TWI516596B - A composition for promoting cell regeneration and a preparation method thereof - Google Patents

Description

Composition for promoting cell regeneration and preparation method thereof

The present invention relates to a composition for promoting cell regeneration and a preparation method thereof. In particular, the present invention relates to a composition comprising human growth factors and promoting cell regeneration by encapsulation using a liposome entrapment technique and a preparation method thereof.

The skin not only has the functions of regulating body temperature and moisture, but also the first line of defense of the human immune system. Once the skin is lost, it is vulnerable to foreign pathogen infection. Patients who have experienced burns, ulcers, inflammation, radiation therapy, surgery, and diabetes are prone to infections and cause great harm to the body when they are exposed to skin defects and abnormalities for a long time. The internal tissues and organs of the human body are caused by aging, diseases caused by apoptosis, functional failure, etc., especially brain damage caused by degenerative diseases of the brain or acute injury, which affects human health. Therefore, tissue wound healing and regeneration are repaired. The protective mechanism is an important area of medical research.

At present, most of the drugs used for treating wounds and promoting healing are fungicides, antibiotics, corticosteroids, etc., which have their own shortcomings for wound healing, and the wound healing rate is poor. Thus, methods such as artificial skin or other skin substitutes, cell therapy, and the like have been developed. Related studies have pointed out that a variety of growth factors play an important role in the wound healing process of the skin. After skin injury, platelets release a variety of growth factors and cytokines, including epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and platelet-derived growth. Factor (platelet-derived growth factor, PDGF), human insulin-like growth factor (IGF-1), vascular endothelial growth factor (VEGF), transforming growth factor-α (TGF-α), transforming growth Factoring β (transformation growth factor-β, TGF-β), interleukin (IL), connective tissue growth factor (CTGF), activin, tumor necrosis factor α (tumour necrosis factor) , TNF-α) and the like. These growth factors attract neutrophils and macrophage to the wound to clear injured tissues, cells and phagocytose infections, activate nearby fibroblasts and keratinocytes, and induce peripheral blood vessels. Perform an angiogenesis effect.

On the other hand, fibroblasts at the wound site are transformed into myofibroblasts under the influence of certain growth factors, particularly PDGF and TGF-β. These myofibroblasts express a large amount of alpha-actin to contract the wound and synthesize collagen fibers. The arrangement of these collagen fibers deposited on the wound is affected by the contraction of the myofibroblasts and is different from the collagen fibers of the original normal tissue, thereby forming scar tissue. From the fourth week after the injury, to about six months, the collagen in the scar tissue will be rearranged, making the shape and function of the scar tissue close to the original normal tissue. Recently, studies have indicated that TGF-β regulates wound healing not only for macrophages and fibroblasts, but also stimulates the transcription of mRNA of type I and type II collagen to promote wound healing. The accumulation of collagen to improve the healing rate of the wound. Therefore, TGF-β has been shown to produce innocent epithelial regeneration and accelerate healing of skin wounds formed by radiation exposure. Studies have shown that a large amount of TGF-β can be secreted during the embryonic period of mammals. However, when the epidermis of adult mammals is injured, the amount of TGF-β secreted is very small. It is a wound after birth and is easy to have muscle fibers. Cell contraction scar.

Since growth factors play an important role in the regeneration and repair mechanism of wounds, various factors such as G-CSF, EGF, bFGF, IGF-1, PDGF-BB and VEGF growth factors have been widely used in the pharmaceutical and beauty care industries. For G-CSF, it has long been used for medical treatment of bone marrow transplantation for many years. At present, it is also clinically injected into patients with embolic stroke, which can help the recovery of blood stasis in the brain. This patient can recover part of the muscle strength if he receives general rehabilitation treatment for half a year to one year, but may not be able to stand up. However, after injecting G-CSF for 5 consecutive days in 3 days of stroke, he can stand within 3 months. Even someone can run and jump, and recover quite quickly. Because G-CSF pushes stem cells in the bone marrow to the peripheral blood vessels, there will be protein expression in the stroke, attracting stem cells to the affected area to repair and differentiate into cells such as cranial nerves and blood vessels, thus helping the stroke patients to recover quickly. In addition, G-CSF protects the nerves, prevents the brain from necrosis due to stroke ischemia, and has anti-inflammatory effects, which can inhibit the inflamed tissues and cells in the stroke. However, G-CSF injection therapy is only effective for embolic stroke, and hemorrhagic stroke is still mainly for clot removal surgery. The current scientific report confirms that the combination of SCF and G-CSF is used in Alzheimer's disease and embolic-type stroke animal experiments, and the effect is much better than G-CSF alone. Therefore, a variety of growth factor compounds will have market value as medical applications for different purposes.

However, when genetic engineering is currently used to prepare a large amount of the above growth factors, genes expressing the above human growth factors are individually inserted into a vector to multiply and further purify the human growth factor expressed thereby. Although this method can reduce the cost, the disadvantage is that the purified protein has different purity and may contain endotoxin or other undesirable proteins, which may affect the therapeutic effect or the concurrent allergic reaction; in addition, one plastid is inserted at the same time or more. Growth factor protein genes, all of which are active, are very difficult, if you want to operate multiple selected plastids at the same time It takes time and labor; in addition, when prokaryotic or fungi are used to express human growth factor proteins, the activity of most of the produced proteins is reduced.

On the other hand, stem cell therapy utilizes human adult stem cells, such as stem cells located in the bone marrow, epidermis, and intestines, to treat various diseases. Human mesenchymal stem cell (hMSC) isolated from human bone marrow is an adult stem cell with various differentiation potentials, which can be differentiated into bone, cartilage, muscle, skin and other tissues in different culture environments. The characteristics of separation, culture and rapid proliferation in vitro have been confirmed by many studies to strengthen the mechanism of regenerative healing of tissue cells. However, transplantation into the human body by stem cell injection or surgery requires long-term clinical trials to assess whether allogeneic stem cell transplantation produces immune rejection. Furthermore, the medical resources and money spent on stem cell therapy are also higher than general therapy.

Therefore, there is a need to propose a method that combines the advantages of genetic engineering and stem cell therapy and improves its disadvantages. That is, there is a need for a method for simultaneously providing three or more growth factor proteins expressed by human cells, and at the same time, the growth factor can be administered by a general external or oral route to promote regeneration of tissue cells.

In view of the above problems, the present invention provides a composition containing human growth factor and embedded in a microlipid embedding technique to promote cell regeneration and a preparation method thereof.

Accordingly, one aspect of the present invention provides a composition for preparing a human growth factor comprising a human stem cell factor (SCF), a granulocyte colony-stimulating factor (G-CSF), and a sputum extract. The concentration of the human stem cell growth factor, white blood cell growth factor and alfalfa extract is 5 to 100 ng/ml.

In another aspect, the invention provides a method for preparing a human growth factor. First, the primary culture of human bone marrow mesenchymal stem cells was carried out in the primary culture medium. Next, the suspension cells in the primary culture solution were removed and the adherent cells were selected and subcultured with the subculture medium. Thereafter, the adherent cells are selected and cultured in the last culture medium prepared by the above composition, wherein the last culture solution is a serum-free DMEM (Dulbecco's Modified Eagle Medium) culture solution, and the human stem cell growth by microbial genetic engineering is added. Factor (SCF), white blood cell growth factor (G-CSF) and sputum extract make human bone marrow mesenchymal stem cells can express a variety of human growth factor components in the last day of culture and secretion in the fourth day, and finally water-in-oil Water-in-Oil-in-Water (W/O/W) was embedded in a liposome-embedded technique.

The specially formulated final culture solution enables the adherent bone marrow stem cells to secrete a variety of human growth factors into the final culture secretion to form a solution containing various human growth factors. That is, the above various human growth factor solutions contain at least a composition of a plurality of human growth factors secreted by human bone marrow stem cells, that is, the "human growth factor composition" referred to in the present invention. The above composition comprises at least three growth factors selected from the group consisting of SCF, EGF, VEGF-D, VEGF, bFGF, neutrophil activating peptide 78 (ENA-78), growth regulating oncogene (Growth-regulated) Oncogene, GRO), interferon gamma (interferon-γ, IFN-γ), IGF-1, angiogenin (Angiogenin), monocyte chemotactic protein 1 (MCP-1), PDGF-BB, placental growth factor (placenta growth factor, PIGF), TGF-β1, tissue inhibitor of metalloproteinase (TIMP-1; TIMP-2), leptin, thrombopoietin (Tpo), chemotaxis 21 cytokines such as factor (RANTES), IL-6, IL-8.

The method for preparing human growth factors according to the embodiment of the present invention can be easily obtained. A human growth factor composition in which the type, activity, and safety of human growth factors are higher than those produced by genetic engineering. Moreover, the growth factor composition obtained by the method according to the embodiment of the present invention contains at least a plurality of growth factors related to cell regeneration, which can effectively enhance the ability of cell regeneration.

In addition, the specially formulated final culture solution according to the embodiment of the present invention can not only provide additional nutrients to the human bone marrow mesenchymal stem cells during the preparation of the human growth factor composition, but also contribute to the promotion of the embodiment of the present invention. The ability of the human growth factor composition contained in the composition of cell regeneration to promote cell regeneration.

In still another aspect, the present invention provides a composition for promoting cell regeneration, the composition comprising at least the human growth factor composition of the present invention, a nutrient component, an emulsifier solution, an oil phase liquid, and an increase in stability and gastric acid tolerance. colloid. The above nutrient component comprises a portion of the nutrient solution used in the preparation of the human growth factor composition according to an embodiment of the present invention. The above emulsifier solution and the oil phase liquid can embed the composite composition made by human bone marrow stem cells into a stable water-in-oil-in-water (W/O/W) manner. Highly acidic micro-lipid inclusions. It not only uses the epidermal coating method, but also utilizes the characteristics of easy osmotic absorption of the phospholipid layer membrane to transport the anti-wrinkle activating factor to the bottom layer of the skin, and stabilizes the stem cell environment from the epidermal layer of the skin to achieve the effect of protecting stem cells from planting, effectively resisting wrinkles and avoiding aging, and promoting Cell regeneration; oral administration, through the digestive tract to the blood circulation, still has the biological activity of repairing epidermal trauma.

According to a specific embodiment of the present invention, the liposome-embedded body embedded in the composition for promoting cell regeneration can be applied to an artificial wound of an experimental animal. Apply to artificial wounds, it can be observed that the composition can promote wound healing, reduce the chance of wound inflammation and infection, and reduce the occurrence of scars. In addition, the composition is applied to the skin of the subject, and wrinkles are observed to be significantly reduced. Less, the number of acne is reduced, and the production of acne scars is reduced, so that the composition can be applied to a pharmaceutical composition or an auxiliary that promotes wound healing and slows wound inflammation and infection.

A specific embodiment of the present invention is a gray hair animal application, which uses the gray hair animal model of the present invention to simulate the white hair symptoms caused by the death of hair follicle melanocytes caused by aging. By applying the composition for promoting cell regeneration of the present invention, the white hair symptom of the experimental animal is obviously improved, indicating that the composition can be applied to activate the hair follicle melanocytes, and improve the pharmaceutical composition of white hair caused by aging or Auxiliary.

One embodiment of the invention is the use of a bald animal, using the bald animal model of the present invention to mimic a baldness pattern in humans. By applying the composition for promoting cell regeneration of the present invention, it is apparent that the alopecia symptoms of the experimental animals are significantly improved, and on the scalp of the subject, a significant increase in the hair volume of the subject, the density of the hair follicles can be clearly observed. The phenomenon of improvement, darkening of the hair color, etc., indicates that the composition of the present invention can be applied to a pharmaceutical composition or an auxiliary which stimulates hair follicle growth and activates hair follicle melanocytes.

One embodiment of the present invention is to delay the application of neurodegeneration in the brain. Parkinson is one of the most common neurodegenerative diseases in the elderly. Therefore, the present invention takes the Parkinson animal test as an example, and the neurotoxic substance 1-methyl- 4-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes symptoms of Parkinson's disease in mice. The mice were then orally administered with the composition for promoting cell regeneration of the present invention. The results show that after oral administration of the composition of the present invention, the Parkinson's disease model mice can significantly slow down the degradation of dopaminergic neurons in the substantia nigra of the brain, increase the secretion of dopamine in the brain, and promote the proliferation of hippocampal neurons in the brain. The composition of the present invention can be applied to a pharmaceutical composition or an adjuvant that delays degenerative brain diseases of the brain.

A specific embodiment of the present invention is for promoting the application of blood hematopoietic stem cells, and the oral administration of the composition for promoting cell regeneration of the present invention can significantly increase the number of hematopoietic stem cells in the blood, indicating that the composition of the present invention can be used for promotion. A pharmaceutical composition or aid for blood hematopoietic stem cells.

The above and other aspects of the present invention will be apparent from the following description of the preferred embodiments and the appended claims. Spirit and scope.

For the purpose of illustrating the invention, the presently preferred embodiments are shown in the drawings. However, it should be understood that the invention is not limited to the preferred embodiment of the drawings.

Figure 1 shows a microarray of human growth factor antibodies, wherein (A) is a human growth factor species listed in the human growth factor antibody microarray wafer; (B) is in the presence of stem cell growth factor (SCF) and white blood cell growth factor (G- Human growth factor secreted from the last culture medium of CSF); (C) human growth factor secreted in the final culture medium containing stem cell growth factor (SCF), white blood cell growth factor (G-CSF) and alfalfa extract .

Figure 2 shows the concentration of the growth factor of the present invention obtained by using a standard in an ELISA test, wherein (A) shows the concentration of stem cell growth factor and (B) shows the concentration of vascular endothelial growth factor.

Figure 3 is a schematic view showing the structure of the liposome prepared by the present invention, wherein (A) shows the structure of the liposome under the optical microscope; (B) shows the structure of the liposome under the fluorescence microscope; and (C) shows the liposome A schematic diagram of the correlation with the oil phase and the aqueous phase liquid during preparation.

Figure 4 shows the liposome growth of oral-embedded white blood cell growth factor (G-SCF) Add the effect of the number of white blood cells in the blood.

Fig. 5 shows a case where the wound healing of the mouse skin is carried out, wherein when the composition for promoting cell regeneration provided by the present invention is used, (A) is the wound image on the 0th day, and (B) is the wound image on the 3rd day; In the case of the composition for promoting cell regeneration provided by the present invention, (C) is the wound image on the 0th day, and (D) is the wound image on the 3rd day.

Figure 6 shows a comparison of wound healing rates in mice.

Figure 7 shows the activation of mouse hair follicle melanocytes, wherein (A), (C), (E) are the positive control group on days 9, 14, and 21, respectively, and (B), (D), ( F) The test group was on the 9th, 14th and 21st days respectively.

Figure 8 shows the case of mouse hair follicle cell proliferation, wherein (A), (C), (E), (G) are the positive control group on the 0th, 7th, 14th, 21st day, respectively, and (B), (D), (F), and (H) are the cases of the test group on days 0, 7, 14, and 21, respectively.

Figure 9 shows the case where the skin wound is healed and the fine lines are lightened, wherein (A), (C), (E), (G) are skin conditions before application of the composition for promoting cell regeneration provided by the present invention, and (B) ), (D), (F), (H) are the skin conditions after 30 days of application.

Figure 10 shows the growth of hair follicle cells in the human head and the activation of melanocytes, wherein (A), (C), (E) are the hair conditions before application of the composition for promoting cell regeneration provided by the present invention, and (B) (D), (F) are the condition of the hair after application.

Figure 11 shows the degeneration of dopaminergic neurons in the substantia nigra region of mice, wherein (A) is a normal mouse; (B) is a mouse administered with MPTP; and (C) is administered with MPTP and administered to the present invention. A mouse treated with a composition that promotes cell regeneration.

Figure 12 shows the situation of neonatal neuronal proliferation in the hippocampus of mice, (A) (Normal) mice; (B) mice administered with MPTP injection; and (C) mice treated with MPTP injection and administered with a composition for promoting cell regeneration provided by the present invention.

Figure 13 shows a comparison of brain dopamine concentrations in chronic Parkinson's mice.

Figure 14 shows a comparison of brain dopamine concentrations in acute Parkinson's mice.

Figure 15 shows the use of flow cytometry to analyze the composition of the cells for promoting cell regeneration provided by the present invention, which affects the proliferation of hematopoietic stem cells in human blood, wherein (A), (C), (E) before administration, and (B), (D), and (F) are the cases after taking.

In the following specific examples, the invention will be more specifically described. It is to be understood that the specific embodiments of the invention described herein are intended to be

Embodiment (1)

Method for preparing human growth factors

The method comprises the steps of: (a) performing primary culture of human bone marrow mesenchymal stem cells in a primary culture solution; (b) removing suspended cells in the primary culture medium and selecting adherent cells, and performing the subsequent culture medium. Subculture; and (c) selection of adherent cells in the subculture medium, and final culture in the final culture medium. The above-mentioned last culture medium is a serum-free medium and contains human growth factors and/or sputum extracts, so that the adherent cells can secrete various growth factors into the final culture solution to form a human growth factor solution.

In the above steps, the healthy human bone marrow mesenchymal stem cells (purchased from The National Disease Research Interchange, NDRI, USA) Primary culture was carried out for 3 days in DMEM medium (Dulbecco's modified eagle medium, available from Gibico, Cat. No. 12100-061) containing 10% fetal bovine serum. Then, the suspension cells in the primary culture solution were removed and the adherent cells were selected, and the DMEM culture solution containing 10% fetal bovine serum was used as the subculture medium, and subculture was carried out for 3 days. After subculture, the last culture medium was cultured for 3 days. The last culture medium was a serum-free medium containing about 5 to 100 ng/ml of SCF and G-CSF, and/or sputum extract of 5 to 100 ng/ Ml. The cockroach extract is prepared by placing the oysters after the shovel in boiling water for a suitable period of time, and then rinsing the mash into a lyophilized powder for extraction. The lyophilized powder is dissolved in about 10 volumes of 100% ethanol solution and stirred for extraction, and then the supernatant is removed by centrifugation, filtered, and concentrated under reduced pressure to obtain a dried product. Finally, the dried product is dissolved in 70% ethanol. .

In the final culture step, the adherent cells in the final culture solution can secrete a plurality of human growth factors into the final culture solution to form a human growth factor composition. The human growth factor composition comprises at least three of the following growth factors: SCF, EGF, VEGF-D, VEGF, bFGF, ENA-78, GRO, IFN-γ, IGF-1, Angiogenin, MCP-1, PDGF-BB, PIGF , TGF-β1, TIMP-1, TIMP-2, leptin, Tpo, RANTES, IL-6 and IL-8.

Subculture steps can be repeated up to 20 times depending on the requirements. The cultured human bone marrow mesenchymal stem cells can be cryopreserved after the subculture step, and the cryopreserved human bone marrow mesenchymal stem cells can be subcultured after thawing, but the human bone marrow mesenchymal stem cells after thawing The number of subcultures can only be repeated up to 3 times.

In a specific embodiment of the present invention, a nutrient solution may be added to the final culture solution, the nutrient solution comprising at least one amino acid and/or at least one vitamin. Amino acid May be alanine, arginine, aspartic acid, aspartic acid, cysteine, cystine, glutamic acid, glycine, histidine, isoleucine, leucine, Amino acid, methionine, phenylalanine, valine, serine, threonine, tryptophan, tyrosine or valine, and each selected amino acid has a concentration of about 5 ~100mg/L. The above vitamins may be vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B12, vitamin C, vitamin D2, vitamin E, vitamin H, vitamin K3, choline chloride, folic acid, or muscle. Alcohol, and each selected vitamin has a concentration of about 0.1 to 10 mg/L.

In one embodiment of the invention, the human growth factor composition prepared using the embodiments of the invention is dialyzed to further purify the human growth factor composition. According to one particular embodiment of the present invention, use of a 3.5KD dialysis membrane (purchased from ^{Pierce, SnakeSkin TM Pleated Dialysis Tubing,} 3,500 MWCO) dialysis composition according to the above-described human growth factors, sustainable dialysis for about 3 to 5 days . By the dialysis step, substances having a molecular weight of less than 3,500 Dalton (D) in the human growth factor solution can be substantially removed, thereby achieving the purpose of purifying the human growth factor solution.

Since the molecular weight of the human growth factor contained in the human growth factor composition to be prepared according to the embodiment of the present invention is much larger than 3.5 KD, for example, the molecular weight of EGF is about 6 KD and the molecular weight of TGF-β1 is as high as 250 KD, according to the present invention. Most of the human growth factors produced by the methods of the examples remain in the dialysis membrane. Finally, the solution remaining in the dialysis membrane is collected, which contains at least three human growth factors. Such a human growth factor composition prepared by the method of the present invention is characterized by comprising at least three detectable growth factors (detection methods will be described in detail below), and the growth factors may be SCF, EGF, VEGF- D, VEGF, bFGF, ENA-78, GRO, IFN-γ, IGF-1, Angiogenin, MCP-1, PDGF-BB, PIGF, TGF-β1, TIMP-1, TIMP-2, leptin, Tpo, RANTES, IL-6 and IL-8.

Example (2)

Qualitative analysis of human growth factor components

Human cytokine antibody to the microarray chip (RayBio ^TM Human Cytokine Antibody Arrays) detects the dialysate composition of growth factors, human growth factor antibody microarray detection results shown in FIG. 1 of FIG. It can be found from Fig. 1(A) that the human growth factor composition and the limit of detection (LoD) contained in the human growth factor composition prepared according to the embodiment of the present invention are as follows: EGF (detection limit 1 pg/ml) , bFGF (detection limit 10000pg/ml), IGF-1 (detection limit 10pg/ml), PDGF (detection limit 1000pg/ml), VEGF (detection limit 100pg/ml), TGF-β1 (detection limit 100pg/ml), Angiogenin (detection limit 10pg/ml), IFN-γ (detection limit 100pg/ml), RANTES (detection limit 2000pg/ml), Thrombopoietin (detection limit 100pg/ml), TIMP (detection) The limit was 1 pg/ml), GRO (detection limit 100 pg/ml), IL-6 (detection limit 1 pg/ml), and IL-8 (detection limit 1 pg/ml).

In the present specification, the term "detection limit" refers to the lowest detection concentration of the detection method used in the qualitative analysis of human growth factors by the method of the embodiment of the present invention. In other words, the concentration of a particular human growth factor contained in the sample to be tested must be at least equal to or greater than the detection limit used by the detection method before it can be detected. In this case, the specific human can also be Growth factors are called "detectable" human growth factors. An additional increase in various growth factors was observed in the final culture medium with additional sputum extract (Fig. 1(B), Fig. 1(C) and Table 1).

In addition, each using SCF RayBio ^TM ELISA Kit kit and VEGF RayBio ^TM ELISA Kit set, and the quantitative analysis of SCF two kinds of growth factors VEGF, and the results shown in FIG 2 (A) and 2 (B). The SCF and VEGF standards attached to each set were used as regression lines to determine the concentration of SCF in the human growth factor composition prepared in the examples of the present invention was about 138 pg/ml, and the concentration of VEGF was about 25130 pg/ Ml.

Compared to the stem cell culture method used in the prior art, the culture solution contains no detectable TGF-β1 (detection limit 100 pg/ml) and SCF (detection limit 10 pg/ml), that is, using the prior art stem cell culture method. The content of TGF-β1 in the culture solution is less than 100 pg/ml, and the SCF content is less than 10 pg/ml. In addition, the concentration of VEGF in the culture solution is only about 200-500 pg/ml using the prior art stem cell culture method. However, the human growth factor composition of the embodiment of the present invention contains detectable TGF-β1, that is, the content of TGF-β1 is 100 pg/ml or more, and the concentration of SCF (138 pg/ml) and VEGF are contained. The concentration (25130 pg/ml) is much higher than previous techniques.

Example (3)

Composition for promoting cell regeneration

In another embodiment of the present invention, a composition for promoting cell regeneration comprising at least a human growth factor composition, a nutrient component, an emulsifier solution, and an oil phase according to an embodiment of the present invention is provided. liquid. The above nutrients are included in accordance with the present invention A portion of the nutrient solution used in the preparation of human growth factors, in a preferred embodiment, the nutrient component comprises at least one or more amino acids and/or vitamins. The above emulsifier solution and oil phase liquid can embed the human growth factor composition and nutrient components into a liposome-embedded material by means of water-in-oil-in-water (W/O/W).

The emulsifier solution comprises from about 0.5% to about 1% Tween 80, and from about 1% to about 10% sorbitan sesquioleate (Arlacel 83). In a particular embodiment of the invention, the emulsifier solution may further comprise from about 80% to about 90% pure water, from about 1-10% hyaluronic acid, and/or from about 1-10% glucose.

The oil phase liquid comprises from about 1% to about 10% lecithin, from about 1% to about 10% cholesterol, and from about 80% to about 90% phospholipids. In an embodiment of the invention, the above phospholipids, such as castor oil and/or mineral oil, can be replaced by other oil and fat components.

According to an embodiment of the present invention, a water-soluble colloid can be added to the composition for promoting epidermal cell regeneration to increase the stability of the liposome embedding. The water-soluble colloid comprises about 1-10% Tween 20, about 1-10% propylene glycol (PG), about 1-10% polyethylene glycol-600 (polyethylene glycol, PEG-600), and about 1-5% of a colloidal substance, wherein the above colloidal substance may be Sanxian gum, a transparent gum, or a combination thereof. In a particular embodiment of the invention, the water soluble colloid may further comprise from about 1% to about 10% hyaluronic acid.

According to one embodiment of the invention, about 2-5 grams of Tween 80, about 2-5 grams of Arlacel 83, about 3-5 grams of hyaluronic acid, and about 2-5 grams of glucose are added to about 10 ml of purified water at about 4 °C. It was dissolved to prepare an emulsifier solution having a total volume of about 20 ml. On the other hand, about 5-10 grams of lecithin, and about 2-5 grams of cholesterol are added to about 20-30 ml of castor oil and about 10-20 ml of mineral oil to prepare an oil phase liquid having a total volume of about 40 ml. In addition, in about 60-80ml of pure In water, about 10-20 grams of Tween 20, about 10-20 grams of PG, about 2-5 grams of PEG-600, about 5-10 grams of Sanxian gum, and about 10-20 grams of hyaluronic acid are added to prepare a total volume. 120 ml of water-soluble colloid.

About 20 ml of the emulsifier solution and an equal volume (i.e., about 20 ml) of the human growth factor composition dialyzed against the 3.5 KD dialyzed membrane were taken at about 4 °C. Next, about 40 ml of the oil phase liquid was slowly added, and the mixture was uniformly stirred at about 4 ° C with a high pressure homogenizer of 8,000 to 10,000 rpm, and the stirring was continued for about 10 to 20 minutes. To form a liposome nanoparticle (W/O/W) structure. Finally, about 120 ml of the water-soluble colloid was added, and the mixture was uniformly stirred at about 4 ° C with a homogenizer of 2,500-3,000 rpm, and the stirring was continued for about 20 minutes. The resulting chylomicron composition (Fig. 3 (C)) was the present invention. A composition for promoting cell regeneration in the examples. The structure of the liposome nanoparticle (W/O/W) was observed under the light microscope (Fig. 3(A)), and when the composition contained green fluorescent protein, it was observed under a fluorescent microscope. It was found that the fluorescent protein was indeed embedded inside the liposome nanoparticle and exhibited green fluorescence (Fig. 3(B)).

In the present embodiment, the purpose of adding the water-soluble colloid is to increase the stability of the liposome nanoparticle, and the water-soluble protein in the liposome nanoparticle can be kept active at room temperature for 2 to 3 years. In addition, in the oral test of mice, G-CSF was used as a test embedding material, which promoted the proliferation of white blood cells in the blood. Compared with the injection control group, the G-CSF can be administered orally through the gastrointestinal tract. The cell, the effect of the injection (Figure 4), shows that the embedding method can effectively protect the growth factor and avoid decomposition by the gastric protein enzyme.

Example (4)

Mouse skin wound healing / epidermal cell regeneration test

In a specific embodiment of the invention, the effect of the composition of the invention to promote cell regeneration on wound healing rate is tested in a mouse skin wound healing test. The test was divided into two groups, a group of experimental groups (treated with microlipids embedded with growth factors) and a group of control groups (treated with microlipids embedded with physiological saline), each using 6 eight BALB/C mice of Zhoulingda, each mouse was cut into a 0.5×0.5 cm ² wound on the back of the mouse after shaving with a sterilized scalpel, and then the mouse skin wound was observed under a microscope and taken. The wound image on day 0 was recorded and the wound area on day 0 was calculated in software.

One hour after the wound was caused, about 100 μl of the liposome was evenly spread on the wound of each group of test mice. Thereafter, about 100 μl of the liposome was applied in the same manner every 24 hours, and applied a total of three times. Twenty-four hours after the third application, that is, on the third day after the wound was produced, the skin wound of the mouse was observed under a microscope, and the wound image of the third day was taken and recorded, and the wound area on the third day was calculated by software.

The microscope used in this embodiment is connected to a charge coupled device (CCD) to capture the image of the mouse skin wound, and after capturing the image, the image is transmitted to a computer for image processing and wound area calculation and statistics. The software used for image processing and wound area calculation is the Northern Eclipse image system; the data is analyzed by the software SigmaStat program (2002), which is a one-way analysis of Variance (ANOVA) of Duncan's The calculation is performed by Duncan's New Multiple Range Test (DMRT).

The effect of mouse skin wounds is shown in Figure 5. Fig. 5(A) is the wound image of the 0th day of the test group mice; Fig. 5(B) is the wound image of the 3rd day of the mice of the test group; Fig. 5(C) is the wound of the 0th day of the control group mice. Image; and Figure 5 (D) is the wound image of the control mice on day 3. It can be found from the experimental results that the composition (test group) for promoting cell regeneration by the embodiment of the present invention is The skin wound area of the mice after three days was significantly smaller than that of the control group.

The skin wound area on day 0 and day 3 of each group of mice was calculated by image processing software to convert the wound healing rate as follows: wound healing rate (%) = (day 0 skin wound area - 3rd The skin wound area of the day) / The area of the skin wound on the 0th day × 100%.

The average wound healing rate of each group of mice is shown in Fig. 6. The wound healing rate of the test group was 77.9±7.8%, and the wound healing rate of the control group was 58.7±4.4%. From Fig. 6, it can be found that the wound healing rate of the test group was significantly different (p < 0.05) from the control group.

Example (5)

Mouse gray hair test

In a specific embodiment of the invention, the effect of the present invention is improved in a gray-haired animal model using F1 progeny of C3H and BALB/C hybrid mice, after removal of the F1 progeny, After 7 days of continuous application with 1-20% hydroquinone and 1-20% of glycic acid, the regenerated hair of the treated area appears white, due to the melanocytes when the hair follicles are aged. It is easy to lack tyrosinase activity, tyrosinase is an important key enzyme that mainly forms melanin, and the whitening gum inhibits the activity of tyrosinase. In the gray-haired model animal established by the present invention, one of the parental BALB/C mice has a white color due to the homotypic tyrosinase-deficient gene (Tyr ^- /Tyr ^- ), and the other parental C3H mouse has the same type of cheese. The gene (Tyr ⁺ /Tyr ⁺ ) appears brown, so its F1 hybrid progeny has a heterotypic tyrosinase gene (Tyr ⁺ /Tyr ^- ), which is extremely susceptible to lack of cheese after treatment of whitening drugs. Lysin produces white hair, which is similar to white hair produced by human aging; therefore, if the growth factor-containing liposome can make the hair color of the gray animal model darker, the liposome should also be It has the same effect on human gray hair. In this test, a total of two groups were included, including: whitening gel and gray hair model animals treated with phosphate buffered saline (PBS) as a positive control group; and using whitening gum and containing Growth factors of the liposome-treated gray hair model animals were used as test groups. Each group had 6 mice and the entire experiment was repeated 3 times. Before topical application of the drug on day 0, the back hair of the test animal (about 2 x 2 cm ² size) was depilated with tweezers, and on the second day, the hair removal site was treated with 50 μl of liposome, once a day for 3 weeks, in the afternoon. It was treated with whitening gel and treated once a day for one week. During the experiment, the liposome was treated more than whitening gel for 2 weeks.

The effect of the gray-haired test in mice is shown in Fig. 7. On the 9th day, compared with the control group, the mice in the test group were observed to have melanin deposition on the back skin (Fig. 7(B)); At 14 days, the back hair of the mice began to appear, while the mice in the test group showed grayish black on the back (Fig. 7(D)), while the mice in the control group showed grayish color (Fig. 7). (C)), and on the 21st day, it was apparent that the mice in the test group had dark black hair (Fig. 7(F)), while the control group still showed grayish white (Fig. 7(E)). .

This experiment demonstrates that the composition of the present invention does activate hair follicle melanocytes and improves white hair symptoms due to aging.

Example (6)

Mouse alopecia test

In a specific embodiment of the invention, the effect of the invention is improved in a bald animal model using the F1 progeny of C57BL/6J and CBA/J hybrid mice, the F1 progeny After treatment with testosterone, it causes alopecia mode, C57BL/6J spontaneously The mutated mouse has a bald-like appearance with the same type of jb/jb gene. The mutant mouse is used as the female parent, and the CBA/J mouse is used as the male parent. The progeny obtained by the hybridization are called B6CBAF1/j hybrid mice; the hybrid has been confirmed. Rats can cause alopecia by injection of testosterone (1 mg/mice/day) for 3 weeks. Since the crossed rat is more sensitive to ketone than the crossed mouse, the male mouse is used as the male baldness of the present invention. (Androgenetic Alopecia, AGA) model animal.

In this trial, a total of two groups were included, including a male alopecia model using a testosterone and a liposome-embedded PBS as a positive control group, and a male alopecia model animal treated with a growth factor-containing liposome after using a testosterone. Take the test group. Each group had 6 mice and the entire experiment was repeated 3 times. Before topical application of the drug on day 0, the back hair of the test animals (about 2 x 2 cm ² size) was depilated with tweezers, and the second day of the afternoon was treated with testosterone, once a day for 3 weeks, at the same time, at 50 μl in the morning. The microlipids were used to treat the hair removal site and were treated once a day for 3 weeks.

The effect of the male male alopecia test is shown in Fig. 8. From the positive control group, it can be observed that the back hair of the experimental mice is still stopped growing on the 21st day, and the mice in the test group can be found until the 14th day. (Fig. 8(F)), the skin of the back of the mouse began to have melanin production, and by day 21 (Fig. 8 (H)), compared with the control group (Fig. 8 (G)), the hair of the mouse It has completely recovered to the state before depilation. This experiment demonstrates that the composition of the present invention does stimulate hair follicle cell proliferation and improve alopecia.

Example (7)

Human skin test

The data of the test subjects are shown in Table 2. The test sites of each test subject were photographed before the test, as shown in Figures 9(A), 9(C), 9(E), and 9(G).

Table 2 Information on human skin test subjects

The growth factor-containing microlipids prepared in Example 3 were applied to the test sites of each tester, applied twice a day, and 2 ml was applied once, and after 30 days of continuous application, the test sites of each test subject were photographed, such as 9(B), 9(D), 9(F), and 9(H) are shown. As can be seen from Figs. 9(A) and 9(B), after 30 days from the continuous use of the composition for promoting cell regeneration provided by the present invention, facial wrinkles can be diluted. Further, as can also be seen from Figs. 9(D), 9(F) and 9(H), the composition for promoting cell regeneration provided by the present invention can also dilute the scar of the skin of the face (e.g., acne).

Example (8)

Human alopecia test

The data of the test subjects are shown in Table 3. The test sites of each test subject were photographed before the test, as shown in Figures 10(A), 10(C) and 10(E).

The growth factor-containing microlipids prepared in Example 3 were applied to the test sites of each tester, applied twice a day, 2 ml once, and continuously applied for 4 to 6 months, and photographed and recorded for each test subject. The test sites are shown in Figures 10(B), 10(D) and 10(F). As can be seen from Fig. 10(B) and Fig. 10(D), the use of the composition for promoting cell regeneration provided by the present invention can improve hair gray hair and promote hair follicle growth and hair density after 4 to 6 months. increase. In addition, As can also be seen from Fig. 10(F), the composition for promoting cell regeneration provided by the present invention can also activate the growth of hair follicle cells and increase the amount of the forehead portion of the user.

Example (9)

Delaying brain degeneration test in mice

In a specific embodiment of the invention, the composition for promoting cell regeneration of the embodiments of the invention is tested in a mouse Parkinson animal model to delay the effects of neuronal cell degeneration in the brain. Eight to 12 weeks old C57BL/6 male rats were divided into three groups. The experimental group was given 10 days of continuous growth of lipid-containing lipids, twice a day, each dose was 0.2 ml, the aforementioned liposome At the 6th day of administration, MPTP injection was started at a dose of 18 mg/kg and administered continuously for 5 days by subcutaneous injection; the liposome of the MPTP group was embedded with PBS; and the control group was treated with PBS. Liposomes and subcutaneous injection of PBS. On the 10th day after MPTP treatment, the mice were sacrificed, the brain was sliced, the dopamine neurons were labeled with antibodies to analyze the number of dopamine neurons in the substantia nigra region, and the new cells were labeled with antibodies to analyze the microlipids for newborns. The influence of cells. As can be seen from Figure 11, after treatment with MPTP, the number of dopaminergic neurons in the substantia nigra of mice was reduced (Fig. 11(B)), while mice fed with micronucleus containing growth factors were evident. The number of dopamine neurons in the substantia nigra region was reduced (Fig. 11(C)). As is apparent from Fig. 12, the number of newborn cells in the hippocampus of the mice fed with the growth factor-containing liposome significantly increased (Fig. 12(C)). In addition, after separating the striatum of the mouse brain, the dopamine content in the brain was analyzed by a high pressure liquid chromatography, and it can be seen from Fig. 13 that the mice in the test group had dopamine in the brain as compared with the MPTP group. There is a tendency for the content to increase significantly.

In the acute test, 8 to 12 weeks old C57BL/6 male rats were divided into five groups, one of which was changed to PBS without the growth factor-containing microlipids in the PBS group. The group received 6 days of growth factor-containing liposome, which was fed twice a day at a dose of 0.2 ml. On the second day, 4 times of MPTP (18 mg/kg ip) was administered continuously for 2 hours. Then, on the 7th, 14th, 21st and 28th day after the injection of MPTP, the mice were sacrificed to measure the dopamine concentration in the brain of the brain, and it can be seen from Fig. 14 that the dopamine in the brain increased with time. In the trend, it is presumed that the composition for promoting cell regeneration of the present invention has an effect of improving Parkinson's disease.

It can be explained from the above examples that mice treated with MPTP may cause degeneration of nerve cells in the brain, thereby causing a decrease in brain dopamine secretion, resulting in Parkinson's symptoms in mice, and administration of the promoting cells of the present invention. After the reconstituted composition, it can delay the degeneration of nerve cells in the brain, stimulate the number of new cells in the hippocampus of the brain, and restore the secretion of dopamine in the brain.

Example (10)

Increase human hematopoietic stem cell test

The human blood hematopoietic stem cell test is carried out by using the composition for promoting cell regeneration of the present invention, and the test subject uses oral growth factor-containing liposome, 20 ml each time, once a day, and blood is taken before the test and after the test. . After separating the white blood cells from the blood of the subject, the hematopoietic stem cells were labeled with the antibody, and then the proportion of hematopoietic stem cells in the blood was analyzed by flow cytometry. As a result, as shown in Fig. 15, after taking the liposome containing growth factor for 1 month, the proportion of hematopoietic stem cells in the blood of the two subjects was 0.11% and 0.19%, respectively (Fig. 15(A) and (C)). , increased to 0.28% and 0.34% (Figure 15 (B) and (D)), and after taking lipids containing growth factors for 12 months, the proportion of hematopoietic stem cells in the blood of the subjects was clearly observed from 0.22%. The increase to 4.59% indicates that the composition for promoting cell regeneration of the present invention can stimulate the proportion of hematopoietic stem cells in the blood by oral administration, thereby achieving the function of promoting cell and tissue regeneration.

It will be apparent from the above-described preferred embodiments of the present invention that the present invention has the following advantages. First, the human bone marrow mesenchymal stem cells can produce a human growth factor composition by the method for preparing a human growth factor according to an embodiment of the present invention, and the human growth factor composition comprises at least a plurality of growth factors associated with wound healing/cell regeneration. In addition, since these growth factors are expressed by human mesenchymal stem cells, the components are the same as the human body's own growth factors and do not contain prokaryotic endotoxin. On the other hand, the activity and species of these growth factors are also superior to those of human adult cells and/or genetically modified xenogenic cells.

Furthermore, according to a specific embodiment of the present invention, a nutrient solution containing one or more amino acids and/or vitamins may be added to the last culture solution to provide additional nutrients to the human bone marrow during the culture process. Leaf stem cells. Further, when the composition for promoting cell regeneration is prepared according to the present invention, it is not necessary to remove the nutrient solution additionally added in the last culture, and thus the nutrient solution contained in the composition for promoting cell regeneration can additionally provide the epidermis, Regeneration of dermal tissue, and nutrients required for collagen fiber reforming, and the human growth factor composition of the present invention produces a multiplying effect to further enhance wound healing and/or collagen fiber reforming ability of epidermal and dermal tissues .

Finally, the above-mentioned composition for promoting cell regeneration can be formed not only by a liposome entrapment method to form a growth factor liposome which can be used internally or externally, but also can be added to an artificial skin or a skin substitute. Promotes epidermal cell regeneration, or as an active or auxiliary ingredient in dermatological or cosmeceutical products.

While the present invention has been described above by way of a preferred embodiment, it is not intended to limit the invention, and the present invention may be modified and modified without departing from the spirit and scope of the invention. The scope of protection is defined by the scope of the patent application Prevail.

Claims (9)

A method for preparing a human growth factor, the method comprising: performing a primary culture in which a human bone marrow mesenchymal stem cell is cultured in a primary culture solution; and removing one of the adhesive cells in the primary culture solution and substituting The culture medium is subjected to a subculture; and the adhesive cells in the subculture medium are taken out, and a final culture is performed in a final culture medium, so that the adhesive cells in the final culture medium secrete various growth factors to The last culture solution is used to form a human growth factor solution, wherein the final culture solution is a serum-free medium and contains an ethanol extract of hydrazine. The method of claim 1, wherein the primary culture fluid and the secondary culture fluid are DMEM culture fluid containing one of fetal fetal bovine serum. The method of claim 1, wherein the step of the primary culture is carried out for 3 days. The method of claim 1, wherein the subculture step is performed for 3 days. The method of claim 1, further comprising the step of repeating the subculture, wherein the number of repetitions is at most 20 times. The method of claim 1, wherein the step of cultivating the last generation is carried out for 3 days. The method of claim 1, wherein the ethanol extract has a concentration of 5 to 100 ng/ml. The method of claim 1, further comprising removing the human growth factor solution for dialysis to purify the human growth factor solution. The method of claim 8, wherein the dialysis is performed using a dialysis membrane of 3,500 Daltons for a period of 3-5 days.