CN112156061A

CN112156061A - Genetically modified mesenchymal stem cell extract, extraction method and application in aspects of skin tightening and aging resistance

Info

Publication number: CN112156061A
Application number: CN202010845617.9A
Authority: CN
Inventors: 刘明录; 王立新; 韩庆梅; 万磊; 冯建海; 金海锋; 强邦明; 卢永灿; 刘媛媛
Original assignee: Shandong Xinrui Biotechnology Co ltd
Current assignee: Shandong Xinrui Biotechnology Co ltd
Priority date: 2020-08-20
Filing date: 2020-08-20
Publication date: 2021-01-01

Abstract

The invention discloses a gene modified mesenchymal stem cell extract, an extraction method and application in the aspects of skin tightening and anti-aging, wherein the gene modified mesenchymal stem cell extract at least contains one of SDF-1, VEGF, CXCR4 and klotho, and is verified by adopting a detection method comprising the following steps: extracting total RNA of cells; removing the genomic DNA; carrying out reverse transcription reaction; digesting and cracking the genetically modified mesenchymal stem cells to obtain the extract of the genetically modified mesenchymal stem cells. The genetically modified mesenchymal stem cell extract of the present invention can promote the expression of hyaluronic acid, elastin, collagen or reticulin in the skin.

Description

Genetically modified mesenchymal stem cell extract, extraction method and application in aspects of skin tightening and aging resistance

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a genetically modified mesenchymal stem cell extract, an extraction method and application in the aspects of skin tightening and anti-aging.

Background

In human life, improving the appearance has taken a greater focus than all other daily life related problems add up. Consumer products for beautifying the human body are far more numerous than consumer products for treating human diseases. Improving body tone and appearance is a growing multi-billion dollar industry that includes cosmetics, nutraceuticals, pharmaceuticals, and physical therapy. Consumer attention has focused on the latest ingredients for a combination of anti-aging, anti-wrinkle, body shaping, varicose reduction, acne elimination, and weight loss. Of all these cosmetic and cosmetic concerns, improvement of facial appearance is a concern that is of greatest concern and concern to humans, and especially women. Therefore, selective treatment of the face is of vital importance to consumers.

The visual appearance, physical characteristics and physiological function of skin can change undesirably with age and prolonged exposure to adverse environmental factors. The most significant changes include the development of fine lines and wrinkles, loss of elasticity, increased sagging, loss of firmness, loss of color uniformity, rough surface texture, and mottled pigmentation. Less obvious but measurable changes include general reduction in cell and tissue viability, reduced cell replication rates, reduced skin blood flow, reduced moisture content, accumulation of structural and functional errors, and reduced ability of the skin to self-remodel and repair.

Many of the changes in appearance and function described above are caused by changes in the outer epidermal layer of the skin, while other changes are caused by changes in the lower dermis. Regardless of the stimulus for skin damage, when damage occurs, many natural and complex biochemical mechanisms are initiated in an attempt to repair the damage. When the epidermis is damaged, the basal cells of the epidermis divide at a more frequent rate in response to the damage. This increase in replication rate results in the damaged epidermis being more rapidly replaced by new epidermis and stratum corneum, a process known as "epidermal cell renewal". Common injuries that increase the rate of epidermal cell renewal include abrasion, chemical injury, excessive solarization, allergic or non-allergic contact stimulation.

On a molecular level, these aging changes are associated with biochemical changes in the content and structure of the extracellular matrix in which the major cells of the dermis are located. Collagen is highly cross-linked, inelastic, the amount of elastin is reduced, distribution is incorrect, the amount of hyaluronic acid is reduced, all of which result in a reduction in intercellular water. Due to this change, the number and distribution of metal ions, growth factors, hormones and cytokines are changed, resulting in a decrease or cessation of the metabolic activity of fibroblasts.

Although these cells have a natural mechanism for repairing themselves and the matrix, their ability to repair the injury is weaker and weaker with the increase of age and the injury is aggravated, and the disease condition is worsened. If resting fibroblasts are capable of being metabolically activated and stimulated to divide, they will synthesize new extracellular matrix, and the old damaged matrix will be enzymatically degraded and replaced. This balanced process of synthesis and degradation is known as "dermal remodeling". Activation can be accomplished in a number of different ways, including chemical stimulation with selected hormones, growth factors, cytokines, vitamins, plant extracts and retinoids, or by increasing the nutrient supply to the tissue.

There are two types of factors that cause skin aging, endogenous and exogenous. Intrinsic aging is the process of natural aging of the human body. The decrease of epidermal stem cells with age may lead to a decrease of basal layer cell dysfunction, hyaluronic acid and moisturizing factors. In addition, a reduction in secretion of hormones (such as sebaceous glands) may slow down the metabolism of the skin, thereby causing the skin to lose tension and elasticity. Deterioration of dermal fibroblast function may also result in a slow decrease in collagen and elastin production, leading to degradation of dermal connective tissue, leading to skin laxity and even wrinkling. Dermal connective tissue degeneration can reduce the water-storing function of the skin, resulting in dry skin and water shortage. Finally, atrophy of the epidermis and dermis may reduce the protective capacity of the skin or result in loss of elasticity of the blood vessels, resulting in subcutaneous bleeding.

Extrinsic aging is caused by direct contact of the skin with the environment, where the skin is exposed to external factors such as sunlight, pollution, free radicals, and smoking. Extrinsic aging of the skin includes inhibition of cell renewal and repair, reduction of protective capacity, and accumulation of melanin. The main factor causing skin aging is damage by ultraviolet rays (UV) from the sun, which may accelerate skin aging 5 to 10 times. Long-wave Ultraviolet (UVA) and medium-wave Ultraviolet (UVB) are the most common ultraviolet rays in daily life. Chronic exposure to UVB stimulates dermal fibroblasts to express collagenase to degrade collagen, resulting in a decrease in collagen density, collagen denaturation, and loss of skin elasticity. In addition, melanocytes may begin to deteriorate, resulting in uneven skin color. Therefore, free radicals are excessively generated in the skin, resulting in the formation of skin darkness and black spots, and even various skin cancers.

To date, there are many methods for preventing and repairing skin aging, including extracellular matrix (ECM) supplementation therapy, botulinum injection, and exfoliative physical therapy. ECM supplementation includes coating the skin surface with collagen, elastin, hyaluronic acid or vitamin E. However, the structure of these molecules is too large to be efficiently absorbed by the skin and enter the skin. The botulinum injection can improve skin relaxation, but the effect can only be maintained for 3-6 months, and side effects such as ptosis, facial expression stiffness, muscle weakness at the injection part and the like can be caused. Although exfoliation therapy can remove aged keratin and stimulate skin proliferation, it has many side effects including weakened skin, slow repair, and many postoperative complications.

Cell therapy is another option for treating skin aging, including the use of stem cells to improve damaged skin tissue. For example, adipose-derived stem cells (ADSCs) are pluripotent stem cells isolated from adipose tissue, and the ADSCs have high plasticity and can differentiate into various tissue cells such as nerve cells, vascular endothelial cells, cardiac muscle cells, liver cells, cartilage cells, and muscle cells. Previously, most of the adipose tissue obtained from liposuction procedures was discarded as waste. However, studies have found that adipose tissue is rich in mesenchymal stem cells, with proliferation in vitro and multiple differentiation potential. The cells have the advantages of low invasiveness and small harm to human bodies. In addition, these cells can be cultured in vitro and can be widely used for various types of body tissues. These cells can spontaneously migrate to the injured area and repair the wound.

Currently, skin cell therapy must be performed by injecting cells into a subject. The most common method of injecting a quantity of stem cells is subcutaneous injection. However, this treatment is invasive and carries a risk of causing many wounds that may affect the appearance of the skin. In addition, the use of such conventional cell therapy has many limitations because the safety of living cells should be strictly evaluated before injection, since living cells are used in such therapy.

In view of the limited curative effect and side effect of the ECM supplementation therapy and the botulinum injection, and the potential risk and limitation of cell therapy, the development of the genetically modified mesenchymal stem cell extract, the extraction method and the application in the aspects of skin tightening and anti-aging have urgent research values, and also have good economic benefits and large-scale medical application potential, which is the basis and the motivation for the completion of the invention.

Disclosure of Invention

The present inventors have conducted intensive studies to overcome the above-identified drawbacks of the prior art, and as a result, have completed the present invention after having made a great deal of creative efforts.

Specifically, the technical problems to be solved by the present invention are: provides a gene modified mesenchymal stem cell extract, an extraction method and application in the aspects of skin tightening and anti-aging, and promotes the expression of hyaluronic acid, elastin, collagen or reticulin in the skin.

In a first aspect, there is provided a genetically modified mesenchymal stem cell extract comprising at least one of SDF-1, VEGF, CXCR4, klotho.

In the present invention, as a preferred embodiment, the genetically modified mesenchymal stem cell is a mesenchymal stem cell modified with a secretory Klotho gene.

In the invention, as a preferable technical scheme, the nucleic acid sequence of the secretory Klotho gene is shown as SEQ ID NO. 1.

In the present invention, as a preferred technical scheme, the genetically modified mesenchymal stem cell is obtained by a preparation method comprising the following steps:

obtaining pAV-EF1 alpha-Klotho plasmid by using a plasmid preparation technology;

transfecting 293T cells with good state by using plasmids;

preparing umbilical cord mesenchymal stem cells;

infecting the umbilical cord mesenchymal stem cells by using adenovirus.

In a second aspect, the present invention provides a method for extracting a genetically modified mesenchymal stem cell extract, comprising the steps of:

digesting and cracking the genetically modified mesenchymal stem cells to obtain the extract of the genetically modified mesenchymal stem cells.

In a third aspect, the present invention provides the use of a genetically modified mesenchymal stem cell extract for skin tightening and anti-aging.

In the present invention, as a preferred embodiment, the genetically modified mesenchymal stem cell extract of the present invention may be used in the form of cosmetics or pharmaceuticals.

In the present invention, as a preferable technical solution, in the application, the cosmetic or pharmaceutical includes other additives such as a flavoring agent, perfume, toner or coloring agent for enhancement.

In the invention, as a preferable technical scheme, in the application, a proper amount of preservative, conservative agent and antifungal agent can be added into the cosmetics or the medicines.

In addition, the skin care product, cosmetic or pharmaceutical agent may be supplemented with one or more other active ingredients to further enhance the efficacy of the skin care product, cosmetic or pharmaceutical agent, or to increase the flexibility of the formulation, as long as the other active ingredients do not adversely affect the intended effect of the mesenchymal stem cell extract.

In the invention, as a preferable technical scheme, in application, the cosmetic comprises a facial mask, a neck mask, an emulsion, a spray and an essence.

In the invention, as a preferable technical scheme, in the application, the application form of the genetically modified mesenchymal stem cell extract adopts a cosmetic facial mask.

Due to the adoption of the technical scheme, the invention has the following beneficial effects:

the product provided by the invention can easily penetrate the skin when applied to the surface of the skin, promote the expression of hyaluronic acid, elastin, collagen or reticulin in the skin, stimulate the proliferation of cells in the management area, stimulate the secretion of trophic factors by the cells in the management area, or stimulate the accumulation and proliferation of endogenous hematopoietic progenitor cells in the management area.

The composition of the invention contains components for tightening the face, reducing the 'double chin' and neck thinness, and has the effects of resisting aging and wrinkles. A facial mask and neck mask product can be applied to the face, chin and neck area to provide components specific to the body area for reducing fat, slimming, firming, anti-aging and anti-wrinkle to achieve optimal effects. In addition, the mask is believed to have an adsorptive effect, which removes unwanted oils, contaminants, impurities and dead cells from the stratum corneum. Removing the mask ensures deep cleansing of the skin, especially the stratum corneum. It also provides a hyperhydrated state of the epidermis, thereby improving skin tone and texture.

The invention can enhance the effects of skin whitening, acne removing, senile plaque removing and fat reducing by adding the mesenchymal stem cell extract modified by the gene. It also has effects in promoting the formation of collagen and elastin in skin, resisting oxidation and inflammation.

The genetically modified mesenchymal stem cell extract of the present invention is considered to be an effective method for repairing skin aging, including, but not limited to, promoting wound healing, improving skin quality and skin laxity, reducing skin wrinkles and stripes, improving skin firmness, whitening skin, reducing skin dullness and skin spots, and removing acne.

Various cosmetic delivery means are available in the present invention, such as masks, neck masks, sprays, serums, etc., which are used to provide beneficial ingredients and compositions to the facial area. Cosmetic masks provide an optimal delivery system for delivering beneficial skin ingredients and compositions to the facial skin.

Drawings

FIG. 1 shows the expression levels of SDF-1, VEGF, CXCR4 and klotho in the genetically modified mesenchymal stem cells of the present invention.

Detailed Description

The invention is further illustrated by the following specific examples. The use and purpose of these exemplary embodiments are to illustrate the present invention, not to limit the actual scope of the present invention in any way, and not to limit the scope of the present invention in any way.

Example 1

The secretory Klotho gene of the modified mesenchymal stem cell has a nucleic acid sequence shown as SEQ ID NO. 1. The plasmid pAV-EF1 alpha-Klotho is obtained by using a plasmid preparation technology, and is subjected to accurate sequencing by Shanghai bioengineering GmbH, and then is extracted in large quantity for later use.

Example 2

Preparation of umbilical cord mesenchymal stem cells

Collecting umbilical cord of newborn donated in hospital, sterilizing with 75% alcohol twice in clean bench, placing in culture dish, peeling off Fahrenheit gelatin tissue with forceps, and cutting with scissors to 0.5mm²Small pieces of size. The minced Fahrenheit tissue is transferred to a culture flask and cultured in UltraCULTURE medium containing plasma substitutes, and observed with a microscope every day. When 80% of the bottom of the bottle is covered by the climbed stem cells, passage is carried out, the growth speed of the cells is accelerated after passage, and the cells are transmitted to P4 for substitution for experiments every 2-3 days.

Example 3

The 293T cells are recovered by a conventional technical method, and when the culture state is good, the cells are used for transfection experiments. Mixing pAV-EF1 alpha-Klotho, packaging plasmid and helper plasmid at a ratio of 1:1:1, transfecting 293T cell at 37 deg.C and 5% CO₂Culturing in incubator for 48h, collecting supernatant, centrifuging at 4 deg.C for 10min at 2000g, transferring to new EP tube, filtering with 4.5 μm filter, and storing at-80 deg.C. The virus titer determination was carried out, and the virus titer obtained in the present invention was 3.3X 10⁷TU/mL。

Example 4

Adenovirus infected umbilical cord mesenchymal stem cells

1. Digesting and counting the mesenchymal stem cells with good growth state, and diluting to 1 × 10⁵Perml, add to six well plate, and supplement medium to 3 mL. Put at 37 ℃ with 5% CO₂Culturing in an incubator for 24 h.

2. 2mL of the virus solution was removed from-80 ℃ and polybrene (purchased from Sigma) was added to a final concentration of 8. mu.g/mL.

3. Taking out the six-hole plate from the incubator, removing the culture medium in the six-hole plate, adding the prepared virus solution, and putting the virus solution into the incubator to continue culturing for 24 hours.

4. After 24h, the virus solution in the six-well plate was removed, complete medium was added for culture, and 3ug/mL puromycin was added for selection at 37 ℃ with 5% CO₂Culturing in an incubator, changing the culture medium every two days, and culturing for 5 days to obtain viable cells, namely the stem cells infected by the adenovirus.

Example 5

(I) extraction of Total RNA from cells

1. Respectively taking 1 × 10⁷Normal and Klotho genetically modified stem cells, 300g, centrifuged for 5 min.

2. Discard the supernatant, add 600. mu.L Buffer RLT (10. mu.L. beta. -mercaptoethanol to 1 mL), reverse mix, vortex, and lyse the cells thoroughly.

3. Adding 600 μ L70% anhydrous alcohol, quickly blowing, and mixing.

4. The sample was transferred to a pink RNeasy spin column with a 2mL recovery tube, centrifuged at 8000g for 15s, and the filtrate was discarded.

5. Add 700. mu.L Buffer RW1 to column, centrifuge at 8000g for 15s, and discard the filtrate and collection tube.

6. Transfer column to new collection tube, add 500 u L Buffer RPE, 8000g centrifugation 15s, discard filtrate.

7. An additional 500. mu.L of Buffer RPE, 8000g, was centrifuged for 2 minutes and the tube and filtrate were discarded.

8. Centrifuging 8000g of new collecting tube for 1min, and air drying.

9. RNeasy spin column was transferred to a 1.5mL collection tube, 30-50. mu.l RNase-free water was added to the filter, and the mixture was allowed to stand for 5min, centrifuged at 8000g for 1min, and RNA was eluted.

10. The concentration of RNA was determined by measuring the absorbance of the filtrate at a wavelength of 260nm using a spectrophotometer.

(II) Synthesis of cDNA

Removal of genomic DNA:

in RNA specific region operation, using DEPC water treated EP tube, pipette gun. The reaction mixture was prepared on ice according to the following composition, and the mixture was dispensed into each reaction tube, and finally the RNA sample was added.

5×gDNA Eraser Buffer	2.0μL
		gDNA Eraser	1.0μL
Total RNA	1.0μL
		RNase free water	6.0μL
Total volume	10.0μL

Mixing, at 42 deg.C for 2min, 4 deg.C to completely remove gDNA from RNA.

Reverse transcription reaction:

the reaction solution was prepared on ice. In order to ensure the accuracy of the preparation of the reaction solution, the Master Mix should be prepared in the amount of the reaction number +2, and then 10. mu.L of the Master Mix should be dispensed into each reaction tube. After gentle mixing, reverse transcription reaction is carried out immediately.

Reaction solution of the above step	10.0μL
		PrimeScript RT Enzyme Mix I	1.0μL
RT Primer Mix	1.0μL
		5×PrimeScript Buffer 2	4.0μL
RNase free water	4.0μL
		Total volume	20μL

Mixing, heating at 37 deg.C for 15min, at 85 deg.C for 5sec, and at 4 deg.C, and storing at-20 deg.C for long term storage.

(III) RT-qPCR (reverse transcription-quantitative polymerase chain reaction) for detecting contents of SDF-1, VEGF, CXCR4 and klotho

PCR reaction solution is prepared according to the following components, and the whole process is carried out on ice. The PCR primer is synthesized corresponding gene primer and has clear mark.

SYBR premix Ex TaqⅡ2×	10μL
		PCR Forward Primer(10μM)	0.8μL
PCR Reverse Primer(10μM)	0.8μL
		ROX Reference DyeⅡ50×	0.4μL
RT reaction solution (cDNA solution)	2.0μL
		ddH2O	6μL
Total volume	20μL

And mixing the reaction solution uniformly and putting the mixture into a fluorescent quantitative PCR instrument. The Real Time PCR reaction was run in a two-step process with the following reaction conditions:

after the reaction was completed, the amplification curve and the lysis curve of Real Time PCR were confirmed, and the PCR was quantitatively analyzed, with the results shown in FIG. 1. The expression level of SDF-1, VEGF and CXCR4 in the gene modified mesenchymal stem cell is about 5 times higher than that of the normal stem cell, while the expression level of Klotho gene is about 10 times higher.

Example 6

Digesting the gene modified mesenchymal stem cells by using pancreatin digestive juice, transferring the cells into a 50mL centrifuge tube, centrifuging the cells at 400g for 5min at room temperature, and discarding the supernatant. Adding 1% NP40 lysate, 1% PMSF, 1% beta-glycerophosphate, and 1% Na₃VO₄Resuspend the cells. Shaking the shaker at 4 deg.C for 20min to fully lyse the cells, and centrifuging at 12000rpm for 10 min. Collecting supernatant, namely the extract of the genetically modified mesenchymal stem cells.

Example 7

The skin care products, cosmetics and pharmaceuticals of the present invention may be presented in any suitable form without particular limitation. For example, skin care, cosmetics and pharmaceuticals may be applied topically in the form of, but not limited to, creams, sprays, masks or neck masks. The skin care product, cosmetic or pharmaceutical product manufactured by the genetically modified mesenchymal stem cell extract of the present invention may further include other additives such as a flavoring agent, perfume, toner or coloring agent for enhancement. Appropriate amount of antiseptic, conservant, antifungal agent, etc. can also be added to improve storage property of skin care product, cosmetic or medicament. In addition, the skin care product, cosmetic or pharmaceutical agent may be supplemented with one or more other active ingredients to further enhance the efficacy of the skin care product, cosmetic or pharmaceutical agent, or to increase the flexibility of the formulation, as long as the other active ingredients do not adversely affect the intended effect of the mesenchymal stem cell extract.

Example 8

The skin care product, the cosmetic or the medicine prepared by the mesenchymal stem cell extract can be used at different administration frequencies according to the requirements of a subject. Such as once a day, several times a day, or once in a few days. For example, when used for application to the skin surface to repair aged skin, the dosage of the skin care product, cosmetic or pharmaceutical may range from 0.01 to 1ml/cm²Preferably 0.05-0.5ml/cm². However, for the subject with more serious skin aging, the dosage can be increased by several times or dozens of times according to the actual requirement.

Selecting subjects to use the genetically modified mesenchymal stem cell extract and the normal mesenchymal stem cell extract, using the spray in the morning and at night respectively for each group of subjects, and using the mask in the evening to measure the moisture content of the stratum corneum of the skin, the elasticity of the skin, the depth of wrinkles, pores and pigmentation within 4 weeks. The measurement method is as follows: the skin was measured on the left and right cheek areas using the a-ONE SMART facial diagnostic system from korean england. The measurement results are as follows: after the subjects used the product for 4 weeks, the skin showed different degrees of pore reduction, increased skin moisture content, reduced wrinkles, lighter color spots and skin elasticity. Moreover, the effect of using the genetically modified mesenchymal stem cells is more obvious than that of using the mesenchymal stem cells.

It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should also be understood that various alterations, modifications and/or variations can be made to the present invention by those skilled in the art after reading the technical content of the present invention, and all such equivalents fall within the protective scope defined by the claims of the present application.

Sequence listing

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Claims

1. A genetically modified mesenchymal stem cell extract characterized by: the extract contains at least one of SDF-1, VEGF, CXCR4, klotho.

2. The genetically modified mesenchymal stem cell extract of claim 1, wherein: the genetically modified mesenchymal stem cell is a mesenchymal stem cell modified by utilizing a secretory Klotho gene.

3. The genetically modified mesenchymal stem cell extract of claim 2, wherein: the nucleic acid sequence of the secretory Klotho gene is shown in SEQ ID NO. 1.

4. The genetically modified mesenchymal stem cell extract of claim 3, wherein: the gene modified mesenchymal stem cell is obtained by adopting a preparation method comprising the following steps:

transfecting 293T cells with good state by using plasmids;

preparing umbilical cord mesenchymal stem cells;

infecting the umbilical cord mesenchymal stem cells by using adenovirus.

5. The extraction method of the gene modified mesenchymal stem cell extract is characterized by comprising the following steps: digesting and cracking the genetically modified mesenchymal stem cells to obtain the extract of the genetically modified mesenchymal stem cells.

6. Use of a genetically modified mesenchymal stem cell extract for skin tightening and anti-aging.

7. The use of claim 6, wherein: the use form of the genetically modified mesenchymal stem cell extract comprises cosmetics or medicines.

8. The use of claim 7, wherein: the cosmetic or pharmaceutical comprises other additives.

9. The use of claim 7, wherein: the cosmetic or medicine is added with proper amount of antiseptic, conservant, and antifungal agent.

10. Use according to claim 8 or 9, characterized in that: the cosmetic comprises facial mask, neck mask, emulsion, spray and essence.