SEC12 protein expression promoter

Abstract

To provide a collagen secretion promoter and also provide an SEC12 protein expression promoter to promote the secretion of collagen.SOLUTION: The present invention provides a collagen secretion promoter containing ectoine, and an SEC12 protein expression promoter containing ectoine.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to an SEC12 protein expression promoter.

Background Art

[0002] Collagen is an important tissue in the dermis that gives the skin firmness and elasticity, and maintaining the function of collagen has been required. In previous approaches, attention has often been paid to the amount of collagen produced by dermal fibroblasts. However, in recent years, the biosynthetic pathway involved in the transport and secretion of collagen peptides and the process of forming collagen fibers are considered to be important. The skin has a structure of the stratum corneum, epidermis, and dermis from the outside, and collagen exists in the dermis part. Collagen is produced by fibroblasts in the dermis. Collagen is first made in cells and then becomes collagen fibers by being correctly secreted, giving the skin firmness and elasticity. Since collagen exists in an environment where it is constantly damaged by factors such as ultraviolet rays and reactive oxygen species due to stress, it is important to produce high-quality collagen earlier than it is damaged. For this purpose, it is necessary to increase the rate at which collagen is secreted from cells. The collagen secretion promoting effect of the extract of Selaginella tamariscina is known (Patent Document 1). Also, a collagen secretion promoter containing berberine is known (Patent Document 2). Further development of a collagen secretion promoter is required.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0004] The objective is to provide a collagen secretion promoter. Furthermore, to provide an SEC12 protein expression promoter in order to promote collagen secretion. [Means for solving the problem]

[0005] The main components of this invention are as follows: 1. A collagen secretion promoter containing ectoin. 2. An SEC12 protein expression promoter containing ectoin. [Effects of the Invention]

[0006] The collagen secretion promoter of the present invention causes collagen produced within cells to be rapidly secreted outside the cells. When the collagen secretion promoter of the present invention acts on fibroblasts in the dermis, the secretion of collagen into the dermis is promoted, collagen fibers are constructed in the dermis, and an improvement in skin firmness and elasticity can be expected. The SEC12 protein expression promoter of the present invention is expected to promote the secretion of collagen produced within cells to the extracellular space. [Brief explanation of the drawing]

[0007] [Figure 1] The collagen secretion-promoting effect when various raw materials are added to NIH3T3 cells. [Figure 2] Increased collagen secretion over time when ectoin was added to NIH3T3 cells. [Figure 3] Ectoin's effect on promoting SEC12 protein expression in NIH3T3 cells. [Modes for carrying out the invention]

[0008] The collagen secretion promoter and SEC12 protein expression promoter of the present invention contain ectoin. Ectoin is a type of cyclic amino acid obtained from the halogensiophytic bacterium (Halomonas elongata) that inhabits salt lakes in Egypt. Because ectoin has the property of holding onto water molecules, it has high water retention capacity and protects protein structures by stopping the movement of hydration water. In addition, ectoin has the effect of increasing heat shock proteins.

[0009] The concentration of ectoin contained in the collagen secretion promoter and SEC12 protein expression promoter of the present invention is preferably 0.0001% by mass or more and 5% by mass or less.

[0010] The collagen secretion-promoting agent of the present invention can be in the form of a topical skin preparation or an oral preparation. Examples of topical skin preparations include lotions, emulsions, creams, gels, ointments, etc., while examples of oral preparations include tablets, powders, granules, beverages, etc. [Examples]

[0011] <Confirmation of ectoin's collagen secretion-promoting effect and SEC12 protein expression-promoting effect> Visualization was performed on NIH-3T3 cells introduced with human type I procollagen by adding a material, and the amount of collagen secreted after material addition was evaluated over time by measuring the amount of GFP bound to collagen. In addition, cells were collected at the time of addition, and the SEC12 protein expression level was measured.

[0012] <Vector Construction> A vector was constructed containing nucleic acid in which EGFP was bound to the inside of human preprocollagenIα1 cDNA and mCherry was bound to the C-terminus, as described in Example 1 of Publication WO2016 / 152882. EGFP was inserted into the BamHI site of the preprocollagenIα1 cDNA, and mCherry was inserted into the EcoR1 site of the C-propeptide. The vector construction method is as follows: A DNA fragment was created by adding restriction enzyme BamHI sites to both ends of EGFP cDNA, and this was inserted into the BamHI site inside human preprocollagen1α1 cDNA. During this process, adjustments were made to ensure that the collagen protein and EGFP protein were linked during protein translation. Next, a DNA fragment was created by adding EcoR1 sites to both ends of mCherry cDNA, and this was inserted into the EcoR1 site of human preprocollagen1α1-EGFP fusion cDNA. Again, adjustments were made to ensure that the collagen protein and mCherry protein were linked during protein translation, and the base sequence was verified at each step of the procedure. Competent cells from cryopreserved TOP10F' were thawed, and 100 ng of plasmid DNA was immediately added and mixed. After standing on ice for 30 minutes, the mixture was heated at 42°C for 1 minute 30 seconds and immediately cooled on ice. 1 mL of culture medium was added, and the mixture was spread onto an LB plate supplemented with 100 μL of ampicillin. The plate was incubated overnight at 37°C, and the resulting colonies were used. This procedure was repeated to obtain the vector. The obtained vector contained nucleic acid of the desired length. All clones could be cleaved with EcoRI.

[0013] <Fluorescence observation of collagen within cells> The vectors obtained using the above procedure were introduced into mouse NIH3T3 cells, and fluorescence was detected using a confocal fluorescence microscope. The fluorescence signals of EGFP and mCherry were observed 48 hours later using the fluorescence microscope. Because EGFP is inserted into the collagen protein and mCherry is inserted into the C-propeptide, it was confirmed that the procollagen immediately after synthesis was present in the cytoplasm, indicated by a yellow fluorescence signal, while the collagen protein cleaved by processing was present, indicated by a green fluorescence signal.

[0014] <Measurement of the amount of collagen secreted extracellularly> NIH3T3 cells of mice transfected with the vector obtained above (EGFP-Collagen transfected cells) were seeded in a 96-well plate at a cell density of 3,500 cells / well. After culturing for 3 days at 37°C in the presence of 5% CO2, the evaluation raw material solution (Table 1) was diluted using 1% FBS / PS / phenol red free DMEM, and each raw material was added to reach the concentration of mass% described in Table 1, followed by culturing in a CO2 incubator at 37°C. The culture supernatant was collected in a round-bottom 96-well plate 8, 24, and 30 hours after addition. 100 μl of the centrifuged (1,000 rpm, 5 min, RT) supernatant was transferred to a black-bottom 96-well plate immediately before measurement, and EGFP fluorescence was measured using a fluorescence photometer EnSpire (PerkinElmer). A purified standard product of EGFP (0 - 20 ng / μl) was used for the preparation of the standard curve. The measured values of each evaluation raw material 8, 24, and 30 hours after addition were divided by the measured values 8, 24, and 30 hours after addition of the evaluation raw material without addition (None) to obtain a ratio, and the amount of EGFP-Collagen secreted into the culture supernatant was relatively evaluated. The results are shown in Table 1 and Figure 1. Ectoin showed a remarkable effect of promoting collagen secretion.

[0015]

Table 1

[0016] NIH3T3 cells of mice transfected with the vector obtained above (EGFP-Collagen transfected cells) were seeded in a 12-well plate at a cell density of 100,000 cells / well. After culturing at 37°C in the presence of 5% CO2 for 24 hours, ectoine was diluted using 1% FBS / PS / phenol red free DMEM and added to a concentration of 0.1% by mass, and then cultured in a CO2 incubator at 37°C. The culture supernatant was collected in a round-bottom 96-well plate 2, 4, 16, 24, 48, 72, and 96 hours after the addition. After collection, the supernatant was centrifuged (1,000 rpm, 5 min, RT) to prepare samples for WB (western blot). Using the prepared samples, WB was performed, and EGFP-Collagen was detected with an anti-GFP antibody (2956, CST). The band intensity of approximately 180 kDa derived from EGFP-Collagen was measured, and the value was normalized with the protein concentration of each sample. Based on the normalized measured value 2 hours after the addition of ectoine, the ratio with the normalized measured values 4, 16, 24, 48, 72, and 96 hours after the addition was determined to examine the change over time in the amount of collagen secretion (Table 2, Figure 2). The addition of ectoine increased the amount of collagen secretion over time.

[0017]

Table 2

[0018] <Measurement of the expression level of SEC12 protein produced intracellularly> NIH3T3 mouse cells (EGFP-Collagen-transformed cells) into which the vector obtained above was introduced were seeded in a 12-well plate at a cell density of 100,000 cells / well and cultured for 24 hours at 37°C in the presence of 5% CO2. Then, the evaluation raw material solution (Table 3) was diluted with 1% FBS / PS / phenol red-free DMEM and added, and the cells were cultured in a CO2 incubator at 37°C. After 24 hours, the medium was removed, and 50 μl of Laemmli buffer (0.09 M Tris-HCl, pH 6.8, 3% SDS, 10% glycerol) / protease inhibitor cocktail (Sigma Aldrich) was added to each well to prepare cell lysate. The cell lysate was lysed by freezing and thawing three times at -80°C. The protein concentration of the cell lysate was measured by BCA, and then prepared for Western blotting (WB), and WB was performed. SEC12 was detected using the anti-Preb (SEC12) antibody (10146-2-AP, Proteintech). The band intensity of 45 kDa derived from SEC12 was measured, and the value was standardized by the protein concentration of each sample. The expression level of SEC12 protein was evaluated by determining the ratio of the standardized measurement values ​​with each evaluation material added to the standardized measurement value of the sample without any evaluation material added (None) (Table 3, Figure 3). As a result, SEC12 protein expression was more promoted with the addition of ectoin compared to TGF-β1, which was added as a positive control.

[0019] [Table 3]

Claims

[Claim 1] An SEC12 protein expression promoter containing ectoin.

Images