CN116725948B - Adipose-derived stem cell-mediated gel and preparation method and application thereof - Google Patents
Adipose-derived stem cell-mediated gel and preparation method and application thereof Download PDFInfo
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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
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/498—Pyrazines or piperazines ortho- and peri-condensed with carbocyclic ring systems, e.g. quinoxaline, phenazine
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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
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
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- A61K9/0014—Skin, i.e. galenical aspects of topical compositions
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/06—Antipsoriatics
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
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Abstract
The invention discloses a gel mediated by adipose-derived stem cells, a preparation method and application thereof, wherein the gel is prepared by mixing mesenchymal stem cell supernatant, carbomer gel, 1, 4-dioxo-2, 3-dibromomethylquinoline reagent and sterile deionized water. The gel mediated by the adipose-derived stem cells acts on the affected part of psoriasis in a smearing way, and can reduce skin scales, erythema, skin thickening and other psoriasis-like skin lesions and regulate inflammatory reaction caused by psoriasis by promoting regeneration and repair of damaged tissues, reducing inflammatory reaction and regulating normal generation of keratinocytes to treat the psoriasis. The gel prepared from the human adipose-derived mesenchymal stem cells is safe, efficient, free of side effects, simple in production process and has the potential of realizing medical transformation.
Description
Technical Field
The invention belongs to the technical field of biopharmaceuticals, and discloses a preparation method of a gel mediated by adipose-derived stem cells, the gel mediated by adipose-derived stem cells prepared by the preparation method, and application of the gel in treating psoriasis.
Background
Psoriasis is commonly called psoriasis, and is a chronic and easily recurrent inflammatory skin disease. The common skin damage is marked by erythema scaling, the skin damage has clear boundary, thick-layer scales can be higher than the surface of the skin, obvious itching can be accompanied, great pain is caused to patients, and the life quality of the patients is reduced. Psoriasis is mostly in the period of young and strong, has longer course of disease, and has the characteristics of heavy winter and light summer, namely aggravation in winter and obvious alleviation in summer. The pathogenesis of psoriasis is complex, and the specific mechanism is still under study, and it is clear that psoriasis is a genetic disease caused by polygenic change, mainly caused by T lymphocyte immune mediation and under the combined action of various immune cells, and finally can cause obvious characteristics of psoriasis such as keratinocyte hyperproliferation.
The clinical treatment center thought at present mainly starts from the aspects of controlling inflammation, relieving symptoms and the like. The main medicines for treatment are mainly hormone and immunosuppressant, and have large side effect, poor curative effect and easy recurrence. For autoimmune diseases, stem cell therapy can replace and repair damaged or dead tissues, and can also intervene in the onset link of immune disorder, so that the therapeutic effect is considerable. Mesenchymal stem cells (mesenchymal stromal cells, MSCs) are adult stem cells derived from mesoderm, have self-renewal and multidirectional differentiation potential, can promote regeneration and repair of damaged tissues, have important immune regulation and control effects, and in addition, MSCs regulate and control inflammatory responses of organisms mainly by influencing proliferation, differentiation and inflammatory factor secretion of immune cells involved in inflammatory responses. MSCs are used for treating psoriasis in recent years, can obviously improve psoriasis-like skin lesions, and are safe and efficient.
Currently, MSCs are derived from a variety of tissues including bone marrow, umbilical cord, placenta, and fat, and there are four major problems with clinical trials and treatments of MSCs: 1. the cell demand is huge, and adipose tissue is easier to obtain than bone marrow, umbilical cord, placenta and other tissues, so the adipose-derived mesenchymal stem cell preparation has more market prospect. 2. The MSCs from different sources may have heterogeneity, and the acquisition of autologous stem cells may cause damage to the autologous, but autologous adipose tissue is more abundant, and the acquisition of adipose-derived mesenchymal stem cells from autologous tissue is easy and has lower influence on the autologous. 3, there may be heterogeneity in MSCs of different origin; 4, it is difficult to unify standards for MSCs of different sources, and these problems seriously hamper clinical treatment of MSCs.
Therefore, the carbomer gel for the adipose-derived mesenchymal stem cells, which has strong safety and can effectively relieve the psoriasis symptoms, meets the market demand, has wide market value and application prospect, and has very important significance for promoting the treatment of psoriasis skin repair by adding the 1, 4-dioxo-2, 3-dibromomethylquinoline reagent into the adipose-derived mesenchymal stem cells.
Disclosure of Invention
In view of the above-mentioned shortcomings, the invention provides a adipose-derived stem cell-mediated gel, and a preparation method and application thereof, and the invention is realized by the following technical means:
in a first aspect of the invention there is provided an adipose stem cell-mediated gel comprising:
25mL of the adipose-derived mesenchymal stem cell supernatant concentrate, 75mL of carbomer gel and 100mL of the adipose-derived stem cell-mediated gel were added with 200. Mu.L of 50mg/mL of 1, 4-dioxo-2, 3-dibromomethylquinoline reagent.
In a second aspect of the present invention, there is provided a method for preparing a adipose-derived stem cell-mediated gel for preparing the human adipose-derived stem cell gel, comprising the steps of:
(1) Preparing a supernatant of the adipose-derived mesenchymal stem cells;
(2) Preparing gel;
(3) Preparing a 1, 4-dioxo-2, 3-dibromomethylquinoline reagent;
mixing the adipose-derived mesenchymal stem cell supernatant, carbomer gel and 1, 4-dioxo-2, 3-dibromomethylquinoline reagent according to a proportion.
Further, the mesenchymal stem cells in the step (1) are human adipose-derived mesenchymal stem cells, and the preparation method of the supernatant is as follows:
A. resuscitates 2 tubes of human adipose mesenchymal stem cells (Huatuo organism, cat# HTX 2309), inoculates into 2 10cm dishes, cultures in DMEM medium containing 10% FBS, 100U/mL penicillin and 100U/mL streptomycin at 37℃in a carbon dioxide cell incubator containing 5% by volume;
B. when the cell density reaches more than 90%, replacing serum-free stem cell culture medium to starve for nine hours, and recovering supernatant;
C. dialyzing to remove salt in the supernatant;
D. recovering human adipose mesenchymal stem cells, and temporarily preserving in a refrigerator at-80 ℃;
E. dissolving cells with supernatant after dialysis and desalting, then performing cell disruption with an ultrasonic cell disrupter, centrifuging for 10min at 10000r, and filtering and sterilizing;
the prepared human adipose-derived mesenchymal stem cell supernatant is freeze-dried for 24 hours in a freeze dryer to prepare a powdery preparation for convenient use.
Further, the gel in the step (2) is carbomer gel, and the preparation method is as follows:
A. weighing 0.2g carbomer in weighing paper, and placing in an ultra-clean bench for ultraviolet sterilization for 30min;
B. 0.2g carbomer was poured into a centrifuge tube, 55ml DDW (ultra light water), 20ml0.1mol/L NaOH was added;
C. after standing overnight, the tube was jelly-like.
Further, the preparation method of the 1, 4-dioxo-2, 3-dibromomethylquinoline reagent in the step (3) comprises the following steps:
A. 10mg of 1, 4-dioxo-2, 3-dibromomethylquinoline powder was weighed and dissolved in 200. Mu. LDMSO (dimethyl sulfoxide) to prepare 50mg/mL of 1, 4-dioxo-2, 3-dibromomethylquinoline solvent.
The invention has the beneficial effects that:
1. the gel mediated by the adipose-derived stem cells acts on the affected part of psoriasis in a smearing way, and treats the psoriasis by promoting regeneration and repair of damaged tissues, reducing inflammatory reaction, regulating normal generation of keratinocytes and the like, so that the gel prepared by the adipose-derived stem cells is safe, efficient and free of side effects, has a simple production process, and has the potential of realizing medical transformation.
2. The gel mediated by the adipose-derived stem cells can reduce skin scales, erythema, psoriasis-like skin lesions such as epidermis thickening and the like, and regulate inflammatory reaction caused by psoriasis. According to experimental results, the drug smearing group and the stem cell gel smearing group can obviously relieve the phenotypes of psoriasis-like skin scaling increase, erythema coverage and epidermis thickening caused by IMQ, the effect of the stem cell gel smearing group is better than that of the drug smearing group, the scaling, erythema and epidermis thickening symptoms are lighter, the dysplastic cells are obviously reduced, and the epidermis layer thickness is obviously reduced. The gel is effective in improving psoriasis scaling, erythema and epidermis thickening symptoms, and has antiinflammatory effect.
Drawings
FIG. 1 is a morphological observation of back skin lesions after a period of treatment in each group of mice;
FIG. 2 is a histological observation of back skin lesions after a period of treatment in each group of mice;
figure 3 shows the results of the skin thickness measurement after a period of treatment for each group of mice.
Detailed Description
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Further, it is understood that various changes and modifications may be made by those skilled in the art after reading the teachings of the present invention, and such equivalents are intended to fall within the scope of the claims appended hereto.
Example 1
An adipose-stem-cell-mediated gel comprising:
mesenchymal stem cell supernatant, carbomer gel, 1, 4-dioxo-2, 3-dibromomethylquinoline reagent and sterile deionized water.
Example 2
A method of preparing a adipose-derived stem cell-mediated gel, comprising:
(1) Preparing a adipose-derived mesenchymal stem cell supernatant:
(2) Preparing gel;
(3) Preparing a 1, 4-dioxo-2, 3-dibromomethylquinoline reagent;
(4) Mixing the adipose-derived mesenchymal stem cell supernatant, carbomer gel and 1, 4-dioxo-2, 3-dibromomethylquinoline reagent according to a proportion.
Further, the mesenchymal stem cells in the step (1) are human adipose-derived mesenchymal stem cells, and the preparation method of the supernatant is as follows:
A. resuscitates 2 tubes of human adipose-derived mesenchymal stem cells, inoculates the cells into 2 10cm dishes, and cultures the cells in a carbon dioxide cell incubator with a volume fraction of 5% at 37 ℃ by using a normal stem cell culture medium;
B. when the cell density reaches more than 90%, replacing serum-free stem cell culture medium to starve for nine hours, and recovering supernatant;
C. dialyzing to remove salt in the supernatant;
D. recovering human adipose mesenchymal stem cells, and temporarily preserving in a refrigerator at-80 ℃;
E. dissolving cells with supernatant after dialysis and desalting, then performing cell disruption with an ultrasonic cell disrupter, centrifuging for 10min at 10000r, and filtering and sterilizing;
the prepared human adipose-derived mesenchymal stem cell supernatant is freeze-dried for 24 hours in a freeze dryer to prepare a powdery preparation for convenient use.
Further, the gel in the step (2) is carbomer gel, and the preparation method is as follows:
A. weighing 0.2g carbomer in weighing paper, and placing in an ultra-clean bench for ultraviolet sterilization for 30min;
B. 0.2g carbomer was poured into a centrifuge tube and 55ml DDW,20ml NAOH at 0.1mol/L was added;
C. after standing overnight, the tube was jelly-like.
Further, the preparation method of the 1, 4-dioxo-2, 3-dibromomethylquinoline reagent in the step (3) comprises the following steps:
A. 10mg of 1, 4-dioxo-2, 3-dibromomethylquinoline powder was weighed out and dissolved in 200. Mu. LDMSO to prepare 50mg/mL of 1, 4-dioxo-2, 3-dibromomethylquinoline solvent.
Application example 1
The experimental method comprises the following steps:
1. animal experiment group
The mice were randomly assigned to the following 3 groups of 3:
model group (IMQ): the backs of the mice were coated with 40mg of 5% imiquimod cream daily;
drug application group (imq+calcipotriol): the backs of the mice were coated with 40mg of 5% imiquimod cream daily, and from day 4 on calcipotriol cream;
stem cell gel smear group (IMQ+Stem cell gel): the backs of the mice were coated with 40mg of 5% imiquimod cream daily, and from day 4 on human adipose mesenchymal stem cell gel.
2. Mouse psoriasis model establishment
Each group of mice was shaved on the back the day prior to the experiment. The back Mao Ti of each mouse was removed by electric hair clippers, and the remaining hair on the back of the mouse was removed by applying a depilatory cream to expose the skin having an area of about 3cm by 2 cm. After one day, the back skin of each mouse was applied with 40mg of 5% imiquimod ointment daily for 7 consecutive days. On day 4, the mice in each group were observed to develop significant erythema, scaling and thickening of the back skin, i.e. successful modeling.
Detecting items:
1. morphological observation of skin lesions on the back of mice
The skin condition of the back of the mice was recorded daily using a digital camera, and the detailed results are shown in fig. 1.
2. Histological observation of skin lesions on the back of mice
The skin tissue of the mice is stained with hematoxylin-eosin (H & E), after 7 days of molding, the animals are sacrificed, the skin of the mice is taken, the mice is fixed for 24 hours by formalin solution, the liver of the mice is cut into slices with the thickness of 3mm, and the slices are placed in an embedding box and washed with tap water with slow flow rate for 1.5 hours. Immersing the washed tissue embedding box in a full-automatic animal tissue dehydrator for dehydration, and setting an automatic program: 40min for 70% ethanol, 50min for 80% ethanol, 1h for 90% ethanol, 1h for 95% ethanol, 1h for absolute ethanol, 30min for 100% xylene, 1h for 100% xylene, 30min for high melting paraffin, and 1h for high melting paraffin. The dehydration procedure is about 9.5 hours, paraffin embedding is carried out on the liver tissue, the embedded sample is rapidly solidified at the freezing table of minus 20 ℃, wax blocks are trimmed into trapezium, and the trapezium is frozen for more than 12 hours in a refrigerator of minus 20 ℃. Cutting the tissue sample into 5 mu M slices by using a paraffin slicer, putting the slices into a 42 ℃ slice spreading machine, fully spreading the slices, fishing out pathological-grade glass slides, and drying at room temperature for 4-6 hours.
The H & E staining procedure was as follows, 100% xylene 10min, absolute 1.5min,90% ethanol 1min,80% ethanol 1min,50% ethanol 1min, water wash 3min, hematoxylin 5min, water wash 3min,1% acetic acid ethanol 2s, water wash 3min,0.8% ammonia 2s, water wash 5min, eosin 2s,95% ethanol 1min, absolute 30s,100% xylene 1min. The tissue is soaked in 100% xylene solution, and is rapidly sealed with neutral resin glue and cover glass, and is horizontally placed on a airing frame. After the neutral resin is solidified, observing pathological morphological changes of the liver tissue of the mouse by utilizing a multifunctional fluorescence microscope, picking up images at different positions of a tissue sample, and analyzing the results.
3. Mouse epidermis thickness determination
Experimental results:
fig. 1: morphological observation of skin lesions on the back of mice
From fig. 1, it can be observed that the drug-coated group and the stem cell gel-coated group can significantly reduce the phenotype of psoriasis-like skin scaling, erythema coverage and epidermis thickening caused by IMQ, and the effect of the stem cell gel-coated group is superior to that of the drug-coated group, and the scales, erythema and skin thickness symptoms are lighter.
Fig. 2: histological observation of skin lesions on the back of mice
The staining results of fig. 2h & e show that the phenomena of psoriasis-like skin epidermis thickening and the like caused by IMQ can be obviously reduced by the medicine smearing group and the stem cell gel smearing group, the keratinized incomplete cells are obviously reduced, and the effect of the stem cell gel smearing group is better than that of the medicine smearing group.
Fig. 3: mouse epidermis thickness determination
As can be seen from fig. 3, the drug-coated group and the stem cell gel-coated group showed significantly reduced mice keratinized cells compared to the model group, and the thickness of the epidermis layer was significantly lower than that of the model group, wherein the thickness of the cortex layer of the stem cell gel-coated group was the smallest. The thickness of the skin layer of the mice of the stem cell gel smearing group is obviously lower than that of the skin layer of the mice of the medicine smearing group (P is less than 0.05), and the difference has statistical significance.
In conclusion, the drug smearing group and the stem cell gel smearing group can obviously relieve the psoriasis-like skin scaling, erythema coverage and epidermis thickening phenotype caused by IMQ, the effect of the stem cell gel smearing group is better than that of the drug smearing group, the scaling, erythema and epidermis thickening symptoms are lighter, the cells with insufficient keratinization are obviously reduced, and the epidermis layer thickness is obviously reduced. The gel is effective in improving psoriasis scaling, erythema and epidermis thickening symptoms, and has antiinflammatory effect.
While the invention has been described in detail with reference to the preferred embodiments thereof, it will be understood by those skilled in the art that various changes and modifications may be made therein without departing from the spirit of the invention, and it is intended that the invention be limited only by the scope of the appended claims. Comprising the following steps:
1. for the human adipose mesenchymal stem cell freeze-dried powder used in the technical scheme of the invention, if other convenient and easily-obtained stem cells are used, or other concentration technologies or permissions can be used for achieving the purpose of the invention;
2. the carbomer gel in the technical scheme of the invention can be replaced by chitosan gel and the like, and the aim of the invention can also be achieved.
Claims (5)
1. A method of preparing a adipose-derived stem cell-mediated gel, comprising:
(1) Preparing a adipose-derived mesenchymal stem cell supernatant:
resuscitating human adipose-derived mesenchymal stem cells in a carbon dioxide cell incubator containing 5% by volume at 37 ℃ using DMEM medium containing 10% fbs, 100U/mL penicillin and 100U/mL streptomycin; after the cell density reaches more than 90%, changing a serum-free stem cell culture medium, starving, and recovering supernatant; dialyzing to remove salt in the supernatant; recovering human adipose mesenchymal stem cells, and temporarily preserving in a refrigerator at-80 ℃; dissolving cells with supernatant after dialysis and desalination, then performing cell disruption with an ultrasonic cell disruption instrument, centrifuging, filtering and sterilizing; lyophilizing the supernatant of the prepared human adipose-derived mesenchymal stem cells for 24 hours in a lyophilizing machine to obtain a powder preparation;
(2) Preparation of 1, 4-dioxo-2, 3-dibromomethylquinoline reagent:
10mg of 1, 4-dioxo-2, 3-dibromomethylquinoline powder was weighed and dissolved in 200. Mu.L of DMSO to prepare 50mg/mL of 1, 4-dioxo-2, 3-dibromomethylquinoline reagent;
(3) 25mL of adipose-derived mesenchymal stem cell supernatant, 75mL of carbomer gel and 200 mu L of 1, 4-dioxo-2, 3-dibromomethylquinoline reagent are mixed according to a proportion to obtain the adipose-derived mesenchymal stem cell.
2. The method of manufacturing of claim 1, wherein:
the starvation time was 9 hours.
3. The method of manufacturing of claim 1, wherein:
the centrifugation conditions are as follows: centrifuge 10000r for 10min.
4. The method of manufacturing of claim 1, wherein:
the carbomer gel is prepared by the following method:
A. weighing 0.2g carbomer in weighing paper, and placing in an ultra-clean bench for ultraviolet sterilization for 30min;
B. 0.2g carbomer was poured into a centrifuge tube and 55ml DDW,20ml0.1mol/L NaOH were added sequentially;
C. standing overnight for 12 hr.
5. A adipose-derived stem cell-mediated gel prepared by the preparation method according to any one of claims 1 to 4.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103191154A (en) * | 2012-01-06 | 2013-07-10 | 上海交通大学医学院 | Application of mesenchymal stem cells in preparation of psoriasis treatment medicines, and extraction method of mesenchymal stem cells |
| KR20170065447A (en) * | 2015-12-02 | 2017-06-13 | 중앙대학교 산학협력단 | Pharmaceutical compositions for contraception containing Conoidin A as an active ingredient |
| CN107488627A (en) * | 2017-09-11 | 2017-12-19 | 上海亚睿生物科技有限公司 | A kind of biological gel for treating intractable skin injury and its application |
| DE202018003814U1 (en) * | 2018-08-19 | 2018-11-09 | Mohamed Shehata Ali Mohamed | Antipsoriatic gel for skin application II |
| CN111420034A (en) * | 2019-01-08 | 2020-07-17 | 上海莱馥医疗科技有限公司 | Mixed stem cell preparation for treating psoriasis and preparation method thereof |
| CN112704687A (en) * | 2019-10-08 | 2021-04-27 | 上海赛比曼生物科技有限公司 | Preparation containing mesenchymal stem cells and application thereof in psoriasis treatment |
| CN113304103A (en) * | 2021-04-23 | 2021-08-27 | 西北农林科技大学 | Preparation method and application of human mesenchymal stem cell supernatant dry powder gel |
| CN113491713A (en) * | 2021-06-24 | 2021-10-12 | 四川大学华西医院 | Adipose-derived mesenchymal stem cell culture supernatant gel and preparation method thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR102108567B1 (en) * | 2017-06-27 | 2020-05-08 | 이화여자대학교 산학협력단 | Pharmaceutical composition for colorectal cancer with inhibiting the activity of Peroxiredoxin |
-
2023
- 2023-07-14 CN CN202310865153.1A patent/CN116725948B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103191154A (en) * | 2012-01-06 | 2013-07-10 | 上海交通大学医学院 | Application of mesenchymal stem cells in preparation of psoriasis treatment medicines, and extraction method of mesenchymal stem cells |
| KR20170065447A (en) * | 2015-12-02 | 2017-06-13 | 중앙대학교 산학협력단 | Pharmaceutical compositions for contraception containing Conoidin A as an active ingredient |
| CN107488627A (en) * | 2017-09-11 | 2017-12-19 | 上海亚睿生物科技有限公司 | A kind of biological gel for treating intractable skin injury and its application |
| DE202018003814U1 (en) * | 2018-08-19 | 2018-11-09 | Mohamed Shehata Ali Mohamed | Antipsoriatic gel for skin application II |
| CN111420034A (en) * | 2019-01-08 | 2020-07-17 | 上海莱馥医疗科技有限公司 | Mixed stem cell preparation for treating psoriasis and preparation method thereof |
| CN112704687A (en) * | 2019-10-08 | 2021-04-27 | 上海赛比曼生物科技有限公司 | Preparation containing mesenchymal stem cells and application thereof in psoriasis treatment |
| CN113304103A (en) * | 2021-04-23 | 2021-08-27 | 西北农林科技大学 | Preparation method and application of human mesenchymal stem cell supernatant dry powder gel |
| CN113491713A (en) * | 2021-06-24 | 2021-10-12 | 四川大学华西医院 | Adipose-derived mesenchymal stem cell culture supernatant gel and preparation method thereof |
Non-Patent Citations (4)
| Title |
|---|
| Depletion of peroxiredoxin II promotes keratinocyte apoptosis and alleviates psoriatic skin lesions via the PI3K/AKT/GSK3β signaling axis;Ying-Hao Han 等;cell death discovery;20230727;第1-11页 * |
| Mesenchymal Stem Cell Conditioned Media Ameliorate Psoriasis Vulgaris: A Case Study;Rajasekar Seetharaman 等;Case Reports in Dermatological Medicine;20190502;第2019卷;摘要、第2.2项 * |
| Proteomic analysis of psoriatic skin tissue for identification of differentially expressed proteins: Up-regulation of GSTP1, SFN and PRDX2 in psoriatic skin;JOOHYUN RYU 等;INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE;20111231;第785-792页 * |
| 干细胞分泌因子凝胶剂对老化皮肤创伤愈合的影响;冯琳 等;黑龙江八一农垦大学学报;20201031;第32卷(第5期);第64页第1.3.2项、第68页右栏 * |
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