KR20110126307A - A method for detecting agent inducing endoplasmic reticulum stress - Google Patents

Abstract

PURPOSE: A method for detecting an ER stress inducer using SEAP expression epithelial cell line is provided to accurately induce ER stress. CONSTITUTION: An alkali phosphatase expression vector of a secretory type contains SV40 promoter, hygromycin gene, and alkali phosphatase gene. A human epithelial cell line which always expresses SEAP(secreted alkaline phosphatase) contains the expression vector. The epithelial cell line is an intestine epithelial cell. A composition for detecting the ER stress inducer contains the epithelial cell line. The ER stress inducer is fungal toxin, 8-keto trichothecen. A method for detecting the ER stress inducer comprises: a step of adding a sample to the composition for reaction; and a step of observing secretion change of the SEAP.

Description

 A method for detecting agent inducing endoplasmic reticulum stress

The present invention relates to a method for detecting vesicle stress-inducing substances using secretory alkaline phosphatase (SEAP) expression vector, SEAP expressing epithelial cell line and SEAP expressing epithelial cell line. More specifically, the present invention relates to a method for detecting 8-keto trichothecene, which is a endoplasmic reticulum stressor, using a human epithelial cell line expressing SEAP at all times.

Mycotoxins are defined as secondary metabolites produced by fungi that have some detrimental effects on humans or animals through food or feed and are distinguished from bacterial toxins and the like. Unlike bacterial toxins, mycotoxins are low molecular weight substances and include many known substances. Their chemical structure and harmful effects on living organisms vary considerably. Some of these are not harmful to humans, some exhibit high acute toxicity in the human body, and others are recognized to be carcinogenic or tumorigenic in the kidneys and liver. Due to the low molecular weight, mycotoxins are difficult to degrade or remove under conventional food processing and cooking conditions.

In addition, mycotoxins have high stability and, when ingested into food animals through feed, remain in meat or dairy products from animals, and finally exhibit toxicity after ingestion into the human body.

Mycotoxin contamination can be caused by a variety of pathways, but livestock and aquatic products (e.g., caused by primary contamination of crops and supply of contaminated fodder, caused by fungal invasion during crop cultivation, harvesting, storage and processing) For example, meat, milk and eggs) are largely classified as secondary pollution. Primary pollution represents regional characteristics depending on factors such as the type of crop (fungal substrate), the type of fungus occurring in the growing environment and the environmental conditions (eg temperature, humidity and precipitation).

Tricortesene mycotoxins are representative mycotoxins that contaminate major grains such as barley, wheat, rye, oats and corn. Representative tricortesene mycotoxins include deoxynivalenol (DON), nivalenol (NIV), and T-2 toxin (T-2). The fungi that produce them, Fusarium gramminearum, F. culmorum, and F. sporotrichioides, are commonly present in soil, and are commonly found in temperature, humidity, and precipitation. Satisfaction of such natural conditions can easily infect crops, causing the mycotoxin contamination of the crops to spread.

For the purpose of preventing contamination of foods and feeds, regulations on the residual concentrations of tricortesene mycotoxin in foods and feeds are adopted in many countries. Therefore, it is important to quickly and accurately quantify trichothecene mycotoxin in foods and feeds. In addition, since the first cells to be exposed to the highest concentration of trichothecene are intestinal mucosal epithelial cells, the quantitative analysis method of trichothecene based on human epithelial cells is urgently needed.

Recognizing the necessity of the above, the present inventors found that tricortesene induces vesicle stress in human epithelial cells, and based on this, a vesicle stress-inducing substance is detected using a human epidermal cell line expressing secretory alkaline phosphatase at all times. By developing a bio-monitoring method that can be achieved, the present invention has been completed.

It is therefore an object of the present invention to provide a secreted alkaline phosphatase (SEAP) expression vector.

Another object of the present invention is to provide a human epithelial cell line expressing SEAP at all times.

Still another object of the present invention is to provide a composition and a method for detecting the vesicle stress-inducing substance.

In order to solve the above problems, the present invention provides a secretion type alkaline phosphatase expression vector consisting of the SV40 promoter, hygromycin gene and alkaline phosphatase gene.

The expression vector preferably has the structure of Figure 2A.

In another aspect, the present invention provides a human epithelial cell line expressing secreted alkaline phosphatase (SEAP), wherein the expression vector is introduced.

The epithelial cell line is preferably a human small intestinal epithelial cell.

In another aspect, the present invention provides a composition for detecting the endoplasmic reticulum stress-inducing substance comprising the human epidermal cell line.

The endoplasmic reticulum stressor is preferably a fungal toxin.

The fungal toxin is preferably 8-keto tricortesene, more preferably deoxynivalenol (DON), 3-acetyl DON, 15-acetyl DON, nivalenol or 4-acetyl nivalenol.

The composition of the present invention can be used in the form of a kit or microarray.

In another aspect, the present invention (a) adding a sample to be tested in the composition of claim 4 to react; And (b) observing secretion changes of the secreted alkaline phosphatase (SEAP); It provides a method for detecting the endoplasmic reticulum stress containing a.

In the method, if the secretion of SEAP is suppressed may further comprise determining that the sample is contaminated with the endoplasmic reticulum stress causing material.

The sample may be food, grain or feed to determine whether it is contaminated with the endoplasmic reticulum stress causing material.

The endoplasmic reticulum stressor is preferably a fungal toxin, and more preferably 8-keto tricortesene.

According to the present invention, since the human epidermal cell line, which is the cell which is first exposed to the fungal toxin at the highest concentration, is used, it is possible to detect a substance that induces vesicle stress more accurately. In addition, by establishing a toxic mechanism of endoplasmic reticulum stress inducing substances, and by analyzing biological quantitative activity of toxins, it is possible to detect contaminated grains in advance to prevent diseases caused by toxins. have. In particular, the analysis of SEAP secreted out of the cell, it is possible to continuously measure the activity in real time without dissolving epithelial cells, and through the cell-based quantitative bioassay similar structure 8-keto trichothecene (8-keto trichothecene) Can be used to calculate the toxic equivalent factor (TEF), which indicates the relative degree of toxicity of toxins.

Figure 1 shows the endoplasmic reticulum stress induced results of 8-keto trichothecenes in HCT-8 human small intestinal epithelial cells. 1 for vehicle treatment, 2 for 8-keto tricortesene DON treatment, 3 for 15-acetyl DON treatment, 4 for 3-acetyl DON treatment, 5 for NIV treatment, 6 for 4-acetyl NIV treatment for 2 or 10 minutes It was. Figure 1 A shows the Western blot analysis of cell lysates, which is a graph quantifying phosphorylated eIF2α. B extracts the total RNA and shows the RT-PCR result of each mRNA.
FIG. 2A shows the SEAP gene expression plasmid construct comprising the SV40 promoter and hygromycin resistance marker.
Figure 2b shows the result confirmed by RT-PCR whether the prepared plasmid is introduced into HCT-8 cells and stably expresses SEAP. Figure 2c confirms the result of SEAP secretion by treating thapsigargin known as endoplasmic reticulum stress-inducing material.
Figure 3 shows the effect on the SEAP mRNA and enzymatic activity of 8-keto tricortesene in vitro. 1 is vehicle treatment, 2 is 8-keto tricortesene DON treatment, 3 is 15-acetyl DON treatment, 4 is 3-acetyl DON treatment, 5 is NIV treatment, and 6 is 4-acetyl NIV treatment for 24 hours. Among them, A is the result of measuring RNA by RT-PCR, and B is the result of the activity of SEAP obtained by treating recombinant SEAP with 8-ket tricortesene in an amount of ID 50 or 2 × ID 50.
4 shows the activity of SEAP by treating SEAP expressing cells with DON, 15-acetyl DON, 3-acetyl DON, NIV and 4-acetyl NIV, respectively, in 8-keto trichothecene. Relative SEAP activity was calculated as a percentage of SEAP activity in the medium relative to the total protein, and all experiments were repeated three times.
Figure 5 shows the grain matrix effect in the SEAP-based DON monitoring method. Corn (A) or rice (B) containing DON was reacted with SEAP expressing cell lines to confirm the correlation between actual DON amount and DON amount measured by SEAP-based biomonitoring.

The present invention relates to a secreted alkaline phosphatase expression vector consisting of the SV40 promoter, hygromycin gene and alkaline phosphatase gene.

Preferably, the expression vector is based on the pGL3 control vector (Promega company), the SV40 promoter (position: 48-250bp), inserted SEAP (position: 280-1854bp), inserted hygromycin ( Hygromycin) resistance gene (position: 2362-3960bp), Amp resistance gene (position: 5044-5904bp) and other elements, SE40 is constantly expressed by the action of the SV40 promoter, in the vector itself, especially high Vectors can be designed to facilitate the generation of constitutively expressed epithelial cell lines by incorporating a gromycin resistance gene for 2 weeks.

The most preferred expression vector structure of the present invention is the same as the structure shown in Figure 2A.

In the present invention, "vector" refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in a suitable host. Vectors can be plasmids, phage particles or simply potential genomic inserts. Once transformed into the appropriate host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. Since plasmids are the most commonly used form of current vectors, "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. Plasmid vectors include (a) the initiation of replication to efficiently replicate to include hundreds of plasmid vectors per host cell, (b) antibiotic resistance genes to allow selection of host cells transformed with plasmid vectors, and (c ) It has a structure including restriction enzyme cleavage site where foreign DNA fragment can be inserted. Although no appropriate restriction enzyme cleavage site is present, the use of synthetic oligonucleotide adapters or linkers according to conventional methods facilitates ligation of the vector and foreign DNA.

In one embodiment of the present invention, the hygromycin gene (SalI / BamHI) was transferred to pGL3 control vector in pGL4.14, and this plasmid L was used to make a cell line for intracellular luciferase assay. Alkaline phosphatase genes were isolated from pSEAP2-basic vector and cloned back into plasmid L at HindIII / HpaI position (Plasmid P, pSEAP, FIG. 2) to establish a secretory reporter system. Therefore, all of the vectors of the present invention possess a hygromycin gene, thus enabling the construction of a stable cell line.

The present invention also relates to a human epithelial cell line which always expresses the secreted alkaline phosphatase (SEAP) into which the expression vector is introduced.

Transformations injecting expression vectors are described in Sambrook, et. al., it can be readily accomplished using the calcium chloride method described in section 1.82 of supra. Alternatively, electroporation (Neumann, et. Al., EMBO J., 1: 841, 1982) can also be used for transformation of these cells.

In one embodiment of the present invention, lipofectamine 2000 was used to inject the plasmid expressing the SEAP at all times into HCT-8 cells.

Preferably, the cell line of the present invention is preferably a human small intestinal epithelial cell.

The present invention also relates to a composition for detecting endoplasmic reticulum stress-inducing substance comprising the epithelial cell line.

In one embodiment of the present invention, as a result of testing whether phosphorylation of 8-keto tricortesene in the HCT-8 human small intestine epithelial eIF2α which is a representative method for identifying the ER stress-induced response, eIF2α phosphorylation was increased in a few minutes, GRP78 expression Decreased, and ATF6 increased in expression. In addition, the present invention has developed a cell-based assay system using the SEAP reporter to monitor the endoplasmic reticulum stress controlled by tricortesene. The vesicle stress-inducing substance detection composition and system were developed by confirming that secretion of SEAP is reduced when the vesicle stress-inducing substance 8-keto tricortesene is treated with SEAP-expressing human small intestinal epithelial cell line.

The vesicle stressor of the composition is preferably a fungal toxin, and more preferably 8-keto trichothecene. 8-keto tricortesene includes deoxynivalenol (DON), 3-acetyl DON, 15-acetyl DON, nivalenol or 4-acetyl nivalenol, and the like.

The composition can be used in the form of a kit or microarray.

In addition, the present invention comprises the steps of (a) reacting by adding a sample to be tested to a composition comprising a human epithelial cell line stably expressing SEAP; And (b) observing the degree of inhibition of secretion of secreted alkaline phosphatase (SEAP); It relates to a method for detecting endoplasmic reticulum stress containing a.

The level of SEAP expression of a composition containing a cell culture containing a human epithelial cell line stably expressing SEAP is measured. After adding the sample to test whether the cell culture medium is contaminated with the endoplasmic reticulum stressor, and reacting, the expression change of SEAP can be observed to detect whether the endoplasmic reticulum stressor is infected. If the secretion of SEAP is inhibited, it can be determined that the sample is contaminated with the ER stressor. The SEAP expression change can be confirmed by a known ELISA method.

The detection method of the present invention can be easily detected only through the cell culture. The sample to be tested is not limited in kind, and may be, for example, food, cereal or feed. The endoplasmic reticulum stressor is preferably a fungal toxin, more preferably 8-keto tricortesene.

Hereinafter, the present invention will be described in detail through examples. The following examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples according to the gist of the present invention to those skilled in the art. Will be self-evident.

Cell Culture and Sample Preparation

HCT-8 human epithelial cells were obtained from the human assembly site, which was purchased from the American Type Culture Collection (Rockville, MD, USA). Cells were 10% (v / v) fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, Mo., USA) thermally inactivated in a 5% CO ₂ humidified incubator at 37 ° C., 50 units / Incubated with RPMI medium (Invitrogen, Carlsbad, CA. USA) added ml penicillin (penicillin (Sigma-Aldrich)) and 50 μg / ml streptomycin (Sigma-Aldrich).

Cell number and viability were confirmed by standard assays by excluding trypan blue (Sigma-Aldrich) using a hemacytometer.

Construction and Transfection of Plasmids

A SEAP reporter plasmid (pSV40-SEAP-hyg) with hygromycin resistance and SV40 as a promoter was prepared based on the pGL3 control vector (Promega, Madison, WI, USA). More specifically, the hygromycin gene (SalI / BamHI) was transferred to the pGL3 control vector in pGL4.14. The plasmid L was used to make a cell line for intracellular luciferase assay, and the alkaline phosphatase gene was isolated from pSEAP2-basic vector and cloned back into the plasmid L at the HindIII / HpaI position to establish a secretory reporter system. (Plasmid P, pSEAP). As a result, since both vectors possess the hygromycin gene, it was possible to construct a stable cell line.

The plasmid was transfected into HCT-8 cells using Lipofectamine 2000. The plasmid injection efficiency was about 50% to 60% as determined by the pMX-enhanced green fluorescent protein (GFP) vector. Cells were selected in complete medium containing 400 μg / ml hygromycin B (Invitrogen) to obtain cell lines expressing pSV40-SEAP-hyg safely. Selection was continued until monolayer colonies were produced. A single transfectant was maintained in complete medium with 10% fetal bovine serum and 200 μg / ml hygromycin B added.

SEAP assay

Transformed HCT-8 cells were seeded in 24-well plates at a concentration of 5 × 10 ⁴ cells / well, and then treated with various concentrations of trichothecenes at 37 ° C. for 24 hours. The collected culture solution (400 μl) was heated to 65 ° C. for 5 minutes, and 100 μl of the supernatant of the heated culture medium was added to 100 μl of 2 × SEAP assay buffer (2M diethanolamine, 1 mM MgCl ₂ , 20 mM 1-homoal). (Including homoarginine).

After the mixture was incubated at 37 ° C. for 10 minutes, the reaction was completed by adding 20 μl of 120 mM p-nitrophenyl phosphate dissolved in 1 × SEAP assay buffer. The final mixture was further incubated at 37 ° C. for 15 minutes. The absorbance of the reaction mixture was measured at 405 nm with an ELISA (Enzyme-linked immunosorbent assay) reader (Molecular devices, Sunnyvale, CA, USA).

Western immunoblot analysis

Protein expression levels were compared using Western blot. Cells were washed with cold phosphate buffer and lysed with boiling lysis buffer [1% (w / v) sodium dodecyl sulfate, 1.0 mM sodium ortho-vanadate, and 10 mM Tris, pH 7.4] and sonicated for 5 seconds.

Lysates containing proteins were quantified using a BCA protein assay kit (Pierce, Rockford, IL, USA). 50 μg of protein was isolated by mini-gel electrophoresis (Bio-Rad, Hercules, Calif., USA). The protein was transferred to a polyvinylidene fluoride membrane (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and the blots were transferred from Tris-buffered saline plus Tween 0.05% (TBST) to 5% skim milk for 1 hour. bloking. Each antibody was probed for 2 hours at room temperature or overnight at 4 ° C.

After washing three times with TBST, the blots were incubated with horseradish-conjugated secondary antibody for 1 hour and washed three more times with TBST. Proteins were detected with enhanced chemiluminescent substrates (Amersham Pharmacia Biotech).

Reverse transcription-polymerase chain reaction (RT-PCR)

RNA was extracted with RNeasy kit (Qiagen, Valencia, CA, USA). RNA (100 ng) of each sample was transcribed into cDNA by BD Sprint PowerScript (Clontech, Mountain View, CA, USA). The following primers were used to amplify with Takara HS ExTaq DNA Polymerase (Takara Bio, Shiga, Japan) in a Mycycler thermal cycler (Bio-Rad).

<Primer used>

Forward primer of SEAP: 5'-GGTGAACCGCAACTGGTACT-3 '; SEQ ID NO: 1

Reverse primer of SEAP: 5'-CATGAGATGGGTCACAGACG-3 '; SEQ ID NO: 2

Forward primers of human g lyceraldehyde- 3-phosphate dehydrogenase (GAPDH): 5'-TCA ACG GAT TTG GTC GTA TT-3 '; SEQ ID NO: 3

Reverse primer of human glyceraldehyde-3-phosphate dehydrogenase: 5'-CTG TGG TCA TGA GTC CTT CC-3 '; SEQ ID NO: 4

Data obtained through experiments in the present invention was statistically analyzed using SigmaStat for Windows (Jandel Scientific, San Rafael, CA, USA). Student ' t test was performed to compare two groups of data. To analyze several groups, pairwise comparisons and ANOVA by Student-ewman-euls (SNK) method were performed.

Example 1: Confirmation of vesicle stress induction of 8-keto trichothecenes in HCT-8 cells

In the present invention, the detected 8-keto trichothecenes (deoxynivalenol (DON), 15-acetyl DON (15-acetyl DON), 3-acetyl DON (3-acetyl DON), The main effects of nivalenol (NIV) and 4-acetyl NIV on eIF2α phosphorylation were identified in HCT-8 human small intestinal epithelial cells All tricotenes increased eIF2α phosphorylation in minutes (Fig. 1A) HCT-8 used in the experiment is a human epithelial cell model mainly used for microbial infection and inflammatory response. (Avantaggiato et al. , 2004; Obremski et al. , 2008) As other representative markers of endoplasmic reticulum stress, GRP78, ATF6 and IRE1α were used to assess the presence of each 8-keto trichothecenes. Vesicle-specific chaperone GRP78 was applied to 8-keto tricortesene. Was confirmed that it inhibited (Fig. 1B). In addition, as an ER sensor, was ATF6 is induced by all toxin, was expressed in IRE1α also vary in the HCT-8 cells.

Example 2: Identification of Cell Lines Stably Expressing Secretory Protein-Based Reporters

Reduction of intracellular secretion mechanisms is a major process of endoplasmic reticulum stress because cells react to endoplasmic reticulum stress by disrupting protein synthesis, folding, secretion and degradation. In the present invention, a cell-based assay system was developed using the SEAP reporter to monitor vesicle stress regulated by tricortesene. Plasmids were constructed with the SEAP reporter gene activated with the SV40 promoter. In addition, hygromycin selection markers were inserted for stable expression in HCT-8 cells (FIG. 2A).

RT-PCR with specific primers for the amplification of the SEAP gene introduced confirmed that SEAP mRNA was always produced in the transfected cells (FIG. 2B). As a positive control of the bio-monitoring assay, thapsigargin material produced by endoplasmic reticulum stress was used to confirm the effect of inhibiting SEAP secretion. Reduced SEAP protein reduction correlated strongly with the chemical amount of the endoplasmic reticulum stressor in the biomonitoring assay (FIG. 2C).

Tricotesene is known to inhibit protein biosynthesis by disrupting ribosomal function, so the change in SEAP in this monitoring method may be due to inhibition of translation by toxin. Therefore, in order to rule out this possibility, the degree of activity of SEAP on the total protein of the positive control group was measured in% ratio.

SEAP activity can also be influenced by various factors such as transcription, altered enzyme activity. In the present invention 8-keto trichothecenes (8-keto trichothecenes) was used to measure the inhibitory capacity of SEAP mRNA levels (Fig. 3A).

However, the newly synthesized SEAP transcripts were not inhibited by tricortesene treatment. Tricortesene, such as nivalenol or 4-acetyl nivalenol, rather raised SEAP mRNA levels. Tricortesene (DON and its derivatives) also did not alter the enzymatic activity of SEAP itself (FIG. 3B).

In other words, the human small intestinal epithelial cell line secreting SEAP was made to monitor external endoplasmic reticulum stress, and the transcriptional and enzymatic activity of SEAP was not affected by 8-keto trichothecenes fungal toxin. Could.

Example 3: 8-keto trichothecenes endoplasmic reticulum stress mediated SEAP biomonitoring

8-keto trichothecene induces endoplasmic reticulum stress and toxin-induced cellular responses were analyzed by measuring SEAP reporter activity. DON, 3-acetyl DON, and 15-acetyl DON inhibited secretion of SEAP in a concentration-dependent manner, with a negative correlation between the amount of toxin and SEAP activity in HCT-8 cells. Had a relationship (FIGS. 4A-4C). The correlation of DON exposure was shown by a strong regression coefficient (r = -0.9978), with higher results according to 3-acetyl DON and 15-acetyl DON. Cell-based bio-monitoring could also be used to identify endoplasmic reticulum stress by NIV and its derivative 4-acetyl nivalenol (fusarenon-X, fusarenon X) having a pattern similar to DON and its derivatives (FIGS. 4D and 4E). ).

Since the bioassay was performed using tricothecene in the control solution dimethylsulfoxide (DMSO), it was necessary to use toxins in the grain in consideration of the matrix effect in order to accurately measure the SEAP activity.

Control corn, rice grain extract and DON were mixed and used for SEAP monitoring. Since the matrix of the organism itself has SEAP activity, the basal SEAP activity level in grain extracts was higher than in DMSO solution.

Although SEAP activity was negatively correlated with DON concentration in the grain matrix, the amount of DON measured in the standard matrix was different from that of the actual toxin, and the matrix effect equation could be derived (FIG. 5). The relative matrix effect of corn or rice on the DMSO standard matrix in the SESAP assay system was 0.70 and 0.90, respectively. Considering the matrix effect, 8-keto tricortesene in contaminated grains can be detected using the SEAP monitoring method. In addition, the minimum sensitivity of the assay is 4 ng / ml of tricortesene, which corresponds to 20 ppb DON in cereals.

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Claims (14)

A secreted alkaline phosphatase expression vector consisting of the SV40 promoter, hygromycin gene and alkaline phosphatase gene. The expression vector of claim 1, wherein the expression vector has the structure of FIG. 2A. The type of vector is introduced claim 1. expressed, secreted alkaline phosphatase human epithelial cell line which constantly expresses a (SEAP). 4. The human epithelial cell line of claim 3, wherein said epithelial cell line is a small intestine epithelial cell. A composition for detecting a vesicle stress-inducing substance comprising the epithelial cell line of claim 3 or 4. 6. The composition of claim 5, wherein the endoplasmic reticulum stressor is a fungal toxin. 7. The composition of claim 6, wherein the fungal toxin is 8-keto tricortesene. 8. The composition of claim 7, wherein said 8-keto tricortesene is deoxynivalenol (DON), 3-acetyl DON, 15-acetyl DON, nivalenol or 4-acetyl nivalenol. 6. The composition of claim 5, wherein the composition is used in the form of a kit or microarray. (a) reacting by adding a sample to be tested to the composition of claim 5; And
(b) observing secretion changes of secreted alkaline phosphatase (SEAP);
Method for detecting endoplasmic reticulum stress comprising a. The method of claim 10, further comprising determining that the sample is contaminated with the endoplasmic reticulum stressor when secretion of SEAP is inhibited. The detection method according to claim 10, wherein the sample is food, grain or feed. 13. The detection method according to any one of claims 10 to 12, wherein the endoplasmic reticulum stressor is a fungal toxin. The method of claim 13, wherein the fungal toxin is 8-keto tricortesene.

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