阅读说明：本技术 一种激活毛囊再生的干细胞制剂 (Stem cell preparation for activating hair follicle regeneration ) 是由 张正亮 于 2021-09-30 设计创作，主要内容包括：本发明公开了一种激活毛囊再生的干细胞制剂,涉及生物制剂技术领域,其原料包括：间充质干细胞外泌体、黄连提取物、洋甘菊提取物、丹参提取物、生理盐水。相较于单一的间充质干细胞外泌体的生物制剂,本发明的生物制剂能够有效促进毛囊发育再生,促进细胞增殖,再生出更多的毛发,且制剂中采用的间充质干细胞外泌体,性能稳定,降低免疫排斥。(The invention discloses a stem cell preparation for activating hair follicle regeneration, which relates to the technical field of biological preparations and comprises the following raw materials: mesenchymal stem cell exosome, coptis extract, chamomile extract, salvia miltiorrhiza extract and normal saline. Compared with a single biological preparation of the mesenchymal stem cell exosome, the biological preparation can effectively promote development and regeneration of hair follicles, promote cell proliferation and regenerate more hairs, and the mesenchymal stem cell exosome adopted in the preparation has stable performance and reduces immunological rejection.)

1. A stem cell preparation for activating hair follicle regeneration is characterized in that the raw materials comprise: mesenchymal stem cell exosome, coptis extract, chamomile extract, salvia miltiorrhiza extract and normal saline.

2. The stem cell preparation for activating hair follicle regeneration according to claim 1, wherein in the stem cell preparation, the mesenchymal stem cell exosome is 30-40 μ g/mL, the coptis extract is 5-10 μ g/mL, the chamomile extract is 3-6 μ g/mL, and the salvia miltiorrhiza extract is 2-4 μ g/mL.

3. The preparation of stem cells for activating hair follicle regeneration according to claim 1 or 2, wherein the mesenchymal stem cell exosomes are prepared as follows: washing umbilical cord, removing artery and vein, cutting, adding α -MEM culture medium containing 10% FBS, 100U/mL penicillin and 100U/mL streptomycin, and culturing at 37 deg.C under 5% CO₂Culturing in an incubator, replacing fresh culture solution every 3d, digesting with 0.25% trypsin when cell fusion reaches 80-90%, and subculturing; subculturing to P3 or P4 generation, culturing for 48 hr, and centrifuging to extract exosome.

4. The preparation of stem cells for activating hair follicle regeneration according to claim 3, wherein the specific operation of extracting exosomes by centrifugation is as follows: centrifuging at 4 deg.C at 500g for 5min, and collecting supernatant; centrifuging at 2000g for 10min, and collecting supernatant; centrifuging at 10000g for 20min, and collecting supernatant; and finally, centrifuging for 2 hours at a rotating speed of 100000g, and taking the precipitate to obtain the product.

5. The preparation of stem cells for activating hair follicle regeneration according to claim 1 or 2, wherein the extract of coptidis rhizoma is prepared by an alcohol extraction method, which comprises the following steps: pulverizing Coptidis rhizoma, adding 70-75% ethanol solution, and reflux extracting.

6. The stem cell preparation for activating hair follicle regeneration according to claim 1 or 2, wherein the chamomile extract is prepared by steam distillation, and the specific operation is as follows: pulverizing flos Matricariae Chamomillae, adding deionized water, ultrasonic dispersing, heating to boil, maintaining slightly boiling, separating oil and water, dissolving oil layer in appropriate amount of anhydrous ethanol, and concentrating under reduced pressure to remove ethanol.

7. The preparation of stem cells for activating hair follicle regeneration according to claim 1 or 2, wherein the Salvia miltiorrhiza Bunge extract is prepared by an alcohol extraction method, and the specific operation is as follows: pulverizing Saviae Miltiorrhizae radix, adding 75-80% ethanol solution, and reflux-extracting.

8. The preparation of stem cells for activating hair follicle regeneration according to any one of claims 1 to 7, which is prepared by the following method: suspending the mesenchymal stem cell exosome in physiological saline, adding the coptis extract, the chamomile extract and the salvia extract, mixing and dispersing to obtain the medicine.

Technical Field

The invention relates to the technical field of biological agents, in particular to a stem cell preparation for activating hair follicle regeneration.

Background

With the increasing social pressure and the increasing pace of life, the continuous deterioration of the environment and the eating habits of poor diet, along with the increasing unhealthy and sub-healthy state of people, people generally show that the hair of people has health problems, and more alopecia patients are younger. Alopecia not only affects the quality of life, but also brings great negative effects to the psychology of patients.

In the prior art, the treatment for alopecia mainly comprises drug therapy and hair follicle transplantation, wherein the drug therapy adopts minoxidil, finasteride, dutasteride and other drugs for treatment, the effect is effective, but only hair shedding can be prevented, and certain side effects are caused; the hair follicle transplantation is to collect hair follicles from hair follicle-enriched hair areas of patients through operation and transplant the hair follicles to specified scalp areas, however, the method has limited hair follicles of donors, cannot transplant the hair follicles in large areas, and transplanted hair is generally difficult to grow for a long time and needs to be operated again after a period of time.

In recent years, the application of stem cell technology for hair follicle regeneration has been widely used. However, the single application of stem cell biologics has limited ability to regenerate hair follicles, and further improvement of the ability of cells in hair follicles to resist alopecia has been desired.

Disclosure of Invention

Based on the technical problems in the background art, the invention provides a stem cell preparation for activating hair follicle regeneration, which has stable performance, and can effectively promote the development and regeneration of hair follicles, promote cell proliferation and regenerate more hairs.

The invention provides a stem cell preparation for activating hair follicle regeneration, which comprises the following raw materials: mesenchymal stem cell exosome, coptis extract, chamomile extract, salvia miltiorrhiza extract and normal saline.

Preferably, in the stem cell preparation, the mesenchymal stem cell exosome is 30-40 mug/mL, the coptis chinensis extract is 5-10 mug/mL, the chamomile extract is 3-6 mug/mL, and the salvia miltiorrhiza extract is 2-4 mug/mL.

Preferably, the mesenchymal stem cell exosomes are prepared as follows: washing umbilical cord, removing artery and vein, cutting, adding α -MEM culture medium containing 10% FBS, 100U/mL penicillin and 100U/mL streptomycin, and culturing at 37 deg.C under 5% CO₂Culturing in an incubator, replacing fresh culture solution every 3d, digesting with 0.25% trypsin when cell fusion reaches 80-90%, and subculturing; subculturing to P3 or P4 generation, culturing for 48 hr, and centrifuging to extract exosome.

Preferably, the specific operation of extracting exosomes by centrifugation is as follows: centrifuging at 4 deg.C at 500g for 5min, and collecting supernatant; centrifuging at 2000g for 10min, and collecting supernatant; centrifuging at 10000g for 20min, and collecting supernatant; and finally, centrifuging for 2 hours at a rotating speed of 100000g, and taking the precipitate to obtain the product.

Preferably, the coptis chinensis extract is prepared by an alcohol extraction method, and the specific operation is as follows: pulverizing Coptidis rhizoma, adding 70-75% ethanol solution, and reflux extracting.

Preferably, the chamomile extract is prepared by adopting a steam distillation method, and the specific operation is as follows: pulverizing flos Matricariae Chamomillae, adding deionized water, ultrasonic dispersing, heating to boil, maintaining slightly boiling, separating oil and water, dissolving oil layer in appropriate amount of anhydrous ethanol, and concentrating under reduced pressure to remove ethanol.

Preferably, the salvia miltiorrhiza extract is prepared by an alcohol extraction method, and the specific operations are as follows: pulverizing Saviae Miltiorrhizae radix, adding 75-80% ethanol solution, and reflux-extracting.

Preferably, the preparation method is as follows: suspending the mesenchymal stem cell exosome in physiological saline, adding the coptis extract, the chamomile extract and the salvia extract, mixing and dispersing to obtain the medicine.

Has the advantages that: the invention provides a stem cell preparation for activating hair follicle regeneration, which is obtained by compounding a mesenchymal stem cell exosome, a coptis extract, a chamomile extract and a salvia miltiorrhiza extract. The coptis chinensis extract is matched with the mesenchymal stem cell exosomes, so that BMP signals can be improved, the induction capability of hair follicles can be improved, a Wnt/beta-catenin/channel can be activated, the formation of a substrate required by the development of the hair follicles and the division of basal cells of the hair follicles can be promoted, and more hairs can be regenerated; the salvia miltiorrhiza extract is added, the expression of TGF-beta signals is promoted in a cell proliferation stage, the cell proliferation is effectively promoted, the activation and maintenance of hair follicle stem cells are induced, the hair growth is induced and regulated, and the salvia miltiorrhiza extract and the hair follicle stem cells are matched with each other, so that when the hair follicle enters a growth cycle, the epidermal cell proliferation in the hair follicle is promoted, the development perfection of the hair follicle structure is promoted, the hair follicle resting period can be inhibited, and the hair follicle is promoted to enter the next growth cycle; in addition, the salvia miltiorrhiza bunge has the effects of activating blood circulation, stimulating the local blood circulation of the scalp, improving the metabolism capability of the scalp, well conveying nutrient substances to the scalp, promoting cell regeneration, activating hair follicles and ensuring the normal growth of the hairs; the added substances of the bicyclic sesquiterpenoids in the chamomile extract can obviously improve the bioavailability of the functional components in the coptis chinensis after being compatible with the coptis chinensis. Compared with a single biological preparation of the mesenchymal stem cell exosomes, the biological preparation can effectively promote development and regeneration of hair follicles and regenerate more hairs, and the mesenchymal stem cell exosomes adopted in the preparation have stable performance and reduce immunological rejection.

Detailed Description

The technical solution of the present invention will be described in detail below with reference to specific examples.

Example 1

A stem cell preparation for activating hair follicle regeneration comprises the following raw materials: the exosome of the mesenchymal stem cell is 35 mug/mL, the extract of the coptis is 7 mug/mL, the extract of the chamomile is 4.5 mug/mL, the extract of the salvia miltiorrhiza is 3 mug/mL and normal saline;

wherein the coptis chinensis extract is prepared by an alcohol extraction method, and the specific operation is as follows: crushing coptis, and mixing according to the weight ratio of 1: adding 75% ethanol solution into the mixture with a solid-to-liquid ratio of 10, extracting under reflux twice for 1.5h each time, mixing the filtrates obtained by the two extractions, concentrating under reduced pressure, and drying to obtain the final product;

the chamomile extract is prepared by adopting a steam distillation method, and the specific operation is as follows: crushing chamomile, adding a proper amount of deionized water, performing ultrasonic dispersion for 30min, and continuously adding deionized water to adjust the solid-to-liquid ratio to be 1: 15, heating to boil and keeping slight boiling for 3h, stopping heating, separating oil from water, dissolving the oil layer into a proper amount of absolute ethyl alcohol, and then carrying out reduced pressure concentration to remove the ethyl alcohol to obtain the oil-water separation oil-water-phase;

the salvia miltiorrhiza extract is prepared by an alcohol extraction method, and the specific operations are as follows: pulverizing Saviae Miltiorrhizae radix, and mixing according to the weight ratio of 1: adding 80% ethanol emulsion into the mixture with a solid-to-liquid ratio of 10, extracting under reflux twice, each time for 1.5h, mixing the filtrates obtained by the two extractions, concentrating under reduced pressure, and drying to obtain the final product;

the preparation of mesenchymal stem cell exosomes was as follows: washing umbilical cord, removing artery and vein, cutting, adding α -MEM culture medium containing 10% FBS, 100U/mL penicillin and 100U/mL streptomycin, and culturing at 37 deg.C under 5% CO₂Culturing in an incubator, replacing fresh culture solution every 3d, digesting with 0.25% trypsin when cell fusion reaches 90%, and subculturing; when subculturing to P3 generation, centrifuging and extracting exosome after culturing for 48h, and specifically operating as follows: centrifuging at 4 deg.C at 500g for 5min, and collecting supernatant; centrifuging at 2000g for 10min, and collecting supernatant; centrifuging at 10000g for 20min, and collecting supernatantLiquid; and finally, centrifuging for 2 hours at a rotating speed of 100000g, and taking the precipitate to obtain the product.

The preparation method of the stem cell preparation for activating hair follicle regeneration comprises the steps of firstly suspending the mesenchymal stem cell exosome in physiological saline, then adding the coptis extract, the chamomile extract and the salvia extract, mixing and dispersing to obtain the stem cell preparation.

Example 2

Compared with example 1, the difference is only that: the raw materials comprise: 30 mug/mL of mesenchymal stem cell exosome, 10 mug/mL of coptis extract, 3 mug/mL of chamomile extract, 4 mug/mL of salvia miltiorrhiza extract and normal saline; the preparation and preparation methods of other raw materials are the same.

Example 3

Compared with example 1, the difference is only that: the raw materials comprise: the exosome of the mesenchymal stem cell is 40 mug/mL, the extract of the coptis is 5 mug/mL, the extract of the chamomile is 6 mug/mL, the extract of the salvia miltiorrhiza is 2 mug/mL and normal saline; the preparation and preparation methods of other raw materials are the same.

Comparative example 1

Compared with example 1, the difference is only that: the raw materials do not contain coptis chinensis extract; the composition, the dosage and the preparation method of the other raw materials are the same.

Comparative example 2

Compared with example 1, the difference is only that: the raw materials do not contain chamomile extract; the composition, the dosage and the preparation method of the other raw materials are the same.

Comparative example 3

Compared with example 1, the difference is only that: the raw materials do not contain the salvia miltiorrhiza extract; the composition, the dosage and the preparation method of the other raw materials are the same.

Comparative example 4

Compared with example 1, the difference is only that: the raw materials only comprise: mesenchymal stem cell exosomes and physiological saline; the composition, the dosage and the preparation method of the other raw materials are the same.

Hair follicle regeneration test in mice

1. Establishing a alopecia areata model: uniformly coating 0.05g of imiquimod cream on the back skin of a C3H/HEJ mouse, wherein the area is about 1.5 multiplied by 1.5cm, the imiquimod cream is applied once every other day, the maximum continuous application time is not more than 10 weeks, and the part of the imiquimod cream is cleaned by normal saline before each administration so as to remove the residual medicine in the previous time; the area of alopecia areata with the size of more than 1 multiplied by 1cm can be formed at the administration part after about 5 weeks, which indicates that the molding is successful.

2. And (3) injection of the preparation: mice with successful alopecia areata modeling were randomly divided into 8 groups, wherein 7 groups were injected with the stem cell preparations prepared in examples 1-3 and comparative examples 1-4, respectively, and recorded as treatment groups 1-7, and 1 group was injected with physiological saline and recorded as blank group, and 4 mice per group, and the specific injection operation was as follows: mice were anesthetized with sodium pentobarbital prior to injection, and then injected with the stem cell preparation at alopecia areata with a microsyringe at 1mL per square centimeter of alopecia areata skin and a penetration depth of 2 mm.

3. And (4) observing results: the mice were sacrificed 3 weeks after daily observation after injection, hair follicle formation was observed under a dissecting microscope and the average number and length of hairs were counted.

4. And (3) test results: see table 1.

It was observed that the number of hair follicles was increased in the treatment groups 1-7 compared to the blank group. The number of hairs and the length of hairs were increased in the treatment group 1 compared to the treatment groups 4 to 6, and the treatment groups 5 to 6 were also superior to the treatment group 4, probably because the combination of chamomile extract, salvia miltiorrhiza extract and coptis chinensis extract could improve the bioavailability and further improve the efficacy.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.