JP2017521406A - Stem cell anti-senescence and / or stem cell rejuvenation method, preparation using trans-cinnamaldehyde, and kit used for treatment with stem cells - Google Patents

Abstract

The use of trans-cinnamaldehyde is provided. The stem cell anti-senescence and / or stem cell rejuvenation method according to the present invention includes treatment of a stem cell with trans-cinnamaldehyde (TC), and the stem cell is not a human totipotent stem cell. [Selection] Figure 4

Description

  The present invention relates to the use of trans-cinnamaldehyde (TC), and more particularly to the use of trans-cinnamaldehyde in stem cells, including stem cell anti-aging and / or stem cell rejuvenation and treatment with stem cells.

  Stem cells are divided into totipotent stem cells, pluripotent stem cells, multipotent stem cells and unipotent stem cells according to their self-renew and differentiation ability. Stem cells are roughly classified into two types, embryonic stem cells (ES cells) and adult stem cells, depending on the order and distribution before and after appearing in the development process of stem cells. As evidenced by research, cell differentiation is reversible, and when a specific gene is introduced into a fully differentiated mature somatic cell, the mature somatic cell is reprogrammed into a pluripotent cell. It is possible to become. Among such reprogrammed pluripotent cells, those having the characteristics and functions of embryonic stem cells are referred to as induced pluripotent stem cells (iPS cells). The induced pluripotent stem cells differentiate into any body tissue and are used for disease research and treatment.

  In addition, there are many diseases for which effective treatment methods have not yet been established in the current medical research field, such as diabetes, autoimmune rejection, cerebral infarction, myocardial infarction, renal failure, leukemia, muscle atrophy, severe anemia, There are diseases such as Alzheimer's disease, Parkinson's disease and cancer. By applying stem cells having pluripotency to regenerative medicine, it has become possible to solve the treatment difficulties faced for many years and treat intractable diseases. Stem cell therapy has become one of the hopes for patients with certain intractable diseases.

  The success or failure of stem cell therapy, in which stem cells are transplanted into the body to treat diseases, depends on the quality of the stem cells.

  However, there are several causes for reducing the quality of stem cells. For example, cellular senescence that occurs due to various stresses such as stagnation of the cell cycle and a decrease in the proliferation rate can be cited as one of the main causes. It is done. Once cells age, stem cells or progenitor cells that are essential for tissue repair, tissue regeneration, and turnover are deficient, and the therapeutic effect of stem cells cannot be improved. For this reason, if the anti-aging of stem cells can be achieved effectively, the success rate of treatment with stem cells can be increased.

  The present invention has been made in view of such conventional problems. In order to solve the above-mentioned problems, the present invention is the result of research and development for the above-mentioned therapeutic demand, and the inventors of the present plan have found that trans-cinnamaldehyde has been shown to have anti-aging of stem cells, rejuvenation of stem cells, etc. In addition to being effective, it has been found that it can be applied to treatment with stem cells and can enhance the effect of treatment with cells.

  Therefore, an object of the present invention is to provide a method for preventing aging of stem cells and / or rejuvenating stem cells. That is, the stem cells are treated with trans-cinnamaldehyde. For example, about 0.1 to about 50 μg / mL of trans-cinnamaldehyde is injected into the stem cell culture solution to treat the stem cells.

  Another object of the present invention is to provide a composition used for preventing aging of stem cells and / or rejuvenating stem cells. This composition contains, for example, trans-cinnamaldehyde as an example.

  Still another object of the present invention is to provide a preparation using trans-cinnamaldehyde. This preparation is used for preventing aging of stem cells and / or rejuvenating stem cells, and is preferably a health food or a drug.

Furthermore, still another object of the present invention is used for treatment with stem cells, and
(1) a first portion having stem cells;
(2) a second portion having trans-cinnamaldehyde;
(3) To provide a kit comprising a third part and / or a stem cell culture medium present in the first part and / or the second part.
When using this kit, first, the stem cells are treated with trans-cinnamaldehyde in a stem cell culture solution, and then the treated stem cells are used for treatment with stem cells.

  Still another object of the present invention is to provide a method of treatment with stem cells in which an effective amount of stem cells is administered to an individual in need of treatment. This stem cell is a stem cell that has been previously treated with trans-cinnamaldehyde.

  Hereinafter, detailed technical contents and specific embodiments of the present invention will be described so that those who have ordinary knowledge in the technical field to which the present invention belongs can clearly understand the features of the present invention.

The flow cytometry analysis figure which shows the surface antigen expression condition of the fat origin stem cell which performed various processes. The flow cytometry analysis figure which shows the surface antigen expression condition of the fat origin stem cell which performed various processes. The figure which shows the osteogenic differentiation capability of the fat origin stem cell which performed various processes in the osteogenic differentiation test. The figure which shows the osteogenic differentiation capability of the fat origin stem cell which performed the various process in the osteogenic differentiation test. The figure which shows the osteogenic differentiation capability of the fat origin stem cell which performed the various process in the osteogenic differentiation test. The figure which shows the adipogenic differentiation capability of the fat origin stem cell which performed various processes in the adipogenic differentiation test. The figure which shows the adipogenic differentiation capability of the fat origin stem cell which performed the various process in the adipogenic differentiation test. The figure which shows the adipogenic differentiation capability of the fat origin stem cell which performed the various process in the adipogenic differentiation test. The figure which shows the relative number of the osteogenic differentiation cell among the fat origin stem cells which performed various processes. Here, the vertical axis indicates the absorbance value at a wavelength of 450 nm, and the horizontal axis indicates adipose-derived stem cells (***: P value is less than 0.01, indicating that there is a significant difference) after various treatments. Show. The figure which shows the relative number of the adipogenic differentiation cell among the fat origin stem cells which performed various processes. Here, the vertical axis indicates the absorbance value at a wavelength of 510 nm, and the horizontal axis indicates fat-derived stem cells (***: P value is less than 0.01, indicating that there is a significant difference) that has been subjected to various treatments. Show. The Ki67 dyeing | staining photograph which shows the proliferation rate of the fat origin stem cell which performed various processes. The Ki67 dyeing | staining photograph which shows the proliferation rate of the fat origin stem cell which performed various processes. The Ki67 dyeing | staining photograph which shows the proliferation rate of the fat origin stem cell which performed various processes. The figure which shows SA- (beta) -gal activity in the fat origin stem cell processed by trans- cinnamaldehyde at different density | concentration. Here, the vertical axis represents SA-β-gal activity percentage, and the horizontal axis represents adipose-derived stem cells subjected to various treatments (***: P value is less than 0.01, indicating that there is a significant difference). . It shows the SA-β-gal activity in adipose derived stem cells previously after inducing senescence in H ₂ O ₂ was subjected to various treatments. Here, the vertical axis indicates the percentage of SA-β-gal activity, and the horizontal axis indicates each group of adipose-derived stem cells subjected to various treatments (***: P value is less than 0.01 and there is a significant difference) Represents). The photograph which shows the activity of SA- (beta) -gal in the fat origin stem cell which performed various processes. The photograph which shows the activity of SA- (beta) -gal in the fat origin stem cell which performed various processes. The photograph which shows the activity of SA- (beta) -gal in the fat origin stem cell which performed various processes. The figure which analyzed the expression level of the SIRT1 gene in the fat origin stem cell which performed the trans- cinnamaldehyde treatment by different density | concentration (quantitative reverse transcription Polymerase chain reaction (quantitative RT-PCR)). Here, the vertical axis indicates the expression level of the SIRT1 gene, and the horizontal axis indicates adipose-derived stem cells subjected to various treatments (***: P value is less than 0.01, indicating that there is a significant difference). . By quantitative reverse transcription polymerase chain reaction, it was analyzed the expression levels of SIRT1 gene in adipose-derived stem cells were previously H ₂ O ₂ at different concentrations after inducing senescence trans- cinnamaldehyde process FIG. Here, the vertical axis indicates the expression level of the SIRT1 gene, and the horizontal axis indicates adipose-derived stem cells subjected to various treatments (***: P value is less than 0.01, indicating that there is a significant difference). . The figure which analyzed the expression level of SIRT1 gene in the fat origin stem cell which performed various processes by quantitative reverse transcription polymerase chain reaction. Here, the vertical axis represents the expression level of the SIRT1 gene, and the horizontal axis represents adipose-derived stem cells that have been subjected to various treatments (ns: P value is 0.417, indicating no significant difference). The photograph which shows the result of the semiquantitative reverse transcription polymerase chain reaction (semiquantitative RT-PCR) which shows the expression level of a senescence related gene, a cell cycle regulation related gene, and a pluripotency related gene in the fat origin stem cell which performed various processes. The statistical bar graph which shows the result of the semiquantitative reverse transcription polymerase chain reaction which shows the expression level of an aging related gene, a cell cycle regulation related gene, and a pluripotency related gene in the fat origin stem cell which performed various processes. The statistical bar graph which shows the telomerase (telomerase) activity in the fat origin stem cell which performed various processes. Here, the vertical axis indicates telomerase activity, and the horizontal axis indicates cells that have undergone various treatments (***: P value is less than 0.01, indicating that there is a significant difference). The photograph which shows the activity of SA- (beta) -gal in the human bone marrow mesenchymal stem cell which performed various processes. The photograph which shows the activity of SA- (beta) -gal in the human bone marrow mesenchymal stem cell which performed various processes. The photograph which shows the activity of SA- (beta) -gal in the human bone marrow mesenchymal stem cell which performed various processes. The figure which shows SA- (beta) -gal activity in the human bone marrow mesenchymal stem cell which performed various processes. Here, the vertical axis indicates the percentage of SA-β-gal activity, and the horizontal axis indicates human bone marrow mesenchymal stem cells subjected to various treatments (***: P value is less than 0.05, and there is a significant difference. Represents). The photograph which shows SA- (beta) -gal activity in the Wharton's jelly stem cell which performed various processes. The photograph which shows SA- (beta) -gal activity in the Wharton's jelly stem cell which performed various processes. The photograph which shows SA- (beta) -gal activity in the Wharton's jelly stem cell which performed various processes. The figure which shows SA- (beta) -gal activity in the Wharton's jelly stem cell which performed various processes. Here, the vertical axis represents the percentage of SA-β-gal activity, and the horizontal axis represents a Wharton's jelly stem cell subjected to various treatments (***: P value is less than 0.05, indicating that there is a significant difference. ). The figure which analyzed the expression level of SIRT1 gene in the human bone marrow mesenchymal stem cell which performed various processes by quantitative reverse transcription polymerase chain reaction. Here, the vertical axis represents the expression level of SIRT1 gene, and the horizontal axis represents human bone marrow mesenchymal stem cells (***: P value of less than 0.05, with significant difference) after various treatments. ). The figure which analyzed the expression level of SIRT1 gene in the Wharton's jelly stem cell which performed various processes by quantitative reverse transcription polymerase chain reaction. Here, the vertical axis represents the expression level of SIRT1 gene, and the horizontal axis represents Wharton's jelly stem cells (***: P value is less than 0.05, indicating that there is a significant difference) after various treatments. Show. A Masson's trichrome staining photograph showing the progress of fibrosis in the liver of a “TAA group” rat (rat with liver fibrosis). Hematoxylin and eosin staining (H & E staining) showing the progress of hepatitis symptoms in the liver of “TAA group” rats (rats with liver fibrosis). Photographs of immunohistochemical staining of α-fetoprotein (α-fetoprotein, AFP) in rat liver tissue sections on day 7 subjected to various treatments. Immunohistochemically stained photographs of α-fetoprotein in rat liver tissue sections on day 7 after various treatments. Immunohistochemically stained photographs of α-fetoprotein in rat liver tissue sections on day 7 after various treatments. Immunohistochemically stained photographs of α-fetoprotein in rat liver tissue sections on day 7 after various treatments. Immunohistochemically stained photographs of α-fetoprotein in rat liver tissue sections on day 7 after various treatments. Photographs of immunohistochemical staining of α-fetoprotein in rat liver tissue sections on day 14 after various treatments. Photographs of immunohistochemical staining of α-fetoprotein in rat liver tissue sections on day 14 after various treatments. Photographs of immunohistochemical staining of α-fetoprotein in rat liver tissue sections on day 14 after various treatments. Photographs of immunohistochemical staining of α-fetoprotein in rat liver tissue sections on day 14 after various treatments. Photographs of immunohistochemical staining of α-fetoprotein in rat liver tissue sections on day 14 after various treatments. Fig. 4 is a Masson's trichrome staining photograph showing that the degree of fibrosis varies with time in the liver of variously treated rats. Fig. 4 is a Masson's trichrome staining photograph showing that the degree of fibrosis varies with time in the liver of variously treated rats. Hematoxylin and Eosin Staining (H & E staining) photographs showing that the symptoms of hepatitis vary with time in the liver of variously treated rats. Hematoxylin and eosin staining (H & E staining) showing that the symptoms of hepatitis vary with time in the liver of variously treated rats. Statistical bar graph showing the number of liver fibrosis in rats subjected to various treatments (***: P value is less than 0.01, indicating a significant difference). Statistical bar graph showing activation scores of rat liver (***: P value is less than 0.01, indicating significant difference). Statistical bar graph showing GPT expression level in rat liver (***: P value is less than 0.01, indicating significant difference). Statistical bar graph showing GOT expression level in rat liver (**: P value is less than 0.05, indicating that there is a significant difference). Statistical bar graph showing albumin expression level in rat liver (***: P value is less than 0.01, indicating significant difference). Statistical bar graph showing prothrombin time of rat liver (***: P value is less than 0.01, indicating significant difference). Statistical bar graph showing total bilirubin expression in rat liver.

  Hereinafter, specific embodiments of the present invention will be described. However, the present invention can be implemented in many different forms without departing from the spirit of the present invention, and the protection scope of the present invention should not be construed as being limited to the scope described in the specification. In addition, unless otherwise specified in the text, terms used in the present specification (and claims) to which “one”, “the above”, and similar terms are attached are singular and plural. Should be understood to include both. “Effective amount” or “therapeutically effective amount” refers to the amount of an effective compound that is entrusted to an individual to at least partially improve the condition of the individual. An “individual” refers to a mammal, which is a human or a non-human animal.

  Stem cells are widely used in modern medicine, and their applications include, for example, treatment of autoimmune diseases (eg, diabetes, autoimmune rejection), treatment of gastrointestinal diseases (eg, anal / gastrointestinal fistula), Treatment of liver diseases (eg, cirrhosis, liver fibers), treatment of kidney diseases (eg, renal failure), treatment of cardiovascular diseases (eg, cerebral infarction, myocardial infarction), nervous system diseases (eg, Alzheimer's disease, Parkinson's disease) ), Treatment of blood diseases (eg leukemia), treatment of dysplasia (eg osteoarthritis, knee osteoarthritis), treatment of periodontal disease, treatment of tendonitis, treatment of spinal injury, It is used for the treatment of head wounds, plastic surgery (eg unilateral atrophy, small depression scars), alopecia, skin whitening and / or wrinkle removal. This is described, for example, in Stem Cells: innovations in clinical applications. Stem Cells int. Volume 2014, Article ID 516278, 9 pages, the full text of which is referred to here.

  However, when stem cells are subjected to various stresses due to several generations of subculture and / or transplantation into the body, the therapeutic effect of stem cells may be affected. This is described, for example, in Replicative senescence-associated gene expression changes in mesenchymal stromal cells are similar under different culture conditions.Haematologica. 95 (6): 867-74 (2010) .See the full text of this document here. To do. As is clear from the study, with stem cell aging, for example, the proliferation rate of stem cells decreases, senescence associated-β-galactosidase (SA-β-gal) activity increases in stem cells, Phenomena such as decreased expression of SIRT1 gene and decreased telomerase activity in stem cells occur. If these phenomena can be reversed and / or recovered, it will be effective in preventing aging of stem cells, as already reported, and will be beneficial for application of stem cells to disease treatment. For example, Four faces of cellular senescence. J Cell Biol. 192 (4): 547-556 (2011), A biomarker that identifies senescent human cells in culture and in aging skin in vivo.Proc Natl Acad Sci US A. 92 (20): 9363-9367 (1995), SIRT1 overexpression antagonizes cellular senescence with activated ERK / S6k1 signaling in human diploid fibroblasts.PLoS One. 5; 3 (3): e1710 (2008), Aging, tumor suppression and cancer: high wire-act! Mech Ageing Dev. 126 (1): 51-8 (2005) and Telomeres shorten during ageing of human fibroblasts.Nature. 345 (6274): 458-460 (1990), The full text of these documents is referenced here.

  The inventor of the present proposal conducted trans-synergization on senescent stem cells exhibiting aging phenomena such as a decrease in proliferation rate, an increase in SA-β-gal activity, a decrease in the expression level of SIRT1 gene, and a decrease in telomerase activity. It has been discovered that treatment with mualdehyde can effectively reverse and / or recover the aging phenomenon in these stem cells. Further studies have shown that trans-cinnamaldehyde effectively reduces SA-β-gal activity in common stem cells (ie, unaged stem cells) and increases SIRT1 gene expression in common stem cells. I also found out.

  Therefore, the present invention relates to stem cell anti-senescence and stem cell rejuvenation and applications thereof, and includes provision of compositions, preparations and methods used for stem cell anti-senescence and / or stem cell rejuvenation. Trans-cinnamaldehyde is used in the preparation of the formulation, the composition includes trans-cinnamaldehyde, and the method includes treating stem cells with trans-cinnamaldehyde.

  Moreover, the composition, formulation and method of the present invention can be applied to any stem cells, for example, embryonic stem cells, adult stem cells, and induced pluripotent stem cells. Adult stem cells include, for example, hematopoietic stem cells and mesenchymal stem cells (including adipose-derived stem cells, bone marrow mesenchymal stem cells, Wharton's jelly stem cells, and the like). They are classified into stem cells, neural stem cells, epidermal stem cells, muscle stem cells, adipose-derived stem cells, pancreatic stem cells, corneal stem cells, liver stem cells, intestinal epithelial stem cells, and the like.

  Based on the method of the present invention, when stem cells are pretreated with trans-cinnamaldehyde prior to treatment with stem cells, at least one of the following effects can be obtained. That is, an effect of increasing the proliferation rate of stem cells, decreasing SA-β-gal activity in stem cells, increasing SIRT1 gene expression in stem cells and improving telomerase activity in stem cells can be obtained. Stem cell rejuvenation can be achieved. Thereby, the therapeutic effect by a stem cell increases. trans-Cinnamaldehyde is used in the form of a composition or formulation.

  In the stem cell anti-aging and / or stem cell rejuvenation method according to the present invention, the stem cells can be treated with trans-cinnamaldehyde under any conditions as long as they are harmless to the survival of the stem cells. In one embodiment of the invention, this treatment is performed in a stem cell culture medium. trans-Cinnamaldehyde and / or a composition and / or formulation of trans-cinnamaldehyde is added to a stem cell culture containing the stem cells to be treated or added to a stem cell culture containing, for example, cryopreserved stem cells Is done.

  Any culture solution can be used in the present invention as long as it corresponds to the stem cells to be treated. The stem cell culture medium usually contains essential components for providing nutrients and conditions (eg, hydrogen ion index) necessary for stem cell growth and differentiation. In general, stem cell culture media are basal media, animal serum (eg, fetal bovine serum), growth factors (eg, human recombinant epidermal growth factor, fibroblast growth factor), nutrients (eg, bovine Pituitary extracts), non-essential amino acids (eg L-glutamine, N-acetyl-L-cysteine), redox agents (eg L-ascorbic acid), antibiotics (eg penicillin, streptomycin), etc. Including. The basic culture medium used in the method of the present invention is, for example, K-SFM medium (keratinocyte-serum free medium), DMEM culture medium (Dulbecco's Modified Eagle's Medium), IMDM culture medium (Iscove's modified Dulbecco's medium, IMDM, Invitrogen) , MEM medium (Minimum Essential Medium), α-MEM medium, BME medium (Basal Media Eagle), MEM / F12 medium, Ham's F10 medium, Ham's F12 medium and RPMI medium (Rosewell Park Memorial Institute) Including, but not limited to. For example, when the method of the present invention is used for anti-aging of adipose-derived stem cells and / or rejuvenation of adipose-derived stem cells, treatment with trans-cinnamaldehyde can be performed using a K-SFM medium as a basic culture solution. it can.

  Preferably, the stem cells are treated by adding about 0.1 μg / mL to about 50 μg / mL of trans-cinnamaldehyde in the stem cell culture medium. More preferably, the stem cells are treated by adding about 2 μg / mL to about 20 μg / mL of trans-cinnamaldehyde in the stem cell culture solution, thereby providing an anti-aging effect of the stem cells. Alternatively, stem cells are treated by adding about 4 μg / mL to about 20 μg / mL of trans-cinnamaldehyde in the stem cell culture solution, thereby providing a rejuvenating effect of the stem cells.

  The present invention provides stem cell anti-aging and / or stem cell rejuvenation compositions and formulations using trans-cinnamaldehyde. These compositions and preparations are compositions and preparations containing trans-cinnamaldehyde of the method according to the present invention described above, and are used for the treatment of stem cells in vitro, thereby enhancing the effect of using stem cells. And can be used, for example, in the human body, thereby providing a rejuvenating or anti-aging effect of stem cells in vivo.

  The composition or preparation used for anti-aging of stem cells and / or rejuvenation of stem cells according to the present invention includes a medium when used in vitro. The medium may be any medium that does not adversely affect the stem cells to be treated and does not affect the treatment effect of trans-cinnamaldehyde, and includes, for example, dimethyl sulfoxide, ethanol, or 0.5% methylcellulose. Consists of a phosphate buffer solution.

  When the composition or formulation used for stem cell anti-aging and / or stem cell rejuvenation according to the present invention is used in a human individual, the composition or formulation is formed in any suitable form. It does n’t have any special restrictions. By way of example, the composition or formulation is a drug or drinkable food, such as a food additive, beverage additive, dietary supplement, food composition, beverage component, health food, nutritional product, pharmaceutical food, nutritional product. It may be provided in the form or in the form of a drug, but is not limited thereto. Preferably, the composition or formulation is provided in the form of a health food or drug.

  When a composition and / or formulation according to the present invention is provided as a drug, the drug is provided in a suitable drug form corresponding to the required dosage regime. For example, the drug may be used for an individual in need of a dosage mode such as oral or parenteral (eg, subcutaneous, intravenous, muscle, abdominal cavity or nasal cavity), but is not limited thereto. In addition, the medicine can be provided after selecting and using an appropriate medium according to the usage type and application.

  Taking the case where the drug is in a form that is applied for oral administration, the drug provided by the present invention includes any pharmaceutically acceptable medium that does not adversely affect the effect of trans-cinnamaldehyde, for example, solvent (water, Saline, dextrose, glycerin, ethanol or the like and combinations thereof), oily solvents, diluents, stabilizers, absorption delaying agents, disintegrating agents, emulsifiers, antioxidants, adhesives, lubricants, Hygroscopic materials, solid carriers (eg starch, bentonite) and the like. This drug is provided in a drug form applicable to oral administration using any method, for example, tablets (including sugar-coated tablets), pills, capsules, granules, powders, fluid extracts, solutions, syrups. It may be provided in the form of an agent, suspension, emulsion, tincture and the like.

  When the drug is in the form of an injectable drug or a drip drug applied to subcutaneous, intravenous, intramuscular or intraperitoneal injection, the drug provided by the present invention contains one or more solvents, such as isotonic solutions, base buffers. (For example, phosphate buffer or citrate buffer), solubilizer, emulsifier, 5% sugar solution and other media, and other ingredients are added to intravenous injection, emulsion intravenous injection, powder injection You may provide as pharmaceutical forms, such as an agent, a suspension injection, or a powder suspension injection. In addition, the drug may be prepared in a drug form dissolved in another solution or suspension to form a solid pre-injection drug, and the solid pre-injection drug may be made into an emulsifiable drug form. May be provided. Then, before administering the solid pre-injection drug to an individual, first, the solid pre-injection drug is dissolved or emulsified in another solution or suspension to obtain an injection. It becomes possible to provide. In addition, the form of the external medicine which can be put in via the nasal cavity or the skin is, for example, emulsion, cream, gel (for example, gel water), salve (for example, dispersion ointment or ointment), spray or solution (for example, Cleaning fluid and suspension).

  If necessary, the drug provided in the present invention may further contain an appropriate amount of an additive, such as a seasoning, a toning agent, a coloring agent, etc. for enhancing the mouth feel and visual effect when taking the drug. And media, binders, buffers, thickeners, lubricants, disintegrants, stabilizers, emulsifiers, powders, suspensions, preservatives, preservatives, antibacterial agents that improve the stability and storage stability of the drug Or you may further contain an antifungal agent etc. Also, depending on the situational demand, the drug may further contain one or more other active ingredients, or may be used in combination with a drug containing one or more other active ingredients. In this way, it is possible to further enhance the effect of the drug, and it is also possible to increase the operational flexibility and responsiveness related to the preparation and the formulation. Other active ingredients may be used as long as they do not cause an adverse effect on the effect of trans-cinnamaldehyde.

  The agent provided in the present invention can be used at different frequencies such as once a day, several times a day, or once every few days, and such frequency depends on the age and weight of the individual to be cast. It depends on the health condition. For example, when a drug is orally administered to an individual for stem cell anti-aging and / or stem cell rejuvenation, the daily dosage calculated with trans-cinnamaldehyde is about 10 mg / kg body weight to about 500 mg / body weight 1 kg-day may be used, preferably about 60 mg / kg body weight to about 240 mg / kg body weight, more preferably about 100 mg / kg body weight to about 160 mg / kg body weight. Here, “mg / kg body weight” as a unit refers to a dosage required per kg body weight of an individual. However, the dosage for individuals with severe aging of stem cells can be increased according to actual demand, and may be increased several times or several tens of times, for example.

  When the composition and / or formulation of the present invention is provided in the form of a health food, this form may be, for example, dairy products, meats, breads, vegetables, feeds, juices, teas, sports drinks, nutrition Contains drinks, but is not limited to these. The addition process of trans-cinnamaldehyde varies depending on the type of health food, but it is sufficient that the environment in the addition process does not affect the treatment effect of trans-cinnamaldehyde. For example, trans-cinnamaldehyde may be added at the raw material stage in the manufacturing process (ie, as part of a health food) or added at the finished product stage after manufacturing is complete (ie, health care). Food additive). The form of trans-cinnamaldehyde in the addition step may be as it is, or trans-cinnamaldehyde may be other various components (for example, protein, saccharide, fat, trace element or vitamin, etc. It may be added and mixed as a tablet, capsule, granule, powder, suspension, or emulsified liquid preparation produced by mixing with (but not limited to).

  The health food provided by the present invention further comprises an appropriate amount of an excipient (for example, lactose, sucrose, starch or mannitol), a disintegrant (for example, calcium carbonate or calcium fibrinoglycolate), binding, depending on demand. Agents (eg pregelatinized starch, gum arabic, carboxymethylcellulose, polyvinylpyrrolidine or hydroxypropylcellulose), thickeners (natural rubber), lubricants (eg talc powder, magnesium stearate or polyethylene glycol 6000) and / or other Additives (for example, flavoring agents, toning agents, coloring agents, etc. that enhance the mouthfeel and visual effects when taking health foods, and media, buffers, stabilizers, emulsifiers, powders that improve the stability and storage stability of health foods Agents, suspending agents, preservatives, preservatives, antibacterial agents or antifungal agents). The health food may further contain one or more other active ingredients according to demand, and may be used in combination with a health food containing one or more other active ingredients. Thereby, the effect of health food can be further strengthened, and the flexibility and responsiveness of the formulation operation can be enhanced. Other active ingredients may be used as long as they do not cause adverse effects on the effect of trans-cinnamaldehyde.

  The health food provided in the present invention can be edible at different frequencies such as once a day, several times a day or once every few days, and this frequency depends on the age and weight of an individual who eats the health food. And changes according to health status. The content of trans-cinnamaldehyde in the health food provided by the present invention is adjusted according to a predetermined group group, and is preferably adjusted to a predetermined dose. For example, the daily dose is about 500 mg / day per individual, and if it contains 250 mg trans-cinnamaldehyde per serving of health food, it is recommended that the individual eat about 2 health foods daily. Is done. Recommended dietary doses for the health foods of the present invention, dietary standards and conditions for certain family groups (eg, pregnant women, patients) or recommendations for taking with other foods or medicines are marked on the exterior. Then, based on the displayed contents, the user can safely take health foods at home or himself without guidance from a doctor, pharmacist or other specialists.

  According to the present invention, the stem cells are treated with trans-cinnamaldehyde before treatment with the stem cells, so that anti-aging and rejuvenation of the stem cells are induced, so that the therapeutic effect by the stem cells is enhanced. The present invention relates to the discovery and application that trans-cinnamaldehyde can be used for treatment with stem cells, and includes the provision of kits and methods used for treatment with stem cells.

  The kit used for the treatment with stem cells according to the present invention comprises (1) a first part having stem cells, (2) a second part having trans-cinnamaldehyde, (3) a third part and / or a first part and And / or a stem cell culture medium present in the second part. When using this kit, first, the stem cells are treated with trans-cinnamaldehyde in a stem cell culture solution, and then the treated stem cells are used for treatment with stem cells. Regarding the selection of stem cells, the selection of the culture medium, and the use conditions and method of trans-cinnamaldehyde, all are performed as described above. trans-Cinnamaldehyde is preferably used at a concentration of about 0.1 μg to about 50 μg per mL of culture medium.

  In the kit of the present invention, each component is usually packaged or stored separately and then transported or sold independently. However, the components may be combined together to form a kit and then delivered or sold. In addition, the method of using the kit is described in the instruction manual. When using the kit, the user mixes each component on site according to the order and process described in the instruction manual, and then the stem cell is added. It can be cultured, treated and applied.

For example, when each component in the kit of the present invention is packaged or stored separately and transported or sold separately, it is desirable that the stem cells are stored in a cryopreservation solution in an environment of −80 ° C. trans-Cinnamaldehyde is preferably stored in an environment avoiding light at 4 ° C. or lower, for example, in a solvent such as phosphate buffered saline containing dimethyl sulfoxide, ethanol and 0.5% methylcellulose, and The stem cell culture solution is stored in an environment of −20 ° C.
For example, when the components in the kit of the present invention are combined and delivered and sold, the inside is a container at −80 ° C. (eg, liquid nitrogen container), the inside is lower than 4 ° C., and the inside is −20 ° C. It is desirable to put stem cells, trans-cinnamaldehyde, and a stem cell culture solution in each of the containers (eg, ice box).
Although the shape and size of each container are not particularly limited, each container has a function of isolating from the external temperature, and is configured so that the storage temperature does not affect each other when each component is delivered and sold together. Has been. When an appropriate cryopreservation solution is used for the preservation of stem cells, the cryopreservation solution effectively maintains stem cell viability and does not affect the chromosomal composition, proliferation and differentiation ability of the stem cells.
Usually, the composition of the cryopreservation solution is adjusted according to the stem cells. The cryopreservation solution generally contains cell nutrients such as dimethyl sulfoxide and glucose, a pH adjuster, and the like, but does not contain serum or animal protein.

  The treatment method using stem cells according to the present invention is characterized by administering an effective amount of stem cells to an individual in need of treatment, and the stem cells are pretreated with trans-cinnamaldehyde. For example, in a specific embodiment according to the method of the present invention, the stem cells exhibit an anti-aging function by being pre-treated with trans-cinnamaldehyde before being used for treatment of fibrotic liver. Enhance the therapeutic effect on the fibrotic liver by stem cells. Stem cell treatment and related substance selection, trans-cinnamaldehyde dose range and therapeutic applications are performed as described above.

  In the following examples, the invention is further illustrated by examples. These examples are provided for the purpose of illustration and are not intended to limit the protection scope of the present invention. The protection scope of the present invention is set forth in the appended claims.

A. Example of cell experiment 1: Preparation of stem cells (1) Adipose derived stem cell (ADSC)
Adipose-derived stem cells are subcultured 5 to 7 times in serum-free keratinocyte medium (purchased from keratinocyte-serum free medium, K-SFM, Invitrogen-Gibco, etc.) at 37 ° C. and 5% carbon dioxide did. This K-SFM medium is composed of human recombinant epidermal growth factor (purchased from Gibco), bovine pituitary extract (purchased from bovine pituitary extract, Invitrogen-Gibco, etc.), 10% Fetal bovine serum (FBS, purchased from Hyclone), 1% penicillin / streptomycin (P / S, purchased from Biowest), 0.2 mM L-ascorbic acid (L-ascorbic acid, purchased from Sigma) and Contains 2 mM N-acetyl-L-cysteine (purchased from Sigma).

The culture solution containing the adipose-derived stem cells subcultured as described above is divided into four equal parts, each of which is added with a final concentration of 100 μM H ₂ O ₂ (purchased from Sigma-Aldrich), treated for 2 hours, and stem cells Induced aging. Thereafter, the old culture medium is removed and a fresh culture medium is added, and trans-cinnamaldehyde (trans-cinnamaldehyde, TC, purchased from Sigma-Aldrich) at a final concentration of 0, 2, 4 or 6 μM is added, respectively. Time treatment, “H ₂ O ₂ set (treatment with H ₂ O ₂ for 2 hours, no treatment with TC)”, “H ₂ O ₂ + TC2 set (treatment with H ₂ O ₂ for 2 hours, Treatment with TC at a concentration of 2 μM was performed for 6 hours), “H ₂ O ₂ + TC4 group (treatment with H ₂ O ₂ was performed for 2 hours, and treatment with TC at a concentration of 4 μM was performed for 6 hours)” and “H ₂ An adipose-derived stem cell called “O ₂ + TC6 group (treated with H ₂ O ₂ for 2 hours and treated with TC at a concentration of 6 μM for 6 hours)” was obtained.

The culture solution containing the adipose-derived stem cells subcultured as described above was divided into 5 equal parts, and H ₂ O ₂ having a final concentration of 100 μM was added to each, and the cells were treated for 2 hours to induce stem cell senescence. Thereafter, the old culture solution was removed and a fresh culture solution was added, and a TC with a final concentration of 6 μM was added to the first aliquot and treated for 6 hours to obtain adipose-derived stem cells called “TC6H group”. The second to fifth aliquots were each treated with 6 μM final concentration of 6 μM and treated for 6 hours, after which the cells were transferred into a DMEM (Dulbecco's modified essential medium) culture medium, respectively 12, 24, 48 and The culture was continued for 72 hours, and adipose-derived stem cells called “TC12H group”, “TC24H group”, “TC48H group” and “TC72H group” were obtained.

  The culture solution containing the adipose-derived stem cells subcultured as described above is divided into 4 equal parts, and TCs having final concentrations of 0, 2, 4, and 6 μM are added to each, and treated for 6 hours. No treatment was performed ”,“ TC2 group (treatment with TC at a concentration of 2 μM was performed for 6 hours) ”,“ TC4 group (treatment with TC at a concentration of 4 μM was performed for 6 hours) ”and“ TC6 group (concentration of 6 μM) The adipose-derived stem cells, which were treated with TC for 6 hours, were obtained.

(2) Wharton's jelly stem cell (WJSC)
Wharton's jelly stem cells were subcultured 5 to 7 times in IMDM culture medium (Iscove's modified Dulbecco's medium, purchased from Invitrogen) at 37 ° C. and 5% carbon dioxide. The IMDM culture solution is 10% fetal bovine serum (purchased from Hyclone), 10 μg / mL alkaline fibroblast growth factor (bFGF, purchased from R & D system), L-glutamine (concentration 2 mM) Invitrogen-Gibco, etc.) and 100 units / mL penicillin / streptomycin (purchased from Invitrogen).

The culture solution containing Wharton's jelly stem cells subcultured as described above is divided into three equal parts, and H ₂ O ₂ having a final concentration of 100 μM is added to each of the first and second equal parts and treated for 2 hours. Induced stem cell senescence. Then, it added fresh culture medium to remove the old culture, a final concentration of 0 or 6μM of TC was added to 6 hours, respectively, performed "H ₂ O ₂ pairs (H ₂ O ₂ by treatment for 2 hours However, the treatment with TC was not performed) and “W ₂ O ₂ + TC6 group (treatment with H ₂ O ₂ was performed for 2 hours and treatment with TC at a concentration of 6 μM was performed for 6 hours)” Stem cells were obtained. No treatment was performed on the third aliquot, and Wharton's jelly stem cells called “untreated group” were obtained.

(3) Human bone marrow mesenchymal stem cell (hBMMSC)
Human bone marrow mesenchymal stem cells (hBMMSC) were subcultured 5 to 7 times in IMDM medium (purchased from Invitrogen-Gibco, etc.) at 37 ° C. and 5% carbon dioxide. IMDM culture solution is 10% fetal bovine serum (purchased from Hyclone), 10 μg / mL alkaline fibroblast growth factor (purchased from R & D system), 2 mM L-glutamine (purchased from Invitrogen-Gibco, etc.) ) And 100 units / mL penicillin / streptomycin (purchased from Invitrogen-Gibco, etc.).

The culture solution containing human bone marrow mesenchymal stem cells subcultured as described above is divided into 3 equal parts, and H ₂ O ₂ having a final concentration of 100 μM is added to each of the first and second equal parts for 2 hours. Treated and induced stem cell senescence. Then, it added fresh culture medium to remove the old culture, a final concentration of 0 or 6μM of TC was added to 6 hours, respectively, performed "H ₂ O ₂ pairs (H ₂ O ₂ by treatment for 2 hours However, treatment with TC was not performed) and “H ₂ O ₂ + TC6 group (treatment with H ₂ O ₂ was performed for 2 hours and treatment with TC at a concentration of 6 μM was performed for 6 hours)” Leaf stem cells were obtained. No treatment was performed on the third aliquot, and human bone marrow mesenchymal stem cells called “untreated group” were obtained.

Example 2: Effect of H ₂ O ₂ and trans-cinnamaldehyde on surface antigen expression of stem cells Example 1 (1) “Untreated group”, “H ₂ O ₂ group” and “H ₂ O ₂ + TC6 group” Different antibodies were allowed to act on adipose-derived stem cells. The antibodies are human leukocyte differentiation antigen 14 (CD14) antibody purchased from Dako, human leukocyte differentiation antigen 29 (CD29) antibody, human leukocyte differentiation antigen 44 (CD44) antibody, Human leukocyte differentiation antigen 45 (CD45) antibody, human leukocyte antigen-ABC (human leukocyte antigen-ABC, HLA-ABC) antibody, human leukocyte differentiation antigen 34 (CD34) antibody purchased from Becton Dickinson Human leukocyte differentiation antigen 49b (CD49b) antibody, human leukocyte differentiation antigen 73 (CD73) antibody, human leukocyte differentiation antigen 90 (CD90) antibody, and human leukocyte antigen-DR (HLA-DR) antibody And including. Thereafter, expression of each group of cell surface antigens was analyzed by flow cytometry (Beckman Coulter, model number: FC500). The results are shown in FIGS. 1A and 1B.

As can be seen from FIGS. 1A and 1B, there is no clear change in the expression pattern of the surface antigens of “H ₂ O ₂ group” and “H ₂ O ₂ + TC6 group” as compared to “untreated group”. The set of adipose-derived stem cells all express mesenchymal stem cell markers (eg, CD29, CD44, CD73, CD90 and HLA-ABC), but hematopoietic stem cell markers (eg, CD34, CD45 and HLA-DR). ) Was not expressed. As this result shows, neither H ₂ O ₂ treatment nor TC treatment affects the expression of stem cell surface antigens.

Example 3: Effect of trans-cinnamaldehyde on the differentiation ability of stem cells (1) Osteogenic differentiation test In the DMEM culture solution, (1) “untreated group”, “H ₂ O ₂ group” and “ The adipose-derived stem cells of “H ₂ O ₂ + TC6 set” were cultured and induced so that the adipose-derived stem cells were directed to osteogenic differentiation. This culture is 10% fetal bovine serum, 20 nM dexamethasone, 100 units / mL penicillin, 100 μg / mL streptomycin, 2.5 μg / mL amphotericin, 10 mM b-glycerin. Contains phosphate (b-glycerophosphate) and 0.05 mM L-ascorbic acid 2-phosphate. On the 7th day after culturing, staining was carried out using an alkaline phosphatase detection kit (purchased from EMD Millipore, model number: CSCR004), and the results taken with a microscope are shown in FIGS. 2A to 2C. Cells after osteogenic differentiation have a purple color when viewed under a microscope. After imaging, an absorbance value (OD450 nm) at a wavelength of 450 nm for each group was measured (this absorbance value corresponds to the number of purple cells), and H ₂ O ₂ and / or the osteogenic differentiation ability of adipose-derived stem cells and / or The effect of TC was examined. The result is shown in FIG. 2G.

As can be seen from FIGS. 2A to 2C and FIG. 2G, the number of cells after osteogenic differentiation of “H ₂ O ₂ group” is clearly reduced compared to “untreated group”, and “H ₂ O ₂ + TC6 group” The number of cells after osteogenic differentiation of "" was comparable to that of the "untreated group". As a result, as is shown, osteogenic differentiation potential of stem cells was reduced by treatment with H ₂ O _2, the of H ₂ O ₂ adverse effects normally osteogenic differentiation potential is in stem cells counteracted by treatment with TC Recovered.

(2) Adipogenic differentiation test Adipose-derived stem cells of “untreated group”, “H ₂ O ₂ group” and “H ₂ O ₂ + TC6 group” of Example 1 (1) were cultured in DMEM culture medium. Adipose-derived stem cells were induced to adipogenic differentiation. This culture solution contains 10% fetal bovine serum, 10 μg / mL insulin, 1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) and 100 μM indomethacin. (Indomethacin) is included. The results of oil red O staining (Oil Red O staining) on the seventh day from the culture and taken with a microscope are shown in FIGS. 2D to 2F. Stem cells produce oil droplets in the cells during adipogenic differentiation, and after oil red O staining, the oil droplets in the cells appear orange-red when viewed under a microscope. The process of oil red O staining is described in Coptis chinensis alkaloids acting anti-adipogenic activity on 3T3-L1 adipocytes by downregulating C / EBP-α and PPAR-γ. Fitoterapia. 98: 199-208 (2014). The full text of is here referenced.

  After imaging, each set of cell oil droplets was extracted with isopropanol (4% Nonidet P-40 detergent), and each set of absorbance values at wavelength 510 nm (OD 510 nm) (this absorbance value corresponds to orange-red cells. , Corresponding to the number of cells after adipogenic differentiation). The result is shown in FIG. 2H.

As seen from FIG. 2D to 2F and FIG 2H, the number of cells after adipogenic differentiation when compared to "unprocessed assembly", "H ₂ O ₂ sets" are clearly reduced, "H ₂ O ₂ The number of cells after adipogenic differentiation of “+ TC6 group” was similar to that of the “untreated group”. As a result, as is shown, adipogenic differentiation potential of stem cells was reduced by treatment with H ₂ O _2, the of H ₂ O ₂ adverse effects normally adipogenic differentiation potential is in stem cells counteracted by treatment with TC Recovered.

Example 4: Effect of stem cell rejuvenation and stem cell anti-aging by trans-cinnamaldehyde (1) Proliferation rate analysis Example 1 (1) “Untreated group”, “H ₂ O ₂ group” and “H ₂ O ₂ ” The “+ TC6 group” adipose-derived stem cells were each washed twice with phosphate buffered saline (PBS). Thereafter, the mixture was fixed with 3.7% formaldehyde, and a blocking reaction was performed with 10% fetal calf serum (diluted in PBS). Then, the cells are washed once with PBS, Ki67 antibody (purchased from Novus biological) is reacted with the cells at 4 ° C., one day later, the cells are further washed once with PBS, and the secondary antibody (goat anti rabbit IgG (purchased from Merk Millipore) was allowed to react with the cells for 2 hours. Finally, light-shielding staining was performed with a DAPI stain (purchased from Invitrogen), and then observed with a fluorescence microscope. The results are shown in FIGS. 3A to 3C. Nuclear proteins in the G1, S, G2, and M phases of proliferating cells are stained with the Ki67 antibody, but cells in the resting G0 phase are not stained. Therefore, it can be seen that the more cells that are stained with Ki67, the more cells are proliferating (that is, the proliferation rate is faster).

As can be seen from FIG. 3A to FIG. 3C, the cells stained with Ki67 in “H ₂ O ₂ group” are clearly decreased as compared with “untreated group”, while in “H ₂ O ₂ + TC6 group”. The number of Ki67-stained cells was clearly increased and increased so as to correspond to the “untreated group” (the pointed by the arrow is a proliferating cell). As this result, the stem cells are aged by treatment with H ₂ O _2, the adverse effects of H ₂ O ₂ is counteracted by treatment with TC, the growth rate of the aged stem cells were recovered to normal (i.e., stem cells Anti-aging).

(2) Activity measurement test of senescence associated β-galactosidase (SA-β-gal) Senescent cells are stained by SA-β-gal staining under acidic conditions (eg, pH 6.0). Therefore, it is possible to know the degree of aging of cells by detecting the expression level of SA-β-gal in the cells.

The fat-derived stem cells of Example 1 (1) were each washed twice with PBS. Thereafter, the cells were fixed with 3% formaldehyde and washed once with PBS. Each set of cell staining was then performed for 12 hours in a prepared fresh staining solution at 37 ° C. in a CO ₂ free environment. This staining solution comprises 1 μg / mL 5-bromo-4-chloro-3-indolyl β-D-galactosidase (X-gal), Containing 40 mM citrate / sodium phosphate (pH 6.0), 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM sodium chloride, and 2 mM magnesium chloride.

  The staining solution was removed and each set of cells was washed with PBS. Finally, it was observed and photographed at a magnification of 10 times with a microscope, and the percentage of cells showing SA-β-gal activity was calculated. The results are shown in FIGS. 4A to 4E.

  As shown in FIG. 4A, the number of cells showing SA-β-gal activity increased in TC concentration in all of “TC2 group”, “TC4 group” and “TC6 group” as compared with “untreated group”. It decreased with it. This indicates that TC has stem cell rejuvenation ability.

As seen from FIG. 4B to FIG. 4E, compared with "untreated group", the number of cells exhibiting SA-β-gal activity in the "H ₂ O ₂ sets" are obviously increased, which H ₂ O ₂ indicates that stem cells are aged. Also shows a comparison with the _"H 2 _{O 2} pairs", _"H 2 _O 2 + TC2 pairs", all in SA-β-gal activity _"H 2 _O 2 + TC4 sets" and _"H 2 _O 2 + TC6 sets" The cell number is clearly reduced, indicating that the adverse effects of H ₂ O ₂ -induced stem cell aging were counteracted by TC.

(3) Gene expression analysis (3-1) Preparation of cDNA Using RNA purification kit (RNeasy Mini kit, purchased from Qiagen), each set of fat-derived stem cells, bone marrow mesenchymal stem cells and Wharton's jelly stem cells of Example 1 Total RNA was extracted, and then the total RNA obtained using a reverse transcription kit (RT Pre Mix, purchased from iNtRON Biotechnology) was reverse transcribed into cDNA.

(3-2) Quantitative RT-PCR
Take 1 μL of each fat-derived stem cell cDNA obtained in (3-1) above, 0.6 μL of the corresponding primer of SIRT1 gene shown in Table 1 (5 pM / μL), 3.4 μL of deionized water and 10 μL SYBR Green PCR master mix (purchased from Thermo Scientific) was added, and the reaction was then carried out in a synchronous quantitative polymerase chain reactor (ABI prism 7700 sequence detection system) to quantify the gene expression level. The reaction conditions are as follows. i) 50 ° C, 2 minutes, 1 cycle. ii) 95 ° C., 10 minutes, 1 cycle. iii) 95 ° C., 15 seconds, 60 ° C., 30 seconds, 72 ° C., 30 seconds, 40 cycles of the above steps. iv) 72 ° C., 10 minutes, 1 cycle. v) Finally, the reaction is terminated at 4 ° C. The results are shown in FIGS. 5A to 5C.

As can be seen from FIG. 5A, SIRT1 (silent mating type information regulation 2 homolog 1 and silent mating type information regulation 2 homolog 1) of “TC 2 set”, “TC 4 set” and “TC 6 set” as compared with “H ₂ O ₂ set”. 1) Gene expression clearly increased with increasing TC concentration. This indicates that TC has stem cell rejuvenation ability.

As can be seen from Figure 5B, compared to the _"H 2 _{O 2} pairs", _"H 2 _O 2 + TC2 pairs", the expression of SIRT1 gene _"H 2 _O 2 + TC4 sets" and _"H 2 _O 2 + TC6 pairs" are It clearly increased as the TC concentration increased. This indicates that TC has the anti-aging ability of stem cells.

  As can be seen from FIG. 5C, compared to the “TC6H group”, there was no clear difference in the expression of the SIRT1 gene in the “TC12H group”, “TC24H group”, “TC48H group”, and “TC72H group”. This shows that the effect of increasing SIRT1 gene expression of stem cells by TC is maintained for at least 72 hours.

(3-3) Semi-quantitative RT-PCR
Take 2.5 microliters of the adipose-derived stem cell cDNA of “untreated group”, “H ₂ O ₂ group” and “H ₂ O ₂ + TC6 group” obtained in (3-1) above, and prepare a 2.5 μL table. 1. Add corresponding primers (concentration 10 μM) for each gene shown in 1, 7.5 μL of deionized water and 12.5 μL of EconoTaq® PLUS GREEN 2X Master Mix (Lucigen, Middleton, Wisconsin, USA). The reaction was carried out in a polymerase chain reactor. The reaction conditions were set as follows. i) 94 cycles of 30 ° C., 30 seconds, 55 ° C., 30 seconds, 94 ° C., 60 seconds, 30 cycles of reverse transcription polymerase chain reaction (RT-PCR) were performed. ii) 72 ° C, 10 minutes. iii) Finally, the reaction was terminated by cooling to 4 ° C. The obtained polymerase chain reaction (PCR) product was electrophoresed with 2% agaropectin for 30 minutes (voltage: 100 V), agaropectin was put into ethidium bromide (EtBr), and stained for 10 minutes. Images were taken using a gel imaging system (DOC PRINT DP-001FDC, Vilber Lourmat France). The results are shown in FIG. 6A. Finally, the expression level of each gene was quantified by software (Image J). The result is shown in FIG. 6B.

As can be seen from FIGS. 6A and 6B, as compared to the "untreated group", the expression of SIRT1 gene _"H 2 _{O 2} sets" are clearly reduced, compared to the _"H 2 _{O 2} sets" The expression of SIRT1 gene of “H ₂ O ₂ + TC6 pair” was clearly increased. Compared with "untreated group", the expression of p21 and p53 genes, _"H 2 _{O 2} sets" are obviously increased, compared to the _"H 2 _{O 2} pairs", _"H 2 _O 2 + TC6 sets" The expression of p21 and p53 genes was clearly decreased. Compared to _"H 2 _{O 2} pairs", _"H 2 _O 2 + TC6 pairs" of OCT4 (octamer-binding transcription factor 4 ) and no clear difference in the expression of NANOG gene.

  It is known that SIRT1 gene expression is related to aging, p21 and p53 gene expression is related to cell cycle regulation, and OCT4 and NANOG gene expression is related to stem cell pluripotency. Yes. Thus, the above results indicate that TC has the ability to regulate stem cell anti-aging and cell cycle, while not affecting stem cell pluripotency.

(3-4) Microarray Analysis
Using the reverse transcription kit (RT Pre Mix, purchased from iNtRON Biotechnology) and the corresponding primers for each gene shown in Table 2 and the corresponding primers for the SIRT1 gene and p53 gene shown in Table 1, they were obtained in (3-1) above. About 20 or 30 cycles of reverse transcription polymerase chain reaction was performed on the total RNA of “H ₂ O ₂ group” and “H ₂ O ₂ + TC6 group” adipose-derived stem cells to obtain single-stranded cDNA of each gene. .

The above cDNA was hybridized with a gene chip (affymetrix GeneChip Human Genome U133 plus 2.0 Array, Affymetrix), and then the microarray was scanned with a microarray scanner system (Gene Chip (registered trademark) Scanner 3000) to analyze the data. As shown in Table 3, by comparison with the _"H 2 _{O 2} sets" of gene expression (1 time gene expression in _"H 2 _{O 2} sets" setting), gene expression _"H 2 _O 2 + TC6 sets" Is shown in multiples.

As can be seen from Table 3, compared to the _"H 2 _{O 2} sets" cellular growth-related genes in adipose-derived stem cells _"H 2 _O 2 + TC6 sets" (i.e., NYFA, CCND1, EIF5, SIRT1 , E2F6, POLR3H, The expression of hTERT and PCNA) was all clearly increased and the expression of cellular apoptosis related genes (ie GADD45B, PIM1, p53 and p16) was all clearly decreased. As this result shows, TC increases cell growth rate by increasing transcription, translation-related gene expression and SIRT1 gene expression in the cell.

(4) Test for measuring telomerase activity Telomerase activity is a key regulator of cell senescence. Therefore, analysis of telomerase activity in adipose-derived stem cells of “untreated group”, “H ₂ O group ₂ ” and “TC6H group” of Example 1 (1) by the telomerase repeat sequence amplification method (TRAP). Went. First, a primer at the 3 ′ end of telomere (namely, Telo TAGGG) is extended by the catalysis of telomerase by polymerase chain reaction (PCR), and a TTAGGG repeat sequence is amplified, and then each set by enzyme immunoassay (ELISA). The product signal of was detected. The results are shown in FIG. In addition, telomerase activity in HEK293 cells (no treatment was performed) was used as a positive control.

As can be seen from FIG. 7, the telomerase activity of “H ₂ O ₂ group” is clearly decreased as compared with “Untreated group”, and compared with “H ₂ O ₂ group”, “TC6H group” Telomerase activity was clearly improved. This result indicates that stem cells were aged by treatment with H ₂ O ₂ and that telomerase activity of stem cells aged by treatment with TC was improved, which indicates that TC has the anti-aging ability of stem cells. .

(5) Test of bone marrow mesenchymal stem cell and Wharton's jelly stem cell (5-1) Activity measurement test of SA-β-gal Using each set of bone marrow mesenchymal stem cell and Wharton's jelly stem cell of Example 1, The step (2) was repeated. The results are shown in FIGS. 8A to 8D and FIGS. 9A to 9D, respectively.

As can be seen from FIGS. 8A to 8D, cells exhibiting SA-β-gal activity in human bone marrow mesenchymal stem cells of “H ₂ O ₂ group” as compared to “untreated group” human bone marrow mesenchymal stem cells. the number is clearly increased, as compared with human bone marrow mesenchymal stem cells "H ₂ O ₂ pairs", the SA-β-gal activity in human bone marrow mesenchymal stem cells "H _{2 O 2} + _TC6 sets" The number of cells shown was clearly reduced. This result is consistent with that of adipose-derived stem cells.

As can be seen from FIGS. 9A to 9D, the number of Wharton's jelly stem cells exhibiting SA-β-gal activity in the “H ₂ O ₂ ” Wharton's jelly stem cells as compared to the “untreated” Wharton's jelly stem cells. has increased clearly compared with Wotonzu jelly stem cells _"H 2 _{O 2} sets" cells exhibiting SA-β-gal activity in Wotonzu jelly stem cells _"H 2 _O 2 + TC6 sets" clearly It was decreasing. This result is also consistent with the result of adipose-derived stem cells.

(5-2) SIRT1 gene expression analysis Using the cDNAs of human bone marrow mesenchymal stem cells and Wharton's jelly stem cells obtained in (3-1) in the same manner as in the above step (3-2). Quantitative RT-PCR was performed. The results are shown in FIGS. 10A and 10B.

As can be seen from FIG. 10A, the expression of SIRT1 gene in human bone marrow mesenchymal stem cells of “H ₂ O ₂ group” is clearly reduced compared to “untreated group” human bone marrow mesenchymal stem cells, This indicates that the cells were aged by H ₂ O ₂ treatment. On the other hand, compared to human bone marrow mesenchymal stem cells "H ₂ O ₂ pairs", the expression of the SIRT1 gene in human bone marrow mesenchymal stem cells "H _{2 O 2} + _TC6 sets" are obviously increased, which , Indicating that TC has the anti-aging ability of stem cells. This result is consistent with that of adipose-derived stem cells.

As can be seen from FIG. 10B, the expression of the SIRT1 gene in the “H ₂ O ₂ ” Wharton's jelly stem cells is clearly reduced compared to the “untreated” Wharton's jelly stem cells, which is H _2. It shows that the cells were aged by O ₂ treatment. On the other hand, compared with Wotonzu jelly stem cells _"H 2 _{O 2} pairs", the expression of the SIRT1 gene in Wotonzu jelly stem cells _"H 2 _O 2 + TC6 sets" are obviously increased, which, TC stem cells It has anti-aging ability. This result is also consistent with the result of adipose-derived stem cells.

B. Animal Experiment Example 5: Construction of Liver Fibrosis Rat Model Eight weeks of male Wister rats (total 35 animals) were given 200 mg / kg body weight of thioacetamide (TAA) every 3 days by intraperitoneal injection. A single injection was made for a total of 60 days (ie a total of 20) to obtain a liver fibrosis rat model referred to as the “TAA set” rat. Separately, normal “comparison group” rats (total of 12 rats) that were not injected with thioacetamide and were injected with the same volume of normal saline were also prepared. Four days after the last injection was completed, four “comparison group” rats and three “TAA group” rats were sacrified with ether and analyzed as follows.

(1) Histopathological score evaluation Rat tissue samples of rats of “Comparative group” and “TAA group” were taken, and then the tissue samples were cut and stained into sections, and histopathological score evaluation was performed. The results are shown in FIGS. 11A and 11B. The staining methods are Hematoxylin and Eosin Staining (H & E Staining) and Masson's trichrome staining, which are Adipose-derived stem cells can abrogate chemical-induced liver fibrosis and facilitate recovery of liver function. Cell Transplant. 21 (12): 2753-2764 (2012), the full text of this document is referred to here. The results of hematoxylin and eosin staining and Masson trichrome staining were analyzed by Metavir staging system, and as shown in Table 5 below, staging was performed to obtain the activity score and fibrosis score, respectively. . The higher the active score, the greater the degree of hepatitis. The higher the fibrosis score, the greater the degree of fibrosis.

  As can be seen from FIG. 11A (Masson trichrome staining result), accumulation of collagen, which is characteristic of liver fibrosis, and the connection of two blood vessels were observed in the liver tissue of the “TAA group” rat. Further, when the results shown in FIG. 11A were analyzed by the Metavir staging system, the fibrosis score of the “TAA group” rat was 4 while the fibrosis score of the “comparison group” rat was 0. From this result, it was found that the rat liver became fibrotic severely after long-term injection of TAA (and thus developed cirrhosis).

  As can be seen from FIG. 11B (hematoxylin and eosin staining results), the liver tissue of the “TAA group” rat is clearly infiltrated with macrophages. Further, when the results shown in FIG. 11B were analyzed by the Metavir staging system, the number of active sites of the “TAA” rat was 3 while the number of active sites of the “comparison group” rat was 0. From this result, it was found that the liver of rats exhibited severe inflammatory symptoms by long-term injection of TAA.

(2) Biochemical function index analysis Collect the heart blood of “comparison group” and “TAA group” rats and use the biochemical module (purchased from Roche, model number: Integra 800) to determine the biochemical function index of the liver. Analyzes were made to assess the extent of liver injury. Biochemical function index includes glutamate oxaloacetate transaminase (GOT) expression level, glutamate pyruvate transaminase (GPT) expression level, albumin expression level, total bilirubin (total bilirubin) ) Expression level and prothrombin time. The results are shown in Table 4.

  As can be seen from Table 4, in comparison with the “comparison group”, the expression levels and prothrombin time in the blood of GOT, GPT and total bilirubin in the “TAA group” rats all increased clearly. The expression level in blood was clearly reduced. From this result, it was reconfirmed that the rat liver was damaged by long-term injection of TAA.

Example 6: Strengthening effect of trans-cinnamaldehyde on the therapeutic effect of stem cells The remaining 32 “TAA group” rats in Example 5 were treated with “pseudo-injection group”, “stem cell group”, “H ₂ O group ₂ ” and “TC”. Each group was divided into “groups” (eight groups for each group), and different treatments (the day of treatment was called “Day 0 after treatment”) were performed. The “pseudo-injection set” was injected with only 300 microliters of normal saline into the fibrotic liver of rats. The “stem cell set” was injected into the fibrotic liver of rats with 300 microliters (containing 1 × 10 ⁶ cells) of the “untreated set” adipose-derived stem cells obtained in Example 1 (positive control set). . “H ₂ O ₂ set” was injected into the fibrotic liver of rats with 300 microliters (containing 1 × 10 ⁶ cells) of “H ₂ O ₂ set” adipose-derived stem cells obtained in Example 1. (Negative control group). “TC set” was injected into the fibrotic liver of rats with 300 microliters (containing 1 × 10 ⁶ cells) of the “H ₂ O ₂ + TC6 set” adipose-derived stem cells obtained in Example 1.

  On the 7th day, each group of rats was treated differently, each group of 4 rats was sacrified with ether, and after the treatment, on the 14th day, the remaining 4 rats in each group were sacrified. . Samples of heart blood and liver tissue from each set of rats were collected and analyzed as follows.

(6-1) α-fetoprotein (α-fetoprotein, AFP) immunohistochemical staining “Comparison group (untreated group)”, “Pseudo-injection group”, “Stem cell group”, “H ₂ O group ₂ ” and “TC” Α-fetoprotein immunohistochemical staining analysis was performed on a section of liver tissue of the “set” rat with a human α-fetoprotein antibody (Calbiochem, ST1673). The results of “day 7” after stem cell transplantation are shown in FIGS. 12A to 12E, and the results of “day 14” after stem cell transplantation are shown in FIGS. 12F to 12J. The α-fetoprotein antibody is directed against human α-fetoprotein. Therefore, cells with α-fetoprotein signal (brown cells indicated by arrows) differentiate after transplantation and become hepatocyte-like cells (hepatocyte- like cells).

As can be seen from FIGS. 12A to 12J, the “stem cell group”, “H ₂ O ₂ group”, and “TC group” liver tissue of “seventh day” and “14th day” after stem cell transplantation There are many cells with α-fetoprotein signals (where the arrows point). This result shows that each set of stem cells survives and differentiates after being transplanted into the rat liver. Below, the therapeutic effect which each group of stem cells gives to a liver fibrosis rat is explained in full detail.

(6-2) Histopathological score evaluation “Comparative group”, “Pseudoinjection group”, “Stem cell group”, “H ₂ O group ₂ ” and “TC group” The histopathological score of 1) was evaluated. The results are shown in FIGS. 13A to 13D, 14A and 14B. The results of Masson trichrome staining are shown in FIGS. 13A and 13B, and the results of hematoxylin and eosin staining are shown in FIGS. 13C and 13D. 14A and 14B are converted into data and shown in Table 6 below.

As can be seen from FIG. 13A to FIG. 13D, FIG. 14A, FIG. 14B, and Table 6, a severe inflammatory reaction occurs in the liver of rats injected with TAA for a long time, and severe fibrosis due to macrophage infiltration in the liver tissue ( Furthermore, he suffered from cirrhosis). However, in the liver of rats treated with the “H ₂ O ₂ + TC6 pair” adipose-derived stem cells, the degree of the inflammatory response is reduced and the fibrosis of the liver is improved.

(6-2) Biochemical function index analysis “Comparative group”, “Pseudoinjection group”, “Stem cell group”, “H ₂ O group ₂ ” and “TC group” The biochemical function index analysis of 2) was performed. The results are shown in FIGS. 15A to 15E.

As can be seen from FIG. 15A, the GPT expression level of the “stem cell group” is clear compared with the GPT expression level of the “H ₂ O ₂ group” (93 ± 13 units / L) on the “day 14” after the treatment. Decreased (72 ± 14 units / L). In addition, compared with the “stem cell group”, the GPT expression level of the “TC group” was also clearly reduced (66 ± 14 units / L). This result indicates that the treatment with the “H ₂ O ₂ + TC6 pair” adipose-derived stem cells effectively reduces the amount of GPT expression in the blood of liver fibrosis rats.

As can be seen from FIG. 15B, the GOT expression level of the “stem cell group” is clearly decreased as compared to the GOT expression level (208 ± 12 units / L) of the “pseudo-injection group” on “day 14” after the treatment. (162 ± 60 units / L). In addition, compared with the “stem cell group”, the GOT expression level of the “TC group” was also clearly reduced (137 ± 18 units / L). This result shows that the treatment with fat-derived stem cells of “H ₂ O ₂ + TC6 group” effectively reduces the GOT expression level in the blood of liver fibrosis rats.

As can be seen from FIG. 15C, the albumin expression level of the “pseudo-injection group” (3 × 3) is compared with the albumin expression level of the “stem cell group” (3.52 ± 0.28 g / dL) on “day 14” after the treatment. .23 ± 0.01 g / dL) was clearly low. In addition, the albumin expression level of the “TC group” was clearly increased as compared to the “pseudo-injection group” (3.48 ± 0.03 g / dL). This result indicates that the treatment with the “H ₂ O ₂ + TC6 pair” adipose-derived stem cells effectively increases the expression level of albumin in the blood of liver fibrosis rats.

As can be seen from FIG. 15D, the prothrombin time of the “TC group” (10.88 ±±) compared to the prothrombin time of the “pseudo-injection group” (23.10 ± 2.5 seconds) on the “day 14” after the treatment. 0.67 seconds) clearly decreased. This result indicates that prothrombin time in liver fibrosis rats is effectively reduced when treated with the “H ₂ O ₂ + TC6 pair” adipose-derived stem cells.

As can be seen from FIG. 15E, compared to the total bilirubin expression level of “stem cell group” (0.05 ± 0.02 mg / dL) on “day 14” after treatment, the total bilirubin expression level of “TC group” ( (0.034 ± 0.03 mg / dL) clearly decreases, and approaches the total bilirubin expression level (0.02 ± 0.02 mg / dL) of the “comparison group”. This result shows that the treatment with adipose-derived stem cells of “H ₂ O ₂ + TC6 group” effectively reduces the total bilirubin expression level in the blood of liver fibrosis rats.

  The above results indicate that treatment of stem cells with trans-cinnamaldehyde (TC) is effective for stem cell anti-aging, thereby increasing the therapeutic effect of stem cells.

  The embodiments disclosed in the present specification are for the purpose of explaining, not limiting the present invention, and the spirit and scope of the present invention are not limited by such embodiments. The protection scope of the present invention should be construed according to the claims, and all technologies within the equivalent scope should be construed as being included in the scope of the present invention.

Claims (10)

  Anti-senescence of stem cells and / or stem cells, characterized in that the method comprises treatment of stem cells with trans-cinnamaldehyde (TC), wherein the stem cells are not human totipotent stem cells How to rejuvenate.   The anti-senescence of stem cells according to claim 1, wherein the stem cells are embryonic stem cells, adult stem cells or induced pluripotent stem cells. And / or stem cell rejuvenation methods.   The stem cell anti-senescence and / or stem cell rejuvenation method according to claim 1, wherein the stem cells are treated with 0.1 to 50 µg / mL trans-cinnamaldehyde in a stem cell culture medium.   A preparation using trans-cinnamaldehyde, which is used for anti-aging of stem cells and / or rejuvenation of stem cells, wherein the stem cells are not human totipotent stem cells.   The preparation using trans-cinnamaldehyde according to claim 4, wherein the preparation is a health food or a medicine.   The preparation using trans-cinnamaldehyde according to claim 4, wherein the stem cells are embryonic stem cells, adult stem cells or induced pluripotent stem cells. (1) a first portion having stem cells;
(2) a second portion having trans-cinnamaldehyde;
(3) In a kit comprising a third part and / or a stem cell culture medium present in the first part and / or the second part,
The stem cell is not a human totipotent stem cell, and when the kit is used, the stem cell is treated with trans-cinnamaldehyde in the stem cell culture solution, and then the treated stem cell is treated with the stem cell. The kit used for the treatment by the stem cell characterized by using.   The kit used for treatment with stem cells according to claim 7, wherein the stem cells are at least one selected from embryonic stem cells, adult stem cells, and induced pluripotent stem cells.   The kit used for treatment with stem cells according to claim 7, wherein the stem cells are at least one selected from adipose-derived stem cells, bone marrow mesenchymal stem cells, and Wharton's jelly stem cells.   The kit used for treatment with stem cells according to claim 7, wherein trans-cinnamaldehyde is used at a concentration of 0.1 to 50 µg per mL with respect to the stem cell culture medium.