CN111358749B

CN111358749B - Composition for promoting skin wound healing and preparation method thereof

Info

Publication number: CN111358749B
Application number: CN202010386308.XA
Authority: CN
Inventors: 刘明录; 王立新; 刘媛媛; 卢永灿; 强邦明; 张传鹏; 王亮; 许淼
Original assignee: Shandong Xinrui Biotechnology Co ltd
Current assignee: Shandong Xinrui Biotechnology Co ltd
Priority date: 2020-05-09
Filing date: 2020-05-09
Publication date: 2023-01-03
Anticipated expiration: 2040-05-09
Also published as: CN111358749A

Abstract

The invention provides a composition for promoting skin wound healing, which comprises the following active ingredients: the preparation method comprises the following steps of mesenchymal stem cell culture supernatant, 2-deoxy-D-ribose, estradiol, vaseline oil, butanediol, citric acid, shea butter and xanthan gum. The composition spray containing the mesenchymal stem cell culture supernatant, the 2-deoxy-D-ribose, the E2 and various active substances, provided by the invention, is used for treating the burned and scalded surfaces of mice, and each composition component has no natural extract, so that no side effect is generated in the whole treatment process, and the safety is high; the wound surface of the mouse can be completely healed only by two weeks, the healing rate reaches 100 percent, and the total effective rate reaches 100 percent; the composition for promoting the healing of the skin wound can reduce inflammatory reaction, accelerate the healing of the skin wound and avoid the phenomenon of scar hyperplasia, and has obvious effect on treating chronic wounds, deep burns, diabetic ulcers and large-area wound surfaces.

Description

Composition for promoting skin wound healing and preparation method thereof

Technical Field

The invention relates to the technical field of medical wound repair, in particular to a composition for promoting skin wound healing and a preparation method thereof.

Background

The skin is the largest organ of the human body and is the first barrier against pathogens. The healing of skin wound is a physiological process of regeneration and repair of skin tissue after being damaged, and although the burn and scald wound surface has a great progress in clinical treatment, the skin wound healing method still has a plurality of problems of insufficient donor skin source, difficult deep wound repair, difficult healing of chronic wound, functional disorder caused by scar hyperplasia and the like.

In recent years, with the development of regenerative medicine, stem cell therapy has shown a wide application prospect in the field of trauma as one of effective methods for biological therapy. Mesenchymal Stem Cells (MSCs), an important member of the stem cell family, have a multipotent differentiation, a strong self-renewal ability, a low immunogenicity, and an immunoregulatory function, and are considered as one of the seed cells that are ideal in the field of regenerative medicine. Multiple studies show that MSCs have strong ability to promote wound healing and function to promote tissue repair and regeneration. MSCs are capable of expressing and paracrine various cytokines such as: fibroblast Growth Factor (FGF), endothelial growth factor (VEGF), epidermal Growth Factor (EGF), interleukin (IL), tumor necrosis factor-alpha (TNF-alpha), and the like. In the process of wound healing, FGF migration can accelerate wound healing, VEGF can promote angiogenesis, IL and TNF-alpha can reduce inflammatory response, and the factors play positive roles in improving microenvironment around the wound and promoting wound healing.

In the existing stem cell technology for treating wounds and scalds, bone mesenchymal stem cells are mostly adopted to promote wound healing, but the number of the bone mesenchymal stem cells is small, operation is required, and extraction is difficult. Adipose-derived stem cells are also used for treating burn and scald wounds, but the extraction of adipose-derived stem cells also requires surgical operations. At present, mesenchymal stem cells are used for treating wounds, a method of local injection of the wounds is mostly adopted, new wounds are brought to large-area wounds and burns, and the capability of the stem cells for repairing the wounds to heal is limited.

When the existing medicine is used for treating wounds, the healing rate is low, the effective rate is low, the treatment time is long, inflammatory reaction is easy to cause, and the phenomenon of scar hyperplasia is caused.

Disclosure of Invention

In order to solve the defects in the prior art, the invention provides a composition for promoting skin wound healing and a preparation method thereof, and the following aims are achieved:

(1) The composition provided by the invention uses natural extracts, does not contain any non-natural additive, and has high safety and no side effect.

(2) The composition provided by the invention can reduce inflammatory reaction, promote wound healing and avoid scar hyperplasia.

(3) The composition provided by the invention can promote the healing of skin wounds, and has the advantages of high healing rate, high effective rate and short healing period.

In order to solve the technical problems, the invention adopts the following technical scheme:

a composition for promoting healing of a skin wound, the composition comprising the following active ingredients: the preparation method comprises the following steps of mesenchymal stem cell culture supernatant, 2-deoxy-D-ribose, estradiol, vaseline oil, butanediol, citric acid, shea butter and xanthan gum.

The following is a further improvement of the above technical solution:

in the composition, the mass percent of active ingredients is 65.4-80.6%, and the mass ratio of the mesenchymal stem cell culture supernatant to the 2-deoxy-D-ribose to the estradiol to the vaseline oil to the butanediol to the citric acid to the shea butter to the xanthan gum is 18-22:18-22: 18-22: 4.5-5.5: 4.5-5.5: 0.8-1.2: 0.8-1.2: 0.8-1.2.

The composition comprises the following components in percentage by mass: 18-22% of mesenchymal stem cell culture supernatant, 18-22% of 2-deoxy-D-ribose, 18-22% of estradiol, 4.5-5.5% of vaseline oil, 4.5-5.5% of butanediol, 0.8-1.2% of citric acid, 0.8-1.2% of shea butter, 0.8-1.2% of xanthan gum and the balance of auxiliary agents to 100%.

The composition comprises the following components in percentage by mass: 20% of mesenchymal stem cell culture supernatant, 20% of 2-deoxy-D-ribose, 20% of estradiol, 5% of vaseline oil, 5% of butanediol, 1% of citric acid, 1% of shea butter, 1% of xanthan gum and the balance of auxiliary agents to 100%.

The mesenchymal stem cell culture supernatant is collected when the human umbilical cord mesenchymal stem cells are cultured for 3-4 generations.

The mesenchymal stem cell culture supernatant is an UltraCULTURE culture medium containing EGF factors, FGF factors, VEGF factors and IL-10 factors, wherein the concentration of the EGF factors is 74-76ng/ml, the concentration of the FGF factors is 90-95ng/ml, the concentration of the VEGF factors is 83-87ng/ml, and the concentration of the IL-10 factors is 88-92ng/ml.

When the composition is a spray, the auxiliary agent is water.

In view of the important role of mesenchymal stem cells in wound healing treatment, the optimal administration mode is found, so that the migration, immunoregulation, angiogenesis and repair capabilities of MSCs are maximized, the healing time of skin wounds is shortened, the healing rate and the effective rate are improved, the inflammatory response is reduced, and the scar hyperplasia phenomenon is avoided.

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

(1) The composition spray containing the mesenchymal stem cell culture supernatant, the 2-deoxy-D-ribose, the E2 and various active substances, provided by the invention, is used for treating the burned and scalded surfaces of mice, and each composition component has no natural extract, so that no side effect is generated in the whole treatment process, and the safety is high; the wound surface of the mouse can be completely healed in 14 days, the healing rate is up to 100 percent, and the total effective rate is up to 100 percent;

(2) The composition for promoting the healing of the skin wound can reduce inflammatory reaction, accelerate the healing of the skin wound and avoid the phenomenon of scar hyperplasia, and has obvious effect on treating chronic wounds, deep burns, diabetic ulcers and large-area wound surfaces.

Drawings

FIG. 1 is a microscopic view of the morphology of P3 generation umbilical cord mesenchymal stem cells;

FIG. 2 is a flow chart of flow cytometry to identify the mesenchymal stem cell surface associated antigen CD 90;

FIG. 3 is a flow chart of flow cytometry to identify the mesenchymal stem cell surface associated antigen CD 73;

figure 4 flow cytometry to identify the mesenchymal stem cell surface associated antigen CD 105;

FIG. 5 is a flow chart of flow cytometry to identify the mesenchymal stem cell surface associated antigen CD 44;

FIG. 6 is a flow chart of flow cytometry to identify the mesenchymal stem cell surface associated antigen CD 34;

FIG. 7 is a flow chart of flow cytometry for identifying HLA-DR as a surface associated antigen of mesenchymal stem cells.

The specific implementation mode is as follows:

in order to make those skilled in the art better understand the technical solutions of the present invention, the technical solutions in the embodiments of the present invention are described in detail below with reference to specific examples.

Example 1 preparation of culture solution of umbilical cord mesenchymal stem cell supernatant

Sterilizing umbilical cord of clinical healthy newborn in term of month twice with 75% alcohol in a clean bench, placing in a culture dish, removing Fahrenheit gum with forceps, and cutting with scissors to 1mm ³ The tissue blocks with the size are pasted on the bottom wall of a 50ml CULTURE bottle at a lattice spacing of 1 cm, an Ultra CULTURE medium containing a serum substitute is added for CULTURE, the CULTURE is carried out in an incubator with 5% CO2 and 37 ℃,1 liquid is changed every 3d, passage and expansion are carried out when the cells reach 70-80% fusion, and the cell morphology is observed under an inverted microscope. When the cell culture supernatant reaches P3, adding a culture medium to continue culturing for 48h, collecting the culture supernatant, centrifuging at 3000g/10min to obtain a supernatant, and filtering with a 220-mesh filter membrane to remove cell debris, wherein the obtained filtrate is the mesenchymal stem cell culture supernatant; 0.25% trypsin digests the cells at the bottom of the flask for P3 passages, adjusting the cell density to 1X 10 ⁶ And (4) putting the mixture into a flow tube, and respectively adding an anti-mouse anti-human monoclonal antibody: CD29, CD44, CD90, CD105, igG1-FITC and HLA ⁃ DR, incubated at 4 ℃ for 30min in the dark. And (3) washing twice with PBS (phosphate buffer solution), performing detection analysis by using a flow cytometer, and identifying the umbilical cord mesenchymal stem cells when the expression rates of CD73, CD44, CD90 and CD105 are more than 98 percent and the expression rates of IgG1-FITC and HLA ⁃ DR are less than 1 percent.

The mesenchymal stem cell culture supernatant contains Vascular Endothelial Growth Factor (VEGF), insulin-like growth factor (IGF-1), epithelial Growth (EGF), fibroblast growth factor-2 (FGF-2), granulocyte macrophage stimulating factor (GM-CSF), basic fibroblast growth factor (bFGF), HGF, interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta).

And (3) measuring the content of main factors of the mesenchymal stem cell culture supernatant by using an ELISA quantitative kit, wherein the concentration of the EGF factor is 75ng/ml, the concentration of the FGF factor is 92ng/ml, the concentration of the VEGF factor is 85ng/ml, and the concentration of the IL-10 factor is 90ng/ml.

Example 2A composition for promoting healing of skin wounds

The composition comprises an active ingredient and an auxiliary agent;

the active ingredients comprise mesenchymal stem cell culture supernatant, 2-deoxy-D-ribose, estradiol, vaseline oil, butanediol, citric acid, shea butter and xanthan gum;

the auxiliary agent is water;

the composition comprises the following components in percentage by mass:

TABLE 1

The mesenchymal stem cell culture supernatant is an UltraCULTURE culture medium containing EGF factors, FGF factors, VEGF factors and IL-10 factors, wherein the concentration of the EGF factors is 75ng/ml, the concentration of the FGF factors is 92ng/ml, the concentration of the VEGF factors is 85ng/ml, and the concentration of the IL-10 factors is 90ng/ml.

Mixing the components uniformly according to the proportion, and filling the mixture into a spray bottle to obtain the composition containing the mesenchymal stem cell supernatant.

Example 3 in vitro wound healing experiments

(1) Preparation of mouse scald model

Selecting a plurality of nude mice with age of 16 weeks and weight of about 200, fasting for 12 hours before operation without water supply. Anaesthetizing the nude mouse, fixing the nude mouse on a foam board, and carrying out conventional disinfection after the back is unhaired; boiling in water, respectively sticking to the skin of the right side of the spinal column of the back of the mouse with a cylindrical hollow tube (diameter of 2 cm), adding 10ml of boiling water, maintaining for 20s to form a 2cm round scald wound surface, and sterilizing with iodophor.

(2) Observation of wound healing in mice

The scalding mice are randomly divided into 3 groups, and each group comprises 20 mice, which are respectively:

blank (saline);

control group (commercially available scald medication);

experimental group (i.e. spray prepared in example 2).

Experimental group animals: spraying the composition spray 1 times every day in the morning, at noon and evening on the scald wound surface, and respectively spraying normal saline and test spray on the wound surface in the same manner for blank group and control group.

The day of cell injection was day 1 (1 d), and the local wound area of mice was measured at different time points (3 d, 7d, and 14 d) after injury for each group, and the wound healing rate was calculated according to the following formula.

Healing rate/% = (original wound area-non-healed wound area)/original wound area × 100%

Effectiveness evaluation criteria:

the healing rate is more than or equal to 75 percent for healing, more than or equal to 50 percent for obvious effect, more than or equal to 25 percent for effective effect, and less than or equal to 25 percent for ineffective effect.

Total effective rate/% = (number of effective cases + number of effective cases)/total number of mice in each group x 100%

As can be seen from tables 2 and 3, the wound healing rate of the mice in the experimental group reaches 100% and the mice are completely healed when the 14 th day is treated; the wound healing rate of the mice is only 60% and 80% in the blank group and the control group. The total effective rate of the experimental group is 100%, the total effective rate of the control group is 80%, the total effective rate of the experimental group is higher than that of the control group, and the difference of the total effective rates of the two groups has statistical significance (P < 0.05). The results show that the treatment effect of the composition of the experimental group on the wound surface of the mouse is obviously superior to that of the scald medicaments purchased on the market.

In the experimental process, 2 mice in the blank group and the control group respectively have severe inflammatory reaction and enlarged ulcer surface when the treatment is carried out for 3-7d, and the healed position has scar hyperplasia. The wound surface of the mouse in the experimental group is the fastest in healing speed, and the wound is basically healed after about 10 days of treatment; on the 14 th day of treatment, the patients were completely recovered, and no scar hyperplasia occurred in 20 mice. Therefore, the composition containing the mesenchymal stem cell supernatant provided by the invention can effectively treat skin wounds and scald wounds, accelerate skin wound healing and shorten the course of disease.

TABLE 2 comparison of wound healing rates in mice

TABLE 3 comparison of Total effective rates of mouse wounds

The 2-deoxy-D-ribose, the estradiol, the vaseline oil, the butanediol, the citric acid, the shea butter, the xanthan gum and the mesenchymal stem cell culture supernatant in the composition have a synergistic effect, and the 2-deoxy-D-ribose, the estradiol, the vaseline oil, the butanediol, the citric acid, the butter resin and the xanthan gum can improve and promote the wound healing capacity of the mesenchymal stem cell culture supernatant and shorten the treatment period.

The composition can also be prepared into paste, powder and the like by compounding the active ingredients described in the example 2 with other conventional auxiliary agents, and different product forms have no influence on the effect of the composition on treating pregnancy and scars.

Claims

1. A medicament for promoting healing of skin wounds, comprising:

the medicine comprises the following components in percentage by mass: 20% of mesenchymal stem cell culture supernatant, 20% of 2-deoxy-D-ribose, 20% of estradiol, 5% of vaseline oil, 5% of butanediol, 1% of citric acid, 1% of shea butter, 1% of xanthan gum and 100% of auxiliary agent;

the mesenchymal stem cell culture supernatant is collected when the human umbilical cord mesenchymal stem cells are cultured for 3-4 generations;

the mesenchymal stem cell culture supernatant is an UltraCULTURE culture medium containing EGF factors, FGF factors, VEGF factors and IL-10 factors, wherein the concentration of the EGF factors is 74-76ng/ml, the concentration of the FGF factors is 90-95ng/ml, the concentration of the VEGF factors is 83-87ng/ml, and the concentration of the IL-10 factors is 88-92ng/ml;

the medicine is a spray, and the auxiliary agent is water.