatong plastic tissue, be cut into 1mm3Piece of tissue.
Further, by with piece of tissue described in pancreatin and collagenase digesting separating.Time is 1-2 hours or so.
Further, with 200 mesh sieve net filtrations after digestion, then with brine, gained cell adds stem cell
Culture is based on containing 5%CO237 DEG C of incubator cultures.
Further, Secondary Culture when cell length is to 80% fusion, obtains the mescenchymal stem cell.
Further, in S2 with stem cell media come mescenchymal stem cell described in amplification cultivation, until the mesenchyme
Stem cell high expressing cell surface marker CD29 and CD105.
Further, the mescenchymal stem cell is removed from the culture by the method for centrifugation in S4.
Further, the removal of impurity is gone by ammonium sulfate precipitation in S5 and then passes through desalination of dialysing, thus solution described in purification.
Present invention also offers a kind of compositionss comprising stem cell secretion factor prepared by said method.
Present invention also offers a kind of mouth sprays comprising stem cell secretion factor prepared by above-mentioned composition.
The invention has the beneficial effects as follows：The present invention prepare mouth sprays by respiratory tract by oral cavity, pharynx, larynx, trachea, prop up
Trachea, pulmonary etc. are partially completed absorption process.Mainly through oral mucosa and pulmonary absorption, the sublingual mucosal epithelium of human mouth is not
By keratinization, most beneficial for the systemic Absorption of biologically effective molecule；Next to that gums and hard palate, in general, human oral mucosa
Permeability to biomolecule：Hypoglossis mucous membrane>Buccal mucosa>Gingiva.Oral mucosal absorption does not have the first pass effect of liver, effectively into
Divide and blood circulation is entered by the vein enriched in neck directly, play a role rapidly.It is short the time required to working.To biologically effective molecule
Compliance it is good, absorption process and assimilation effect it is easily controllable；Mucosa not easy damaged, it is easy to repair；Gastrointestinal is avoided to digest
The Digestion of enzyme；Can play and locally or systemically act on.
Pulmonary has a huge surface area, and blood capillary enriches, can be rapidly and efficiently absorb the larger albumen of molecular weight
Polypeptides matter, effective absorbance are high, and alveolar cyst wall is made up of adhesion, and these cells abut capillary network, gas
Alveolar-capillary barrier is less, and albumen and polypeptide are short by the distance that air blood is exchanged, and speed is fast；The distribution of pulmonary enzyme concentrate and
Biological activity is low, and proteins and peptides class material can still keep biological activity by pulmonary absorption；Pulmonary absorption biologically effective point
Son can avoid gastrointestinal tract to the harmful effect of biologically effective molecule and can avoid the first pass effect of liver, improve biological utilisation
Degree.
Cytokine comprising mescenchymal stem cell secretion in the compositionss of the present invention and mouth sprays, mescenchymal stem cell
At least secretory immune regulatory factor (HGF, LIF), chemotactic factor (RANTES, SDF-1 α, fractalkine, MIP-1 α, MCP-
1st, MCP-2), support the trophic factors (IL-6, FGF-2, PDGF-AA, PDGF-BB, EGF) of CFU-GM, the revascularization factor
(VEGF165, FGF-2, PDGF-AA, PDGF-BB, EGF), suppress the cicatrix factor (HGF, FGF-2), anti-apoptosis factor
(VEGF165, FGF-2, HGF), wound healing correlation factor (IL-6, IL-8, TGF-β 1, MCP-1, VEGF, GM-CSF),
TIMP-1), each collagen type (I, II, III, IV collagen type) and various lysozyme etc..Stem cell secretion these
Bioactive molecule can support the bioactive functions of stem cell itself, while improving body holistic health state, adjust whole
Body or the microenvironment of local, repair cell, the function of improving cellular metabolism, activate senile cell, promote body tissue regeneration
Recover with organ dysfunction, stimulate the anti-aging effects of other adult cells of body.
Specific embodiment
The principle and feature of the present invention are described below in conjunction with example, example is served only for explaining the present invention, and
It is non-for limiting the scope of the present invention.
1. the preparation of stem cell mouth sprays
1) healthy human body umbilical cord is collected, umbilical cord is further cleaned, then cut with aseptic operation umbilical cord cutting is divided into carefully
Cell；
2) fine tissue after shredding is by adding the digestion umbilical cord tissue block 1-2 such as pancreatin, collagenase of debita spissitudo little
When, to differentiate cell.The cell that collection is differentiated carries out adhere-wall culture with stem cell media, obtains umbilical cord mesenchyma dry thin
Born of the same parents；
3) amplification cultivation of mescenchymal stem cell, is entered in the ranks by way of convenient stem cells culture with stem cell media
The culture of mesenchymal stem cells, carries out changing liquid, fluid infusion according to cell growth condition；
4) observation of cell growing state, takes the expression of cell detection cell surface marker CD29, CD105, high expression
During above-mentioned detection material, centrifugation removes cell, obtains the cell culture fluid containing mescenchymal stem cell excreted factor；
5) taking cell culture fluid carries out ammonium sulfate precipitation, and after dialysis desalination, Jing vacuum lyophilizations are made comprising stem cell
Then said composition is prepared into mouth sprays by the compositionss of excreted factor.
2. in culture fluid some stem cell secretion factors content
More solito culture mescenchymal stem cell, observes growth of mesenchymal stem cells situation, when cell reaches 80%-
During 90% fusion, part supernatant is taken, IL-6, IL-8, MCP-1, TIMP-1 in cells and supernatant is quantitative determined with ELISA method
Content, if 3 parallel holes, the result for obtaining is as shown in table 1
The content of some cytokines in 1 culture fluid of table
In from table 1, in culture fluid, contain certain density stem cell secretion factor, therefore, the combination for especially preparing
Also contain stem cell secretion factor in thing and mouth sprays.Can be used for sub-health population, need to improve the crowd of immune state, need
The crowd for improving body anti-inflammatory power, the crowd for having defying age demand.
The foregoing is only presently preferred embodiments of the present invention, not to limit the present invention, all spirit in the present invention and
Within principle, any modification, equivalent substitution and improvements made etc. should be included within the scope of the present invention.
Claims (8)
1. a kind of method for preparing the compositionss comprising stem cell secretion factor, it is characterised in that comprise the following steps：
S1：The separating mesenchymal stem cell from embryonal connective tissue；
S2：Mescenchymal stem cell described in amplification cultivation, obtains the culture comprising stem cell secretion factor；
S3：Mescenchymal stem cell is removed from the culture obtains the solution comprising stem cell secretion factor；
S4：Solution described in purification, and carry out vacuum lyophilization and obtain the compositionss comprising stem cell secretion factor.
2. method according to claim 1, it is characterised in that the embryonal connective tissue is from umbilical cord.
3. method according to claim 2, it is characterised in that the embryonal connective tissue is the huatong plastic group by made by umbilical cord
Knit.
4. method according to claim 1, it is characterised in that with stem cell media filling between described in amplification cultivation in S2
Matter stem cell, until mescenchymal stem cell high expressing cell the surface marker CD29 and CD105.
5. method according to claim 1, it is characterised in that removed from the culture by the method for centrifugation in S3
The mescenchymal stem cell.
6. the method according to any one of claim 1-5, it is characterised in that the removal of impurity is gone by ammonium sulfate precipitation method in S4
Then by desalination of dialysing, thus carry out solution described in purification.
7. a kind of compositionss comprising stem cell secretion factor, it is characterised in that by any one of claim 1-6
Method is prepared.
8. a kind of mouth sprays comprising stem cell secretion factor, it is characterised in that prepared by the compositionss described in claim 7
Obtain.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610962034.8A CN106511381A (en) | 2016-11-04 | 2016-11-04 | Composition and oral spray which contain stem cell secreted factors, and preparation method thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610962034.8A CN106511381A (en) | 2016-11-04 | 2016-11-04 | Composition and oral spray which contain stem cell secreted factors, and preparation method thereof |
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| CN106511381A true CN106511381A (en) | 2017-03-22 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111394304A (en) * | 2020-03-27 | 2020-07-10 | 北京臻溪谷医学研究中心（有限合伙） | Stem cell conditioned culture supernatant for repairing respiratory system injury and preparation thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103266081A (en) * | 2012-01-21 | 2013-08-28 | 中国人民解放军军事医学科学院附属医院 | Efficient method for isolating and culturing mesenchymal stem cells from umbilical cord |
| CN104099294A (en) * | 2013-04-03 | 2014-10-15 | 北京京蒙高科干细胞技术有限公司 | Medium based on stem cell-excreted factor and its preparation method and use method |
| CN106214620A (en) * | 2016-08-10 | 2016-12-14 | 美国安泰健康投资管理公司 | A kind of preparation method of the facial film lyophilized powder containing stem cell secretion factor |
-
2016
- 2016-11-04 CN CN201610962034.8A patent/CN106511381A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103266081A (en) * | 2012-01-21 | 2013-08-28 | 中国人民解放军军事医学科学院附属医院 | Efficient method for isolating and culturing mesenchymal stem cells from umbilical cord |
| CN104099294A (en) * | 2013-04-03 | 2014-10-15 | 北京京蒙高科干细胞技术有限公司 | Medium based on stem cell-excreted factor and its preparation method and use method |
| CN106214620A (en) * | 2016-08-10 | 2016-12-14 | 美国安泰健康投资管理公司 | A kind of preparation method of the facial film lyophilized powder containing stem cell secretion factor |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111394304A (en) * | 2020-03-27 | 2020-07-10 | 北京臻溪谷医学研究中心（有限合伙） | Stem cell conditioned culture supernatant for repairing respiratory system injury and preparation thereof |
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