KR102648399B1 - Method for manufacturing animal model of sentinel lymph node using fluorescent screening - Google Patents

Abstract

The present invention relates to a method for producing a sentinel lymph node animal model using fluorescence screening, and specifically, the steps of anesthetizing an animal other than a human; Injecting tumor cells into the axilla of the anesthetized animal; Periodically checking whether sentinel lymph nodes are formed using ultrasound; Four to five weeks after injecting the tumor cells, injecting a near-infrared fluorescent material around the sentinel lymph nodes formed by the tumor cells; and observing the accumulation of the fluorescent material in the sentinel lymph nodes and tumor cells using a light sensing system. It relates to a method for manufacturing a sentinel lymph node animal model using fluorescence screening, including.

Description

Method for manufacturing sentinel lymph node animal model using fluorescence screening {METHOD FOR MANUFACTURING ANIMAL MODEL OF SENTINEL LYMPH NODE USING FLUORESCENT SCREENING}

The present invention relates to a method for producing a sentinel lymph node animal model using fluorescence screening, and specifically, the steps of anesthetizing an animal other than a human; Injecting tumor cells into the axilla of the anesthetized animal; Periodically checking whether sentinel lymph nodes are formed using ultrasound; Four to five weeks after injecting the tumor cells, injecting a near-infrared fluorescent material around the sentinel lymph nodes formed by the tumor cells; and observing the accumulation of the fluorescent material in the sentinel lymph nodes and tumor cells using a light sensing system. It relates to a method for manufacturing a sentinel lymph node animal model using fluorescence screening, including.

The incidence of breast cancer continues to increase, but with the spread of health checkups and the development of diagnostic techniques, the rate of early breast cancer diagnosis has also increased. Axillary lymph node surgery has changed from the past lymph node dissection to sentinel lymph node biopsy. In the case of lymph node enucleation, the entire lymph node is removed, causing various side effects. Currently, two methods are commonly used for sentinel lymph node biopsy: radioactive isotopes and biological dyes.

When using a biological sample, direct confirmation is possible with the naked eye, but it is somewhat inaccurate, and complications such as permanent skin discoloration or a decrease in oxygen saturation may occur when the biological sample is locally injected, and the lymph node transit time is short, so several lymph nodes after the sentinel lymph node. This has the disadvantage of being colored. The use of radioactive isotopes has high reproducibility as it reacts sensitively regardless of the depth of the body, but has the disadvantage of not being able to be used in medical institutions that do not have nuclear medicine facilities.

Republic of Korea Patent No. 10-2226485

Recently, much research has been conducted on fluorescent labels using indocyanine green (ICG) and methods using near-infrared fluorescence cameras. Therefore, we would like to develop a method for sentinel lymph node biopsy that is accessible, safe, and highly accurate by applying the ICG marker. Using ICG, you can directly check the path of lymphatic vessels and lymph nodes without having to cut the skin.

Prior to this, when developing and applying a diagnosis and treatment system for breast cancer or sentinel lymph nodes, animal testing is inevitable for various verifications, and for this, the development of a sentinel lymph node animal model must be preceded.

Although various animal models of tumors have been developed, it is believed that there is no sentinel lymph node animal model. Therefore, we plan to develop a sentinel lymph node animal model that can be used in future sentinel lymph node-related research or experiments.

In one embodiment of the present invention, anesthetizing animals other than humans; Injecting tumor cells into the axilla of the anesthetized animal; Periodically checking whether sentinel lymph nodes are formed using ultrasound; Four to five weeks after injecting the tumor cells, injecting a near-infrared fluorescent material around the sentinel lymph nodes formed by the tumor cells; and observing the accumulation of the fluorescent material in the sentinel lymph nodes and tumor cells using a light sensing system. Provided is a method for producing a sentinel lymph node animal model using fluorescence screening, including a method.

The number of tumor cells may be 1×10 ⁶ to 1×10 ⁸ , but is not limited thereto.

The tumor cells are injected as a suspension of 0.2 to 1 cc, but are not limited thereto.

The near-infrared fluorescent substance may be selected from the group consisting of indocyanine green (ICG), cy3.5, cy5, cy5.5, cy7, Cypate, ITCC, NIR820, NIR2, IRDye78, IRDye80, and IRDye82, but is limited thereto. It doesn't work.

The light sensing system includes a modulator, a lock-in amplifier that receives the modulation signal information, and a light source, and excites near-infrared fluorescent material accumulated in the sentinel lymph node among the light emitted from the light source. An excitation filter that transmits only the excitation light, a probe, and an emission filter that is connected to the probe and selectively transmits only the fluorescence emitted from the near-infrared fluorescent material accumulated in the sentinel lymph node, passes through the emission filter. It may include, but is not limited to, a near-infrared sensor that senses fluorescence and converts it into an electrical signal, and a speaker that generates an alarm through the electrical signal.

According to another embodiment of the present invention, a sentinel lymph node animal model manufactured by the above manufacturing method is provided.

The present invention applies the ICG marker to enable an accessible, safe, and highly accurate sentinel lymph node biopsy, and allows the lymphatic vessel travel path and lymph nodes to be directly confirmed without the need to incise the skin, making it a diagnostic and treatment system for breast cancer or sentinel lymph nodes. It can be applied to the development of etc.

Figure 1 is a photograph showing the step of inoculating tumor cells into the axillary region in the method of manufacturing a sentinel lymph node animal model of the present invention.
Figure 2 is a photograph showing the step of injecting indocyanine green (ICG) around the sentinel lymph node and the step of detecting fluorescence of the sentinel lymph node in the method of manufacturing the sentinel lymph node animal model of the present invention.
Figure 3 is a photograph of a sentinel lymph node produced by the sentinel lymph node animal model manufacturing method of the present invention and a fluorescence detection photograph of the extracted sentinel lymph node.
Figure 4 is a photograph of the sentinel lymph node produced by the sentinel lymph node animal model manufacturing method of the present invention observed through a fluorescence detector.

In this specification, when a part “includes” a certain element, this means that it may further include other elements rather than excluding other elements, unless specifically stated to the contrary.

In one embodiment of the present invention, anesthetizing animals other than humans; Injecting tumor cells into the axilla of the anesthetized animal; Periodically checking whether sentinel lymph nodes are formed using ultrasound; Four to five weeks after injecting the tumor cells, injecting a near-infrared fluorescent material around the sentinel lymph nodes formed by the tumor cells; and observing the accumulation of the fluorescent material in the sentinel lymph nodes and tumor cells using a light sensing system. Provided is a method for producing a sentinel lymph node animal model using fluorescence screening, including a method.

The non-human animals may include non-human primates such as guinea pigs, hamsters, rodents other than rats, cats, dogs, pigs, rabbits, sheep, goats, deer, horses, cows, baboons, chimpanzees and monkeys. , small and medium-sized animals that are larger than rats, so that lesions can be observed with an endoscope, etc. may be more desirable, but may not be limited to this.

The animal may be a rabbit, preferably a New Zealand white rabbit, but is not limited thereto.

The animal anesthesia can be performed according to a method commonly performed in the art, and the type is not limited, but includes, but is not limited to, zoletyl/xylazine chloride; Ketamine/Xylazine; ketamine/medetomidine; ketamine/xylazine/acepromazine; Sudium pentibarbital administered alone; Alternatively, a method of anesthesia can be used by administering isoflurane, preferably by administering zoletyl/xylazine chloride.

According to one embodiment of the present invention, the tumor cells may be cancer cells or lung cancer cells isolated from a lung cancer patient.

The tumor cells include kidney cancer cells (e.g., RENCA), stomach cancer cells, brain cancer cells, lung cancer cells, breast cancer cells, ovarian cancer cells, liver cancer cells, bronchial cancer cells, nasopharyngeal cancer cells, laryngeal cancer cells, pancreatic cancer cells, bladder cancer cells, colon cancer cells, and uterus cancer cells. It may be any one selected from the group consisting of cervical cancer cells. Most preferably, it may be liver cancer cells or lung cancer cells, which are cells of the same respiratory system. If medium-sized animals such as rabbits are used as recipients of cancer cells, it is most preferable to use VX2 cells.

The VX2 cell line used in the present invention is a squamous cell cancer cell line developed from the skin of a rabbit. When injected in a certain amount or more depending on the weight of the animal, a tumor is formed at the transplant site even without the injection of a special immunosuppressant.

The number ^of tumor cells may be ¹ For a New Zealand White rabbit weighing 2.5 to 3 kg, preferably 1 x 10 ⁷ tumor cells can be inoculated.

The suspension may contain a thickening agent, and the thickening agent makes it possible to implant and fix tumor cells in a more precise location. This thickener may preferably be commercially available Metrigel (product name: BD MatrigelTM Basement Membrane Matrix, 5ml vial) or Cultrex Basemetn Membrane Extract (BME).

The mixture of tumor cells and thickener may contain 1 to 500 parts by weight of tumor cells, preferably 50 to 350 parts by weight, based on 100 parts by weight of the thickener. When the mixed weight ratio of the tumor cells and the thickener mixture satisfies the above range, the viscosity of the tumor cells and the thickener mixture can meet the condition of 3-25 cps, preferably 5-15 cps.

The tumor cells are injected as a 0.2 to 1 cc suspension, and are preferably injected as a 0.5 cc suspension.

The tumor cell suspension is prepared by adding PBS (Phosphate buffered saline) to the tumor cells, and the injection dose of the suspension can be adjusted depending on the type and weight of the animal to be inoculated. In the case of rabbits, the dosage is based on 2.0 to 2.5 kg rabbit. 0.5 cc can be injected.

After injecting tumor cells into the axillary region, it is desirable to measure the blood pressure and oxygen saturation of the animal inoculated with the tumor cells.

If the monitored blood pressure and oxygen saturation are normal, a step of checking whether sentinel lymph nodes are formed using ultrasound may be performed periodically after tumor cell inoculation, preferably every 1 to 2 weeks.

Thereafter, a step of injecting a near-infrared fluorescent material around the sentinel lymph node formed by the tumor cells can be performed 4 to 5 weeks after the tumor cells are injected.

Generally, sentinel lymph nodes can be formed 4 weeks after tumor cells are injected.

The near-infrared fluorescent substance may be selected from the group consisting of indocyanine green (ICG), cy3.5, cy5, cy5.5, cy7, Cypate, ITCC, NIR820, NIR2, IRDye78, IRDye80, and IRDye82, preferably is indocyanine green (ICG).

The indocyanine green (ICG) is a material for biological imaging and is a safe chemical with a long wavelength that has already been approved by the FDA. It has the property of being decomposed and eliminated or excreted through feces and urine about an hour after being injected into the human body. It is a fluorescent dye that can be used in animals and is advantageous for clinical application.

The ICG is accumulated in cancer tissue by the EPR (Enhanced permeability and retention) effect, and the accumulated ICG receives near-infrared rays of 730-770 nm and emits fluorescence in the near-infrared region of 800-850 nm, which is a longer wavelength, so it can be used with a CCD camera, It can be measured using instruments such as laser scanning and microscope detectors.

The near-infrared fluorescent material can be injected at 0.01 to 0.2 cc each, preferably at 0.1 cc, at 4 to 6 sites spaced 2 to 3 mm apart in radius around the tumor mass.

The near-infrared fluorescent material includes Water for Injection, Purified Water, Sterile Purified Water, Sterile Water for Injection, Bacteriostatic Water for Injection with preservatives, and distilled water ( It can be injected dissolved in either distilled water or normal saline.

After injecting the near-infrared fluorescent material, a step of observing the accumulation of the fluorescent material in sentinel lymph nodes and tumor cells using a light sensing system may be included.

The light sensing system may be a light sensing camera. The light sensing camera used at this time includes a band-pass filter of 820 nm and is used with a 780 nm collimated LED. When filming, lights off are necessary for accurate fluorescence observation.

According to another embodiment of the present invention, the light sensing system may be a fluorescence detector that can detect the fluorescence of ICG by converting it into sound. When using the fluorescence detector, unlike a light sensing camera, the light sensing system can detect the fluorescent substance without turning off the light. It was confirmed that fluorescence can be detected by converting it into sound, and that sensitivity is improved compared to cameras.

The fluorescence detector includes a modulator, a lock-in amplifier that receives the modulation signal information, and a light source, which excites near-infrared fluorescence accumulated in the sentinel lymph node among the light emitted from the light source. An excitation filter that transmits only excitation light, a probe, and an emission filter that is connected to the probe and selectively transmits only the fluorescence emitted from the near-infrared fluorescent material accumulated in the sentinel lymph node, and the emission filter that passes through the emission filter It may include, but is not limited to, a near-infrared sensor that senses fluorescence and converts it into an electrical signal, and a speaker that generates an alarm through the electrical signal.

The fluorescence detector is a fluorescence detector incorporating a heterodyne detection technique and can detect very weak fluorescence.

To put it simply, a modulation signal of a specific frequency is generated using a modulator, and then the modulation signal is input to the light source controller to modulate the intensity of the light source. Additionally, the modulated signal is input to a lock-in amplifier so that the modulated fluorescence signal can be separated from the noise environment later. When excited through a near-infrared light source, fluorescence modulated at the same frequency is generated in the sentinel lymph node by the modulated light source, and this optical signal is sensed by a near-infrared sensor through an emission filter and converted into an electrical signal.

The fluorescence detector may further include a micro-controller that converts the electrical signal into an electrical signal that can be recognized by the speaker. The microcontroller changes the electrical signal into an electrical signal that can be recognized by the speaker, causing the speaker to emit an alarm sound, and the microcontroller notifies the detection of fluorescence in numbers, graphs, or color changes through a provided output device. It may be possible.

More detailed information about the fluorescence detector is specifically disclosed in Republic of Korea Patent No. 10-1852403, and for descriptions of each component, refer to the patent.

According to another embodiment of the present invention, a sentinel lymph node animal model manufactured by the above manufacturing method is provided.

According to another embodiment of the present invention, a method for screening anticancer drugs using the sentinel lymph node animal model is provided.

The anticancer drug screening method includes administering an anticancer drug candidate to a tumor animal model; And it may include observing changes in the tumor.

The anticancer drug candidate can be administered orally or parenterally, and the dosage is not limited thereto, but is determined by those skilled in the art based on conditions such as the nature of the substance, toxicity, type of administration target, age, weight, and health. You can make an appropriate decision based on your judgment.

The step of observing changes in the tumor may be performed by methods commonly used in the art, including observation using the naked eye, a microscope or an endoscope, ultrasound, surgery, biopsy or tissue staining, preferably It can be performed using ultrasound or the above-mentioned optical sensing system.

<Example> Manufacture of sentinel lymph node animal model

1) After intramuscular injection of 5 mg/kg Rompun, a 2.0-2.5 kg New Zealand White rabbit male is administered general anesthesia through inhalation anesthesia using isoflurane.

2) Using an injection, inject 0.5 cc of a suspension of 1 x 10 ⁷ VX2 tumor cells into the muscle, respectively, into the axillary region. Before vaccination, shave the area to confirm the exact location. Add PBS (Phosphate buffered saline) to the tumor cells to create a 0.5 cc suspension. The injection volume of the suspension needs to be adjusted according to the body weight of the rabbit, and 0.5 cc is injected for a 2.0 to 2.5 kg rabbit.

3) Animals are monitored for blood pressure and oxygen saturation after the procedure.

4) After the procedure, the formation of sentinel lymph nodes is checked using ultrasound every 1 to 2 weeks.

5) When it is determined that sentinel lymph nodes have been formed (usually after the 4th week), a small amount of indocyanine green (ICG) is injected. That is, after injecting 0.1 cc each into 4 to 6 areas 2 to 3 mm apart around the tumor mass, the ICG is observed to accumulate in the lymph nodes and tumor and emit fluorescence using a light sensing (fluorescence) system. The above indocyanine green (ICG) is used by dissolving 25 mg in 10 mL of sterile water for injection.

6) After fluorescence screening, lymph nodes are removed and pathological examination is performed.

<Experimental example> Observation of tumor cells

Four weeks after the tumor cells were injected, sentinel lymph nodes formed in the axillary region of the experimental group were removed. Referring to Figure 3, it was confirmed that the injected fluorescence was detected in the extracted sentinel lymph node.

Figure 4 is a fluorescence detector photograph of the extracted sentinel lymph node. It was confirmed that even when the sentinel lymph node was not clearly observed with a fluorescence camera, it could be observed more easily through a fluorescence detector.

So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

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Claims (8)

Anesthetizing animals other than humans;
Injecting tumor cells into the axilla of the anesthetized animal;
Periodically checking whether sentinel lymph nodes are formed using ultrasound;
4 to 5 weeks after injecting tumor cells, injecting a near-infrared fluorescent material around the sentinel lymph node formed by the tumor cells; and
It includes observing the accumulation of fluorescent substances in sentinel lymph nodes and tumor cells using a light sensing system,
The tumor cells are VX2 tumor cells,
Method for producing sentinel lymph node animal model using fluorescence screening.
According to paragraph 1,
A method of producing a sentinel lymph node animal model using fluorescence screening, characterized in that the tumor cells are 1 × 10 ⁶ to 1 × 10 ⁸ .
According to paragraph 1,
A sentinel lymph node animal model manufacturing method using fluorescence screening, characterized in that the tumor cells are injected in a suspension of 0.2 to 1 cc.
According to paragraph 1,
The near-infrared fluorescent substance is selected from the group consisting of indocyanine green (ICG), cy3.5, cy5, cy5.5, cy7, Cypate, ITCC, NIR820, NIR2, IRDye78, IRDye80, and IRDye82. Method for producing sentinel lymph node animal model using screening.
According to paragraph 1,
A method of producing a sentinel lymph node animal model using fluorescence screening, characterized in that the near-infrared fluorescent material is injected into 4 to 6 sites 2 to 3 mm apart in radius around the tumor mass.
According to paragraph 1,
The light sensing system includes a modulator, a lock-in amplifier that receives modulation signal information, and a light source, which excites near-infrared fluorescent substances accumulated in the sentinel lymph nodes among the light emitted from the light source. An excitation filter that transmits only excitation light, a probe, and an emission filter that is connected to the probe and selectively transmits only the fluorescence emitted from the near-infrared fluorescent material accumulated in the sentinel lymph node, and the emission filter that passes through the emission filter A method of manufacturing a sentinel lymph node animal model comprising a near-infrared sensor that senses fluorescence and converts it into an electrical signal, and a speaker that generates an alarm through the electrical signal.
A sentinel lymph node animal model prepared by any one of the methods of claims 1 to 6.
Anticancer drug screening method using the sentinel lymph node animal model of paragraph 7.