阅读说明：本技术 一种具有防脱生发作用的干细胞因子复合物的制备方法 (Preparation method of stem cell factor compound with anti-hair loss and hair growth effects ) 是由 王哲 明磊国 王清霞 李阿峰 于 2019-10-08 设计创作，主要内容包括：本发明公开了一种具有防脱生发作用的干细胞因子复合物的制备方法,属于生物制剂领域,具体包括以下步骤：(1)脐带干细胞、脂肪干细胞、成纤维细胞的分离与扩大培养；(2)脂肪干细胞与成纤维细胞混合条件培养基的准备；(3)脐带干细胞的处理；(4)干细胞因子复合物的制备；(5)干细胞因子复合物的功效验证。在步骤(3)中,用步骤(2)所得混合条件培养基与UVB共同处理脐带干细胞。本发明制备的干细胞因子复合物能从本质上解决问题,防脱生发效果显著,适用于雄激素脱发、烫染损伤等多种脱发疾病。(The invention discloses a preparation method of a stem cell factor compound with the functions of preventing hair loss and growing hair, which belongs to the field of biological preparations and specifically comprises the following steps: (1) separating and expanding culture of umbilical cord stem cells, adipose-derived stem cells and fibroblasts; (2) preparing a mixed conditioned medium of adipose-derived stem cells and fibroblasts; (3) processing the umbilical cord stem cells; (4) preparing a stem cell factor complex; (5) and (4) verifying the efficacy of the stem cell factor complex. In the step (3), the umbilical cord stem cells are treated by the mixed conditioned medium obtained in the step (2) and UVB. The stem cell factor compound prepared by the invention can essentially solve the problems, has obvious effects of preventing alopecia and growing hair, and is suitable for various alopecia diseases such as androgen alopecia, scald and infection injury and the like.)

1. A preparation method of a stem cell factor compound with the effects of preventing hair loss and growing hair is characterized by comprising the following steps:

(1) the method specifically comprises the following steps of:

a: primary isolation and expanded culture of human adipose-derived stem cells;

b: primary isolation and expanded culture of human fibroblasts;

c: primary separation and expanded culture of human umbilical cord stem cells;

(2) the preparation of the adipose-derived stem cell and fibroblast mixed conditioned medium specifically comprises the following steps:

d: respectively arranging the adipose-derived stem cells/fibroblasts obtained in the step (1) according to the ratio of 3-7 x 10⁵Inoculating to 75cm per bottle²In a culture bottle;

e: normally culturing for 3-5 days, and collecting the supernatant to obtain two conditioned media;

f: filtering with 0.22 μm filter to remove cell debris and other larger particle size particles;

g: the BCA method is used for determining the protein concentration of the culture medium under two conditions, and mixing is carried out according to the protein content of the culture medium under two conditions to obtain a mixed condition culture medium;

(3) treating the umbilical cord stem cells, namely treating the umbilical cord stem cells obtained in the step (1) by using 2-6 multiplied by 10⁵Inoculating the cells/dish into a 10cm culture dish, normally culturing for 24h, discarding culture solution, washing with PBS twice, and treating by adding 1-2 ml PBS to cover the cells, and using 10-50 mJ/cm²After UVB irradiation for 10 s-2 min, PBS is discarded, 30-70% of mixed condition culture medium collected in the step (2) and 70-30% of fresh culture medium are added, the temperature is continuously increased to 37 ℃, and 5% CO is continuously added₂Normally culturing;

(4) the preparation method of the stem cell factor compound comprises the following specific steps:

after the treatment of the step (3), normally culturing the cells for 3-5 days, and collecting the supernatant; filtering with 0.22 μm filter to remove cell debris and other larger particles; concentrating the collected conditioned medium by using an ultrafiltration concentration technology to obtain a stem cell factor compound;

(5) and (4) performing efficacy verification on the stem cell factor compound obtained in the step (4).

2. The method for preparing the stem cell factor complex with the effects of preventing hair loss and promoting hair growth according to claim 1, wherein the method comprises the following steps: in the step (2), the adipose-derived stem cell conditioned medium and the fibroblast conditioned medium are mixed according to a certain proportion and then are used for culturing the umbilical cord stem cells, and the protein content ratio range of the adipose-derived stem cell conditioned medium to the fibroblast conditioned medium is 1: 9-7: 3.

3. the method for preparing the stem cell factor complex with the effects of preventing hair loss and promoting hair growth according to claim 1, wherein the method comprises the following steps: the used adipose-derived stem cells, umbilical cord stem cells and fibroblasts are selected from one or more of 3-12 generations.

4. The method for preparing the stem cell factor complex with the effects of preventing hair loss and promoting hair growth according to claim 1, wherein the method comprises the following steps: in the step (3), the UVB irradiation intensity is 10-50 mJ/cm²The irradiation time is 10 s-2 min, and the irradiation cell types include but are not limited to umbilical cord stem cells.

5. The method for preparing the stem cell factor complex with the effects of preventing hair loss and promoting hair growth according to claim 1, wherein the method comprises the following steps: in the step (4), the total protein concentration of the obtained stem cell factor compound is 0.5-2.0 mg/mL; the pH value of the stem cell factor compound is measured by a pH meter and ranges from 6.5 to 8.0.

6. The method for preparing the stem cell factor complex with the effects of preventing hair loss and promoting hair growth according to claim 1, wherein the method comprises the following steps: the verification in step (5) refers to the verification of the hair follicle activation efficacy of the stem cell factor complex obtained in (4).

7. The method for preparing the stem cell factor complex with the effects of preventing hair loss and promoting hair growth according to claim 1, wherein the method comprises the following steps: the verification in step (5) employs a packet verification method.

8. The method for preparing the stem cell factor complex with the effects of preventing hair loss and promoting hair growth according to claim 1, wherein the method comprises the following steps: the stem cell factor compound is suitable for various alopecia diseases such as androgen alopecia, scald injury and the like.

Technical Field

The invention relates to the field of biological preparations, in particular to a preparation method of a stem cell factor compound with the functions of preventing alopecia and growing hair.

Background

In recent years, with intensive research in the field of hair regeneration, it has been found that hair growth is comprehensively regulated by hair follicle stem cells and hair follicle peripheral tissues. In the alopecia disease, a large number of hair follicles are stagnated in a resting period and do not enter a growing period, which is closely related to factors such as reduction of the content of cytokines for regulating the growth of the hair follicles, imbalance of proportions and the like.

Stem cells regulate normal physiological metabolism of tissues and organs in vivo by secreting various factors. These factors include Vascular Endothelial Growth Factor (VEGF), insulin-like growth factor 1(IGF-1), Epidermal Growth Factor (EGF), Keratinocyte Growth Factor (KGF), and the like. Common stem cells include adipose-derived stem cells, bone marrow stem cells, umbilical cord stem cells, gingival stem cells and the like. The umbilical cord stem cells have the characteristics of no harm to donors, no ethical problem, low immunogenicity and the like, and the adipose-derived stem cells have the advantages of wide sources, convenient material taking, small tissue damage and the like, and are two types of stem cells which are widely applied at present. Fibroblasts are the main cells of the dermis layer, are differentiated from cells in the embryonic period, have the characteristics of stem cells, and can synthesize and secrete proteins in a large amount. The hair follicle main body is positioned in the dermis layer and is close to the subcutaneous fat layer, and the dermis and the subcutaneous fat tissue are both involved in the growth, development and cycle regulation of the hair follicle main body. The combination of dermal layer cells and adipose derived cells simulates the living environment of hair follicles in vivo, and can promote the synthesis and secretion of cell factor compounds which are closer to the normal proportion in vivo. Whereas low doses of UVB radiation significantly increase the ability of cells to secrete, migrate and survive.

Therefore, the fibroblast and adipose-derived stem cell conditioned medium is mixed and used for culturing the umbilical cord stem cells, so that the in-vivo growth environment of the stem cells in the hair follicle can be well simulated. Low dose UVB stimulation further promotes the secretion of more cytokine complexes by umbilical cord stem cells that promote entry of the hair follicle into the normal cycle. The stem cell factor compound is enriched and used for supplementing and regulating cell factors of alopecia parts, and can fundamentally repair damaged scalp, regulate hair follicle health and promote hair growth.

Disclosure of Invention

The invention aims to provide a preparation method of a stem cell factor compound with the functions of preventing hair loss and promoting hair growth, and the preparation method is used for solving the problems in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme: a preparation method of a stem cell factor compound with the effects of preventing hair loss and growing hair comprises the following steps:

(1) the primary cell isolation and expanded culture method comprises the following specific steps:

a: primary isolation and expanded culture of human adipose-derived stem cells (ADSCs);

b: primary isolation and expanded culture of human fibroblasts (human fibroplast, hFB);

c: primary isolation and expanded culture of human umbilical cord stem cells (UC-MSC);

(2) the preparation of the adipose-derived stem cell and fibroblast mixed conditioned medium specifically comprises the following steps:

d: the adipose-derived stem cells/fibroblasts obtained in the step (1) are respectively processed into 3-7 × 10⁵Inoculating to 75cm per bottle²In a culture bottle;

e: normally culturing for 3-5 days, and collecting the supernatant to obtain two conditioned media;

f: filtering with 0.22 μm filter to remove cell debris and other larger particle size particles;

g: the BCA method is used for determining the protein concentration of the culture medium under two conditions, and mixing is carried out according to the protein content of the culture medium under two conditions to obtain a mixed condition culture medium;

(3) treating the umbilical cord stem cells, namely treating the umbilical cord stem cells obtained in the step (1) by using 2-6 multiplied by 10⁵Inoculating the cells/dish into a 10cm culture dish, normally culturing for 24h, discarding culture solution, washing with PBS twice, and treating by adding 1-2 ml PBS to cover the cells, and using 10-50 mJ/cm²UVB irradiation is carried out for 10 s-2 min, PBS is discarded, and30 to 70 percent of mixed conditioned medium collected in the step (2) and 70 to 30 percent of fresh medium, and the temperature is continuously 37 ℃ and the CO content is 5 percent₂Normally culturing;

(4) the preparation method of the stem cell factor compound comprises the following specific steps:

after the treatment of the step (3), normally culturing the cells for 3-5 days, and collecting the supernatant; filtering with 0.22 μm filter to remove cell debris and other larger particles; concentrating the collected conditioned medium by using an ultrafiltration concentration technology to obtain a stem cell factor compound;

(5) and (4) performing efficacy verification on the stem cell factor compound obtained in the step (4).

Preferably, in the step (2), the adipose-derived stem cell conditioned medium and the fibroblast conditioned medium are mixed according to a certain proportion and then are used for culturing the umbilical cord stem cells, and the protein content ratio of the adipose-derived stem cell conditioned medium to the fibroblast conditioned medium is in a range of 1: 9-7: 3.

preferably, in the step (2), one or more of adipose-derived stem cells, umbilical cord stem cells and fibroblasts are selected from 3-12 generations.

Preferably, in the step (3), the UVB irradiation intensity is 10-50 mJ/cm²The irradiation time is 10 s-2 min, and the irradiation cell types include but are not limited to umbilical cord stem cells.

Preferably, in the step (4), the total protein concentration of the obtained stem cell factor complex is 0.5-2.0 mg/mL; the pH value of the stem cell factor compound is measured by a pH meter and ranges from 6.5 to 8.0.

Preferably, the verification in step (5) refers to the verification of the hair follicle activation efficacy of the stem cell factor complex obtained in (4).

Preferably, the verification in step (5) employs a packet verification method.

Preferably, the stem cell factor compound is suitable for various alopecia diseases such as androgen alopecia, scald injury and the like.

Compared with the prior art, the invention has the beneficial effects that:

1. the stem cell factor compound prepared by the invention can solve the problem of alopecia from the perspective of recovering the normal growth regulation of hair follicles: the mixed condition culture medium of the adipose-derived stem cells and the fibroblasts is adopted to culture the umbilical cord stem cells, so that the survival metabolic process of the stem cells in the hair follicle in the in-vivo environment can be simulated to a certain extent. Under the stimulation of low-dose UVB, the umbilical cord stem cells are promoted to secrete more cells which are closer to the natural proportion, and the cell factor compound circulating in the normal period of hair follicles is benefited, so that the health of the hair follicles is fundamentally adjusted, the hair regeneration is promoted, and the effect is more durable.

2. The stem cell factor compound prepared by the invention has the advantages that the cell material is convenient to obtain: the neonatal foreskin obtained by fat/circumcision obtained by umbilical cord/liposuction obtained by clinical delivery belongs to medical waste, has wide source and is convenient for industrialization.

3. The stem cell factor compound prepared by the invention is rich in Vascular Endothelial Growth Factor (VEGF), insulin-like growth factor (IGF-1), Epidermal Growth Factor (EGF), Keratinocyte Growth Factor (KGF), basic fibroblast growth factor (bFGF) and the like, and has rich varieties and high content.

Drawings

FIG. 1 is an overall flow chart of the present invention;

FIG. 2 shows the contents of various factors in the stem cell factor complex prepared according to the example of the present invention.

FIG. 3 shows the effect of the stem cell factor complex prepared according to the example of the present invention on the genes involved in hair follicle activation.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive step based on the method of the present invention, belong to the scope of protection of the present invention.

The invention provides a technical scheme that: a preparation method of a stem cell factor compound with the effects of preventing hair loss and growing hair comprises the following steps:

(1) the method specifically comprises the following steps of:

a: primary separation and expanded culture of ADSCs;

removing connective tissue from liposuction obtained fat under aseptic condition, and cutting into 1mm³Left and right tissue mass; transferring the cut tissue into a 50mL centrifuge tube, adding equal volume of normal saline for cleaning, 200g, and centrifuging at normal temperature for 5 min; removing liquid part, adding equal volume of 0.1% collagenase solution into fat particles, performing constant temperature shock digestion for 30min, shaking digestion for 100g, and centrifuging at room temperature for 5 min; removing supernatant, collecting cell precipitate, washing with normal saline, resuspending with alpha-MEM, inoculating to 75cm²Placing in a culture flask at 37 deg.C and 5% CO₂Culturing in an incubator, and carrying out normal passage amplification;

b: hFB primary isolation and expanded culture;

taking clinical neonatal foreskin tissue, transferring to an ultra-clean workbench, soaking for 1min in 75% alcohol, and washing for 3 times with PBS containing double antibodies; removing subcutaneous adipose tissue, and cutting into 1mm³The left and right small blocks; using sterile capillary at 25cm²Dropping a layer of fetal calf serum on the bottom surface of the culture bottle, transferring the foreskin tissue small blocks to the bottom of the cell culture bottle by using forceps, and uniformly inoculating 30 tissue small blocks into each culture bottle; 37 ℃ and 5% CO₂Culturing under the condition; after the tissue blocks adhere firmly, supplementing 4ml of alpha-MEM complete culture medium into the culture bottle, continuously culturing, changing the culture solution for 3 days, and performing passage amplification after the cells grow full;

c: primary isolation and expanded culture of UC-MSC;

cutting the umbilical cord into small sections of about 5cm in a superclean bench, and cleaning residual blood on the surface of the umbilical cord by PBS; after separating umbilical artery and umbilical vein, placing umbilical cord in alpha-MEM culture medium containing double antibody, and cutting into 2mm pieces³Left and right tissue mass; transferring the minced tissue into a 50mL centrifuge tube, and centrifuging at 200 g; the upper medium was carefully discarded, a mixed digestive enzyme equivalent to the tissue volume was added, placed in a 37 ℃ shaker, and digested for 1.5h with shaking. The mixed digestive enzyme contains 2g/L type II collagenase, 0.8g/L neutral protease and 0.03g/L hyaluronidase; centrifuging the digested tissue fluid at 300g for 5min, carefully discarding the supernatant, washing with PBS 1 time, discarding the supernatant, resuspending the precipitate in alpha-MEM complete medium,inoculating at 75cm²In a culture flask, 5% CO at 37 deg.C₂Culturing in an incubator, and carrying out passage amplification;

(2) the preparation of the adipose-derived stem cell and fibroblast mixed conditioned medium specifically comprises the following steps:

d: the adipose-derived stem cells/fibroblasts obtained in the step (1) are processed according to the proportion of 3 multiplied by 10⁵Inoculating to 75cm per bottle²In a culture bottle, the used adipose-derived stem cells and fibroblasts are selected from 6 th generation cells;

e: collecting supernatant after normal culture for 3d to obtain two conditioned media;

f: filtering with 0.22 μm filter to remove cell debris and other larger particle size particles;

g: measuring the protein concentration of the two conditioned media by using a BCA method, and mixing the conditioned media according to the protein content in a ratio of 1:1 to obtain a mixed conditioned medium;

(3) treating umbilical cord stem cells, namely treating the 4 th generation umbilical cord stem cells obtained in the step (1) at a speed of 3X 10⁵Inoculating each cell/dish into 10cm culture dish, culturing for 24 hr, discarding culture solution, washing with PBS twice, and treating by covering cells with 2ml PBS and 30mJ/cm²After UVB irradiation for 40s, PBS was discarded, 40% of the mixed conditioned medium collected in (2) and 60% of fresh medium were added, and the temperature was continued at 37 ℃ with 5% CO₂Normally culturing;

(4) the preparation method of the stem cell factor compound comprises the following specific steps:

after the treatment of the step (3), normally culturing the cells for 3d, and collecting supernatant; filtering with 0.22 μm filter to remove cell debris and other larger particles; concentrating the collected conditioned medium by using an ultrafiltration concentration technology to obtain a stem cell factor compound, wherein the total protein concentration of the obtained stem cell factor compound is 0.6 mg/mL; the pH value of the stem cell factor complex is 7.2 measured by a pH meter; the contents of Vascular Endothelial Growth Factor (VEGF), insulin-like growth factor (IGF-1), Epidermal Growth Factor (EGF), Keratinocyte Growth Factor (KGF) and basic fibroblast growth factor (bFGF) in the stem cell factor complex were measured by Elisa experiments, and the results are shown in fig. 2.

(5) And (4) performing efficacy verification on the stem cell factor compound obtained in the step (4). Performing verification of hair follicle activation efficacy on the stem cell factor compound obtained in the step (4) by adopting a grouping verification method, and enabling hair follicle stem cells (P9 generation) to be 0.5 multiplied by 10⁵The cells/well were inoculated in 6-well plates, incubated overnight, the culture medium was discarded, and after one washing with PBS, the cells were treated in groups including a control group, a UVB group, a stem cell factor complex-added group (CM group), and a UVB + CM group. The control group normally cultures hair follicle stem cells, the UVB group is added with UVB irradiation treatment during culture, the CM group is added with stem cell factor complex treatment, and the UVB + CM group is simultaneously treated with UVB and stem cell factor complex treatment. Wherein the UVB irradiation treatment comprises covering the cells with 1ml PBS and applying UVB30mJ/cm²Irradiating for 1min, discarding PBS, and culturing normally. The stem cell factor compound treatment is to culture the hair follicle stem cells by using 40 percent of the stem cell factor compound collected in the step (4) and 60 percent of fresh culture medium;

after grouping treatment, continuing to culture for 3d, extracting RNA of each group of cells by using a Trizol kit, carrying out reverse transcription to obtain cDNA serving as a template, and detecting the expression quantity of relevant genes activated by hair follicle stem cells through a fluorescence quantitative PCR experiment;

the experimental results are as follows:

detecting expression quantities of related genes such as wnt10b, beta-catenin, LEF-1, cyclin D1, GSK3 beta and c-myc activated by the hair follicle stem cells under different treatment conditions by a fluorescent quantitative PCR method; the result shows that the expression level of UVB + CM group wnt10b, beta-catenin, LEF-1 and cyclin D1 is obviously increased, while the expression level of GSK3 beta, c-myc is reduced, as shown in figure 3. The stem cell factor compound prepared by the invention can solve the problem of alopecia from the perspective of recovering the normal growth regulation of hair follicles.

The above-described embodiments are preferred embodiments of the present invention, and the scope of the present invention is defined by the appended claims and equivalents thereof, without being limited to the above-described embodiments, and various modifications and changes may be made without departing from the spirit and scope of the invention, and therefore, it is intended to cover in the appended claims all such changes and modifications that are within the scope of the invention.