CN105267243B

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Info

Publication number: CN105267243B
Application number: CN201410365950.4A
Authority: CN
Inventors: 李静
Original assignee: Shenzhen Peiyuan Biotechnology Co Ltd
Current assignee: Guojian Jinghua Shenzhen Precision Health Technology Co ltd
Priority date: 2014-07-23
Filing date: 2014-07-23
Publication date: 2020-03-20
Anticipated expiration: 2034-07-23
Also published as: CN105267243A

Abstract

The invention provides a stem cell extract for eliminating skin striae gravidarum, belonging to the technical field of stem cells. The stem cell extract contains a large amount of bioactive factors beneficial to skin regeneration and repair, and the stem cell extract or a product prepared by taking the stem cell extract as a main component can effectively reduce or even eliminate skin striae gravidarum and has no adverse reactions such as stimulation to skin.

Description

Stem cell extract for eliminating skin striae gravidarum

Technical Field

The invention relates to the technical field of stem cells, in particular to a preparation method and application of a stem cell extract.

Background

Stretch marks are pink or purplish red wavy patterns with different widths and different lengths which appear on skins of abdominal walls, inner and outer sides of thighs, buttocks, back waists, chests and other parts of women during pregnancy; after delivery, the colour of these lines gradually fades away, leaving behind white or silvery-white shiny scar lines. The striae gravidarum is mainly formed due to the influence of hormones in the gestation period, and the abdominal distension causes elastic fibers and collagen fibers of the skin to be damaged or broken, so that the skin becomes thinner and thinner. Striae gravidarum is especially obvious for the primiparous and lying-in women, and once appearing, the striae gravidarum cannot naturally disappear, and often accompanied with skin relaxation, so that the postpartum physical state and physical and mental health of the women are affected.

Currently, stretch marks lack effective removal methods. The method of adopting laser, essential oil and oral medicine is adopted in the market, and is not accepted by most people due to the reasons of insufficient obvious effect or obvious side effect and the like.

The placenta is the place for providing various nutrients for the organ development and growth of the fetus during the development period of the fetus, and is also the original hematopoietic organ of the fetus. The placenta has an active ability to synthesize bioactive substances, supporting the development of fetuses, and includes various hormones (protein hormones and steroid hormones) and enzymes. Recent studies have shown that placental tissue contains a variety of stem cells; in addition, stem cells are also embedded in the jelly of the umbilical cord.

Disclosure of Invention

The invention aims to provide a safe and effective preparation for eliminating striae gravidarum and a preparation method thereof.

The preparation of the present invention contains a substance secreted from the stem cells of the placenta or umbilical cord, which is called a stem cell extract. The inventors have found that the extract, by itself, or in combination with other substances, is capable of significantly reducing or eliminating stretch marks. The extract is produced by normal (human) cells, and has no irritation to skin.

The invention provides a method for preparing a stem cell extract. The stem cells used for preparing the stem cell extract are separated from placenta or umbilical cord tissues of normal people and are cultured and amplified in vitro. The stem cells have the common characteristics that: capable of adherent growth, cell surface expresses CD140a, CD105, CD73 and CD29, and does not express CD 45.

The stem cells used for preparing the stem cell extract are obtained by combining the adherent property of the cells and the surface molecular characteristic separation; or directly sorting according to the molecular characteristics of the cell surface.

The stem cell separation method combining cell adherence characteristics and surface molecular characteristics is that, in the first step, placenta or umbilical cord cells are separated according to the cell adherence characteristics. Placental cell adherent separation by cutting placental tissue into small pieces, washing free cells with cold Phosphate Buffer (PBS), sequentially digesting twice with 0.25% trypsin/HBSS in a 37 ℃ water bath for 10 minutes each time, collecting the twice digested fluids, neutralizing with an equal amount of DMEM containing 10% human serum, washing and inoculating in a cell culture dish, and removing suspended cells by changing the fluid after 24 hours. The culture medium is changed every 3 days later, stem cell colonies can be seen in 7-10 days, and the culture is continued until the cells in the culture dish are 70% saturated, and then the cells are collected by trypsinization.

Separating umbilical cord cells by a wall attaching method: the normal human umbilical cord was repeatedly washed with PBS, the blood vessels and adventitia were stripped, the jelly (Wharton's jelly) was collected, cut into small pieces of about 1mm, plated on cell culture dishes, and cultured with DMEM containing 10% human serum at 37 ℃ and 5% CO₂Culturing in incubator, carefully changing the culture solution after 3-5 days, growing cells from the periphery of the tissue after 5-7 days, digesting with trypsin, and collecting the cells.

DMEM in the above culture solution may be replaced with IMDM or α -MEM.

The second step is to separate the placenta and umbilical cord stem cells according to the molecular characteristics of the cell surface: from the placenta or umbilical cord cells obtained by the above method, CD140a positive cells were isolated. The method comprises the steps of digesting cultured placenta or umbilical cord cells by trypsin to form a single cell suspension, and sorting CD140a positive cells by using immunomagnetic beads (Miltenyibittec) carrying anti-CD 140a antibodies or a flow cytometer.

The placenta stem cell can be directly separated according to the surface molecular characteristics of the placenta stem cell, the method is that single cell suspension obtained by digesting the placenta tissue is firstly removed with CD 45-resistant immunomagnetic beads to remove CD45 positive cells, and then CD140a positive cells are separated by the immunomagnetic beads carrying CD140a antibodies; or directly sorting the CD45 negative/CD 140a positive cell population using flow cytometry. The sorted cells were cultured and passaged with the above serum-free medium.

Serum-free culture expansion of placenta or umbilical cord stem cells: the selected placental or umbilical cord stem cells are cultured and passaged in a serum-free medium, which may be a commercially available medium such as MesenGro (StemRD), UltraCULTURE (Lonza), etc., or a self-prepared serum-free medium containing a serum replacement component and a cytokine (e.g., bFGF).

The placenta and stem cells used for preparing the stem cell extract can also be cultured and amplified by a suspension culture method. Isolating the placental or umbilical cord stem cells obtained by the above method to obtain 10⁴The cells were inoculated in a concentration of one ml to a medium containing a gelatin-coated microcarrier (Cultispher), and subjected to amplification culture at 37 ℃ and 5% CO2 in a stirred flask.

According to an embodiment of the invention, the surface of the placental or umbilical cord stem cells express CD140a, CD105, CD73, and CD29, and do not express CD 45.

According to embodiments of the invention, the placental or umbilical cord stem cells, when appropriately induced, can differentiate into skin keratinocyte cells, adipocytes, and the like.

The invention provides a preparation method of a stem cell extract, which is characterized in that human skin keratinocyte conditioned medium is used for inducing human placenta or umbilical cord stem cells to generate substances beneficial to skin regeneration and repair. Adding a certain amount of human skin keratinocyte conditioned medium into human placenta or umbilical cord stem cell culture medium, inducing and culturing for 1-10 days, and culturingThen mixing with culture solution containing 0-20% serum or phosphate buffer under low oxygen (O)₂<5%) for 0.1-120 hr, collecting culture solution, centrifuging at 4 deg.C to remove cell debris, and storing at low temperature.

According to the embodiment of the present invention, the stem cell extract contains dozens of secreted proteins including growth factors promoting skin regeneration and repair such as IGF-1, bFGF, EGF, PDGF, EPO, etc. The inventor finds that the placenta (or umbilical cord) stem cell secretion (stem cell extract) containing various bioactive factors can effectively reduce or eliminate striae gravidarum by itself or a composition formed by the stem cell secretion and other substances. Because these bioactive molecules are produced by normal cells, their concentrations and ratios to each other are more consistent with the physiological needs of the skin, and contribute to skin regeneration and repair.

According to an embodiment of the present invention, the stem cell extract for eliminating striae gravidarum used in the present invention may further have the following additional technical features:

according to the embodiment of the invention, collagen hydrolysate, hyaluronic acid and albumin or one of them are added into the stem cell extract so as to be beneficial to the stabilization of active ingredients in the stem cell extract or exert a synergistic effect.

Drawings

FIG. 1 shows a microscope image of adherent culture of placental and umbilical cord stem cells used in the present invention;

FIG. 2 shows the expression of surface characteristic molecules of placental stem cells used in an embodiment of the invention;

FIG. 3 shows that the placental stem cells used in the examples of the present invention are induced to differentiate to form adipocytes;

FIG. 4 shows that the placental stem cells used in the examples of the present invention are induced to differentiate to form skin epithelial cells.

Detailed Description

Example 1

The separation and amplification culture of the placenta stem cells are as follows:

(1) normal human placenta is selected, the placenta tissue is cut into small pieces, free cells are washed off sufficiently with cold Phosphate Buffer Solution (PBS), then the placenta tissue is digested twice with 0.25% trypsin/HBSS by shaking in a water bath at 37 ℃ for 10 minutes each time, and the digestive juice of the two times is collected and neutralized with an equal amount of DMEM containing 10% human serum. Suspending the cells by 10% human serum DMEM after centrifugation, planting the cells on a cell culture dish, changing the liquid after 24 hours to remove the suspended cells, changing the liquid every 3 days later, observing stem cell colonies after 7-10 days, and continuously culturing until the cells in the culture dish are 70% saturated, digesting and collecting the cells by trypsin.

(2) CD140a positive cells were sorted by immunomagnetic bead method, the basic procedure was as follows (see in particular the method provided by Miltenyibitec). The cells collected above were first filtered through a 40um microwell cell screen (BDbioscience) and centrifuged, the cells were resuspended in PBS containing 1% human serum, the antibody Biotin-anti-human CD140a (Biolegend) was added, and after incubation on ice for 30 minutes, the cells were washed well with PBS (20 volumes of PBS were added, 300g was centrifuged for 10 minutes, and the supernatant was removed). Resuspend cells with 80. mu.l of 1% serum in PBS (10. ltoreq. per liter)⁷Cell), adding 20 μ l of MACS Anti-Biotin MicroBeads (Miltenyibitec), mixing uniformly, incubating at 6-12 ℃ for 15 minutes, washing with PBS, resuspending the cells with 500 μ l of PBS containing 1% serum, and passing through a cell separation column (Miltenyibitec); such as cells>2x10⁸Other types of cell separation columns are selected). After washing with PBS containing 1% human serum, the adsorbed CD140a cells were collected.

(3) And (3) culturing the placental stem cells by using a serum-free culture medium. CD140a positive cells (placental stem cells) were suspended in MesenGro serum-free medium, plated on Corning CellBIND cell culture dishes at 37 ℃ and 5% CO₂Culturing in incubator, changing culture solution every 2-3 days, and digesting with trypsin for passage when cell density reaches 70-80% saturation. The morphology of the cells cultured in the MesenGro serum-free medium was similar to that of the cells cultured in the serum-containing medium (FIG. 1).

(4) Flow cytometry analysis showed that the obtained placental stem cells expressed characteristic surface markers, such as CD105, CD73, CD 29; CD45 was not expressed (fig. 2).

(5) Adipogenic differentiation experiment: inoculating placenta stem cells cultured for 3 generations in six-hole culture plate at lx10 density⁵Pore, change to adipogenic when cells grow to 80% saturationAdipose-induced culture medium (DMEM containing 10% fetal bovine serum, 100. mu.g/ml IBMX, 50. mu.g/ml insulin, 10%^-6M dexamethasone) was added every 3 days, 1 time, and a drop of lipid was evident in the cells at 2-3 weeks (FIG. 3).

(6) Epithelialization differentiation experiment: human foreskin derived skin keratinocyte cells were seeded on two-chamber culture chamber slides, cultured to 80% saturation with K-SFM (Gibco, Life Technologies), and then irradiated with 10Gy of cobalt 60. After 24 hours, inoculate 10 per chamber⁴The human placental stem cells marked by the green fluorescent protein gene are put on the upper surface of the epithelial cell layer of the skin, and are continuously cultured for 7 days by K-SFM containing 1 percent of fetal calf serum, and the liquid is changed for 1 time every 3 days. Finally, cells were fixed with 1% glutaraldehyde, incubated with an antibody against whole keratin, then with a Cy 3-labeled secondary antibody, and observed by confocal microscopy. In addition to green fluorescent protein, some of the placental stem cells also expressed keratin (red fluorescence) (FIG. 4), indicating that the placental stem cells differentiated into keratinocyte epithelial cells.

Example 2

The separation and amplification culture of the umbilical cord stem cells are as follows:

(1) separating umbilical cord cells by a wall attaching method: washing normal human umbilical cord with PBS repeatedly, stripping blood vessel and adventitia, collecting colloid (Wharton's jelly), shearing into small pieces of about 1mm, suspending in DMEM containing 10% human serum, inoculating onto cell culture dish, and culturing at 37 deg.C and 5% CO in an amount not to make tissue pieces suspend₂Culturing in incubator, carefully changing the culture solution after 3-5 days, growing cells from the periphery of the tissue after 5-7 days, digesting with trypsin, and collecting the cells.

(2) CD140a positive cells were sorted by immunomagnetic bead method in the same manner as in (2) of the isolation and expansion culture of placental stem cells in example 1.

(3) Culturing the umbilical cord stem cells in a serum-free culture medium, which is the same as the step (3) of the isolation and expansion culture of the placental stem cells in example 1.

Example 3

Preparation and application of stem cell extract

(1) Preparation of conditioned medium for keratinocyte: human foreskin-derived keratinocytes were cultured in a full K-SFM medium (Life Technologies) to 70% saturation, replaced with a K-SFM medium without additives and cultured for 24 hours, the culture broth was collected and centrifuged at 4 ℃ and 2095G for 30 minutes, and the supernatant was collected by discarding the bottom sediment and stored at-80 ℃ for further use.

(2) Subjecting the placental stem cells to a medium comprising 10% keratinocyte conditioned medium and 90% MesenGro serum-free medium at 37 deg.C and 5% CO₂Culturing in incubator for 3 days until 80-90% saturation, and performing trypsinization and passage.

(3) The cells passaged above were continued in conditioned medium containing 10% keratinocyte cells and 90% MesenGro serum-free medium at 37 ℃ and 5% CO₂Culturing in incubator for 2 days until 70% saturation, removing original culture medium, adding PBS or DMEM corresponding to half amount of original culture medium, culturing at 37 deg.C under 5% CO2 and 1% O2 for 24 hr, collecting culture medium, centrifuging at 4 deg.C under 2095G for 30 min, removing bottom sediment, collecting supernatant (dry cell extract), and storing at 80 deg.C.

(4) Analysis of secreted proteins contained in the extract: the composition and amount of secreted proteins in stem cell extracts were determined using antibody-based protein chips (antibody-based protein array) in conjunction with ELISA. Antibody-based protein chips (

Human Cytokine Antibody Array G Series) from RayBiotech, Inc., USA, ELISA kit from R&The specific assay procedure was performed as provided by the manufacturer. FIG. 5 shows a partially secreted protein contained in the placental stem cell extract.

(5) 1% (w/v) collagen hydrolysate, 1% (w/v) hyaluronic acid and 1% (w/v) albumin were added to the stem cell extract, and stored at 4 ℃ for further use.

(6) Applying stem cell extract (1) or composition (3) prepared by mixing with other components to striae gravidarum skin once a day for one month; can prolong the service time and increase the use effect. Optionally, prior to application, the exfoliating cream is used to remove aged keratinocytes, which facilitates absorption of the active ingredient. Striae gravidarum was generally significantly reduced after 2-3 weeks of use (fig. 6).

Claims

1. Use of a stem cell extract as the sole active ingredient in the manufacture of a product for eliminating or reducing striae gravidarum in the skin; the stem cells are derived from placenta or umbilical cord tissue;

the preparation method of the stem cell extract comprises the steps of adding a certain amount of keratinocyte conditioned medium into a placenta or umbilical cord stem cell culture medium, carrying out induction culture for 1-10 days, then culturing the cells with a culture solution containing 0-20% of serum or a phosphate buffer solution for 0.1-120 hours under a low oxygen condition, collecting the culture solution, and centrifuging at 4 ℃ to remove cell debris to obtain the stem cell extract; the hypoxia is O₂<5 percent; the preparation method of the keratinocyte conditioned medium comprises the following steps: the method comprises the steps of culturing human skin-derived keratinized epithelial cells for a certain time under the condition of a serum-free culture medium, collecting the culture medium, centrifuging and obtaining a supernatant.

2. Use according to claim 1, characterized in that: the stem cells grow in an adherent manner, or grow under suspension culture conditions after attaching microcarriers.

3. Use according to claim 1 or 2, characterized in that: the stem cells express CD140a, CD105, CD73 and CD29 on the surface and do not express CD 45.

4. Use according to claim 1 or 2, characterized in that: the placenta or umbilical cord tissue is derived from a human or an animal.

5. Use according to claim 1 or 2, characterized in that: the stem cells can be genetically modified to be immortalized or have stable properties for a long time.

6. Use according to claim 1 or 2, characterized in that: conditioned medium produced by the stem cells when stimulated by physical or chemical conditions; the stimulation conditions include: chemical inducers, low nutrition, low pH or hypoxia;

the chemical inducer is a cytokine or a substance released from another cell.

7. Use according to claim 1, characterized in that: the stem cell extract is stored and used in a liquid or a powder after being dried at a low temperature.

8. Use of a composition for the manufacture of a product for eliminating or reducing striae gravidarum in skin;

the composition includes a stem cell extract; the stem cells are the only active ingredient in the composition; the stem cells are derived from placenta or umbilical cord tissue;

the preparation method of the stem cell extract comprises the steps of adding a certain amount of keratinocyte conditioned medium into a placenta or umbilical cord stem cell culture medium, carrying out induction culture for 1-10 days, then culturing the cells with a culture solution containing 0-20% of serum or a phosphate buffer solution for 0.1-120 hours under a low oxygen condition, collecting the culture solution, and centrifuging at 4 ℃ to remove cell debris to obtain the stem cell extract; the hypoxia is O₂<5%；

The preparation method of the keratinocyte conditioned medium comprises the following steps: culturing keratinized epithelial cells derived from human skin under the condition of a serum-free culture medium for a certain time, collecting the culture medium, centrifuging and obtaining a supernatant; the composition also comprises a substance which is beneficial to the stabilization of the active ingredients of the stem cell extract.

9. Use according to claim 8, characterized in that: the stem cell extract and the substance which contains active ingredients beneficial to the stabilization of the stem cell extract are glycerol or albumin.

10. Use of a composition for the manufacture of a product for eliminating or reducing striae gravidarum in skin;

The preparation method of the keratinocyte conditioned medium comprises the following steps: culturing keratinized epithelial cells derived from human skin under the condition of a serum-free culture medium for a certain time, collecting the culture medium, centrifuging and obtaining a supernatant; the composition also includes nutrients or bioactives that are beneficial for skin health and repair.

11. Use according to claim 10, characterized in that: the nutrient or bioactive substance beneficial for skin health and repair is collagen and its hydrolysate, hyaluronic acid, fibroin, semen glycines extract, yeast extract, synthetic peptide, epidermal growth factor, fibroblast growth factor or insulin-like growth factor.

12. Use of a composition for the manufacture of a product for eliminating or reducing striae gravidarum in skin;

the composition comprises stem cell extract and substance for promoting percutaneous absorption of bioactive substances and nutrient elements; the stem cells are the only active ingredient in the composition; the stem cells are derived from placenta or umbilical cord tissue;

The preparation method of the keratinocyte conditioned medium comprises the following steps: the method comprises the steps of culturing human skin-derived keratinized epithelial cells for a certain time under the condition of a serum-free culture medium, collecting the culture medium, centrifuging and obtaining a supernatant.