KR20080008072A - The apoptosis sensing cell and the detection method of apoptosis using the same - Google Patents

Abstract

An apoptosis detecting cell transformed by virus vector and a method for detecting apoptosis using the same are provided to measure the apoptosis of an object cell in real time and be usefully used for searching materials including or inhibiting the apoptosis. A virus vector for detecting apoptosis comprises a gene expression cassette in which an NES(nuclear export signal), a caspase-3 cleavage site, a reporter gene, and an NLS(nuclear localization signal) are fused in sequence. A cell for detecting apoptosis is characterized in that the virus vector is transformed into a host cell. A method for measuring apoptosis comprises the steps of: (a) transforming the virus vector into a cell to be tested; and (b) monitoring fluorescent signals by culturing the transformed cell.

Description

Apoptosis sensing cell and the detection method of apoptosis using the same}

1 shows a vector map of a pCaspase3-sensor vector.

2 is a Raw 264.7 cell line transformed with pCaspase3-sensor vector:

    A: Image (60X) of Raw 264.7 cell line observed with phase contrast microscope; And

    B: Fluorescence image of Raw 264.7 cell line transformed with pCaspase3-sensor vector.

Figure 3 is a photograph of monitoring the GFP signal for each time zone after apoptosis induction of the raw 264.7 cell line transformed with the pCaspase3-sensor vector.

Figure 4 is a graph of monitoring the GFP signal by time zone after induction of cell death of the raw 264.7 cell line transformed with the pCaspase3-sensor vector.

Figure 5 is a transformed virus vector for detecting apoptosis into Lett C6 glioma cells:

    A:

      (a): fluorescence image of C6 caspase 3 detection cell;

      (b): fluorescence image of C6 caspase 3 detection cells with caspase 3 activated; And

    B: Diagram showing the action of virus vectors for detecting apoptosis.

6 is a picture of a well plate culture system.

Figure 7 is a photograph of detecting apoptosis by monitoring the GFP signal after processing 500 nM STS to the let C6 glioma cells transformed with a virus vector for detecting apoptosis.

8 is a photograph showing detection of apoptosis by monitoring a GFP signal after 1,000 nM STS treatment to a let C6 glioma cell transformed with a virus vector for detecting apoptosis.

9 is a photograph showing detection of apoptosis by treating 700 nM STS and 750 nM PI in a let C6 glioma cell transformed with a virus vector for detecting apoptosis and monitoring the degree of GFP signal and PI staining.

10 is a photograph showing detection of cell death by monitoring the degree of GFP signal and PI staining after treatment with 1,000 nM STS and 750 nM PI in a let C6 glioma cell transformed with a virus vector for detecting apoptosis.

11 is a cell death was detected by treating 700 nM STS and 750 nM PI to a transformed virus vector for detecting apoptosis virus, and then monitoring the degree of GFP signal and PI staining in one single cell. It is a photograph.

FIG. 12 is a graph illustrating treatment of 700 nM STS and 750 nM PI of transformed Lett C6 glioma cells transformed with a virus vector for detecting apoptosis, followed by monitoring the degree of GFP signal and PI staining in a single cell.

The present invention relates to apoptosis detection cells and apoptosis detection method using the same, and more particularly, a gene expression cassette which is cleaved by caspase-3 activated by apoptosis and moves a fluorescent signal into the nucleus. It relates to a cell death transformed cells transformed with a viral vector comprising a real-time apoptosis detection method using the same.

After the end of the human genome project, functional biology has been emphasized and proteome understanding and functional analysis have been important. Researches on the expression and function of genes and the actions of proteins in living cells are important. For example, researches on cell-based analysis systems that analyze fluorescent images by connecting GFP (green fluorescent protein) to specific proteins or genes Actively done. Increasingly, studies using cell-based techniques to analyze molecular interactions, such as ion channel activity, G-protein-coupled receptor responses, and apoptosis, are increasing.

Apoptosis is a morphological feature that distinguishes it from Necrosis. In cell necrosis, the cell membrane is destroyed, cell lysis is released, and inflammatory factors in the cell are released, causing an inflammatory response. In the case of cell death by apoptosis, the inflammatory reaction does not occur because the cells are formed by apoptotic bodies and are removed by the adjacent phagocytes without the release of the intracellular material. Apoptosis leads to various morphological, biochemical and physiological characteristics, including membrane blebbing, chromatin condensation within the nuclear membrane, formation of apoptotic bodies, and nucleosomal DNA ladders. Formation of DNA ladders, expression of phosphatidyl serine, a phospholipid of cell membranes, and phagocytosis by macrophages or macrophages (Michael, OH. Nature 407: 770-776, 2000).

The major mechanism by which external stimulation causes apoptosis is apoptosis mediated by receptors (Nagata, S. Cell 88: 355-365, 1997). Among them, many studies have been carried out on a method for signaling cell death by Fas. Fas is a receptor that is expressed throughout the surface of various cell types belonging to the Tumor Necrosis Factor (TNF) receptor family, and the cells expressing Fas bind to FasL (Fas ligand, CD95L), which is a ligand of Fas, and signal apoptosis into cells. (aponeosis signal) (Yonehara, S., Ishii, A., Yonehara, M., J. Exp . Med . 169: 1747-1756, 1989; Suda, T., et al ., Cell 75: 1169-1178, 1993). TNF-α is a cytokine structurally similar to FasL and performs various functions to participate in cellular defense mechanisms (Beg, A., Baltimore, D., Science 274: 782-784, 1996).

TNF-α carries a signal that causes apoptosis by its receptor, TNF-R, and a signal that survives cells through NF-κB, the opposite. This balance control is thought to play an important role in maintaining the appropriate number of cells. First, the signaling system that causes apoptosis by TNF-α is as follows (Hsu, H., Shu, H., Pan, MG., Cell 81: 495-504, 1996; Shu, H., Takeuchi, M ., Goeddel, DV., PNAS 93: 13973-13978, 1996; Hw, W., Johnson, H., J. Biol . Chem . 275: 10838-10844, 2000).

As a signal for early apoptosis, when TNF-α binds to its receptor, TNF-R1, and activates, the death domain of TNF-R1 attracts Fas-Associated Death Domain protein (FADD) and TRADD. , Procaspase-8 is cleaved into caspase-8 and activated through the DED portion of FADD (Boldin, MP, et. al . , J. Biol . Chem . 270: 7795-7798, 1995; Bolddin, MP., Et al . , Cell 85: 803-815, 1996). That is, TNF-R1, FADD, and Caspase-8 form DISC ( D eath I n S ignalling C omplex) to prepare for signaling to the next stage (Kischkel, FC., Et al ., EMBO J. 14 : 5579-5588, 1995; Muzio, M., et al ., Cell 85: 817-827, 1996; Muzio, M., et al ., J. Biol . Chem ., 273: 2926-2930, 1998). This activated caspase-8 induces apoptosis by cleaving the caspase species of the next stage and activating the caspase-3, the caspase, which is an executive qualification (Fernande-Alnemri, T., Litwack, G., Alnemri, ES., Cancer Res . 55: 2737-2752, 1997; Villa, P., Kaufmann, SH, Earnshaw WC., Trends Biochem . Sci ., 22: 388-393, 1997; Scaffidi C., et al ., EMBO J 17: 1675-1687, 1998).

Clontech has released the pCaspase3-sensor vector to measure the activity of caspase-3. The pCaspase3-sensor vector can be used to observe apoptosis by cleaving the gas phase-3 cleavage site by the activity of caspase-3 and moving into the nucleus, thereby emitting a fluorescent signal. However, due to the transient transformation of the transformant, low transformation rate, and difficulty in producing a permanently transformed cell line, the sale has been discontinued.

In order to measure the cells exhibiting the activity of caspase-3 (caspase-3) that is activated upon death by receiving apoptosis signal, the present inventors employ a virus expression vector comprising a caspase-3 detection gene cassette. The present invention was completed by confirming that the virus expression vector was transformed into a host cell to monitor cell fluorescence by monitoring a fluorescent signal.

It is an object of the present invention to provide apoptosis detection cells capable of directly analyzing apoptosis in a live state and apoptosis detection method using the same.

In order to achieve the above object, the present invention includes a gene expression cassette in which a neuclear export signal (NES), a caspase-3 cleaveage site, a reporter gene, a neuclear localization signal (NLS) are sequentially fused. It provides a viral vector for detecting apoptosis.

The present invention also provides an apoptosis detection cell transformed into a host cell with the apoptosis detection virus vector.

The present invention also provides a method for measuring apoptosis.

In addition, the present invention provides a method for screening apoptosis-inducing compound.

Hereinafter, the present invention will be described in detail.

Caspase-3, which exhibits activity in the cell upon apoptosis, cleaves the caspase-3 cleavage site of the gene expression cassette of the present invention, and the reporter gene and the NLS move into the nucleus to display a fluorescence signal. Measurement can be made (see FIG. 5B). The present inventors transformed the raw 264.7 cell line with a pCaspase3-sensor vector (see FIG. 1), which is an expression vector including a gene expression cassette in which NES, caspase-3 cleavage site, reporter gene, and NLS were sequentially fused (FIG. 2). Reference). Then, artificially induced apoptosis and observed the fluorescent signal in the cell. As a result, it was observed that the fluorescent signal in the cytoplasm moved into the nucleus after 5 hours (see FIGS. 3 and 4). However, the transformation rate of the vector was very low, 1 × 10 ⁻⁶ . Although the pCaspase3-sensor vector is useful for measuring apoptosis, it is temporarily transformed and has low transformation rate. Therefore, the pCaspase3-sensor vector is troublesome to perform a transformation process before cell death is observed in the cell line to be observed.

Therefore, the present inventors cut a gene expression cassette in which NES, DEVD (Casphase-3 cleavage site), GFP, and NLS were sequentially fused in the pCaspase3-sensor vector, and inserted the lentiviral expression vector. Thereafter, the prepared lentiviral recombinant expression vector was transformed into rat C6 glioma cells (see FIG. 5). The transformed rat C6 glioma cells were named “C6-caspase 3 sensing cells,” and were deposited in the Korea Cell Line Bank located at 28 Yeongeon-dong, Jongno-gu, Seoul (Accession No. KCLRF-BP-00136). Apoptosis was artificially induced by culturing C6 caspase 3 detection cells in a well plate culture system (see Figure 6.) As a result, it was confirmed that GFP fluorescence signal present in the cytoplasm was transferred into the nucleus when apoptosis occurred. (See FIGS. 7 and 8).

The present inventors observed and treated with PI (propidium Iodide), which is a fluorescent pigment that binds DNA to the cells for detection upon apoptosis in order to reconfirm whether apoptosis was measured. As a result, it was confirmed that PI and GFP fluorescence signals coincide in the time zone in which cell death occurs (see FIGS. 9 and 10). The inventors measured apoptosis in a single cell after inducing apoptosis in the detection cells. As a result, it was confirmed that the intracellular PI staining site and the GFP fluorescence signal matched (see FIG. 11), and the GFP fluorescence signal moved from the cytoplasm to the nucleus (see FIG. 12).

Through this, it is possible to measure cell death in real time in living cells using the "C6-caspase 3 sensing cell" for detecting apoptosis of the present invention, which is useful for screening substances that induce or inhibit cell death. It can be confirmed that.

The present invention provides a viral vector for detecting apoptosis, including a gene expression cassette in which NES, caspase-3 cleavage site, reporter gene, and NLS are sequentially fused.

In the viral vector for detecting apoptosis of the present invention, the reporter gene is an enzyme capable of measuring activity by GFP (green fluorescent protein) and fluorescent proteins of various colors, luciferase, fluorescence, luminescence or color development, It is preferable to select from the group consisting of fluorescent proteins using the FRET shape, but all reporter genes with fluorescent signals known to those skilled in the art can be used. In a preferred embodiment of the present invention, GFP is used as a reporter gene, but is not limited thereto. In addition, the viral vector is preferably selected from the group consisting of retroviral vector, lentiviral vector, adenovirus vector, adeno-associated virus vector, herpes virus vector, alpha virus vector, in a preferred embodiment of the present invention Viral vectors were used but are not limited thereto.

In the gene expression cassette of the virus vector for detecting apoptosis of the present invention, caspase-3 cleavage site is cleaved due to caspase-3 activity due to apoptosis, and the reporter gene and NLS are moved into the nucleus. This allows for the measurement of the movement position of the GFP signal in the cell and in real time for cell death.

The present invention also provides an apoptosis detection cell transformed into a host cell with the apoptosis detection virus vector.

In the apoptosis detection cell of the present invention, the host cell is preferably selected from eukaryotic cells including animal cells, insect cells, and yeast, but is not limited thereto. In a preferred embodiment of the present invention, rat C6 glioma cells were used as host cells, and those skilled in the art can use all cells for which cell death is to be observed as host cells.

The present invention also provides a method for measuring apoptosis.

The present invention provides a measuring method as follows: 1) transforming the cell vector for detecting apoptosis into a test cell; And 2) culturing the transformed cells of step 1) to monitor the fluorescence signal.

In the measuring method, the test cell of step 1) is preferably selected from eukaryotic cells including animal cells, insect cells, and yeast, but is not limited thereto. Cell death can be measured using In addition, the method of monitoring the fluorescence signal of step 2) is preferably monitored using a confocal scanning laser microscope, but is not limited thereto, and may be monitored by any method capable of measuring fluorescence signals known to those skilled in the art.

In addition, the present invention provides a method for detecting apoptosis-inducing and inhibitory compounds.

The present invention provides a method for detecting apoptosis-inducing and inhibitory compounds as follows: 1) treating a test compound to the apoptosis detection cell; And 2) monitoring the fluorescence signal while culturing the detection cells treated with the test compound of step 1).

In the screening method of the present invention, the test compound of step 1) may use any substance that a person skilled in the art can expect to cause or inhibit cell death, such as an existing compound, a natural compound, or a drug candidate. In addition, the method of monitoring the fluorescence signal of step 2) is preferably monitored using an inverted onfocal laser scanning microscope, but is not limited thereto. It can be monitored by the method.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

< Example  1> pCaspase3 - Sensor  Transformation Cell Line Construction Using Vector

We constructed a transgenic cell line using the pCaspase3-sensor vector (clontech, USA) (FIG. 1). The pCaspase3-sensor vector is a fusion of a nuclear export signal (NES), a caspase-3 cleavage site (DEVD), a green fluorescent protein (GFP), and a numeric localization signal (NLS). Raw 264.7, a mouse macrophage, contains DMEM medium containing 10% FBS (fetal bovine serum, Gibco ^™ , USA), 100 IU / ml penicillin (Gibco ^™ , USA) and 10 μg / ml streptomycin (Gibco ^™ , USA). (Gibco ^™ , USA) was incubated at 37 ℃ and 5% CO ₂ conditions. Raw 264.7 cells were passaged into 1 × 10 ⁴ cells in 12-well plates and transfection was performed 24 hours later. After 24 hours of passage, CLONfectin ^™ (4 μg in HEPES-buffered saline; Clontech, USA) and 4 μg of the plasmid were mixed, added to Raw 264.7 cell culture, and incubated for 4 hours in an incubator at 5% CO ₂ . After that, the medium containing CLONfectin ^™ and the plasmid was removed and replaced with the culture medium. After incubation for 24 hours, only 50% of neomycin (Sigma, USA) was treated to select the transformed cells (FIG. 2). As a result, the cell transformation rate was very low, 1 × 10 ⁻⁶ .

< Example  2> induction of apoptosis

Example 1 days perfusion chamber for conversion, Raw 264.7 cells transfected with pCaspase3-sensor vector in the (single perfusion chamber; Live Cell Instrument , Korea) to 1X10 passaged into ^four cells, and after 24 h a final concentration of 1 uM STS (staurosporine; Sigma, USA) to induce apoptosis. Cells were cultured at 5% CO ₂ , 37 ° C. Subsequently, the fluorescence signal was observed at each time to investigate whether GFP in the cytoplasm moves into the nucleus. For this purpose, an inverted confocal laser scanning microscope (TCS SL, Leica, Germany) that emits an argon ion laser was used. Fluorescence images of cells after STS treatment were observed at 1 hour intervals from 0 hours to 10 hours, excitation at 488 nm, and emission at 510-600 nm. Images were observed for fluorescence signals using a 100X (NA = 1.3) oil immersion lens at 1 hour intervals for 10 hours.

As a result, it was found that fluorescence shifted from the cytoplasm to the nucleus at 3-4 hours after STS treatment and cell death began to occur. After 5 hours of STS treatment, fluorescence completely shifted to the nucleus, and after 6 hours, cell wall was observed to be destroyed, confirming that cell death completely occurred (FIG. 3). The present inventors graphed the fluorescence intensity profile obtained by monitoring the fluorescence signal in one cell. As a result, after 1 hour of STS treatment, most GFP signals were observed in the cytoplasm and only some of them were observed in the nucleus, whereas after 5 hours of STS treatment, the GFP signal was markedly decreased in the cytoplasm and gradually increased in the nucleus (FIG. 4). ). As a result of the profile, about 50% of the GFP signal shifted to the nucleus between 5-6 hours after STS treatment. After 6 hours of STS treatment, 65% of GFP signals migrated to the nucleus. This experiment confirmed that apoptosis due to caspase-3 activity can be screened by the intensity of the GFP signal migrated from the cytoplasm into the nucleus.

< Example  3> Cascade  3 detection gene containing a cassette Lenti  Virus Expression Vector Preparation

Nhe -NES-DEVD-GFP-NLS Gene Cassette in pCaspase3-sensor Vector of Example 1 Ⅰ and Bcl After cutting into I, a blunt end was prepared using T4 polymerase and inserted into EcoR V of MCS (multi-cloning site) of LentiH1.4 expression vector (Vector Core Inc., Korea), and the lentiviral expression vector was It was named “LentiH1.4-pCaspase sensor expression transfer vector”. The lentiviral expression vector was produced in a 1: 1: 1 molar ratio with VSV-G expression vector (Vector Core Inc., Korea) and gag-pol expression vector (Vector Core Inc., Korea). 293T cells were transformed using Plus (Invitrogen, USA). Then, the culture medium supernatant containing virus vector particles was obtained after 48 hours, filtered through a 0.45 μM membrane filter (Nalgene, USA) and stored at -70 ° C.

< Example  4> Lenti  Transformant Production Using Virus Expression Vector

C6 rat glioma cells (Korea Cell Line Bank) consisted of 10% FBS (fetal bovine serum, Gibco ^™ , USA), 100 IU / ml penicillin (Gibco ^™ , USA) and 10 μg / ml streptomycin (Gibco). ^TM , USA) was cultured in RPMI 1640 medium (Gibco ^TM , USA) at 37 ℃ and 5% CO ₂ conditions. C6 rat glioma cells were passaged into 12-well plates with 1 × 10 ⁴ cells and transfection was performed 24 hours later. After 24 hours of cell passage, the culture medium was removed, and 300 μl of the lentivirus expression vector prepared in Example 3 and 3.4 μg of polyprene (Sigma, USA) were added while adding a new medium, and cultured overnight in a CO ₂ incubator. Thereafter, the culture medium to which the lentiviral expression vector was added was removed, replaced with fresh medium, and cultured for 72 hours. As a result, it was confirmed that the lentiviral expression vector is nearly 100% converted. Thereafter, puromycin (10 nM; Sigma, USA) was added and transformed cells were selected (FIG. 5). The cell line was named “C6-caspase 3 sensing cell” and was located at 28, Yeongun-dong, Jongno-gu, Seoul. It was deposited with the Korea Cell Line Bank on May 30, 2006 (Accession No .: KCLRF-BP-00136).

< Example  5> induction of cell death

The C6 caspase-3 detection cells prepared in Example 4 were passaged to a well plate incubation system (Live Cell Instrument, Korea) (FIG. 6) as 1 × 10 ⁴ cells and the final concentration of STS was 24 hours later. Treatment was performed to 700 and 1,000 nM cell death was observed in the same manner as in Example 2. In addition, the degree of apoptosis was measured in the same manner as in Example 2 by simultaneously treating 750 nM of PI (propidium iodide), which is a fluorescent pigment that binds to DNA upon cell death.

The C6 caspase-3 detection cells were treated with 700 nM STS. As a result of observing the fluorescence signal at 0, 10, 8 and 17 hours, it was confirmed from 17 hours that some fluorescence moved from the cytoplasm into the nucleus (FIG. 7). When the detection cells were treated with 1,000 nM STS, the shape of the cells began to change after 2 hours, and the fluorescence signal was observed to move into the nucleus (FIG. 8). The present inventors measured cell death by treating the fluorescent pigment PI binding to DNA with STS in order to reconfirm fluorescence signal shift according to the degree of cell death. The C6 caspase-3 detection cell was treated with 700 nM STS and 750 nM PI to observe the shift of fluorescence signal. As a result, staining was performed by PI from 9 hours after the treatment. As the movement began, the degree of movement gradually increased (FIG. 9). As a result, apoptosis occurred and the cytoplasmic GFP signal shifted to the nucleus. The present inventors treated 1000 nM STS and 750 nM PI to the C6 caspase-3 detecting cells to observe the fluorescence signal. As a result, the cells were stained by PI from 6 hours after treatment, and the intensity of the fluorescence signal began to move from the cytoplasm to the nucleus and gradually increased. However, at 6 hours, most cells changed shape and cells were destroyed by necrosis. (FIG. 10). In addition, apoptosis occurred at 6 hours, and the position of PI-stained DNA-binding coincided with the position of GFP signal moved from the cytoplasm to the nucleus. The present inventors apoptosis of cells moving from the fluorescent cytoplasm to the nucleus at constant time intervals for 24 hours using a confocal scanning laser microscope targeting one of the cells treated with 700 nM STS and 750 nM PI to the detection cells The fluorescence shift was observed by observing the process (FIG. 11), and a fluorescence intensity profile graph was prepared based on the above results. As a result, fluorescence gradually shifted from the cytoplasm into the nucleus (40%) in the cells treated with STS, and the fluorescence intensity of the nucleus increased by 50% or more after 12 hours, and 80% or more of fluorescence moved into the nucleus after 24 hours. (FIG. 12). Through this, it was confirmed that the prepared C6- caspase 3 detection cell transfers GFP signals from the cytoplasm into the nucleus upon cell death, thereby measuring cell death.

Apoptosis detection cell transformed with the apoptosis detection virus vector of the present invention and apoptosis detection method using the same can measure cell death of a target cell in real time, and is useful for searching for substances that cause or inhibit apoptosis Can be used.

Claims (10)

A viral vector for detecting apoptosis, including a gene expression cassette in which NES (Neuclear export signal), Caspase-3 cleavage site (CES), reporter gene, NLS (Neuclear localization signal) are sequentially fused. The method of claim 1, wherein the reporter gene is GFP (green fluorescent protein) and fluorescent proteins of different colors, enzymes that can measure activity by luciferase (luciferase), fluorescence, luminescence or color development, fluorescent protein using FRET shape Virus vector for detecting apoptosis, characterized in that selected from the group consisting of. The viral vector of claim 1, wherein the viral vector comprises a retroviral vector, a lentiviral vector, an adeno viral vector, an adeno-associated viral vector, and a herpes virus vector. herpes viral vector), alpha viral vector (alpha viral vector) is a viral vector for detecting apoptosis, characterized in that selected from the group consisting of. The viral vector for detecting apoptosis according to claim 1, wherein the viral vector is a lentivirus vector. Apoptosis detection cell which transformed the host cell with the virus vector for apoptosis detection of Claim 1. 6. The apoptosis detection cell according to claim 5, wherein the host cell is a eukaryotic cell including animal cells, insect cells and yeast. The apoptosis detection cell according to claim 5, wherein the host cell is a glioma cell. The apoptosis detection cell according to claim 5, which is a C6-caspase 3 sensing cell (Accession No. KCLRF-BP-00136). 1) transforming a test cell with the virus vector for detecting apoptosis of claim 1; And 2) Apoptosis measurement method comprising the step of culturing the transformed cells of step 1) to monitor the fluorescence signal. 1) treating the test compound to the cell death detection cell of claim 5; And 2) Apoptosis-inducing and inhibitory compound search method comprising the step of monitoring the fluorescent signal while culturing the cell death detection cell of step 1).

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