KR20040025183A - Method And DNA Chip For Monitoring Response of Irradiation on Cancer Patients Using Antioxidant Gene Expression Analysis - Google Patents

Abstract

PURPOSE: A method and a DNA chip for monitoring a response of cancer patients to irradiation therapy using antioxidant gene expression analysis are provided, thereby accurately anticipating the response to irradiation therapy and minimizing adverse side-effects thereof. CONSTITUTION: A method for monitoring a response of cancer patients to irradiation therapy comprises the steps of: collecting a peripheral blood cell from human; irradiating the peripheral blood cell; extracting RNA according to the time period; preparing DNA from the RNA; hybridizing the DNA with antioxidant enzyme cDNA; amplifying the hybridized DNA using one or more pairs of primers selected from (a) DNA fragments of SEQ ID NO: 1 and SEQ ID NO: 2, (b) DNA fragments of SEQ ID NO: 3 and SEQ ID NO: 4, (c) DNA fragments of SEQ ID NO: 5 and SEQ ID NO: 6, (d) DNA fragments of SEQ ID NO: 7 and SEQ ID NO: 8, (e) DNA fragments of SEQ ID NO: 9 and SEQ ID NO: 10, and (f) DNA fragments of SEQ ID NO: 11 and SEQ ID NO: 12; and analyzing expression pattern of the amplified DNA according to the time period. A DNA chip for monitoring a response of cancer patients to irradiation therapy amplifies one or more antioxidant genes corresponding to the following DNA fragments: DNA fragments of SEQ ID NO: 1 and SEQ ID NO: 2 - GPx1; DNA fragments of SEQ ID NO: 3 and SEQ ID NO: 4 - γ-GCS; DNA fragments of SEQ ID NO: 5 and SEQ ID NO: 6 - catalase; DNA fragments of SEQ ID NO: 7 and SEQ ID NO: 8 - CuZn SOD; DNA fragments of SEQ ID NO: 9 and SEQ ID NO: 10 - Mn SOD; and DNA fragments of SEQ ID NO: 11 and SEQ ID NO: 12 - Prx II.

Description

Method and DNA Chip For Monitoring Response of Irradiation on Cancer Patients Using Antioxidant Gene Expression Analysis}

The present invention relates to a method for evaluating the response of cancer patients to radiation therapy by analyzing time-dependent expression patterns of antioxidant enzyme genes specifically expressed from peripheral blood cells of irradiated humans, and a DNA chip therefor.

Ionizing radiation (IR) has been used for cancer treatment for almost a century (Hall et al., Int. J. Radiat. Oncol. Biol. Phys., 46, 1-2, 2000). Since side effects on normal cells are a limiting factor for radiation therapy (Peters et al., Cancer, 77, 2379 ~ 2385, 1996), in order to increase the efficiency of radiation therapy, lethal doses should be delivered to cancer cells and toxic effects on nearby normal cells. (toxic effects) should be reduced (Vijayakumar et al., Lancet, 349, 1-301, 1997). The response of patients exposed to the same dose of radiation varies widely, so for clinicians performing radiation therapy, the development of appropriate test methods to assess the patient's response to radiation therapy is of primary concern. In particular, the development of appropriate test methods to monitor the various reactions that occur after irradiation can be said to be the basis of the recent on-demand chemotherapy. Therefore, various attempts have been made to evaluate the effects of radiation therapy on various animal systems including the human body.

Since non-invasive methods are ideal for examining cancer patients' effects on radiation therapy in a clinically applicable manner, various methods have been developed to measure the response of various body fluids and blood cells. These methods are divided into five groups of indicator changes after irradiation (Walden et al., Biological assessment of damage, TMM Publications, Office of the Surgeon General., 85-103, 1989). The five groups are physical symptomology of prodromal syndrome, changes in white blood cell counts, changes in serum composition, components of urine, and chromosomal variations. Among these, measuring chromosomal variation in human peripheral blood lymphocytes (Peripherial Blood Lymphocyte) has long been considered a useful method for in vivo biodosimetry during radiation therapy. However, studies to find the correlation between radiation damage and chromosomal variation in peripheral blood cells have failed (Bentzen et al., Int. J. Radiat. Biol., 75, 513 ~ 517.1999). Peripheral blood cell metaphase analysis was not adequate to assess radiation damage (Lloyd et al., Phys. Med. Biol., 18, 421-431. 1973). This is mainly due to the postponed cell cycle by radiation (Yamada et al., Cancer Letters, 150, 215-221, 2000; Durante et al., Radiat. Res., 151, 670-676, 1999).

On the other hand, most of the studies aiming to identify molecular markers for the radiation response of cancer cells have examined gene expression through immunostaining, northern blot, or western blot analysis. . It is still difficult to assess the overall radiation effects in this way, and remains a fragmented approach through one or two types of genetic analysis. Assays to assess the response of normal cells to radiation therapy and incidental chemoradiation will be the basis for developing treatment protocols based on the response of treatment patients. In addition, if there is a problem in radiation-responsive gene function during chemotherapy, it may also have an effect on the generation of secondary cancers that appear as a sequelae of treatment. Therefore, further studies to characterize these radio-responsive genes and to investigate their in vivo and in vitro functions using Informatics and functional genomics investigations.

The expression patterns of genes are used to measure the response to radiation in normal cells and cancer cells, and the methods of investigation are DNA chips, differential display PCR (DD-PCR), serial analysis gene expression (SAGE), and EST (expressed). sequencetags). Among them, DNA chips can be applied to various applications such as disease diagnosis and new drug development as well as basic research in molecular biology because they can simultaneously observe the expression patterns of thousands or tens of thousands of gene combinations instead of individual genes. Therefore, measuring mRNA levels on DNA chips is a way to determine the response of different genes to specific stimuli. Although various genes have been reported to be induced by radiation in cancer cells, few studies have investigated this in patients undergoing radiation therapy. In addition, the most appropriate model for studying the in vivo radiation response in patients is to irradiate human blood cells ex vivo to study the genetic response to it, but it is used for radiation therapy. There have been no reports of genes induced by the amount or time to be derived, and many in vitro experiments on gene induction have been performed under supraphysiologic IR doses, which limits their potential for clinical use.

From this point of view, by comparing and analyzing the time-dependent expression patterns of genes in the irradiated sample, it is possible to accurately predict how much radiation can influence the individual's individual constitution from the results before, during and after the radiation treatment. What is needed is a way to minimize the side effects of radiation on patients.

The present invention is to analyze the genes induced by the time elapsed after irradiation as a dose used for general radiation therapy by collecting human peripheral blood cells according to the change of the reactive oxygen species produced by radiation Note the specific expression of antioxidant enzyme genes. In other words, the method of evaluating the response of cancer patients to radiation therapy by time-dependent expression of antioxidant enzyme gene was completed.

Therefore, the present invention is to analyze the time-dependent expression patterns of the antioxidant enzyme gene in the gene obtained from the irradiated human peripheral blood cells according to the elapsed time to evaluate the response of the cancer enzyme to the radiation therapy cancer substrate cDNA on the substrate An integrated DNA chip is provided.

In addition, the present invention is to collect peripheral blood cells from a human, and irradiated with the collected peripheral blood cells in a dose used for general radiation therapy, RNA is extracted from the irradiated peripheral blood cells according to the elapsed time, and the reverse RNA is extracted To generate DNA, the resulting DNA comprising (a) a DNA fragment of SEQ ID NOS: 1 and 2; (b) DNA fragments of SEQ ID NOs: 3 and 4; (c) the DNA fragments of SEQ ID NOs: 5 and 6; (d) DNA fragments of SEQ ID NOs: 7 and 8; (e) DNA fragments of SEQ ID NOs: 9 and 10; And (f) amplifying the antioxidant enzyme gene with one or more primer pairs selected from the group consisting of DNA fragments of SEQ ID NOs: 11 and 12, and analyzing the time-dependent expression patterns of the amplified antioxidant enzyme genes to respond to cancer patients' response to radiation therapy. Provide a method of evaluation.

1 is 1,221 hybridized with DNA fragments reversely transcribed RNA extracted from human peripheral blood cells irradiated with 2Gy, a dose used for general radiation therapy, and RNA extracted from human peripheral blood cells without irradiation. Fluorescence emission profiles of known gene cDNA chips are shown (Panel A: 2 hours after irradiation, Panel B: 6 hours after irradiation, Panel C: 24 hours after irradiation).

FIG. 2 shows 1,221 hybridized DNA fragments reversely transcribed RNA extracted from human peripheral blood cells irradiated with 2Gy, a dose used for general radiation therapy, and RNA extracted from non-irradiated human peripheral blood cells. Hierarchical clustering of known gene cDNA chips shows gene expression patterns.

FIG. 3 shows the results classified by hierarchical clustering of radiation-responsive genes (Panel A: Appears when 2 hours after initial gene-irradiation, Panel B: After 6 hours after gene-irradiation) Panel C: housed 24 hours after irradiation, panel D: housekeeping gene and ungrouped genes-no significant difference, high variation).

FIG. 4 shows six kinds of antioxidants from DNA fragments reverse-transcribed RNA extracted from human peripheral blood cells irradiated with 2Gy, which is a dose used for general radiation therapy, and RNA extracted from human peripheral blood cells without irradiation. The amplification of the enzyme gene is shown on the agarose gel (Fig. 4a: hourly expression pattern of GPx1, Figure 4b: hourly expression pattern of γ-GCS, Figure 4c: hourly expression pattern of Catalase, Figure 4d: CuZn Hourly expression pattern of SOD, 4e: hourly expression pattern of Mn SOD, 4f: hourly expression pattern of Prx II).

As used herein, the term "irradiation" refers to a treatment that kills cancer cells by the action of, for example, electromagnetic radiation including X-rays, ultraviolet rays, alpha particles and gamma rays or alpha or beta particles.

The term "Gy" is the amount of radiation capable of releasing 1 J (Joule) in 1 kg of biological tissue. The radiation used in the present invention is gamma-ray (gamma-ray, IBL 427 Irradiator, CIS Bio International, France), and 2Gy, which is a single dose generally used in chemotherapy, was used.

The term "radiation-responsive gene" refers to a gene tendency that exhibits various responses when exposed to radiation. That is, it shows a reaction such as cell death caused by DNA sequence mutation, abnormal cell circulation, and cell death. These changes differ in the timing of the responses from cell to cell. Genes that show these changes immediately after irradiation, especially early genes, are called genes that change only after some time. In this case, a gene in which no change occurs after irradiation is referred to as a "house keeping gene".

Radiation can affect both cancer cells and normal cells, but many methods and techniques are used to treat cancer by destroying many cancer cells while less affecting normal cells. This does not kill cancer cells immediately, but destroys the function of cancer cells to divide and proliferate, preventing new cancer cells from dividing and producing. Cancer cells that do not divide anymore die at the end of their lifespan. With every treatment, more cells die and dead cells are broken down and transported to the blood, where they are released out of the body, reducing the size of the tumor. Most of the normal cells recover, but some of the normal cells do not recover, resulting in side effects of radiation therapy. Changes in cells, tissues, etc. that may be caused by side effects include changes in cell division timing, cell death and necrosis inhibition, tissue damage, and immune dysfunction.

Biological samples for evaluating the response of cancer patients to radiation therapy can generally be used as a model system by ex vivo irradiation of human normal blood. More preferably, Peripheral Blood Lymphocytes isolated from the blood of cancer patients undergoing radiation therapy may be used. In the present invention, human peripheral blood cells were used. Peripheral blood cells are susceptible to radiation because they are sensitive to radiation, but are known to circulate rapidly throughout the body and to survive and regenerate for a long time in the body. Therefore, measuring chromosomal variation in peripheral blood cells has been used as a useful cell for in vivo biodosimetry during radiation therapy. Therefore, it is most preferable to select peripheral blood cells as a biological sample for measuring the degree of irradiation.

Representative methods of gene expression for radiotherapy include Southern blot, Northern blot, mutation detection and DNA sequencing. However, the results using these methods are very different and in some cases the opposite results. As one of the methods developed to overcome such a problem, a gene search method using a DNA chip capable of analyzing gene expression accurately and quickly is used. A DNA chip is a high density of DNA attached to a huge number of different types of DNA. DNA chips have the advantage of being able to search for at least hundreds of genes at the same time in a short time. In addition, in the case of Southern or Northern blots, a nitrocellulose membrane is used as a medium for attaching a dielectric material, whereas a solid material such as glass can attach a very small amount of genetic material at a high density, and at the same time, a large number of genes There is an advantage to search.

DNA chips are classified into cDNA (complementary DNA) chips and oligonucleotide chips according to the size of the attached genetic material. At least 500 bp of gene (full-length open reading frame or EST) is attached to the cDNA chip, and oligonucleotide consisting of about 15 to 25 bases is attached to the oligonucleotide chip. Table 1 describes general DNA chip fabrication techniques.

Characteristic of DNA chip making technology Production technology Characteristic Chip type Pin microarray Microspotting Using Pins cDNA and oligonucleotides Inkjet Microdropping using the inkjet principle cDNA and oligonucleotides Photolithography Oligonucleotide Direct Synthesis Using Photolithography Oligonucleotide Electronic array Oligonucleotide addressing using electricity Oligonucleotide

Currently, sequencing information from the Human Genome Project is growing rapidly, and this information can be reconfirmed from biomedical experiments. Therefore, the gene expression patterns can be analyzed by the above DNA chip technology to understand the overall cellular event. Therefore, in the present invention, after irradiating peripheral blood cells isolated from humans, a method of selecting time-dependent expression patterns of radiation-responsive genes using 1,221 known gene cDNA chips was used. Genes integrated in the cDNA chip (GenoCheck, Ansan, Korea) used in the present invention is a signal transduction, cell division, metabolism and adaptive / protective response (adaptive / protective response) These genes were produced by amplifying 1,221 known human genes by PCR. Hybridization results with the DNA reversely transcribed from cDNA chip and RNA of irradiated human peripheral blood cells were shown as a set of genes whose genes increased with time (FIG. 1).

Fluorescent materials used to analyze the hybridization results of DNA chips can be used Cy3-dUTP, Cy5-dUTP (Amersham Pharmacia, U.K.). It is a labeling substance that is optionally labeled to compare the intensity of gene expression due to color differences. Thus, when scanning gene expression, Cy3 becomes green at 532nm and Cy5 becomes red at 635nm. Although Cy3-dUTP and Cy5-dUTP were used in this study, Cy3-dCTP and Cy5-dCTP can be used.

Hybridization results of DNA chips were analyzed using a clustering technique to compare various gene samples. This expression of a particular gene was expressed as a vector value, and the degree of similarity according to a certain criterion (called "metric") was called "distance". Grouping the vectors together. Statistically developed statistical cluster analysis includes hierarchicalclustering, K-means, self-organizing maps, principal component analysis (SVD) decomposition, There is a suv vector machine (SVM) method.

In the present invention, hierarchical clustering method was used to cluster genes with similar expression patterns, and standardized by dividing the signal of each spot by the average value of the spot signal of each slide. Clustering results are shown in FIGS. 2 and 3, respectively.

As a result, 62 of the 1,221 genes of the cDNA chip were induced about 2 hours after irradiation (Panel A of FIG. 3), and 16 genes were induced 6 hours after irradiation (FIG. 3). Panel B), 27 genes appeared 24 hours after irradiation (Panel C of Figure 3). There were also 251 genes, including house keeping genes (Panel D of FIG. 3). And 865 genes among 1,221 genes could not be classified into specific categories. These genes have been shown in many normal people. The results show that at least 105 / 1,221 genes change significantly in irradiated peripheral blood cells compared to the control. This shows that the radiation treatment process can be effectively measured by analyzing gene expression patterns of irradiated peripheral blood cells using cDNA chip. Since most anticancer drugs cause DNA damage, stop cell cycles, and cause cell death, the gene sets identified in the present invention can be used for the treatment of various forms of radiation cancer. In addition, the expression patterns of 365 genes identified in irradiated peripheral blood cells will provide valuable information on the response of cancer patients undergoing radiation therapy, and biomarkers to examine patient-specific responses to irradiated radiation. It can be a biomarker. In other words, comparing and analyzing the relative expression patterns of radiation-responsive genes and the bioassay of gene expressions used in the past, measuring the value would be a complementary method to examine the response of cancer patients undergoing radiation therapy. .

Among the radiation-responsive genes derived from the cDNA chip results, the inventors noted the expression of antioxidant enzyme genes after irradiation. The biological significance after irradiation is the generation of reactive oxygen species (ROS) such as superoxide, hydrogen peroxide and hydroxyl radicals (Levinson, Exp. Cell. Res. , 43, 398-413, 1966; Walburg et al., New York, Academic., 5, 145-179, 1975; Simic et al., In Oxygen Radicals in Biology and Medicine, 587-590, 1989). It is also well known that the reaction of radiation with water generates reactive oxygen species. Thus, oxidative stress is induced by reactive oxygen species generated during radiation therapy, which can be an important biological molecule that damages normal tissues (Koh et al., J. Biochem. Mol. Biol. , 32, 440-444, 1999; Cho et al., J. Biochem. Mol. Biol., 33, 137-137, 2000; Park et al., J. Biochem. Mol. Biol., 34, 544-550 , 2001). In order to prove this, the present inventors analyzed specific expression of genes encoding antioxidant enzymes induced in irradiated peripheral blood cells to investigate the expression patterns of antioxidant enzyme genes according to changes in reactive oxygen species.

In order to obtain the antioxidant enzyme gene in the present invention, RT-PCR (Reverse Transcription-Polymerase Chain Reaction) was performed using RNA extracted from irradiated human peripheral blood cells.

RNA extraction can be performed using conventional methods in the art, using Tri reagent, using guanidine, using TRIzol reagent, etc., and using commercially available RNA extraction kits (Qiagen, introgen). Can be used. Preferably TRIzol reagent (GIBCO-BRL Grand Island, NY, U.S.A.) is used.

RT-PCR is a method well known in the art, a technique of synthesizing a corresponding cDNA (complementary DNA) using RNA of a specific site as a template, and then using the PCR amplification, the experimental process is reverse transcriptase (reverse preparing cDNA from RNA using transcriptase); ⑵ is divided into the process of amplifying a specific region using cDNA, ⑵ is the same as a method of amplifying a specific gene region from genomic DNA. Therefore, the antioxidant enzyme gene can be obtained by amplifying RNA extracted from a sample using a reverse transcriptase to generate an antioxidant enzyme gene and amplifying a specific DNA sequence using a primer pair to amplify the antioxidant enzyme gene. there was. Table 2 shows primer pairs for amplifying six kinds of antioxidant enzyme genes specifically changed.

Primer pairs for amplifying antioxidant enzyme genes gene location Sequence SEQ ID NO: GPx1 298-317827-808 5'-CCACCAGGAACTTCTCAAAG-3'5'-TGGCTTCTTGGACAATTGCG-3 ' 12 γ-GCS 413-4301046-1029 5'-GATCGTCTAGAACAGCCCTACGGAGGAAC-3'5'-GATCGGAATTCCTTCAATGGCTCCAGTTCC-3 ' 34 Catalase 617-6361021-1001 5'-CAGATGGACATCGCCACATG-3'5'-AAGACCAGTTTACCAACTGGG-3 ' 56 CuZn SOD 65-85462-445 5'-ATGGCGACGAAGGCCGTGTGC-3'5'-TTGGGCGATCCCAATTAC-3 ' 78 Mn SOD 65-84324-305 5'-GGCATCTGCGGTAGCACCAG-3'5'-TCTCCCTTGGCCAACGCCTC-3 ' 910 Prx II 68-85770-750 5'-GCTCACGCAGTCATGGCC-3'5'-CCCCTTCAGAGAGTGGAGGAA-3 ' 1112 β-actin 226-243843-826 5'-GATCGTCTAGATTCCTATGTGGGCGACGA-3'5'-GATCGGAATTCCAGCGGAACCGCTCATTG-3 ' 1314

The time-dependent expression pattern of the antioxidant enzyme gene obtained using the primer pair of Table 2 is shown in FIG. 4. As shown in FIG. 4, the expression of antioxidant enzyme genes was continuously increased with 2 hours, 6 hours, and 24 hours. Six types of antioxidant enzymes identified in agarose gels were GPx1, γ-GCS, Catalase, CuZn SOD, Mn SOD and Prx II.

In addition, in order to investigate the time-dependent expression patterns of six kinds of antioxidant enzyme genes, peripheral blood cells isolated from different people were irradiated according to the elapsed time. This is to see the reproducibility of the time-dependent expression patterns among different individuals. According to the results, the induction level between individuals was less than 1.5 times, and the expression patterns over time were similar among individuals. This means that these antioxidant enzyme gene mRNAs can be used in each patient as a biomarker when the induced transcript levels have a range of values.

In one embodiment, the present invention provides an antioxidant enzyme cDNA for evaluating the response of cancer patients to radiation therapy by analyzing the expression patterns of antioxidant enzyme genes over time from the irradiated human peripheral blood cells. A DNA chip integrated on a substrate is provided.

As a method for preparing a DNA chip, a conventional method as shown in Table 1 may be used, and preferably, a pin microarray method is used. In general, the method of manufacturing a DNA chip is as follows. Using all possible open reading frames for which sequences have been identified, the computer finds the primers that are required for PCR at the beginning and end of these identified genes, respectively. These primers have similar annealing temperatures, so they must be able to PCR multiple genes at the same time. After PCR, success is examined on an agarose gel and the amplified genes are concentrated to appropriate concentrations. The amplification and concentration genes are then planted on glass slides, which are often used in microscopic experiments, with microarray machines. In addition to the poly-L-lysine glass, a glass coated with silane, silylate, and super-amine may be used. Coating of the glass is necessary for fixing the probe transferred by the microarray. In order to fix the probe, a base capable of complementarily covalently bonding with the material coated on the glass may be inserted at the 3 'or 5' position of the probe. For example, an aldehyde group is inserted by using a linker column at the 3 'or 5' position of the probe to fix the probe to the glass coated with an amine group. In addition, in order to facilitate a covalent bond reaction, the reaction is induced at an appropriate humidity for 1 hour or more, and left for 6 hours or more to fix the DNA probe.

The DNA chip used in the present invention used cDNA (complementary DNA) as a probe, and is characterized in that it contains the antioxidant enzyme gene cDNA. The cDNA probe for detecting the antioxidant enzyme gene can be produced by amplifying genes such as GPx1, γ-GCS, Catalase, CuZn SOD, Mn SOD, Prx II, GST π, Prx I or GR as representative antioxidant enzyme genes.

DNA fragments detected through hybridization with the probe can be produced by a conventional method such as PCR using a gene extracted from a sample as a template. In the present invention, DNA fragments were prepared using primer pairs (Table 1) that specifically amplify antioxidant enzyme genes after obtaining DNA from RT-PCR using RNA extracted from a sample. In order to confirm the hybridization of the prepared DNA fragment and the DNA chip, the fluorescent material such as Cy3-dUTP and Cy5-dUTP (Amersham Pharmacia, U.K.) is added to confirm DNA binding from RNA extracted from a sample. It is a labeling substance that is optionally labeled to compare the intensity of gene expression due to color differences. In the embodiment of the present invention, Cy3-dUTP (green) and Cy5-dUTP (red) were used, but Cy3-dCTP and Cy5-dCTP may be used.

In order to analyze the hybridization result of the DNA chip of the present invention, any of the clustering techniques presented above may be used. Preferably, hierarchical clustering (hierarchical clustering) method was used to cluster the genes with similar expression levels.

As such, the antioxidant enzyme gene can be developed as a molecular marker by showing the difference in expression of the antioxidant enzyme gene in peripheral blood cells of irradiated humans using DNA chip technology combining literature informatics and functional genomics. Will be.

The invention will be more specifically illustrated by the following examples. However, these examples are merely embodiments of the invention and do not limit the scope of the invention.

<Example>

Example 1

Isolation and Irradiation of Human Peripheral Blood Cells

Blood was collected from 25 to 30-year-old normal subjects, and 50 ml of blood was treated with anticoagulants (EDTA, heparin, ACD), and the same amount of PBS (phosphate-buffered saline) was added. A 15 ml tube was filled with 4 ml of Histopaque 1077 (Histopaque 1077, Sigma, USA), and 8 ml of transfused blood (PBS = 1: 1) was lightly stacked thereon. The tube was centrifuged at 500 Xg (1,470 rpm), 25 ° C. for 30 minutes in a swing bucket. Centrifugation creates a white coat in the middle of the tube, which is then carefully shaken with a pasteur pippet, mixed with 10 ml of PBS in a 15 ml tube, followed by 700 Xg (1,800 rpm) at 25 ° C for 10 minutes. Washed. The pellet thus obtained was washed again with 10 ml of PBS. Add 10% heat-inactive fetal calf serum to RPMI 1640 (BRL / life technologies) medium at 56 ° C. for 45 minutes, 100 μl / ml penicillin and 10 μg / ml streptomycin 1 % Was added. The pellet was suspended in RPMI 1640 medium, and the number of cells was measured by a hematocytometer and placed in a T-25 flask to 1 × 10 6 cells / ml, followed by incubation in a 5% CO ₂ , 37 ° C. incubator. After 4 hours only the supernatant was recovered and transferred to a new T-25 flask. Each cell was aliquoted into 4 flasks of 5x106 cells. Each flask was reacted for 24 hours by adding 10 µg / ml 1% hemagglutinin (phytohaemagglutinin, PHA, Sigma). After 2 hours of irradiation at room temperature until 2 Gy was irradiated to a machine (Inha University Medical School) using 137Cs gamma rays (IBL 427 Irradiator, CIS Bio International, France) with 60 cGy / min of peripheral blood cells, 6 hours later, 6 After hours and after 24 hours, non-irradiated control cells and irradiated cells are collected and stored in a -70 ° C cryogenic freezer.

Example 2

Extraction of Total RNA from Human Peripheral Blood Cells

Total RNA was extracted from the peripheral blood cells prepared in Example 1. Total RNA was isolated from the peripheral blood cells of Example 1 using the Trizol (TRIZol, GIBCO-BRL Grand Island, NY, U.S.A.) method. All the solutions used were treated with 0.1% diethyl pryrocarbonate-treated water (DEPC-dH 2 O) to inhibit RNAse activity. Peripheral blood cell cells cultured in Example 1 were completely washed with PBS, and then pitted by adding 1 ml of TRIzol / 1 × 10 6 cells reagent to grind the cells. After standing for 20 minutes on ice, the mill was centrifuged at 14,000 rpm for 15 minutes at 4 ° C. 200 μl of chloroform was added to the supernatant, mixed by vortexing for 15 seconds, and repeated three times. After standing for 20 minutes on ice, the mixture was centrifuged at 14,000 rpm for 15 minutes at 4 ° C. To the new tube was added the same amount of phenol / chloroform, 0.2M sodium acetate (pH 5.2), only supernatant, mixed for 5 seconds and repeated three times. After standing for 20 minutes on ice, the mixture was centrifuged at 14,000 rpm for 15 minutes at 4 ° C. The same amount of isopropanol was added and mixed with the supernatant, and stored for 1 to 12 hours at -70 ° C, followed by centrifugation at 14,000 rpm for 10 minutes at 4 ° C to obtain total RNA. RNA pellets were washed with 1 ml of 75% ethanol treated with DEPC-dH 2 O, dried, re-homogenized with DEPC-dH 2 O and stored at -70 ° C. The RNA concentration was measured for absorbance at 260 nm (RNA; 1 OD 260 = 40 μg of RNA / ml) and 280 nm under the same conditions. 1 μl was run on a 1% TAE agarose gel.

Example 3

Preparation of DNA Fragment Using Total RNA of Human Peripheral Blood Cells

Fluorescently labeled cDNA was prepared using the total RNA of the irradiated control group and the irradiated experimental group irradiated in Example 2. 2 μg of 18-mer oligo dT (Tele-chem) was added to 50 μg of total RNA, and the RNA was denatured at 70 ° C. for 10 minutes and immediately put on ice. Each tube of 50 μg RNA of the control group and the irradiated control group and the irradiated group was irradiated with 10 μM dNTP, 100 μM DTT, 50 μM Cy3-dUTP (control RNA, Amersham Pharmacia, UK) and Cy5-dUTP (treated RNA, Amersham Pharmacia, UK), 200 u reverse transcriptase (4 U / μl, GIBCO-BRL Grand Island, NY, USA) was added and the total volume was 30 μl. At this time, RNA of the peripheral blood cells treated with radiation was labeled with Cy5-dUTP and RNA of the peripheral blood cells without radiation treatment was labeled with Cy3-dUTP. Each tube was incubated at 42 ° C. for 2 hours, and then the nucleotides labeled with each fluorescent material were mixed well and incubated at 65 ° C. for 10 minutes by addition of 1.5 N sodium hydroxide and 2.5 mM EDTA. After adding 470 μl of a pH 8.0 TE solution, the solution was placed in microcon-30 (Millipore, U.S.A.) and centrifuged at 14,000 rpm for 5 minutes. After centrifugation, each of Cy3 and Cy5 was concentrated to 8 μl, and then the column was inverted and centrifuged at 5,000 rpm for 2 minutes.

Example 4

Hybridization with DNA Fragments and cDNA Chips Prepared from RNA of Human Peripheral Blood Cells

In Example 3, hybridization of cDNA prepared by reverse transcription of total blood cells of peripheral blood cells with 1,221 known gene cDNA chips (GenoCheck, Korea) was performed.

Each cDNA prepared in Example 3 was mixed well, and 2.5 µl of 20X SSC and 0.3 µl of 10% SDS were added and boiled at 95 ° C for 2 minutes. In the hybridization chamber (hybridization chamber) was placed on the glass marking the spots on which the cDNA chip was placed. The cDNA was placed well without bubbles in the glass and then covered with a 22 x 22 mm cover glass. Drop 3X SSC to moisten both sides of the glass, add human COT1 DNA, yeast DNA, and polydeoxyadenylic acid to 65 ° C in a humidified chamber (CMT-hybridization chamber, Corning). Hybridization for 16 hours. At the end of hybridization, slides were washed once with 0.5X SSC containing 0.1% SDS at room temperature, washed again with 0.06X SSC for 5 minutes, centrifuged at 500 rpm, 25 ° C for 1 minute 30 seconds, and then laser confocal. Scanning was performed with an Exon scanarray 5000 with a laser confocal microscope (GenePix 5000, USA).

As a result of the experiment, as shown in FIG. 1, the expressed genes were shown in green and red according to the irradiation.

Example 5

Investigation of gene expression patterns by hierarchical clustering of cDNA chip data

Expression patterns of genes induced by radiation in the cDNA chip were compared by measuring the degree of luminescence of the fluorescent material. The fluorescence images (Cy3 and Cy5) of the two fluorescent materials were scanned at wavelengths of 532nm and 635nm, respectively, and laser intensity and PMT sensitivity were set to maximize the signal without increasing the background. . Gene levels with little change for irradiation were set at normal ranges and classified as radiation-responsive genes above normal ranges. In other words,β-actin, GAPDHIf the gene is considered as “1” as a standard and the ratio is more than 2 times higher than that, it is regarded as an up-regulated gene (radiation-responsive gene). If it is 0.5 times or less, it was considered as a down-regulated gene. Eisen (Eisen et al., Proc. Natl. Acad. Sci., 95, 14863-14868. Clustering was performed using GeneSpring software (Silicon Genetics, CA., U.S.A., 2001) according to the method of 1998).

Hybridization results of the cDNA chip were able to cluster between genes with similar gene expression patterns. Clustering results are shown in FIGS. 2 and 3, respectively. The signal of each spot was normalized by dividing by the average value of the spot signal of each slide. Of the 1,221 genes of the cDNA chip, 62 genes were induced 2 hours after irradiation (Panel A of FIG. 3), and 16 genes were induced 6 hours after irradiation (Panel B of FIG. 3). . Twenty-seven genes appeared 24 hours after irradiation (Panel C of Figure 3). In addition, 251 genes were shown, including the house keeping gene (Panel D of FIG. 3). And 865 genes among 1,221 genes could not be classified into specific categories. These genes have been shown in many normal people.

Example 6

Analysis of Hourly Expression of Antioxidant Enzyme Genes Obtained from Human Peripheral Blood Cells Irradiated by Reverse Transcription Chain Polymerization (RT-PCR)

The RNA extracted from the peripheral blood cells of Example 2 was reverse transcribed to identify antioxidant enzyme genes. 1 μl of RNA and 1 μl of 10 × random hexamer were added, DEPC-treated water was added to 20 μl, and then reacted at 70 ° C. for 10 minutes, and immediately cooled with ice. 4 μl of 5 × first strand buffer, 100 μl DTT, 4 μl of 10 mM dNTP mixture, 0.2 μl M-MLV reverse transcriptase, mix well, centrifuge, and react at 37 ° C. for 1 hour 30 minutes. Incubation of residual protein by incubation for 10 minutes at ℃. 30 μl of distilled water was added thereto and stored at −20 ° C. until next use. The DNA thus prepared was 2 μl of 10 × buffer solution, 1.6 μl of 10 mM dNTP, 0.5 μl of primer pair (50 pmol) used to amplify 9 kinds of antioxidant enzyme genes, 1 μl of DNA, 0.1 μg of DNA, Taq polymerase (TaKaRa, 5U / μl) 0.1 After the addition of the solution was adjusted to 20 μl with distilled water, 35 cycles were performed for 5 minutes at 94 ° C., 1 minute at 95 ° C., 1 minute at 54 ° C., and 1 minute at 72 ° C. Elongation at 72 ° C. for 10 minutes was then confirmed on 1% TAE agarose gel. At this time, the DNA obtained by RT-PCR of the peripheral blood cells of the control group irradiated with β-actin and irradiated with the peripheral blood cells after 2 hours, 6 hours, and 24 hours after irradiation was confirmed on an agarose gel.

As a result, the expression of antioxidant enzyme genes increased continuously over 2 hours, 6 hours and 24 hours. The antioxidant enzyme genes identified by agarose gels were GPx1, γ-GCS, Catalase, CuZn SOD, Mn SOD and Prx II, which are six types (Fig. 4). In addition, to investigate the time-dependent expression patterns of six kinds of antioxidant enzyme genes, experiments were conducted on the samples irradiated with peripheral blood cells isolated from normal humans, to see the reproducibility of the time-dependent expression patterns among different individuals. According to the results, the induction level between individuals was less than 1.5 times, and the expression patterns over time were similar among individuals. This means that these antioxidant enzyme gene mRNAs can be used in each patient as a biomarker when the induced transcript levels have a range of values.

The present invention can be useful for comparatively analyzing the response of cancer patients by radiation therapy by providing a method for analyzing the expression patterns of antioxidant enzyme genes in peripheral blood cells of irradiated humans and a DNA chip therefor.

Claims (3)

A DNA chip in which an antioxidant enzyme gene cDNA is integrated on a substrate in order to evaluate a response of cancer patients to radiation therapy by analyzing the expression patterns of antioxidant enzyme genes in the genes obtained according to elapsed time from irradiated human peripheral blood cells. The DNA chip of claim 1, wherein the DNA chip is used to amplify one or more antioxidant enzyme genes corresponding to the DNA fragments and to analyze the time-dependent expression patterns of the amplified antioxidant enzyme genes. DNA fragments of SEQ ID NOs: 1 and 2-GPx1; DNA fragments of SEQ ID NOs: 3 and 4-γ-GCS; DNA fragments of SEQ ID NOs: 5 and 6-Catalase; DNA fragments of SEQ ID NOs: 7 and 8—CuZn SOD; DNA fragments of SEQ ID NOs: 9 and 10—Mn SOD; And DNA fragments of SEQ ID NOs: 11 and 12-Prx II. Peripheral blood cells are collected from humans, the collected peripheral blood cells are irradiated with a dose used for general radiation therapy, RNA is extracted from the irradiated peripheral blood cells according to elapsed time, and the extracted RNA is reverse transcribed to generate DNA. And, the generated DNA (a) DNA fragments of SEQ ID NO: 1 and 2; (b) DNA fragments of SEQ ID NOs: 3 and 4; (c) DNA fragments of SEQ ID NOs: 5 and 6; (d) DNA fragments of SEQ ID NOs: 7 and 8; (e) DNA fragments of SEQ ID NOs: 9 and 10; And (f) amplifying the antioxidant enzyme gene with one or more primer pairs selected from the group consisting of DNA fragments of SEQ ID NOs: 11 and 12, and analyzing the time-dependent expression patterns of the amplified antioxidant enzyme genes to respond to cancer patients' response to radiation therapy. How to evaluate.