AU2017100034A4 - Use of Ethyl p-Methoxycinnamate and Derivatives thereof in Maintaining Self-Renewal and Pluripotency of Stem Cells - Google Patents

Abstract

Abstract Disclosed is a use of ethyl p-methoxycinnamate and derivatives thereof in preparing drugs, culture media, regulating agents or cosmetic and skin caring products for maintaining self-renewal and pluripotency of stem cells. The stem cells comprise adult stem cells, embryonic stem cells and induced pluripotent stem (iPS) cells. Also disclosed is a use of ethyl p-methoxycinnamate and the derivatives thereof in in-vitro amplification of stem cells and preparation of induced pluripotent stem (iPS) cells and a use of ethyl p-methoxycinnamate and the derivatives thereof in preparing drugs for treating cell deletion or injury diseases.

Description

Use of Ethyl p-Methoxycinnamate and Derivatives thereof in Maintaining Self-Renewal and Pluripotency of Stem Cells

Technical FieldThe reference to prior art in this specification is not and should not be taken as an acknowledgment or any form of suggestion that the referenced prior art forms part of the common general knowledge in Australia or in any other country.

The present disclosure belongs to the technical field of biological medicine, and relates to a use of a class of small molecule compounds, i.e. ethyl p-methoxycinnamate and derivatives thereof in maintaining self-renewal and pluripotency of stem cells. The present disclosure also relates to a use of this class of small molecule compounds in preparing drugs for treating cell deletion or injury diseases and cosmetic and skin caring products, as well as a use of in-vitro amplification of stem cells and preparation of induced pluripotent stem (iPS) cells.

Background Art

Stem cells are a class of primitive cells with self-renewal capacity and multi-directional differentiation potential, including embryonic stem cells, adult stem cells and induced pluripotent stem (iPS) cells. Stem cells have a very important theoretical research significance and clinical application value. Using the embryonic stem cells to establish in vitro differentiation systems of a variety of cell types enables people to study the development process of mammals at the levels of cells and molecules; comparing gene transcription and expression of the embryonic stem cells with the stem cells and differentiated cells at different developmental stages can determine the molecular mechanism of embryo development and cell differentiation, meanwhile new genes can be discovered; the research of stem cells provides a new research means for the pharmacology, pharmacodynamics, toxicology, pharmacokinetics and the like of new drugs, overcoming the difficulty that the above experiments cannot be carried out directly in human body, meanwhile improving the effectiveness and safety of drug uses. Stem cells also have important applications in regenerative medicine, and the application of stem cells in the regenerative medicine involve all tissues and organs of human body, and also relate to most of medical problems faced by the mankind, such as cardiovascular diseases, diabetes, Parkinson’s disease, cancer, Alzheimer’s disease, autoimmune diseases, severe burns, spinal cord injury, hereditary diseases, osteoporosis, aging and so on.

In regenerative medicine, stem cell transplantation therapy has become an important means for the treatment of diseases, such as cell transplantation, organ transplantation and so on. Cell transplantation therapy is to transplant healthy stem cells into a patient’s body, to repair or replace injured cells or tissues, so as to achieve the purpose of cure. Organ transplantation therapy takes stem cells as “seeds” to be subjected to directional induced differentiation in vitro to form desired tissues and organs, so as to replace the diseased tissues and organs. To use the stem cells in clinical treatment, first of all there must be sufficient sources, for example, the stem cells are generally required to be 1-4x106/kg body weight for adult transfusion, while the content of the stem cells in each part of cord blood is only 1 x107 or so, therefore the current treatment is merely limited to children. The clinical application shows that the greater the input amount, the more the successful homing after transplantation, and the greater the probability of long-term rooting and reproduction. However, the stem cells in mammals have a very small number and tend to spontaneously differentiate, thus it is quite difficult to culture them in vitro for a long period of time in a large number. If the obtained rate of the stem cells can be tried to be improved during the process of in vitro isolation and culture, it will undoubtedly open up broad prospects for clinical applications of the stem cells.

Although growth factors and bioactive proteins can act as regulating drugs of stem cells, growth factors and bioactive proteins as macromolecular active substances, can intervene in the crisscross physiological processes in body, exhibit extremely complex multiple regulating functions, and are expensive and hard to have medicinal and clinical therapeutic value. Therefore, the potential of small molecule compounds as stem cell regulating drugs has been increasingly attached significance to by people. The small molecule compounds as drugs or culture medium additives have their unique advantages: the small molecule compounds are easy to administrate, and also easy to withdraw after physiological functions are recovered; the small molecule compounds are easy to artificially synthesize and achieve industrialized production, and have a good prospect of medicinal use; the small molecule compounds, especially natural small molecule compounds, having undergone biological metabolic processes, possess good biocompatibility, especially fewer toxic and side effects. Therefore, to search for small molecule compound regulating agents for stem cells has become a research focus of stem cell drugs.

Disclosure

An object of the present disclosure is to provide a class of small molecule compounds, namely, ethyl p-methoxycinnamate and derivatives thereof, that in some aspects may the effect of maintaining self-renewal and pluripotency of stem cells.

The present disclosure is specifically realized through the following technical solution:

The present disclosure provides a use of ethyl p-methoxycinnamate and derivatives thereof in preparing drugs, culture media, regulating agents in maintaining self-renewal and pluripotency of stem cells.

The general structural formula of the ethyl p-methoxycinnamate and the derivatives thereof is as follows:

wherein Ri is 4-OCH3, R2 is H, R3 is H, and R4 is -CH2CH3; or R1 is 4-OCH3, R2 is H, R3 is H, and R4 is -CH2CH2CH(CH3)CH3; or R1 is 4-OCH3, R2 is H, R3 is COOCH2CH3, and R4 is -CH2CH3; or R1 is 4-OCH3, R2 is H, R3 is COCH3, and R4 is -CH2CH3; or R1 is 4-OCH3, R2 is H, R3 is CN, and R4 is -CH2CH3; or R1 is 4-OCH3, R2 is CH3, R3 is H, and R4 is -CH2CH(CH2CH3)CH2CH2CH2CH3; or R1 is 3,4-OCH3, R2 is H, R3 is COCH2CH3, R4 is -CH2CH3; or R1 is 3,4-OCH3, R2 is H, R3 is CN, and R4 is -CH2CH3; or R1 is 2,4-OCH3, R2 is H, R3 is CN, and R4 is -CH2CH3; or R1 is 3,4,5-OCH3, R2 is CH3, R3 is H, and R4 is -CH2CH3; or R1 is 2-OCH2CH3, R2 is H, R3 is H, and R4 is -CH2CH3; or R1 is 4-OCH2CH3, R2 is H, R3 is H, and R4 is -CH2CH3; or R1 is 4-OCH2CH2CH2CH3, R2 is H, R3 is CN, and R4 is -CH2CH3; or R1 is 3-COOCH3, R2 is H, R3 is H, and R4 is -CH2CH3; or R1 is 2-CH3-5-COOCH3, R2 is H, R3 is H, and R4 is -CH2CH3; or R1 is 4-OCOCH3, R2 is H, R3 is COCH2CH3, and R4 is -CH2CH3; or R1 is 3-OCH3-4-OCH2COOCH2CH3, R2 is H, R3 is H, and R4 is -CH2CH3; or R1 is 2-OH, R2 is H, R3 is H, and R4 is -CH2CH3; or R1 is 3,4-OH, R2 is H, R3 is H, and R4 is -CH2CHC(CH3)CH3; or R1 is 3,4-OH, R2 is H, R3 is H, and R4 is -CH2CH2C6H5;

or R1 is 3,4-OH, R2 is H, R3 is H, and R4 is or R1 is 3,4-OH, R2 is H, R3 is H, and R4 is or R1 is H, R2 is H, R3 is H, and R4 is -CH2CH3; or R1 is H, R2 is H, R3 is H, and R4 is -CH(CH3)CH3; or R1 is H, R2 is H, R3 is H, and R4 is -CH2CH2CH2CH3; or R1 is H, R2 is H, R3 is H, and R4 is -CH2CH(CH3)CH3; or Ri is H, R2 is H, R3 is H, and R4 is -CH(CH3)CH2CH3; or Ri is H, R2 is H, R3 is H, and R4 is -CH2CH2CH(CH3)CH3; or Ri is H, R2 is H, R3 is H, and R4 is -CH2CH2CH2OOCCH=CHC6H5; or Ri is H, R2 is H, R3 is H, and R4 is -CH2CH2C6H5; or Ri is H, R2 is H, R3 is H, and R4 is -CH2CH(CH3)C6H5; or Ri is H, R2 is H, R3 is CN, and R4 is -CH2CH3; or Ri is H, R2 is CH3, R3 is H, and R4 is -CH2CH3; or Ri is H, R2 is C6H5, R3 is CN, and R4 is -CH2CH3; or Ri is 2,6-CI, R2 is H, R3 is H, and R4 is -CH2CH3; or Ri is 4-Br, R2 is H, R3 is H, and R4 is -CH2CH3; or Ri is 2-CF3, R2 is H, R3 is H, and R4 is -CH2CH3; or Ri is 2-OH-4-Br, R2 is H, R3 is H, and R4 is -CH2CH3; or Ri is 4-CI, R2 is H, R3 is COOCH2CH3, and R4 is -CH2CH3; or Ri is 4-CI, R2 is H, R3 is OCH2CH3, and R4 is -CH2CH3; or Ri is 3,4-F, R2 is FI, R3 is OCFI2CFI3, and R4 is -CFI2CFI3; or Ri is 2-CI-4-F, R2 is FI, R3 is CN, and R4 is -CFI2CFI3; or Ri is 3-CI-4-OCFI3, R2 is FI, R3 is CN, and R4 is -CFI2CFI3; or Ri is 2-CI-4-OCFI3, R2 is FI, R3 is CN, and R4 is -CFI2CFI3; or Ri is 4-NFI2, R2 is FI, R3 is FI, and R4 is -CFI2CFI3; or Ri is 3-CN, R2 is FI, R3 is FI, and R4 is -CFI2CFI3; or Ri is 4-N02, R2 is FI, R3 is FI, and R4 is -CFI2CFI3; or Ri is 4-(2,4-F-C6FI4), R2 is CFH3, R3 is FI, and R4 is -CFI2CFI3; or Ri is 4-OCFI3, R2 is FI, R3 is FI, and R4 is -CH2-C6H4-CH2-00C-CH=CH-C6H4-0-CH3; or Ri is 4-OCH3, R2 is H, R3 is H, and R4 is -CH2-C-CH2-00C-CH=CH-C6H4-0-CH3; or Ri, R2 and R3 form a heterocyclic structure

, and R4 is -CFI2CFI3; or Ri, R2 and R3 form a heterocyclic structure

and R4 is -CFI2CFI3; or Ri, R2 and R3 form a heterocyclic structure

and R4 is -CH2CH3; or Ri, R2 and R3 form a heterocyclic structure

and R4 is -CH2CH3; or Ri is -N-CH3(CH3), R2 is H, and R3 and R4 form a heterocyclic structure

The ethyl p-methoxycinnamate and the derivatives thereof include: ethyl p-methoxycinnamate and the derivatives thereof include: ethyl p-methoxycinnamate, iso- pentyl methoxycinnamate, diethyl 4-methoxybenzalmalonate, ethyl 2-(4-methoxybenzylidene)acetoacetate, ethyl 2-cyano-3-(4-hydroxyethylphenyl)acrylate, octyl 4-methoxycinnamate, diethyl 2-(3,4-dimethoxybenzylidene)malonate, ethyl 2- cyano-3-(3,4-dimethoxyphenyl)-2-butenoate, ethyl frans-2-cyano-3-(2,4-dimethoxyphenyl)-2-acrylate, ethyl trans- 3- (3,4,5-trimethoxyphenyl)-2-crotonate, ethyl 2-hydroxyethylcinnamate, ethyl trans- 4-ethoxycinnamate, ethyl 3-(4-butoxyphenyl)-2-cyanoacrylate, methyl 3-(frans-3-hydroxyethyl-3-oxo-1 -propenyl)benzoate, methyl 3- (frans-3-ethoxy-3-oxo-1 -propenyl)-4-methylbenzoate, diethyl 4- acetoxybenzylidenemalonate, ethyl 3-(3-methoxy-4-methoxycarbonylmethoxyphenyl)acrylate, ethyl trans- 2-hydroxycinnamate, caffeic acid-1,1-dimethylacrylate, trans-caffeic acid-1-phenylethyl ester, neochlorogenic acid, chlorogenic acid, ethyl cinnamate, /'so-propyl cinnamate, butyl cinnamate, iso-butyl cinnamate, propyl 3-methylcinnamate, iso-pentyl cinnamate, 3-cinnamoyloxypropyl 3-propylacrylate, phenylethyl cinnamate, 3-phenylpropyl cinnamate, ethyl trans- 2-cyanocinnamate, ethyl frans^-methylcinnamate, ethyl 2-cyano-3,3-diphenylacrylate, ethyl 2,6-dichlorocinnamate, ethyl trans- 4-bromo cinnamate, ethyl frans-3-(2-trifluoromethylphenyl)-2-acrylate, ethyl trans-3-(4-bromo-2-hydroxyphenyl)-2-acrylate, diethyl 4-chlorobenzylidenemalonate, ethyl c/'s-3-(4-chlorophenyl)-2-hydroxyethyl-2-acrylate, ethyl c/'s-3-(3,4-difluorophenyl)-2-ethoxy-2-acrylate, ethyl 3-(2-chloro-4-fluorophenyl)-2-cyano-2-acrylate, ethyl 3-(3-chloro-4-methoxyphenyl)-2-cyanoacrylate, ethyl 3-(2-chloro-4-methoxyphenyl)-2-cyanoacrylate, ethyl 4-aminocinnamate, ethyl 3-cyanocinnamate, ethyl 4-nitrocinnamate, 3-[2,4-difluoro(1,1-biphenyl)-4-yl]-2-butenoate, 3- [3-(4-methoxyphenyl)acryloyloxymethyl]benzyl 3-(4-methoxyphenyl)acrylate, 4- [3-(4-methoxyphenyl)acryloyloxy]butyl-2-ynyl 3-(4-methoxyphenyl)acrylate, ethyl indole-2-carboxylate, ethyl 6-methoxy-1-benzofuran-2-carboxylate, ethyl 6-amino-3-methyl-1 -benzofuran-2-carboxylate, ethyl 5-methylindole-2-carboxylate, and frans-3-(4-dimethylaminobenzylidene)dihydro-2(3/-/)-furanone.

The stem cells of the present disclosure comprise adult stem cells, embryonic stem cells or induced pluripotent stem (iPS) cells.

The ethyl p-methoxycinnamate and the derivatives thereof maintain self-renewal capacity and pluripotent state of the stem cells by promoting expression of Oct4 genes.

The pharmaceutical composition of the present disclosure comprises a therapeutically effective amount of the ethyl p-methoxycinnamate and derivatives thereof and a pharmaceutically acceptable carrier, and the carrier is solvent, dispersant, coating, antibacterial and antifungal agent, isotonic agent and absorption delaying agent.

The drugs of the present disclosure can be diluted by a user before use or used directly, and preparation thereof can be conducted by a general processing method commonly known by the person ordinarily skilled in the art.

The drugs of the present disclosure can be prepared into pharmaceutical dosage forms suitable for different administration routes, wherein preferable dosage forms are capsule and tablet. The capsule can comprise any standard pharmaceutically acceptable substances such as gelatin or cellulose. The tablet can be prepared by compressing a pharmaceutical composition with a solid phase carrier and a lubricant in a conventional manner. The solid phase carrier includes starch and sugar bentonite.

The pharmaceutical composition of the present disclosure also can be administrated through parenteral routes. Administration dosage forms for parenteral routes include aqueous solution, isotonic saline solution or 5% sugar solution of the pharmaceutical composition of the present disclosure and other formulations formed with other pharmaceutically acceptable excipients known in the art. A dosage range of formulation of the drugs of the ethyl p-methoxycinnamate and the derivatives thereof of the present disclosure for maintaining self-renewal capacity and pluripotent state of the stem cells is 0.01-100 mg/kg. A use of ethyl p-methoxycinnamate and the derivatives thereof of the present disclosure in in-vitro large-scale preparation of stem cells, which is specifically the use in in-vitro amplification of stem cells and preparation of induced pluripotent stem (iPS) cells.

The present disclosure further provides a use of ethyl p-methoxycinnamate and the derivatives thereof in preparing drugs for treating cell deletion or injury diseases. The injury diseases are injury diseases of cells, tissues or organs, specifically nervous system diseases, immune system diseases, blood system diseases, cardiovascular diseases or skin diseases.

The present disclosure further provides a use of ethyl p-methoxycinnamate and the derivatives thereof in preparing cosmetic and skin caring products.

Ethyl p-methoxycinnamate and the derivatives thereof of the present disclosure can be suspended in a pharmaceutically acceptable carrier (such as a physiological solution), and administrated orally or by intravenous infusion, or by subcutaneous, submuscular, intrathoracic, intraperitoneal, intrarectal, intravaginal, intranasal, intragastric, intratracheal, intrapulmonic injection or infusion and so on.

Ethyl p-methoxycinnamate and the derivatives thereof of the present disclosure can be suspended in the cosmetic and skin caring products, and achieve the efficacy of activating skin stem cells by means of applying.

Ethyl p-methoxycinnamate and the derivatives thereof of the present disclosure can be directly added to a cell culture solution for amplification of stem cells in vitro.

Beneficial effects of the present disclosure are as follows: by studying the small molecule compounds, it is discovered that ethyl p-methoxycinnamate and the derivatives thereof can promote expression of Oct4 genes, so as to maintain the self-renewal capacity and the pluripotent state of stem cells, and they can act as regulating drugs or culture medium additives for maintaining the self-renewal and pluripotency of stem cells, and provide a large number of stem cells for clinical transplantation, and provide a new perspective and a new idea for the treatment of cell loss or injury diseases.

Brief Description of Drawings

Figure 1 shows the effect of ethyl p-methoxycinnamate on the activity of Oct4 promoter;

Figure 2 shows the effect of ethyl p-methoxycinnamate on the expression of Oct4 mRNA;

Figure 3 shows the effect of ethyl p-methoxycinnamate on the expression of Oct4 protein;

Figure 4 shows the effect of ethyl p-methoxycinnamate on the formation of microspheres of suspendedly cultured stem cells;

Figure 5 shows the effect of ethyl p-methoxycinnamate on the formation of microspheres of adherently cultured stem cells; and

Figure 6 shows the effect of derivatives of ethyl p-methoxycinnamate on Oct4 expression; A represents chlorogenic acid, B represents ethyl 2- cyano-3,3-diphenylacrylate, C represents ethyl indole-2-carboxylate, D represents ethyl cinnamate, E represents 4-[3-(4-methoxyphenyl)acryloyloxy]butyl-2-ynyl 3- (4-methoxyphenyl)acrylate, F represents

3- [3-(4-methoxyphenyl)acryloyloxymethyl]benzyl 3-(4-methoxyphenyl)acrylate, G represents 3-cinnamoyloxypropyl 3-propylacrylate, and FI represents diethyl 4- acetoxybenzylidenemalonate.

Detailed Description of Embodiments

Below the present disclosure is further described in conjunction with examples. The following description is merely directed to preferable examples of the present disclosure, rather than limits the present disclosure in other forms, and any person skilled in the art may utilize the technical contents disclosed above and modify them into equivalent examples. Any simple modifications or equivalent variations made to the following examples according to the technical essence of the present disclosure, without departing from the contents of the solutions of the present disclosure, should fall within the scope of protection of the present disclosure.

Example 1 Effect of ethyl p-methoxycinnamate on Oct4 promoter activity

According to a method described in literature (Yang Η M, Do H J, Oh J H, et al. Characterization of putative cis-regulatory elements that control the transcriptional activity of the human Oct4 promoter[J], Journal of cellular biochemistry, 2005, 96(4):821-830.), the promoter of Oct4 gene was picked and cloned into pGL3-Basic, so as to construct pGL3-Oct4P-Basic reporter plasmid. One day before transfection, mouse teratoma cells P19 were inoculated into a 6-well cell culture plate at a density of 2x105 cells/well to make the cells grown to about 80% before transfection. A liposomal transfection reagent EntransterTM-D from Engreen company was used to perform the transfection according to the instructions. The P19 cells were transfected with the target plasmid pGL3-Oct4P-Basic, and the total amount of the plasmid used for the transfection was 3 pg/well. The plasmid was added to 50 pl_ of serum-free DMEM culture medium and fully mixed. 9 μΙ_ of the transfection reagent was then added to another 50 μΙ_ of the serum-free DMEM culture medium, mixed well, and the mixture allowed to stand for 5 min. The DMEM culture medium containing the transfection reagent was then added to the DMEM culture medium containing the plasmid, and the mixture was immediately pipetted more than 10 times, and allowed to stand at room temperature for 30 min. 900 μΙ_ of DMEM culture medium containing 10% fetal calf serum were added to the mixed solution, and gently mixed well. The original cell culture medium in the 6-well plate was discarded, and was replaced by the above mixed solution. The cells were re-suspended in the DMEM culture medium containing 10% fetal calf serum after being cultured for 4-6 h in a 5% C02 cell incubator at 37 eC. The above cells were inoculated in a 96-well plate at a density of 8χ103 cells/well. After being cultured for 24 h, the culture medium was absorbed and removed, the DMEM culture medium containing 3% fetal calf serum was added, and the drug to be screened was added to the culture medium to result in a final concentration of 5 pg/mL, meanwhile DMSO was used as a control. After subsequent continuous culture for 12-24 h, the cells were lysed, and luciferase activity was assayed.

Luciferase Reporter Assay System and FluoStar Optima were used to assay the luciferase activity of the transfected cell lysis solution. To 45 pL of the cell lysis solution was added 5 pL of Assay Cocktail reagent, after they were mixed well, 100 pL of Luciferin reaction solution was added, and fluorescence counting in 2 s was measured using FluoStar Optima instrument, which reflected the activity level of the studied plasmid reporter genes expressing firefly luciferase. To another 20 pl_ of the cell lysis solution was added 37.5 μΙ_ of β-gal Buffer, followed by 12.5 μΙ_ of ONPG (6 mg/ml_), and after they were mixed well, they were allowed to stand at 37 eC for 30 min. An absorption value at 450 nm was assayed using a microplate reader, which reflected the activity level of internal reference plasmid pCMV^-gal expressing β-galactosidase. The ratio between the activity of the firefly luciferase and the activity of the β-galactosidase (β-gal) excluded the difference of the sample transfection efficiency, and could relatively objectively reflect the difference of promoter activity between samples. The relative luciferase activity of different samples was calculated through the following equation:

Results are as shown in Fig. 1. The effect of ethyl p-methoxycinnamate on the activity of Oct4 promoter reached a statistically significant difference (*p < 0.05).

Example 2 Effect of ethyl p-methoxycinnamate on the expression of Oct4 mRNA 1) mRNA extraction and reverse transcription P19 cells were inoculated in a 6-well cell culture plate at a density of 2x105 cells/well. After 24 h, DMEM culture medium containing 3% serum were used for replacement. Ethyl p-methoxycinnamate was added to the experimental group to render its final concentration at 5 pg/mL, and a control group was treated with DMSO. After 12 h, the 6-well plate overgrown with monolayer cells was taken out from a cell incubator, and the cells were washed 3 times with pre-cooled PBS. 1 ml. of a pre-cooled Trizol solution was added. After vigorous pipetting, the mixture was transferred to an EP tube and allowed to stand at room temperature for 5 min. Then 200 μΙ_ of chloroform was added to the mixture, vigorously mixed well for 15 s, and the mixture cotaining chloroform was allowed to stand at room temperature for 2 min and centrifuged at 12000 rpm at 4 eC for 15 min. An upper aqueous phase was pipetted into another EP tube, and an equal volume of pre-cooled isopropanol was added, and the mixture was inverted several times and allowed to stand at room temperature for 10 min to precipitate RNA. After centrifugation at 12000 rpm for 10 min, the supernatant was discarded, and to the precipitate was added 1 ml. of 75% ethanol (prepared by 0.1% DEPC water) for washing, followed by centrifugation at 7500 rpm for 5 min (this step was repeated once). The supernatant was discarded. After natural drying (or oven drying at 37 eC), an appropriate amount of 0.1% DEPC water was added for dissolving at 37 eC for 30 min (or dissolving at 55 eC for 10 min), and the dissolved RNA was stored at -80 eC for standby use.

Reverse transcription was performed using a Reverse Transcription System kit from TaKaRa company, and the reaction system was as follows: RNA template "* ^ 5χReverse Transcription Buffer 4 pl_ 10 mM dNTP mixture 2 μΙ_ RNase inhibitor 20 M (0.5 pl_)

Oligo (dT) 15 primer 50 pmol (1 μΙ_) AMV RTase 5 M (2 μΙ_)

0.1 % DEPC H20 Added to 20 pL

Firstly, the RNA template, 0.1% DEPC water and Oligo (dT) 15 primer were added to a clean EP tube, for water bath at 70 eC for 2 min followed by centrifugation at 1000 rpm for 1 min. The other components of the reverse transcription were added to the EP tube, for water bath at 42 eC for 1 h. The reaction was stopped at 70 eC for 8 min, and finally 0.1% DEPC water was added to the EP tube, until the volume of the solution in the EP tube was 100 μΙ_. 2) RT-PCR reaction

Upstream primer: 5’-CTCGAACCACATCCTTCTCT-3’; downstream primer: 5’-TCGTGCGTGACATTAAGGAG-3. PCR reaction system (25 pL):_

Template DNA (1 pg/pL) 5 pL

Upstream primer (10 pM) 1 pL

Downstream primer (10 pM) 1 μΙ_

10xTaq Buffer 2.5 pL

2.5 mM dNTP 2 pL

Taq DNA polymerase 1 pL

Sterile water Added water to 25 pL PCR reaction conditions:

3) Agarose gel electrophoresis 0.4 g of agarose was weighed and placed in a conical flask. 40 ml. of 1xTAE electrophoresis buffer was added to it, and the mixture was placed in a microwave oven to heat until all the agarose melted, and the resulting mixture was taken out and shaken well. This process was repeated three times. When the solution was cooled to about 50 eC, 20 pl_ of anthocyanin dye was added, so that the final concentration of anthocyanin dye was 0.5 mg/ml_, and the mixture was mixed well. An electrophoresis gel solution was poured into a tank, allowed to stand cool down, and solidified for 30 min. After the gel completely solidified, the comb was pulled out, 1xTAE electrophoresis buffer was poured into the electrophoresis tank until the liquid level was just over the upper surface of a gel plate. PCR products were added to a 10xgel loading buffer in proportion, mixed well, and the resulting mixture was carefully added to sample wells using a micropipettor. After loading, the electrophoresis tank cover was closed, and the power was turned on, so that the voltage was maintained at 100-140 V. When a bromophenol blue band moved to about 2 cm from the gel front, the electrophoresis was stopped, and a gel imaging system was used for observing and photographing.

Results are as shown in Fig. 2. Ethyl p-methoxycinnamate can significantly promote the expression of Oct4 mRNA.

Example 3 Effect of ethyl p-methoxycinnamate on Oct4 protein expression

The effect of ethyl p-methoxycinnamate on Oct4 protein expression was assayed using a Western Blot method. Mouse teratoma cells P19 were inoculated in a 6-well plate. After 24 h, ethyl p-methoxycinnamate with a final concentration of 5 pg/mL was added to continue to act for another 24 h, and the control group was treated with DMSO. After the cells were washed with PBS three times, the cells were scraped with a scraper, transferred to an EP tube, and centrifuged at 4000 rpm for 5 min. The supernatant was discarded. 90 pl_ of lysis solution (50 mM Tris-HCI pH 7.5, 150 mM NaCI, 1 mM NaF, 0.5% NP-40, 2 pg/ml_ Aprotinin, and 1 mM PMSF) was added to each tube of precipitate, and the EP tube was treated on ice for 30 min and vortexed once every 5 min, so that the cells were completely lysed. After centrifugation at 12000 rpm for 10 min, the supernatant was transferred to a new EP tube, 1/3 volume of 4x protein loading buffer was added, mixed well, and boiled for 10 min, and after instantaneous centrifugation, the mixture was subjected to electrophoresis or placed at -80 eC to be frozen for standby use. A transfer membrane buffer was prepared and precooled at 4 eC. A PVDF membrane was soaked in methanol for 40 s, then taken out and soaked in the transfer membrane buffer for standby use. The transfer membrane buffer was placed in a square plate, and a transfer membrane device was installed under the liquid surface. The transfer membrane device was placed in an electrophoresis tank, and the transfer membrane buffer was poured into the electrophoresis tank. The PVDF membrane was taken out after membrane transfer at 100 V for 2 h, and positive and negative surfaces of the membrane and the electrophoretic direction were marked. The PVDF membrane was washed with TBST (0.2% Tween 20), once every 5 min, and twice in total, and blocked with 5% skim milk powder at room temperature for 2 h. After that, the PVDF membrane was washed twice with TBST (0.2% Tween 20), 5 min each time (the membrane washing was conducted on a shaker, the same below). Antibodies of Oct4, after being diluted with TBST (0.2% Tween 20) at 1:500, was placed in a hybridization bag together with the PVDF membrane to stay overnight at 4 eC. The PVDF membrane was taken out and rapidly washed with TBST (0.2% Tween 20) twice, and then washed twice for 5 min, and 3 times for 15 min. HFtP-goat anti-mouse secondary antibody (0.8mg/mL), after being diluted with TBST (0.2% Tween 20) at 1:2000, was placed in a hybridization bag together with the PVDF membrane, to incubate at room temperature for 40 min on the shaker. The PVDF membrane was taken out, and rapidly washed twice with TBST (0.2% Tween 20), and then washed twice for 5 min, and 3 times for 15 min. The PVDF membrane was subjected to ECL color development.

Results are as shown in Fig. 3. Ethyl p-methoxycinnamate can promote the expression of Oct4 protein.

Example 4 Ethyl p-methoxycinnamate and derivatives thereof promote the formation of microspheres of suspendedly cultured stem cells. 5 g agar was weighed and placed in a flask, then 100 ml. of ultra-pure water was added, and the mixture was mixed well, sealed, and then autoclaved for 30 min. In a super clean bench, 9 ml. of pre-heated (50-60 eC) serum-free DMEM culture medium was pipetted into a 10 ml. centrifuge tube, then 1 ml. of uncooled 5% agar was added, and rapidly mixed well with a pipette, resulting in 0.5% agar. 1 ml. of 0.5% agar was pipetted into one well of a 6-well plate and cooled at room temperature for 20-30 min, resulting in a 0.5% soft agar culture medium.

In the super clean bench, D-Hanks was gently added to the surface of the soft agar culture medium, and the D-Hanks was discarded after being gently shaken 2 to 3 times. Cleaning was repeated once. The mouse teratoma cells P19 were gently inoculated on the surface of the soft agar culture medium at a cell density of 4x105 cells/well. After the cell inoculation, ethyl p-methoxycinnamate was added to 1 ml. of DMEM culture medium containing 10% fetal calf serum, mixed well, and then gently added to the experimental group. A final concentration of ethyl p-methoxycinnamate was 5 pg/mL. The control group was treated with DMSO. The next day, half the amount of the solution was changed, through a method that 1 ml. of the culture medium were gently absorbed from one side of the well (note that the cells should not be absorbed), and then fresh culture medium were added, and ethyl p-methoxycinnamate was added thereto, so that the final concentration of the fresh culture medium was 5 pg/mL. After 7 d, 200 pL MTT (5 mg/ml_) was added to the control group and the experimental group respectively, and both of them were placed back into an incubator for continuous culture for 4 h. After successful dyeing, the cells were photographed.

Results are as shown in Fig. 4. Compared with the control group, in the experimental group, the P19 cells can form a higher amount of larger and more typical microspheres. It indicates that ethyl p-methoxycinnamate has the effect of maintaining the self-renewal and pluripotency of stem cells.

Example 5 Ethyl p-methoxycinnamate promotes formation of microspheres of adherently cultured stem cells.

In a super clean bench, mouse teratoma cells P19 were inoculated in a 6-well plate at a density of 1 x104 cells/well, and DMEM culture medium containing 10% fetal calf serum was added to result in a final volume of 2 ml_. The cell culture plate was gently shaken, so that the cells were uniformly distributed in the cell culture plate, and then placed in a cell incubator to be cultured. After 24 h, the old culture medium were discarded, and 3% fetal calf serum DMEM culture medium containing ethyl p-methoxycinnamate was added, so that the final concentration of ethyl p-methoxycinnamate was 5 pg/mL. The original culture medium was discarded every 24 h, and fresh DMEM culture medium containing 3% fetal calf serum was added, and the final concentration of ethyl p-methoxycinnamate therein was made to be 5 pg/ml_ likewise. After continuous culture of 22 d, the cells were photographed.

Results are as shown in Fig. 5. The control group cannot form microspheres, but in the experimental group, the P19 cells can form typical microspheres. It indicates that ethyl p-methoxycinnamate has the effect of maintaining the self-renewal and pluripotency of stem cells again.

Example 6 Derivatives of ethyl p-methoxycinnamate can promote expression of Oct4

The effect of derivatives of ethyl p-methoxycinnamate on Oct4 expression was assayed by a luciferase reporter gene method, the derivatives including diethyl 4-methoxybenzalmalonate, ethyl 3- (3-methoxy-4-methoxycarbonylmethoxyphenyl)acrylate, diethyl 4- acetoxybenzylidenemalonate, chlorogenic acid, ethyl cinnamate, 3-cinnamoyloxypropyl 3-propylacrylate, ethyl 2-cyano-3,3-diphenylacrylate, ethyl 2,6-dichlorocinnamate, diethyl 4-chlorobenzylidenemalonate, ethyl 4-aminocinnate, ethyl 3-cyanocinnamate, ethyl 4-nitrocinnamate, 3- [3-(4-methoxyphenyl)acryloyloxymethyl]benzyl 3-(4-methoxyphenyl)acrylate, 4- [3-(4-methoxyphenyl)acryloyloxy]butyl-2-ynyl 3-(4-methoxyphenyl)acrylate, ethyl indole-2-carboxylate, and ethyl 6-methoxy-1-benzofuran-2-carboxylate.

One day before transfection, mouse teratoma cells P19 were inoculated into a 24-well cell culture plate at a density of 1x105 cells/well, to make the cells grown to about 80% before transfection. A liposomal transfection reagent EntransterTM-D from Engreen company was used to perform the transfection according to the instructions, to co-transfect the P19 cells with target plasmid (PGL3-Oct4P-Basic or pGL3-Basic) and reference plasmid (pCMV^-gal). The total amount of the plasmid used for transfection was 1 pg/well (the target plasmid : the reference plasmid = 2:1). The plasmid was added to 25 pl_ of serum-free DMEM culture medium, and fully mixed. 3 μΙ_ of the transfection reagent was then added to another 25 μΙ_ of serum-free DMEM culture medium, mixed well, and allowed to stand for 5 min. The DMEM culture medium containing the transfection reagent was then added to the DMEM culture medium containing the plasmid, and the mixture was immediately pipetted more than 10 times, and allowed to stand at room temperature for 30 min. 250 μΙ_ of DMEM culture medium containing 10% fetal calf serum was added to the mixed solution, and gently mixed well. The original cell culture medium in the 24-well plate was discarded, and replaced with the above mixed solution. After being cultured for 4-6 h in a 5% C02 cell incubator at 37eC, the original culture medium was absorbed and removed, and replaced with 500 μΙ_ of DMEM culture medium containing 10% fetal calf serum. After being cultured for 24-36 h, the culture medium was absorbed and removed, the DMEM culture medium containing 3% fetal calf serum was added, and ethyl p-methoxycinnamate was added to the culture medium, to result in a final concentration of 5 pg/mL, meanwhile DMSO was used as a control. After subsequent continuous culture for 12-24 h, the cells were lysed, and the luciferase activity was assayed.

Luciferase Reporter Assay System and FluoStar Optima were used to assay the luciferase activity of the transfected cell lysis solution. To 45 pL of the cell lysis solution was added 5 pL of Assay Cocktail reagent, after they were mixed well, 100 pL of Luciferin reaction solution was added, and fluorescence counting in 2 s was measured using FluoStar Optima instrument, which reflected the activity level of the studied plasmid reporter genes expressing firefly luciferase. To another 20 pL of the cell lysis solution was added 37.5 pL of β-gal Buffer followed by 12.5 pL of ONPG (6mg/mL), and after they were mixed well, they were allowed to stand at 37 eC for 30 min. An absorption value at 450 nm was assayed using a microplate reader, which reflected the activity level of internal reference plasmid ρΟΜν-β-gal expressing β-galactosidase. The ratio between the activity of the firefly luciferase and the activity of the β-galactosidase (β-gal) excluded the difference of the sample transfection efficiency, and could relatively objectively reflect the difference of promoter activity between samples. The relative luciferase activity of different samples was calculated through the following equation:

Results are as shown in Fig. 6. All of the eight assayed compounds have the effect of enhancing the activity of the Oct4 promoter, wherein A represents chlorogenic acid, B represents ethyl 2-cyano-3,3-diphenylacrylate, C represents ethyl indole-2-carboxylate, D represents ethyl cinnamate, E represents 4-[3-(4-methoxyphenyl)acryloyloxy]butyl-2-ynyl 3-(4-methoxyphenyl)acrylate, F represents 3-[3-(4-methoxyphenyl)acryloyloxymethyl]benzyl 3-(4-methoxyphenyl)acrylate, G represents 3-cinnamoyloxypropyl 3-propylacrylate, and FI represents diethyl 4-acetoxybenzylidenemalonate. Given all of the 8 compounds have the effect of promoting the expression of Oct4, we consider that it is the common moiety in their structures that exerts this effect, and accordingly we can speculate that other ethyl p-methoxycinnamate derivatives having this common structure, i.e. compounds having the following general formula, should also have the same activity of maintaining the properties of stem cells, and can be used as regulating drugs of stem cells.

Claims (5)

1. Claims:

   1. A use of ethyl p-methoxycinnamate and derivatives thereof in preparing drugs, culture media, regulating agents or cosmetics and skin caring products for maintaining self-renewal and pluripotency of stem cells.
2. 2. The use according to Claim 1, characterized in that a general structural formula of the ethyl p-methoxycinnamate and the derivatives thereof is as follows:

   wherein Ri is 4-OCH3, R2 is H, R3 is H, and R4 is -CH2CH3; or R1 is 4-OCH3, R2 is H, R3 is H, and R4 is -CH2CH2CH(CH3)CH3; or R1 is 4-OCH3, R2 is H, R3 is COOCH2CH3, and R4 is -CH2CH3; or R1 is 4-OCH3, R2 is H, R3 is COCH3, and R4 is -CH2CH3; or R1 is 4-OCH3, R2 is H, R3 is CN, and R4 is -CH2CH3; or R1 is 4-OCH3, R2 is CH3, R3 is H, and R4 is -CH2CH(CH2CH3)CH2CH2CH2CH3; or R1 is 3,4-OCH3, R2 is H, R3 is COCH2CH3, R4 is -CH2CH3; or R1 is 3,4-OCH3, R2 is H, R3 is CN, and R4 is -CH2CH3; or R1 is 2,4-OCH3, R2 is H, R3 is CN, and R4 is -CH2CH3; or R1 is 3,4,5-OCH3, R2 is CH3, R3 is H, and R4 is -CH2CH3; or R1 is 2-OCH2CH3, R2 is H, R3 is H, and R4 is -CH2CH3; or R1 is 4-OCH2CH3, R2 is H, R3 is H, and R4 is -CH2CH3; or R1 is 4-OCH2CH2CH2CH3, R2 is H, R3 is CN, and R4 is -CH2CH3; or R1 is 3-COOCH3, R2 is H, R3 is H, and R4 is -CH2CH3; or R1 is 2-CH3-5-COOCH3, R2 is H, R3 is H, and R4 is -CH2CH3; or R1 is 4-OCOCH3, R2 is H, R3 is COCH2CH3, and R4 is -CH2CH3; or R1 is 3-OCH3-4-OCH2COOCH2CH3, R2 is H, R3 is H, and R4 is -CH2CH3; or R1 is 2-OH, R2 is H, R3 is H, and R4 is -CH2CH3; or R1 is 3,4-OH, R2 is H, R3 is H, and R4 is -CH2CHC(CH3)CH3; or R1 is 3,4-OH, R2 is H, R3 is H, and R4 is -CH2CH2C6H5; or Ri is 3,4-OH, R2 is H, R3 is H, and R4 is

   or Ri is 3,4-OH, R2 is H, R3 is H, and R4 is or Ri is H, R2 is H, R3 is H, and R4 is -CH2CH3; or Ri is H, R2 is H, R3 is H, and R4 is -CH(CH3)CH3; or Ri is H, R2 is H, R3 is H, and R4 is -CH2CH2CH2CH3; or Ri is H, R2 is H, R3 is H, and R4 is -CH2CH(CH3)CH3; or Ri is H, R2 is H, R3 is H, and R4 is -CH(CH3)CH2CH3; or Ri is H, R2 is H, R3 is H, and R4 is -CH2CH2CH(CH3)CH3; or Ri is H, R2 is H, R3 is H, and R4 is -CH2CH2CH2OOCCH=CHC6H5; or Ri is H, R2 is H, R3 is H, and R4 is -CH2CH2C6H5; or Ri is H, R2 is H, R3 is H, and R4 is -CH2CH(CH3)C6H5; or Ri is H, R2 is H, R3 is CN, and R4 is -CH2CH3; or Ri is H, R2 is CH3, R3 is H, and R4 is -CH2CH3; or Ri is H, R2 is C6H5, R3 is CN, and R4 is -CH2CH3; or Ri is 2,6-CI, R2 is H, R3 is H, and R4 is -CH2CH3; or Ri is 4-Br, R2 is H, R3 is H, and R4 is -CH2CH3; or Ri is 2-CF3, R2 is H, R3 is H, and R4 is -CH2CH3; or Ri is 2-OH-4-Br, R2 is H, R3 is H, and R4 is -CH2CH3; or Ri is 4-CI, R2 is H, R3 is COOCH2CH3, and R4 is -CH2CH3; or Ri is 4-CI, R2 is H, R3 is OCH2CH3, and R4 is -CH2CH3; or Ri is 3,4-F, R2 is H, R3 is OCH2CH3, and R4 is -CH2CH3; or Ri is 2-CI-4-F, R2 is H, R3 is CN, and R4 is -CH2CH3; or Ri is 3-CI-4-OCH3, R2 is H, R3 is CN, and R4 is -CH2CH3; or Ri is 2-CI-4-OCH3, R2 is H, R3 is CN, and R4 is -CH2CH3; or Ri is 4-NH2, R2 is H, R3 is H, and R4 is -CH2CH3; or Ri is 3-CN, R2 is H, R3 is H, and R4 is -CH2CH3; or Ri is 4-N02, R2 is H, R3 is H, and R4 is -CH2CH3; or R1 is 4-(2,4-F-C6H4), R2 is CH3, R3 is H, and R4 is -CH2CH3; or R1 is 4-OCH3, R2 is H, R3 is H, and R4 is -CH2-C6H4-CH2-00C-CH=CH-C6H4-0-CH3; or R1 is 4-OCH3, R2 is H, R3 is H, and R4 is -CH2-C-CH2-00C-CH=CH-C6H4-0-CH3; or R1, R2 and R3 form a heterocyclic structure

   , and R4 is -CH2CH3; or R1, R2 and R3 form a heterocyclic structure

   and R4 is -CH2CH3; or R1, R2 and R3 form a heterocyclic structureand R4 is -CH2CH3;

   or R1, R2, R3 form a heterocyclic structure

   , and R4 is -CH2CH3; or R1 is -N-CH3(CH3), R2 is H, and R3 and R4 form a heterocyclic structure
3. 3. The use according to Claim 1 or Claim 2, wherein the ethyl p-methoxycinnamate and the derivatives thereof maintain self-renewal capacity and pluripotent state of the stem cells by promoting expression of Oct4 genes and the stem cells comprise adult stem cells, embryonic stem cells and induced pluripotent stem (iPS) cells.
4. 4. A use of the ethyl p-methoxycinnamate and the derivatives thereof according to any one of Claims 1 to 3 in in-vitro large-scale preparation of stem cellsand in-vitro amplification of stem cells and preparation of induced pluripotent stem (iPS) cells.
5. 5. A use of the ethyl p-methoxycinnamate and the derivatives thereof according to any one of Claims 1 to 4 in preparing drugs for treating cell deletion or injury diseases.