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 collagen fiber formation 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 dermal part. Collagen is produced by fibroblasts in the dermis. Collagen is first produced inside the cell and 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 the cells. The collagen secretion promoting effect of the extract of Drosophila melanogaster is known (Patent Document 1). In addition, a collagen secretion promoter containing berberine is known (Patent Document 2). Further development of collagen secretion promoters 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] To provide a collagen secretion promoter. Also, to provide a SEC12 protein expression promoter for promoting the secretion of collagen.

Means for Solving the Problems

[0005] The main configuration of the present invention is as follows. 1. A collagen secretion promoter containing ectoine. 2. A SEC12 protein expression promoter containing ectoine.

Effects of the Invention

[0006] With the collagen secretion promoter of the present invention, the collagen produced intracellularly is rapidly secreted extracellularly. If the collagen secretion promoter of the present invention is allowed to act 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 the firmness and elasticity of the skin can be expected. It is expected that the SEC12 protein expression promoter of the present invention promotes the extracellular secretion of collagen produced intracellularly.

Brief Description of the Drawings

[0007]

Figure 1

Figure 2

Figure 3

Modes for Carrying Out the Invention

[0008] The collagen secretion promoter and SEC12 protein expression promoter of the present invention contain ectoine. Ectoine is a kind of cyclic amino acid obtained from the halophilic bacteria (Halomonas elongata) inhabiting the salt lakes in Egypt. Since ectoine has the property of incorporating water molecules, it has a high water retention capacity and protects the protein structure by stopping the movement of hydration water. In addition, ectoine has the effect of increasing heat shock protein.

[0009] The concentration of ectoine 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 promoter of the present invention can be made into an external preparation for skin or an oral preparation. Examples of external preparations for skin include lotions, emulsions, creams, gels, ointments, etc., and examples of oral preparations include tablets, powders, granules, beverages, etc.

Example

[0011] <Confirmation of the effect of promoting collagen secretion and promoting SEC12 protein expression of ectoine> The material was added to NIH-3T3 cells transfected with visualized human type I procollagen, and the amount of collagen secretion after the addition of the material was evaluated over time by measuring the amount of GFP bound to collagen. In addition, the cells at the time of addition were collected and the expression level of SEC12 protein was measured.

[0012] <Vector construction> A vector having a nucleic acid in which EGFP was bound inside human preprocollagenIα1 cDNA and mCherry was bound to the C-terminus, as described in Example 1 of WO2016 / 152882, was constructed. EGFP was inserted into the BamHI site of preprocollagenIα1 cDNA, and mCherry was inserted into the EcoR1 site of the C-propeptide. The vector construction method is as follows. A DNA fragment with restriction enzyme BamHI sites added to both ends of EGFP cDNA was prepared and inserted into the BamHI site inside human preprocollagen1α1 cDNA. At this time, in protein translation, regulation was carried out so that the collagen protein and the EGFP protein were connected. Next, a DNA fragment with EcoR1 sites added to both ends of mCherry cDNA was prepared and inserted into the EcoR1 site of human preprocollagen1α1-EGFP fusion cDNA. Also at this time, in protein translation, regulation was carried out so that the collagen protein and the mCherry protein were connected, and the nucleotide sequence was confirmed at each step of the operation. The cryopreserved competent cells of TOP10F’ were thawed, and immediately 100 ng of plasmid DNA was added and mixed. After standing on ice for 30 minutes, it was warmed at 42°C for 1 minute and 30 seconds and immediately ice-cooled. 1 mL of medium was added here and spread on an LB plate supplemented with 100 μL of ampicillin. It was incubated overnight at 37°C, and the colonies that appeared were used. This operation was repeated to obtain the vector. The obtained vector contained the nucleic acid of the target length. All clones could be cut with EcoRI.

[0013] <Fluorescent Observation of Collagen in Cells> The vector obtained by the above procedure was introduced into mouse NIH3T3 cells, and fluorescence was detected with a confocal fluorescence microscope. The fluorescence signals of EGFP and mCherry were observed 48 hours later with a fluorescence microscope. Since EGFP was inserted into the collagen protein and mCherry was inserted into the C-propeptide, it was confirmed that the procollagen immediately after synthesis was present in the cytoplasm by a yellow signal, and the collagen protein cleaved by processing was present in the cytoplasm by a green fluorescence signal.

[0014] <Measurement of the Amount of Collagen Secreted Extracellularly> NIH3T3 cells of mice into which the vector obtained above was introduced (EGFP-Collagen-introduced cells) were seeded in a 96-well plate at a cell density of 3,500 cells / well, cultured for 3 days at 37 °C in the presence of 5% CO2, and then the evaluation raw material solution (Table 1) was diluted using 1% FBS / PS / phenol red free DMEM, and each raw material was added so as to have the concentration of mass% described in Table 1, and the cells were cultured 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 the 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 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 were divided by the measured values 8, 24, and 30 hours after without the evaluation raw material (None) respectively to obtain the 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 (EGFP-Collagen-introduced 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. 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 standardized with the protein concentration of each sample. Using the standardized measured value 2 hours after the addition of ectoine as a reference, the ratio with the standardized 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 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, the evaluation raw material solution (Table 3) was diluted and added using 1% FBS / PS / phenol red free DMEM, and then cultured in a CO2 incubator at 37°C. After 24 hours of addition, 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 placed at -80°C and cell disruption was achieved by freeze-thawing three times. After measuring the protein concentration of the cell lysate by the BCA method, it was prepared for WB (western blot) and WB was performed. SEC12 was detected using an anti-Preb (SEC12) antibody (10146-2-AP, Proteintech). The band intensity of the 45 kDa band derived from SEC12 was measured and the value was normalized by the protein concentration of each sample. Based on the normalized measured value without the evaluation raw material (None), the ratio of the normalized measured value of each evaluation raw material solution added was determined to evaluate the expression level of the SEC12 protein (Table 3, Figure 3). As a result, compared with TGF-β1 added as a positive control, the addition of ectoine promoted the expression of the SEC12 protein more.

[0019]

Table 3

Claims

【Claim 1】 An SEC12 protein expression promoter containing ectoine.

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