CN107137426A - Anthraco-silicosis treatment Alevaire of the body extract of excretion containing human mesenchymal stem cell and preparation method thereof - Google Patents
Anthraco-silicosis treatment Alevaire of the body extract of excretion containing human mesenchymal stem cell and preparation method thereof Download PDFInfo
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Abstract
Alevaire and preparation method thereof is treated the invention discloses a kind of anthraco-silicosis of the body of excretion containing human mesenchymal stem cell extract, wherein main component is the mescenchymal stem cell excretion body extract of high concentration.Mescenchymal stem cell can secrete cytokine profiles in incubation, the deep enough inner membrance basalis of these cell factors energy, promote internal film tissue's differentiation, angiogenesis, granulation tissue growth, promote injured cutaneous tissue structure Regeneration and Repair, the generation of scar connective tissue is reduced, bacterium infection and the inflammatory reaction of wound tissue is reduced.The present invention, within a short period of time with regard to that can obtain human mesenchyme's extract of high concentration, a large amount of stem cell excretion bodies is therefrom isolated by excretion body extractor using the method for continuous hungry mescenchymal stem cell.The Alevaire of the excretion body extract of mescenchymal stem cell containing high concentration of the invention is applied to the reparation and treatment of anthraco-silicosis.
Description
Technical field
The present invention relates to stem cell excretion body extract, the especially a kind of body of excretion containing human mesenchymal stem cell extract
Anthraco-silicosis treatment Alevaire and preparation method thereof.
Background technology
Mescenchymal stem cell (mesenchymal stem cells, MSC) is the one group of foreign cell for coming from matrix group, can
To organize to obtain from most of human body.They have self-renewal capacity, tissue repair, immunoregulation capability, can be to middle embryo
Layer lineage, for example：Fat cell, osteocyte, cartilage cell etc., additionally can to and other germ-layer lineage cells point
Change, for example, break up to epidermal cell, vascular endothelial cell.
Contain a variety of substantial amounts of active components in mescenchymal stem cell cultivating system, such as：Stem cell factor (SCF),
Fibroblast growth factor (FGF), endothelial growth factor (VEGF), epithelical cell growth factor (EGF), cell colony
Stimulating factor, interleukins (IL), collagen, hyaluronic acid etc. more than 80 plants active material.Research has shown that stem cell secretion
Active material be to exist in the form of excretion body mostly, excretion body can be good at and cell membrane fusion, so that will be internal
Active material transports the cytotropic inside of target and played a role.At present, had by mescenchymal stem cell be applied to injury of lungs and
The report that pulmonary fibrosis is repaired, but be due to competent cell viable conditions it is harsher, it is easy to it is dead, therefore by living cells
Role can also have a greatly reduced quality.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of anthraco-silicosis of the body of excretion containing mescenchymal stem cell extract
Alevaire and preparation method thereof is treated, excretion body extract is secreted using the mescenchymal stem cell of high concentration, is controlling for anthraco-silicosis
Treat the Alevaire provided safely, effectively, easy to use.
In order to solve the above-mentioned technical problem, the technical solution adopted by the present invention is：A kind of excretion containing human mesenchymal stem cell
The anthraco-silicosis of body extract treats the preparation method of Alevaire, comprises the following steps：
(1) human mesenchymal stem cell culture：P2-P6 human mesenchymal stem cell recovery, inoculation batch are chosen from cell bank
Secondary to be designated as first batch, second lot, the 3rd batch, inoculum density is respectively 50%-60%, 35%- of maximum degrees of fusion
45%th, 20%-30%；It without phenol red DMEM/DF12 and volume by volume concentration is 10% tire ox that complete medium used in culture cell, which is,
Serum；Cell inoculation is completed, and is put in CO2gas incubator culture, 37 DEG C of temperature, carbon dioxide volume by volume concentration 5%；
(2) when first batch cell fusion degree reaches 70%-80%, cell is cleaned 2 times with physiological saline, is electrolysed with compound
Matter parenteral solution carries out hungry culture；
(3) after starvation is cultivated 12-24 hours, first batch cell fusion degree reaches 90%-95%, collects in cell culture
Clearly, 5-8min is centrifuged under the conditions of 3000-4500g and removes precipitation, take supernatant after centrifugation standby；
(4) the second lot cell of culture is taken, when cell fusion degree reaches 70%-80%, second is cleaned with physiological saline
Batch cell 2 times, hungry culture is carried out with cells and supernatant in step (3)；
(5) after starvation is cultivated 12-24 hours, second lot cell fusion degree reaches 90%-95%, collects in cell culture
Clearly, 5-8min is centrifuged under the conditions of 3000-4500g and removes precipitation, take supernatant after centrifugation standby；
(6) the 3rd batch cell of culture is taken, when cell fusion degree reaches 70%-80%, the 3rd is cleaned with physiological saline
Batch cell 2 times, hungry culture is carried out with cells and supernatant in step (5)；
(7) after starvation is cultivated 12-24 hours, the 3rd batch cell degrees of fusion reaches 90%-95%, collects in cell culture
Clearly, 5-8min is centrifuged under the conditions of 3000-4500g and removes precipitation, take supernatant after centrifugation standby；
(8) the step of collecting after continuous hungry three times (7) supernatant extracts stem cell therein with 0.22um membrane filtrations
Excretion body, prepares into Alevaire with the excretion body that separation acquisition is resuspended in 20-40ml physiological saline, is put in 4 DEG C of refrigerators and preserves.
The human mesenchymal stem cell is human umbilical cord mesenchymal stem cells, fat mesenchymal stem cell, medulla mesenchyma are done
Cell, placenta mesenchyma stem cell, menstrual blood mescenchymal stem cell or dental pulp mescenchymal stem cell.
The anthraco-silicosis treatment Alevaire of the body extract of excretion containing human mesenchymal stem cell made from above-mentioned preparation method.
Beneficial effect of the present invention：Cell is subjected to hungry culture so that it is outer containing various active components that cell is secreted
The concentration for secreting body is improved, and repeats hungry mescenchymal stem cell using hungry supernatant in a short time, carries cell
Taking the concentration of excretion body in thing further increases.And the mescenchymal stem cell excretion body extract of high concentration can promote internal film tissue
Differentiation, angiogenesis, granulation tissue growth, promote injury tissue structure Regeneration and Repair, reduce the fibrosis of tissue, adjust lung
Immune microenvironment, significantly improves tissue fibrosis problem, reduces bacterium infection and the inflammatory reaction of wound tissue.
Brief description of the drawings
Fig. 1 is the mescenchymal stem cell figure of the method culture of the present invention；
Fig. 2 compares for efficiency factor secretory volume in different hungry number of times human umbilical cord mesenchymal stem cells excretion body extracts
Figure；
Embodiment
The present invention is described in further detail with reference to the accompanying drawings and detailed description：
The anthraco-silicosis of the body extract of excretion containing human mesenchymal stem cell of the present invention treats the preparation method of Alevaire, including
Following steps：
(1) human mesenchymal stem cell culture：P2-P6 human mesenchymal stem cell recovery, inoculation batch are chosen from cell bank
Secondary to be designated as first batch, second lot, the 3rd batch, inoculum density is respectively 50%-60%, 35%- of maximum degrees of fusion
45%th, 20%-30%；It without phenol red DMEM/DF12 and volume by volume concentration is 10% tire ox that complete medium used in culture cell, which is,
Serum；Cell inoculation is completed, and is put in CO2gas incubator culture, 37 DEG C of temperature, carbon dioxide volume by volume concentration 5%；
(2) when first batch cell fusion degree reaches 70%-80%, cell is cleaned 2 times with physiological saline, is electrolysed with compound
Matter parenteral solution carries out hungry culture；
(3) after starvation is cultivated 12-24 hours, first batch cell fusion degree reaches 90%-95%, collects in cell culture
Clearly, 5-8min is centrifuged under the conditions of 3000-4500g and removes precipitation, take supernatant after centrifugation standby；
(4) the second lot cell of culture is taken, when cell fusion degree reaches 70%-80%, second is cleaned with physiological saline
Batch cell 2 times, hungry culture is carried out with cells and supernatant in step (3)；
(5) after starvation is cultivated 12-24 hours, second lot cell fusion degree reaches 90%-95%, collects in cell culture
Clearly, 5-8min is centrifuged under the conditions of 3000-4500g and removes precipitation, take supernatant after centrifugation standby；
(6) the 3rd batch cell of culture is taken, when cell fusion degree reaches 70%-80%, the 3rd is cleaned with physiological saline
Batch cell 2 times, hungry culture is carried out with cells and supernatant in step (5)；
(7) after starvation is cultivated 12-24 hours, the 3rd batch cell degrees of fusion reaches 90%-95%, collects in cell culture
Clearly, 5-8min is centrifuged under the conditions of 3000-4500g and removes precipitation, take supernatant after centrifugation standby；
(8) the step of collecting after continuous hungry three times (7) supernatant is with 0.22um membrane filtrations, and with cells and supernatant
Middle excretion body quick-speed extraction apparatus (Chinese application number 201720177150.9) extracts stem cell excretion body therein, with 20-40ml
Physiological saline be resuspended separation obtain excretion body, be put in 4 DEG C of refrigerators and preserve stand-by.The mescenchymal stem cell is between umbilical cord
Mesenchymal stem cells, fat mesenchymal stem cell, mesenchymal stem cells MSCs, placenta mesenchyma stem cell, menstrual blood mesenchyma are done
Cell or dental pulp mescenchymal stem cell.
The culture mescenchymal stem cell of the present invention, is carried out when growth of mesenchymal stem cells degrees of fusion reaches 70%-80%
And Nature enemy, make excretion body of the cell secretion containing a large amount of bioactive substances, and using the multiple starved cells of hungry supernatant,
Short time significantly adds excretion bulk concentration.Stem cell excretion body extract direct lung by way of atomization is given
Medicine, therapeutic effect can be played to greatest extent.
Injury of lungs and the treatment and reparation of pulmonary fibrosis that the anthraco-silicosis that the reparation Alevaire of preparation is used for is caused.
Embodiment 1
(1) human umbilical cord mesenchymal stem cells culture：P4 human umbilical cord mesenchymal stem cells recovery is chosen from cell bank, carefully
Born of the same parents' sum 8.4*107.Inoculation batch is designated as first batch, second lot, the 3rd batch, and inoculum density is respectively maximum degrees of fusion
60% (every bottle of inoculating cell number：9*106, six bottles of inoculating cell), 40% (every bottle of inoculating cell number：6*106, inoculating cell four
Bottle), 20% (inoculating cell number：3*106, two bottles of inoculating cell), cultivating system is 40ml.Cultivated completely used in culture cell
It without phenol red DMEM/DF12 and volume by volume concentration is 10% hyclone that base, which is, and inoculating cell bottle is T-175 blake bottles.Cell connects
Plant and complete, be put in CO2gas incubator culture, 37 DEG C of temperature, carbon dioxide volume by volume concentration 5%；
(2) cultivate 30 hours, when now first batch cell fusion degree reaches 75%, see (Fig. 1), cleaned with physiological saline
Cell 2 times, every bottle with 40ml Multiple electrolytes injections (Otsuka Pharmaceutical (China) Co., Ltd. produce compound electrolyte glucose
MG3 parenteral solutions) carry out hungry culture；
(3) after starvation is cultivated 18 hours, first batch cell fusion degree collects cells and supernatant, 4000g bars up to 95%
Under part centrifuge 8min remove precipitation, take centrifugation after supernatant be put in 4 DEG C it is standby；
(4) the second lot cell of culture is taken, now cell fusion degree reaches 70%, second lot is cleaned with physiological saline
Cell 2 times, every bottle carries out hungry culture with cells and supernatant 40ml in step (3)；
(5) after starvation is cultivated 18 hours, second lot cell fusion degree collects cells and supernatant, 4000g bars up to 95%
Under part centrifuge 8min remove precipitation, take centrifugation after supernatant be put in 4 DEG C it is standby；
(6) the 3rd batch cell of culture is taken, now cell fusion degree reaches 70%, the 3rd batch is cleaned with physiological saline
Cell 2 times, every bottle carries out hungry culture with cells and supernatant 40ml in step (5)；
(7) after starvation is cultivated 24 hours, the 3rd batch cell degrees of fusion collects cells and supernatant, 4000g bars up to 90%
Under part centrifuge 8min remove precipitation, take centrifugation after supernatant be put in 4 DEG C it is standby.
(8) each 40ml of standby supernatant in step (3), step (5) and step (7) is secreted it using excretion body extractor
In stem cell excretion body, and with 10 physiological saline clean 2 times after with 40ml physiological saline be resuspended excretion body, ultrasonication excretion
Cytokine content therein is surveyed in physical examination, is seen (Fig. 2).
(9) each 40ml of standby supernatant in step (3), step (5) and step (7) is secreted it using excretion body extractor
In stem cell excretion body, and with 10 physiological saline clean 2 times after with 30ml physiological saline be resuspended excretion body be stored in sterile west
In woods bottle, be put in 4 DEG C of refrigerators and preserve stand-by, that is, respectively obtain it is hungry once, hungry secondary and hungry three mesenchymas it is dry thin
The extracellular Alevaire for secreting body extract.
(10) healthy male Wistar rat 50 is chosen, body weight 200g or so is randomly divided into 5 groups, respectively blank pair
According to group (normal culture), positive controls (silicosis disease model, with physiological saline nebulae inhalation), group I (silicosis disease model,
The Alevaire nebulae inhalation prepared with a hungry excretion body extract), experimental group II (silicosis disease model, it is secondary outer with starvation
Secrete the Alevaire nebulae inhalation of body extract preparation), (the silicosis disease model, with hungry three excretion body extract systems of experimental group III
Standby Alevaire nebulae inhalation), every group 10.The method for contaminating dirt by tracheal strips prepares silicosis disease model, and every rat contaminates dirt
Nebulae inhalation is given within 1 week after 20mg, dye dirt, treatment time continues 30 days, once, atomization quantity is 1ml/ times for daily atomization.Treatment
Cultivated 30 after end, Pathomorphology inspection is carried out by the full lung dry weight of rat, weight in wet base contrast (table 1), and to lung tissue
(HE dyeing, Azann triple stainings, Gomori Silver stains), Experimental Silicosis In Rats are using level Four classification (table 2) to controlling curative effect
Fruit is evaluated.As a result show, the Alevaire of mescenchymal stem cell excretion body extract prepared by the present invention can be good at pair
Anthraco-silicosis is prevented and treated.
Table 1
The Silicotic Rats of table 1 disease prophylactic treatment evaluation result：T/C is each group index value and the ratio of positive controls；
Examined through Q, compared with positive controls, P<0.05.
Table 2
| Group | Number of animals | Pathological grading |
| Blank control group | 10 | N/A |
| Positive controls | 10 | Ⅱ-Ⅲ |
| Group I | 10 | Ⅰ+-Ⅱ |
| Experimental group II | 10 | Ⅰ-Ⅱ |
| Experimental group III | 10 | Ⅰ |
The Silicotic Rats of table 2 disease pathological grading result：I grade is cellularity silicon tubercle；II grade is fibrous tubercle；III grade is fibre
Dimension property tubercle, in fusion lesion.
In summary, present disclosure is not limited in the above embodiments, and the knowledgeable people in same area can
Can propose other embodiments easily within the technological guidance's thought of the present invention, but this embodiment is included in this hair
Within the scope of bright.
Claims (3)
1. a kind of anthraco-silicosis of the body of excretion containing human mesenchymal stem cell extract treats the preparation method of Alevaire, its feature exists
In comprising the following steps：
(1) human mesenchymal stem cell culture：P2-P6 human mesenchymal stem cell recovery, inoculation batch note are chosen from cell bank
For first batch, second lot, the 3rd batch, inoculum density be respectively the 50%-60% of maximum degrees of fusion, 35%-45%,
20%-30%；It without phenol red DMEM/DF12 and volume by volume concentration is 10% hyclone that complete medium used in culture cell, which is,；
Cell inoculation is completed, and is put in CO2gas incubator culture, 37 DEG C of temperature, carbon dioxide volume by volume concentration 5%；
(2) when first batch cell fusion degree reaches 70%-80%, cell is cleaned 2 times with physiological saline, noted with compound electrolyte
Penetrate liquid and carry out hungry culture；
(3) after starvation is cultivated 12-24 hours, first batch cell fusion degree reaches 90%-95%, collects cells and supernatant,
5-8min is centrifuged under the conditions of 3000-4500g and removes precipitation, takes supernatant after centrifugation standby；
(4) the second lot cell of culture is taken, when cell fusion degree reaches 70%-80%, second lot is cleaned with physiological saline
Cell 2 times, hungry culture is carried out with cells and supernatant in step (3)；
(5) after starvation is cultivated 12-24 hours, second lot cell fusion degree reaches 90%-95%, collects cells and supernatant,
5-8min is centrifuged under the conditions of 3000-4500g and removes precipitation, takes supernatant after centrifugation standby；
(6) the 3rd batch cell of culture is taken, when cell fusion degree reaches 70%-80%, the 3rd batch is cleaned with physiological saline
Cell 2 times, hungry culture is carried out with cells and supernatant in step (5)；
(7) after starvation is cultivated 12-24 hours, the 3rd batch cell degrees of fusion reaches 90%-95%, collects cells and supernatant,
5-8min is centrifuged under the conditions of 3000-4500g and removes precipitation, takes supernatant after centrifugation standby；
(8) the step of collecting after continuous hungry three times (7) supernatant extracts stem cell excretion therein with 0.22um membrane filtrations
Body, prepares into Alevaire with the excretion body that separation acquisition is resuspended in 20-40ml physiological saline, is put in 4 DEG C of refrigerators and preserves.
2. the anthraco-silicosis of the body of excretion containing human mesenchymal stem cell extract treats the preparation side of Alevaire according to claim 1
Method, it is characterised in that the human mesenchymal stem cell is between human umbilical cord mesenchymal stem cells, fat mesenchymal stem cell, marrow
Mesenchymal stem cells, placenta mesenchyma stem cell, menstrual blood mescenchymal stem cell or dental pulp mescenchymal stem cell.
3. the anthraco-silicosis of the body extract of excretion containing human mesenchymal stem cell made from preparation method as defined in claim 1
Treat Alevaire.
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| CN114042029A (en) * | 2021-10-12 | 2022-02-15 | 成都市古月永享生物科技有限公司 | Lyophilized powder composite preparation containing secretion of human pluripotent stem cells and Wharton's jelly-derived mesenchymal stem cells, skin care product and preparation method |
| CN114159550A (en) * | 2021-11-16 | 2022-03-11 | 南京天纵易康生物科技股份有限公司 | Preparation method of microcapsule hair-growing product based on exosome growth factor |
| WO2022105156A1 (en) * | 2020-11-17 | 2022-05-27 | 广州赛莱拉干细胞科技股份有限公司 | Preparation method for and application of mesenchymal stem cell exosome |
| CN116036229A (en) * | 2023-01-05 | 2023-05-02 | 安徽科门生物科技有限公司 | Atomization agent containing stem cell exosomes and application of atomization agent to treatment of lung diseases |
| CN118161450A (en) * | 2024-05-11 | 2024-06-11 | 北京益华生物科技有限公司 | Atomized preparation containing cell active factors and application of atomized preparation in acute pneumonia |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105412153A (en) * | 2015-12-10 | 2016-03-23 | 中国人民解放军第二军医大学 | Application of exosome excreted by mesenchymal stem cells for preparing medicine for preventing and treating hepatitis c virus |
| CN105477016A (en) * | 2015-11-13 | 2016-04-13 | 中国人民解放军第二军医大学 | Application of exosome derived from human mesenchymal stem cells to resistance to tissue fibrosis and scar forming |
| CN106474155A (en) * | 2016-10-19 | 2017-03-08 | 天津普瑞赛尔生物科技有限公司 | External-use gel preparation containing human umbilical cord mesenchymal stem cells extract and its production and use |
-
2017
- 2017-05-17 CN CN201710347031.8A patent/CN107137426A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105477016A (en) * | 2015-11-13 | 2016-04-13 | 中国人民解放军第二军医大学 | Application of exosome derived from human mesenchymal stem cells to resistance to tissue fibrosis and scar forming |
| CN105412153A (en) * | 2015-12-10 | 2016-03-23 | 中国人民解放军第二军医大学 | Application of exosome excreted by mesenchymal stem cells for preparing medicine for preventing and treating hepatitis c virus |
| CN106474155A (en) * | 2016-10-19 | 2017-03-08 | 天津普瑞赛尔生物科技有限公司 | External-use gel preparation containing human umbilical cord mesenchymal stem cells extract and its production and use |
Non-Patent Citations (1)
| Title |
|---|
| MOONHWAN CHOI ET AL.: "Therapeutic Use of Stem Cell Transplantation for Cell Replacement or Cytoprotective Effect of Microvesicle Released from Mesenchymal Stem Cell", 《MOL. CELLS》 * |
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