KR20130122559A - Preparation method of in vitro irradiation-induced cell or tissue injury model - Google Patents

Abstract

The present invention relates to a method for manufacturing a disease model for an in vitro test for developing therapeutic techniques and drugs in medical and pharmaceutical fields and, more specifically, to a method for easily manufacturing a disease model by effectively radiating radiation.

Description

{Preparation method of in vitro irradiation-induced cell or tissue injury model}

The present invention relates to a method for manufacturing a disease model for in vitro cell unit experiments performed for disease treatment and drug development in the pharmaceutical field, and more specifically, to damage models of various cells and tissues by radiation through efficient irradiation It relates to a method that can be produced more easily.

In the field of medicine, various experimental models of cells, tissues or animals are used for the development of disease treatment techniques and drugs. Animal experimental models have been mainly used as a method for easily and quickly confirming disease treatment effects and efficacy of drugs. However, there is a limit to the types of diseases that can be shared between humans and animals, and since the recent opposition to animal experiments due to ethical consideration of animal sacrifices is increasing, there is an alternative to animal experiments. As a result, cell-based experimental models have been in the spotlight.

On the other hand, more than about 50% of all cancer patients are receiving radiation during the course of cancer treatment, which is widely used to treat a variety of benign and malignant tumors, primary and metastatic tumors, and vascular diseases. In particular, radiation therapy is one of the important therapies for the treatment of primary brain tumors, metastatic brain tumors, leukemia or lymphoma, head and neck cancers involving the central nervous system. However, the tolerance of normal cells or tissues to radiation is limited, and it is necessary to adjust the radiation dose to minimize damage to normal cells or tissues due to irradiation. Thus, radiation therapy for cerebral disease is intraoperative radiotherapy, which is irradiated with a large amount of radiation only to the lesion site and minimizes the radiation dose to normal brain tissue, away from conventional whole brain irradiation The area of treatment is expanding with interstitial brachytherapy, gamma knife or linear accelerator radiosurgery. Despite these efforts, however, irradiation of normal tissue around the lesion is inevitable and complications have not diminished (Br J Cancer 85: 1233-1239, 2001). In particular, irradiation of the head in children or adults has a problem that can cause irreversible and severe cognitive impairment several months to years after irradiation.

As such, various drugs are being researched to prevent or treat damage to normal tissues due to irradiation, but drugs or treatments that have excellent effects have not been developed.

Therefore, in the field of radiation therapy, experimental models of cell or tissue damage due to irradiation are very useful for the purpose of studying various mechanisms of cell and tissue damage caused by radiation or to develop techniques and drugs for treating the damage. Can be.

Radiation damage cell or tissue experimental models conventionally used in the field of radiation therapy were radiation damage models based primarily on x-ray instruments. However, in order to prepare the x-ray based cell experimental model, there was a limit to generally culturing the cells in large flasks (25-cm flasks) in order for the cells to be uniformly exposed to radiation (Yamakawa et.al. , 2008, Lin et al., 2010, Mackonis et al., 2011) (see FIG. 14).

When culturing cells in such a large flask, not only is a large time and economic waste because many cells and medium are needed at a time, but also some cells are clustered or some are separated, which makes it difficult to distribute the cells evenly. There is a problem that there is a high possibility that an error occurs in the experimental results because the radiation is not uniformly irradiated.

In addition, when a small culture dish such as a 100 pi cell culture dish or a 6-well or 96-well plate, which is generally used for cell culture, is used, a certain amount of cells can be dispensed relatively uniformly. However, there is a problem that only one dose is applied to one plate, or only one type of cell in one culture dish. For example, to investigate the effects of specific radiation on various cell types or the effects of various radiation on one cell type, separate experiments are performed several times for each condition (by cell type or by radiation dose). There is a problem to be done, there is a big time and cost burden.

Therefore, not only high accuracy results can be obtained by uniform distribution of cells and uniform irradiation of the cells in the culture vessel, but also a highly efficient irradiation system and analysis through the effects of various conditions in one experiment. There is a need to develop a method for producing a cell-level radiation damage model.

Accordingly, the present invention has been made to solve the problems of the conventional x-ray-based irradiation technology, it is possible to uniformly irradiate all the cells that actually exist, not only high accuracy, but also simultaneously for various kinds of cells It is an object of the present invention to provide an efficient irradiation system and a method of manufacturing a cell-based radiation damage model through which radiation can be irradiated, and further, irradiation doses of various conditions can be simultaneously performed by varying the dose of each cell.

Conventional cell damage experiments have been carried out based on x-ray equipment, but as described above, there is a problem that not only is uneconomical but also a high possibility of error in experimental results. In order to solve this problem, the present inventors have found that the high-efficiency radiation damage model, which is used in actual clinical trials, is modified to be optimized for cell-based damage experiments, thereby producing a highly accurate and accurate radiation damage model. It was confirmed that it is possible. In addition, by using a radiation source, such as iridium (Ir), which is used for radiation treatment for actual cancer, an experimental model similar to the actual clinic can be created, and an experimental model can be effectively applied to the development of new disease treatment technology and drugs. The present invention was completed by confirming that it can be easily and simply manufactured.

Hereinafter, the present invention will be described in more detail.

Radiation therapy is largely classified into external beam radiation therapy, which is a method of irradiating radiation from the outside of the human body, and internal radiation or brachytherapy, in which radioactive isotopes are placed directly on the tumor site for treatment. can do.

The key to brachytherapy is to deliver radioisotopes only to and near the target lesion and to reduce transmission to and away from the target lesion, using devices such as linear accelerators to provide high energy radiation or electron beams. Unlike external radiation therapies, which irradiate the patient's outside to allow radiation to reach the tumor site, the radioisotope is inserted directly into the tumor, or a catheter or catheter (into the organ) A mammosite, a catheter with a balloon, is inserted to irradiate a strong dose to the tumor site. Proximity radiation therapy has the advantage of minimizing unwanted exposure to radiation isotopes compared to external radiation therapy, and is widely used in the treatment of cancer in actual clinical practice. In particular, proximity radiotherapy using a catheter aims to directly insert the end of the catheter into the lesion site of the human body to increase the radiation treatment efficiency of the localized area and to minimize the risk of irradiation of surrounding normal tissue.

The present invention is applied by modifying the proximity radiotherapy technology to be optimized for the first cell unit or tissue unit experimental model.

Specifically, the present invention provides radiation by irradiating cells or tissues using a radiation source and a conduit used in proximity radiation therapy, so that x-ray-based experiments that have been used in the manufacture of conventional cell models emit and emit radiation beams. It provides a technique for overcoming the shortcomings of inaccuracy and improving the accuracy of cell experiments. At this time, unlike the conventional proximity radiotherapy technology was used in the manner of inserting the catheter directly into the human body, in the present invention, after placing the catheter on top of a single or a plurality of culture vessels containing cells or tissues, the radiation is specified. Characterized by irradiation under conditions. According to the present invention, it is possible to form a uniform radiation exposure area in the cells, it is possible to manufacture a cell-based radiation damage model with high efficiency in an easier and simpler way.

In one embodiment, the present invention comprises the steps of 1) preparing a plate comprising a plurality of wells, culturing cells or tissue in each well; 2) placing a conduit of the proximity radiation treatment device across the top of each well; And 3) determining the radiation dose and irradiating radiation from the conduit.

In the above, the cell or tissue to be irradiated may be derived from all living organisms, including humans, the kind is not particularly limited. For example, all cells or tissues derived from ectoderm, mesoderm, or endoderm that make up the body of an animal; Pathological cells (eg, tumor cells); All normal cells, including cranial nerve cells; Embryonic cell; Stem cells of all lines, including adult stem cells and dedifferentiated stem cells; Artificially engineered, constructed and cultured cells; The technique of the present invention can be applied to all kinds of cells, from bacteria or viruses to cells constituting plants.

In the above, various well-known culture vessels, such as a culture dish used in the field of a cell or tissue culture, can be used as a plate containing a some well. For example, a cell culture dish consisting of 6, 12, 24, 48, 96, or 384 wells may be used, but this is merely a representative example and the scope of the present invention is not limited thereto. Preferably a 6 well culture dish can be used. A culture dish containing a plurality of wells may be used in single or in plurality.

In the above, as the proximity radiation treatment apparatus, a device including a radiation source and a conduit can be used as a device currently used in the proximity radiation therapy therapy in the clinic. Preferably, the radiation source may be located in the space inside the conduit. As a preferred example, a remotely controlled High Dose Rate (HDR) brachytherapy device that uses a high dose rate such as Gammamed 12i using iridium ( ¹⁹² Ir) as a radiation source for irradiating the planned dose in a short time may be used. . In addition, a variety of radiation irradiation equipment (Ballester et al.) Using various materials capable of emitting radiation, such as ¹³⁷ Cs, ¹⁹² Ir, ¹²⁵ I, ¹⁹⁸ Au, ²⁴¹ Am, ¹⁰³ Pd, ¹⁴⁵ Sm or ¹⁹ Yb as a radiation source It can use without a restriction | limiting in particular. Examples include Microselectron (HDR) Nucletron Engineering BV (Netherlands), Gamma med plus MDS / Nordion (Canada), VariSource Varian associates (United States), and the like.

As the radiation source for irradiation, ¹⁹² Ir for emitting gamma rays, ³² P for emitting beta rays, ⁹⁰ Sr / ⁹⁰ Y, ⁹⁰ Y, ¹⁸⁸ Re, ¹⁶⁶ Ho, and the like, and the radioisotopes are ¹⁹² Ir seeds. ), ³² P coated stents, ⁹⁰ Y wires or coils, ¹⁸⁸ Re solution, ⁹⁰ Sr / ⁹⁰ Y seeds or ¹⁶⁶ Ho-coated balloons It can be used in the form of a stent. Preferably, ¹⁹² Ir may be used in a seed form.

Radiation source wire systems using conduits can make radiation sources such as ¹⁹² Ir, ³² P, ⁹⁰ Sr / ⁹⁰ Y into metallic canisters or seeds with a diameter of 0.014 to 0.040 inches that can enter the wire ends. The radiation source can be advanced downward by entering an iron wire containing the radiation source through the lumen of. This process is called afterloading and can be driven manually or by motor (Teirstein PS, Massullo V, Jani S, Popma JJ, Mintz GS, Russo RJ, Schatz RA, Guarneri EM, Steuterman S, Morris NB, Leon MB). , Tripuraneni P. Catheter-based radiotherapy to inhibit restenosis after coronary stenting.N Engl J Med 1997; 336: 1697-703.).

The conduit is preferably made of a material that does not transmit plastic or vinyl radiation, and the standard and the number of conduits may be appropriately selected and used in accordance with the standard of the irradiation device. Preferably at least one, more preferably from one up to 24 may be used.

The conduit can be configured to arrange a uniform radiation exposure area by placing each cell or tissue across the center of the cultured vessel, for example by attaching it to the cover of the vessel in various ways (tape, glue, etc.) The conduit can be secured to the top of the vessel.

In addition, it may be preferable to include a plurality of cathodes (cathode) that is the point where the radiation stops in the interior of the conduit, preferably one to five cathodes can be arranged so that the top of one vessel (well) have.

The irradiation step may preferably comprise a step of irradiating radiation with 0 to several tens of Gy. All matters concerning the equations and calculations related to the transit time, dose, distance and location of radiation can be applied with reference to a paper published by Supe et al. (Pol J Med Phys Eng 2007; 13 (2): 55-64) .

In addition, even if the type of proximity irradiation apparatus and the type of the radiation source used are different, if the method according to one embodiment of the present invention is used, a plate in which a plurality of wells exist by adjusting the radiation positioning time and distance, preferably 6 Irradiation experiments can be uniformly performed on the well plate.

As a specific example, when using a ¹⁹² Ir as a radiation source and using a 6-well plate having a diameter of about 2 cm and a height of about 1 cm for each well, the radiation is shown in the right conduit of FIG. 1A. As it moves along, the exact amount of radiation at each moving point (cathode) spreads evenly over the entire area inside each well and does not affect other wells around it, resulting in highly accurate results (see FIGS. 3 and 4). .

The manufacturing method of the present invention has an effect that can be adjusted so that the radiation dose independently per each well as described above, according to the purpose of the experiment to apply a single experiment, such as applying a variety of radiation doses or various kinds of cells at the same time Through this, it is possible to manufacture a large number of diverse and accurate experimental models. To this end, in a preferred embodiment, different types of cells or tissues may be cultured in each of the plurality of wells constituting one plate in the present invention. As another example, it is possible to irradiate different rows of wells with different radiation doses from conduits across the top of each well.

As described above, the manufacturing method according to the preferred embodiment of the present invention is capable of uniformly irradiating all cells or tissues in the container by using a plurality of containers and conduits as described above, thereby providing a high accuracy and uniformity of the damage model. In addition to being able to manufacture, it is also possible to irradiate various types of cells or tissues at the same time to prepare a damage model that allows comparative analysis of the effect on a variety of cells or tissues of a specific radiation dose. In addition, it is possible to manufacture a damage model that can compare the effects of various radiation doses by varying the irradiation doses for specific types of cells or tissues, simply and easily through a single experiment, thereby dramatically reducing the number of experiments and the overall cost. Can be reduced. In addition, the method of the present invention can be effectively applied to the development of new disease treatment technology and medicament research results conducted in the laboratory by using a radiation source used for radiation therapy in the clinical field, such as actual cancer treatment.

According to the method of manufacturing a cell-based radiation damage model of the present invention, all cells can be uniformly irradiated, and thus the accuracy of the experiment is remarkably improved, and radiation can be simultaneously irradiated on various cell types, and the dose of each cell can be adjusted. Differently, irradiation experiments under various conditions can be performed at the same time, thereby easily and simply manufacturing high-quality experimental models that can be effectively applied to the development of novel disease treatment technologies and drugs.

1A shows where the radiation conduit is placed (white arrow) and the point where radiation stops (black arrow: indicated using X-ray) in 6 wells.
1B shows the state when the radiation conduit is fixed in 6 wells.
1C shows the radiation machine used. White arrows indicate conduits as in FIG. 1A.
1D shows how the conduit is placed in the cell culture dish and the irradiation distance, respectively.
FIG. 2 shows how the x, y axis was taken according to the position of the conduit and the point was taken from the conduit to the bottom of the vessel to produce the desired three doses. That is, by adjusting only the x, y, z axis and the radiation positioning time using this invention device, the radiation dose is calculated, indicating that the desired and uniform radiation dose can be obtained anywhere within the 6 wells.
Figure 3a shows the calculated time of stay for each point in the radiation dose 1Gy (67 seconds).
3B is a table confirming that the set radiation dose is uniformly emitted (average radiation dose 0.99Gy).
Figure 3c shows the calculated time of stay for each point in the radiation dose 3Gy (203 seconds).
3D is a table confirming that the set radiation dose is uniformly emitted (average radiation dose 3.00Gy).
Figure 3e shows the calculated time of stay for each point in the radiation dose 5Gy (337 seconds).
3F is a table confirming that the set radiation dose is uniformly emitted (average radiation dose 4.98Gy).
FIG. 4A is a graph showing the radiation dose measured according to the distance from the conduit line at 1 Gy based on the average radiation dose obtained in FIG.
FIG. 4B is a graph showing the radiation dose measured according to the distance from the conduit line in 3Gy based on the average radiation dose obtained in FIG. 3.
FIG. 4C is a graph showing the radiation dose measured according to the distance from the conduit line at 5 Gy based on the average radiation dose obtained in FIG.
5 is a graph showing the survival rate of human adipose derived mesenchymal stem cells (MSCs) after 1 to 5 Gy irradiation.
Figure 6 shows a micrograph with a magnification of 200 times the change in MSCs after 1 ~ 5 Gy irradiation.
7 shows Western blot results of cleaved caspase-3 and Bax, which are indicators of apoptosis derived from MSCs after 1 to 5 Gy irradiation.
8 is a graph showing the survival rate of human glioblastoma cells (U87 cells) after 1 to 5 Gy irradiation.
FIG. 9 shows a micrograph at 200 times magnification of changes in U87 cells after 3-10 Gy irradiation.
FIG. 10 shows Western blot results of cleaved caspase-3 and Bax, apoptosis indicators derived from U87 cells after 3 to 10 Gy irradiation.
11 is a graph showing the survival rate of cortical neurons (CN cells) of white paper after 5 to 40 Gy irradiation by MTT assay.
Figure 12 shows a micrograph showing a 200-fold magnification of changes in CN cells after 10-40 Gy irradiation.
Figure 13 shows the micrographs and Hoechst stain staining of the control (non-irradiated group) and 40-fold magnification of CN cells after 40 Gy irradiation.
Figure 14 shows the Western blot results of cleaved caspase-3 and Bax, apoptosis indicators derived from CN cells after 10-40 Gy irradiation.
15 is a graph showing the survival rate of the cells by LDH assay for 12-48 hours after 40Gy irradiation to CN cells.
Figure 16 shows the hourly Western blot results of cleaved caspase-3, cleaved PARP and Bax, which are indicators of apoptosis derived from the cells until 12-48 hours after irradiating 40Gy to CN cells.
FIG. 17 illustrates a problem in that a large flask of 25 cm ² must be used in order to use a radiation source having a wide beam like a photon in a conventional x-ray based experiment, and the beam is not evenly irradiated.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. However, the following examples are merely to illustrate the present invention is not limited to the scope of the present invention.

< Example  1>

1-1. Cell preparation

Each cell was subcultured equally in a plate containing 6 wells and then grown to 100% full. The cultured cells were divided into a control group not irradiated with radiation and an experimental group irradiated with radiation (groups depending on the dose of 1-40 Gy irradiation). Three experiments were performed for each group, and the results for each group were analyzed by Western blot using cleaved caspase-3, PARP, and bax antibodies by extracting proteins from the number of viable cells and the viable cells after 24 hours from the time of irradiation. It was shown by the value obtained by carrying out blot) (refer Example 2). The three substances are proteins that suggest apoptosis of cells.

1-2. Irradiation

Irradiation was performed using GammaMed 12i (MDS / Nordion, formerly Isotopen-technik Dr. Sauerwein GMBH), a high-dose brachytherapy device previously used for brachytherapy, and Ir-192 (seed type) as a source. ) Was used. The activity of the source was 370 GBq (10 Ci), and the reference air kerma rate (RAKR) was 40,84 mGy · m ² / h.

The conduit of the device was fixed on top of the culture dish to cross the center point of the cell culture dish to create a uniform irradiation field around the conduit.

Specifically, in this experiment, three rows of 6-well cell culture plates arranged in two rows of conduits were irradiated with a line of conduits crossing the center of each well, and the radiation was stopped inside the conduit. Cathodes were configured to be placed 3 or 4 per well and the catheter was included in the equipment and used for the actual patient as the specification according to the manufacturer's protocol. The number of cathodes was derived by calculating equal radiation to each well of the 6 well plate (Pol J Med Phys Eng 2007; 13 (2): 55-64) (see FIGS. 1 and 2).

The conduit is placed and the positioning time of the conduit point of the radiation source according to the radiation dose is calculated and set to 67 seconds for 1Gy, 203 seconds for 3Gy, and 337 seconds for 5Gy, respectively. Average radiation doses within 0.99Gy (FIG. 3B), 3.00Gy (FIG. 3D), and 4.98Gy (FIG. 3F) were confirmed to be uniformly irradiated with desired radiation doses (Pol J Med Phys Eng 2007; 13 (2): 55-64).

In addition, as a result of the contour of the radiation dose measured according to the distance away from the conduit line based on the obtained average radiation dose, the radiation exposure area is uniformly equal to a radius of 1 cm without overlapping for each well at any arbitrarily set radiation dose of 1, 3 or 5 Gy. It could be confirmed that it appears. As a result of irradiating the radiation according to the present embodiment, not only the radiation dose may be equally irradiated to each well, but also the radiation is not exposed to other wells besides the corresponding well, so that the irradiation with high accuracy without affecting each other It was confirmed that it is possible (FIGS. 4A to 4C).

< Example  2> Measurement of Cell Change According to the Radiation Dose

2-1. Cell preparation

Brain cortical neurons (CN, directly extracted and cultured by the investigator), human adipose-derived mesenchymal stem cells (MSCs, provided by R & B Bio) and human glioblastoma cells (U87 cell) in Sprague-Dawley rats Cellular change (apoptosis incidence and survival pattern) was measured by exposure to three radiation doses.

First, dispense 3 to 4 MSCs and U87 cells from 1 vial containing 1 X 10 ⁶ into a 100 pi dish, and add 10% FBS (fetal bovine serum) (F0600-050 from GenDEPOT) and 1% penicillin ( DMEM (CA002-010 from GenDEPOT product) (CM002-050 from GenDEPOT product) was incubated at 37 ° C., 5% oxygen conditions for 3-4 days to fill 90%. It was then sub-cultured to contain 1 × 10 ⁵ cells per well of a 6 well culture plate. After stabilizing the cell seeding (Cells seeding) three more days were exposed to radiation dose 1, 3, 5, 10, 20, 40 Gy, respectively. The control group used cells that were not exposed to radiation.

After irradiation, cells are counted after 12, 24, 36 and 48 hours to observe cell apoptosis and recovery, and the survival pattern of living cells is obtained by dissolving surviving cells in lysis solution to extract proteins. Western blot experiments were performed by observing caspase-3 (ca9662 for cell signaling products), PARP (9542 for cell signaling products), Bax, and Bcl-2 (ab7977 for abcam products, ab 7973) (see 2-4 below). Reference).

The cortical neuron of the white paper was a little anesthetized on day E18 of the pregnant white paper, spinal dislocation, and each E18 white paper from the stomach was brought to the clean bench to remove skin and skulls. Only the cortical layers needed by the brain were separated, blood was removed from neurobasal (NB) media (21103-049 from invitrogen) and the tissues were destroyed by treatment with trypsin (25200 from gibco). Crushed tissue was filtered through a micro filter and the cells descended were used in 6 wells.

2-2. Cell counting cell counting )

MSCs and U87 cells were counted using tryphan blue after collecting the wells 24 hours after irradiation, and CN cells were counted by attaching a grid to the microscope eyepiece 24 hours after irradiation. At this time, the ratio of normal cells and dead cells was determined as cell viability.

2-3. Cell viability measurement

Cell viability was measured using LDH (Lactate dehydrogenase) assay and MTT (3- (4,5-dimethylthizol-2-yl) -2,5-diphenyl-tetrazolium bromide) assay. 50 ml of each culture group was transferred to a 96 well plate for LDH assay, and then 100 μl of LDH reaction solution (TOX7 from Sigma) was added and allowed to react at room temperature for 30 minutes. After that, the optical density (OD) value was measured at a wavelength of 490 nm of a microplate reader (Molecular Device). The relative OD value of the irradiated group to the OD value of the non-irradiated control group was expressed as a mean ± standard error (mean ± SEM). Cells that were irradiated for MTT assay were treated with 20 μL of MTT solution (CK04-11 from Dojindo), incubated for 5 hours, and then cultured with 200 μL of DMSO (081-4SK from Honeywell). Thereafter, an optical density (OD) value was measured at a wavelength of 560 nm of a microplate reader (Molecular Device).

2-4. Hoechst  dyeing

Cerebral cortical neurons cultured on cover slips were fixed in 4% formaldehyde solution for 20 minutes, then washed twice with PBS and stained with 5 ug / mL of Hoechst 33342 (Promega H3570) solution. Based on the DNA segmentation and the degree of condensation of the stained cortical neurons, living cells and apoptotic cells were distinguished. Each experimental group counted the number of stained cells in six randomly selected zones under 400 magnification of the fluorescence microscope (OLYMPUS BX61) to determine the ratio of the number of apoptosis cells in the total number of neurons.

2-5. Western Blot ( western blot )

1) Experimental method

Each cell (1 X 10 ⁴ ) was incubated in 50 ml of lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% deoxicholic acid sodium, 1% Triton X-100) for 30 minutes at room temperature. After that, insert the tube containing the cell and the lysis buffer into the ultrasonic machine, shoot the ultrasonic wave 10 times until the intertwined cells are invisible, and break the cells, and centrifuge for 3 minutes (1,2000). g) after, the supernatant was separated. The supernatant was quantitated BCA using the IntRON protein assay kit (21411143 from intron company) according to the manufacturer's method. Western blot analysis of Bcl-2 of aliquoted protein (50 μg) was done with 12% SDS-PAGE. At this time, β-tubulin (ab6046 from Abcam) was compared and analyzed as a house keeping gene. After electrophoresis on the PDVF membrane, blocking was performed at room temperature for 1 hour with 5% skim milk.

After blocking, goat monoclonal to actin (1: 1000 dilution) for actin, rabbit polyclonal to bax (1: 1000 dilution), and caspase-3, respectively, for actin Rabbit polyclonal to caspase-3 (1: 1000 dilution), Bcl-2 (rabbit polyclonal to bcl-2) (1: 1,000 dilution), PARP (rabbit polyclonal to PARP) (1: 1,000 dilution) In order to attach primary antibodies against rabbit polyclonal to β-tubulin (1: 3000 dilution), the cells were incubated overnight at 4 °. After attaching the primary antibody, the cells were washed with TBST 1 × for 1 hour. Monoclonal secondary antibodies against actin (1: 10,000) and goat polyclonal secondary antibodies against rabbit IgG were respectively caspase-3 (1: 10,000), PARP (1: 10,000) and Bax (1: 20,000) , Bcl-2 (1: 10,000) and β-tubulin (1: 20,000) were diluted and attached. Thereafter, the cells were washed with TBST 1X for 1 hour, and then developed by exposure to a film (Agfa) after a detection reaction using an ECL + detection kit (W3651-012 from GenDEPOT company).

<Experimental Results>

1) derived from human fat according to the radiation dose Intermediate lobe  Stem Cells ( MSCs ) Change

FIG. 5 is a graph showing survival rates of MSCs cells divided into four groups, the control group (non-radiated group) and the experimental group (radiation dose 1, 3, 5Gy), and the cell survival rate is 50 when the dose is 3Gy. It was found to be around%.

In addition, when looking at the microscopic photograph of the change of fat stem cells 200 times after irradiation for each group shown in Figure 6 it can be seen that the number of cells decreases as the radiation dose increases, the cell death increases as the radiation dose increases Showed.

On the other hand, the result of Western blot by extracting the protein by the method of Example 2-3 from the cells after irradiation is shown in Figure 7. Figure 7 shows the results of experiments measured by Western blot expression of Bax, cleaved caspase3, which increases expression when apoptosis occurs at each radiation dose, the cells start to die from 1Gy through irradiation From 3Gy, apoptosis occurred, and the higher the radiation dose, the more Bax and cleaved caspase3 expression was observed, resulting in more apoptosis.

This proved that apoptosis of human adipose-derived mesenchymal stem cells increased with increasing radiation dose using the radiation model developed by the present inventors, and proved that the new radiation model is an effective method.

2) human glioblastoma cells according to the radiation dose ( U87 ) Change

8 is a graph showing the survival rate of U87 cells (brain tumor cells), which were divided into four groups, the control group (non-radiated group) and the experimental group (radiation dose 1, 3, 5Gy), and the radiation dose as in the MSCs. When (Dose) is 3Gy, the cell survival rate was about 50%.

In addition, when looking at the microscopic image of 200 times the change of human glioma cells after irradiation (3, 5, 10Gy) for each group shown in Figure 9, it can be confirmed that the number of cells decreases as the radiation dose increases. Cell death was shown to increase.

On the other hand, the result of Western blot by extracting the protein by the method of Example 2-3 from the cells after irradiation is shown in Figure 10. Figure 10 shows the results of experiments measured by Western blot expression of Bax and cleaved caspase3 increased expression when apoptosis occurs at each radiation dose, using a cell count method from 1Gy through irradiation Although apoptotic cells began to die and apoptosis began to occur in earnest from 3Gy, Western blot results showed that the expression of Bax and cleaved caspase3 increased from 5Gy. It is confirmed that a lot occurs.

This proved that the death of human glioblastoma cells (malignant tumor cells of the brain) increases as the radiation dose increased using the radiation model developed by the present inventors, and proved that the new radiation model was an effective method.

3) according to the radiation dose Cortical neurons  ( CN ) Change

11 is a graph showing the survival rate of the CN cells (cortical neuron), which was divided into five groups, the control group (non-radiated group) and the experimental group (radiation dose 5, 10, 20, 40 Gy), and were measured by MTT assay. Unlike stem cells and malignant tumor cells, when the dose was 40Gy, the cell survival rate was about 50%.

In addition, when looking at the microscopic photograph of 200 times the change in the cerebral cortical neurons of the white paper after irradiation of each group shown in Figure 12, the dendrite of the control (dose 0) cells can be observed that the straight and long stretched As the irradiation dose increased, the number of viable cells decreased, the dendrites gradually disappeared, and the fractured form was observed.

FIG. 13 shows micrographs and Hoechst staining of neurons 24 hours after irradiation with control group and 40 Gy radiation. Neurons irradiated with 40 Gy radiation compared to control group showed increased death and many bright blue fragmented nuclei on Hoechst staining. Was observed.

On the other hand, after the protein was extracted from the control group and cortical neurons after 10, 20, 40 Gy irradiation by the method of Example 2-3 shown in Figure 14 to perform the Western blot. FIG. 14 shows the results of experiments using Western blot for the expression level of Bax and cleaved caspase3, the expression of which is increased when apoptosis occurs at each radiation dose. As described above, the higher the radiation dose, the more Bax, cleaved. The expression of caspase3 was increased and apoptosis was confirmed.

This proved that the death of cortical neurons in the rats increased with the increase of the radiation dose using the radiation model developed by the present inventors, and proved that the new radiation model is an effective method.

4) the time after irradiation Cortical neurons  ( CN ) Change

15 is a result of analyzing the degree of apoptosis of cells with time after 40 Gy irradiation to cortical neurons of the white paper by LDH assay. Compared with the control group, the apoptotic cell was observed to be increased in the radiation group and the cell apoptosis was observed as time passed up to 48 hours.

Figure 16 shows the results of verifying the expression of apoptosis-associated protein derived from the cells over time after 40 Gy irradiation to the cortical neurons of the white paper by the method of Example 2-3 by Western blot. The expression of Bax, cleaved caspase3 and cleaved PARP, which increased when apoptosis occurred, increased most at 12 and 24 hours after irradiation, demonstrating that apoptosis was most active at this time. After 24 hours, the degree of suicide was gradually decreased.

In conclusion, as a result of irradiating various types of cells with the new radiation model developed by the present inventors, there was a difference in the radiation dose causing death according to the cell type, and the radiation dose increased It has been demonstrated that the apoptosis of cell types increases proportionally, and that the new irradiation model is a valid method.

Claims (10)

1) preparing a plate including a plurality of wells, and culturing cells or tissue in each well;
2) disposing a conduit of a proximal radiotherapy device including a source of radiation across the top of each well; And
3) determining the radiation dose and irradiating radiation from the conduit,
A method of making radiation damaged cells or tissues.
The method of claim 1, wherein the plate comprising a plurality of wells is a 6, 12, 24, 48, 96 or 384 well plate.
The method of claim 1, wherein the well is a circular well.
The method of claim 1, wherein the radiation source is ¹³⁷ Cs, ¹⁹² Ir, ¹²⁵ I, ¹⁹⁸ Au, ²⁴¹ Am, ¹⁰³ Pd, ¹⁴⁵ Sm, ¹⁹ Yb, ³² P, ⁹⁰ Sr / ⁹⁰ Y, ⁹⁰ Y, ¹⁸⁸ Re, ¹⁶⁶ Ho Radioisotope selected from the group consisting of.
The method of claim 1, wherein the proximity radiation therapy device is a high dose rate proximity radiation therapy device.
The method of claim 1, wherein the conduit is positioned to cross the center point of the top of each well.
The method of claim 1, wherein there are 1 to 24 conduits across the top of each well.
The method of claim 1, wherein the conduit across the top of each well comprises from one to five points per well at which the radiation stops.
The method of claim 1, wherein different kinds of cells or tissues are cultured in each well.
The method of claim 1, wherein each well is irradiated from the conduit with a different radiation dose.

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