WO2015115478A1 - Procédé de détection de cancer employant un fragment de tissu biologique, et procédé pour maintenir des cellules dans un fragment de tissu biologique ex vivo - Google Patents

Procédé de détection de cancer employant un fragment de tissu biologique, et procédé pour maintenir des cellules dans un fragment de tissu biologique ex vivo Download PDF

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WO2015115478A1
WO2015115478A1 PCT/JP2015/052350 JP2015052350W WO2015115478A1 WO 2015115478 A1 WO2015115478 A1 WO 2015115478A1 JP 2015052350 W JP2015052350 W JP 2015052350W WO 2015115478 A1 WO2015115478 A1 WO 2015115478A1
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tissue
tissue piece
cells
minutes
cancer
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PCT/JP2015/052350
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Japanese (ja)
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勝也 山田
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国立大学法人弘前大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

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  • the present invention relates to a cancer detection method using a living tissue-derived tissue piece.
  • the present invention also relates to a method for maintaining cells in a living tissue piece ex vivo in a living state.
  • cancer tissue is not a collection of cells that exhibit uniform properties, but a collection of cells that exhibit various morphological or functional characteristics and degree of differentiation. It may also exist in normal cells. Therefore, in cancer diagnosis, it is necessary to perform pathological diagnosis of biopsy specimens and to evaluate abnormal cells at the cellular level in order to confirm the diagnosis.
  • pathological diagnosis of biopsy specimens the earlier the cancer, the more likely that cancer cells or pre-cancerous cells will be missed, that is, false (False Negative, false negative) is diagnosed. There is a possibility.
  • an abnormality first occurs in epithelial cells generally in the tissue surface layer (mucosal layer) of the digestive tract lumen.
  • tissue surface layer tissue surface layer
  • metastasis becomes difficult after metastasis, it is required to accurately determine “the nature and extent of lesions on the tissue surface” before such a situation occurs.
  • the biopsy tissue is fixed with formalin or the like and then sliced to about 5 microns to prepare a large number of specimens (preparations) and stained with a dye or the like.
  • the pathological specimen obtained in this way can be used by a specialist pathologist to increase the ratio of nucleus to cytoplasm (N / C ratio), abnormal position and shape of the nucleus in the cytoplasm, Pay attention to morphological features such as the appearance of abnormality, and diagnose whether it is normal or indicates an abnormality that is suspected of being cancerous or likely to shift to cancer.
  • the clinical side demands an objective scale that can replace the current pathological diagnosis having such a subjective aspect, or a clear index that can be judged even by a doctor with little experience.
  • 2-NBDG As a means for detecting cancer other than the HE-stained specimen, imaging using 2-NBDG in which a green fluorescent group NBD is bound to D-glucose has been reported.
  • Examples of using 2-NBDG for biopsy of cancer tissue or surgically removed specimens include oral cancer (Non-patent document 1), breast cancer (Non-patent document 2), and Barrett's esophageal cancer (Non-patent document 3). Examples have been reported.
  • 2-NBDG is also taken up by non-cancer cells and images non-cancer cells.
  • a technique has been established for continuously observing the state of uptake into cells by administering a fluorescent glucose derivative while keeping the amorphous cancer tissue removed outside the body with biopsy forceps and the like. In other words, these reports have poor reproducibility and technical reliability. Therefore, these techniques also do not meet the above problems and demands.
  • the present inventors have developed a fluorescent L-glucose derivative (2-NBDLG) in which L-glucose is labeled with a green fluorescent group NBD (Patent Document 1).
  • a method for detecting cancer cells by specific uptake of 2-NBDLG into cancer cells has been reported (Patent Document 2).
  • the present inventors have performed a specific treatment on a tissue piece collected from a living body by biopsy or the like, and thus in the tissue piece for a period sufficient for testing.
  • the present inventors have found that the cells can maintain physiological functions and that cancer cells present in such tissue pieces can be imaged using 2-NBDLG.
  • a method for detecting cancer in a living tissue-derived tissue fragment comprising the following steps: a. Treating a tissue piece collected from a living body at a temperature of 29 to 33 ° C. for at least 30 minutes, b.
  • the tissue piece treated in the above step a has a 7-nitrobenz-2-oxa-1,3-diazole group or a derivative thereof as a fluorescent molecular group in the molecule at a temperature of 35.5 to 37.5 ° C.
  • Treating in the presence of a glucose derivative and c.
  • a detection method comprising: (2) The L-glucose derivative is 2- [N- (7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino] -2-deoxy-L-glucose (2-NBDLG) The detection method according to (1) above. (3) In the step (1) or (2), the step a is a step in which a tissue piece collected from a living body is cooled to about 0 ° C. and then treated at a temperature of 29 to 33 ° C. for at least 30 minutes. The method described.
  • a method for detecting cancer in a tissue-derived tissue piece comprising the following steps: a. Treating a tissue piece collected from a living body at a temperature of 29 to 33 ° C. for at least 30 minutes, b. The tissue piece treated in the step a was treated with 2- [N- (7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-at a temperature of 35.5 to 37.5 ° C.
  • a detection method comprising: (6) The method according to (5) above, wherein the step a is a step of cooling a tissue piece collected from a living body to about 0 ° C. and then treating it at a temperature of 29 to 33 ° C. for at least 30 minutes. (7) The detection method according to the above (5) or (6), wherein the treatment in the step b is performed within 20 minutes.
  • the tissue piece is a tissue piece derived from the group consisting of stomach, esophagus, biliary tract, large intestine, uterus, cervix, vagina, bladder, and prostate. The method according to (1) to (10) above.
  • a method for detecting cancer by detecting an L-glucose derivative present in cells in a living tissue piece comprises the following steps: a. Treating a tissue piece collected from a living body at a temperature of 29-33 ° C. for at least 30 minutes, and b. The tissue piece treated in the step a was treated with 2- [N- (7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-at a temperature of 35.5 to 37.5 ° C.
  • step a is a step of cooling a tissue piece collected from a living body to about 0 ° C. and then treating it at a temperature of 29 to 33 ° C. for at least 30 minutes.
  • a cell maintenance method comprising: (17) The method according to (16) above, wherein the step a is a step of cooling a tissue piece collected from a living body to about 0 ° C. and then treating it at a temperature of 29 to 33 ° C. for at least 30 minutes. (18) The method according to (16) or (17), wherein the tissue piece is selected from the group consisting of a lumen and a lumen of a living body.
  • tissue piece is selected from the group consisting of stomach, esophagus, biliary tract, large intestine, uterus, cervix, vagina, bladder, and prostate.
  • (21) In the detection method of any one of (1) to (15) above, by treating a tissue in the presence of any compound in or after step b, the effect of the compound on cancer cells A screening method comprising evaluating.
  • cancer can be detected by imaging in a state where cells in a tissue are alive using a tissue piece collected from a subject. According to the present invention, it is possible to maintain a state in which cells in a tissue derived from a living body are utilized, and it is possible to observe functional abnormalities of the cells.
  • a transmitted light image (differential interference contrast image, hereinafter referred to as DIC) before administration of a fluorescence-labeled glucose derivative for each of the tissues obtained by biopsy from the central part (A) and the peripheral part (B) in which the tumor is suspected. It is a superposition of the self-fluorescent image of the green channel.
  • the present invention fluorescently labels L-glucose that does not bind to and is not recognized by glucose transporter (GLUT), which is a cell membrane transport mechanism when glucose is transported into normal cells.
  • GLUT glucose transporter
  • the derivative is administered to and contacted with a tissue that may contain a tumor extracted from a human or animal, and the fluorescence emitted by the cells in the tissue is optically detected during contact or after a certain period of time (preferably Relates to a method for selectively distinguishing cancer cells from normal cells and performing selective visualization of cancer by further comparison with that before administration.
  • the fluorescently labeled L-glucose derivative that can be used in the present invention is an L-glucose derivative having a 7-nitrobenz-2-oxa-1,3-diazole group or a derivative thereof as a fluorescent molecular group in its molecule, such as 2- NBDLG, which are specifically disclosed in patent applications WO2010 / 016587 and WO2012 / 133688, which are patent applications by the present inventors.
  • 2-amino-2-deoxy-L-glucose (2-TRLG) having sulforhodamine 101 bonded to sulforhodamine 101 can be used, whereby cancer can be detected with higher accuracy.
  • 2-TRLG is also specifically disclosed in patent applications WO2010 / 016687 and WO2012 / 133688.
  • the target tissue to which the method for detecting cancer of the present invention can be applied is not particularly limited as long as it is a tissue containing a cancer cell population, but the main target tissues include the oral cavity, digestive tract, uterus, cervix, It is a tissue in the lumen of the vagina, bladder, prostate and other organs that may contain a population of cancer cells such as adenocarcinoma that grows two-dimensionally or three-dimensionally.
  • the L-glucose derivative of the present invention has L-glucose in the molecule, so that the interaction with normal cells can be reduced compared to other methods, and cancer in tissues Can be visualized with good contrast.
  • gynecological tumors such as cervical cancer, bladder cancer and prostate cancer
  • other biopsies of lungs and various organs Tissue specimens, cells, and surgically removed tissue are the targets of the method of the present invention.
  • the method of the present invention comprises contacting a tissue piece containing a lesion with a fluorescent-labeled L-glucose derivative for a certain period of time, and then rinsing it into cancer cells in the tissue piece.
  • the present invention relates to a method for incorporating a fluorescently labeled L-glucose derivative and observing fluorescence of cells using an optical device.
  • Specific examples to which the method of the present invention can be applied include, but are not limited to, 1) taking a tissue section, 2) cooling, 3) trimming the tissue as necessary, 4) at a temperature lower than body temperature.
  • Suitable for tissue repair 5) fluorescent glucose uptake process at the same temperature as body temperature, 6) fluorescent glucose washout process, 7) determination of tissue orientation, 8) fluorescence observation, and 9) subsequent histopathological evaluation
  • the feature of the method of the present invention is that if there is an apparatus capable of fluorescence observation of cells, cancer cells can be observed without slicing the tissue.
  • the method of the present invention serves as a guideline for selecting a treatment method as one objective criterion for supplementing pathological diagnosis and can be repeatedly performed, it is expected to be useful as an aid for determining the therapeutic effect of drugs and the like.
  • it should be used as an aid for rapid diagnosis in which the obtained tissue is evaluated by fluorescence without slicing during endoscopic or surgical operations such as endoscopic mucosal removal (EMR). It can also be expected to serve as an aid in determining an appropriate resection range after exposure to the affected area.
  • tissue observation by fluorescence the same process as the preparation of a normal pathological specimen can be carried out. It has been confirmed that it does not affect the evaluation of clinical diagnosis.
  • the method of the present invention is a method for screening a compound having anticancer activity.
  • the present invention if there is an apparatus capable of fluorescence observation of cells, it is possible to observe cancer cells in a living state without slicing the tissue. Can be observed directly. This makes it possible to screen for compounds having anticancer effects.
  • a feature of the method of the invention is that it can distinguish damaged cells and cancer cells in the target tissue. Even if the cells damaged in the cell membrane are cancer cells or non-cancer cells (including normal cells), the fluorescence-labeled glucose derivative enters the cells non-specifically. . In the prior art, the case where the fluorescence-labeled glucose derivative is non-specifically taken into non-cancer cells damaged by such a cell membrane cannot be distinguished from the uptake in cancer cells. In cancer tissue, there is a part where the cell growth rate is higher than that of energy supply, and there are a large number of cells in the intermediate state between completely living cells and completely dead cells. Some of these cells have increased cell membrane permeability, but the cells are not dead.
  • the present invention uses 2-TRLG, a sterically medium bulky and moderately fat-soluble fluorescent group, and L-glucose, a water-soluble molecule that does not bind to GLUT in the molecule, This was made possible for the first time in tissue (piece) diagnosis.
  • the method of the present invention is capable of maintaining a human biopsy tissue or surgically isolated specimen removed outside the body alive.
  • the present invention is not limited to this, and it is not necessary to include all the steps listed below.
  • 1) collection of the tissue 2) Includes the processes of cooling, 3) trimming tissue as needed, 4) repairing tissue damage under mild low temperatures, and 5) maintaining the tissue under body temperature, thereby making the tissue alive Can keep the function of cells in the tissue alive.
  • One embodiment of the present invention is a method for detecting cancer in a living tissue-derived tissue piece, comprising the following steps: a. A process of treating a tissue piece collected from a living body at a temperature of 29 to 33 ° C. for at least 30 minutes or more (preferably, a tissue piece collected from a living body is cooled to about 0 ° C. and then treated at a temperature of 29 to 33 ° C. B.
  • the tissue piece treated in the above step a has a 7-nitrobenz-2-oxa-1,3-diazole group or a derivative thereof as a fluorescent molecular group in the molecule at a temperature of 35.5 to 37.5 ° C. Treating in the presence of a glucose derivative; and c. Detecting the L-glucose derivative present in the cells in the tissue piece.
  • each process will be described.
  • the biological tissue piece used in the method of the present invention is not particularly limited as long as it is a tissue piece that can be collected from an animal, preferably a human. Although not limited to this, for example, it is obtained by collecting the lesion tissue with biopsy forceps or the like while observing the affected area using an endoscope, a laparoscope or other optical means, or other known methods Can do. Although not limited to this, the thickness of the collected tissue is targeted to be about 1 mm or less. Generally, if the thickness is about 600 microns or less, the entire inside of the tissue can be utilized and observation can be performed while maintaining physiological activity.
  • Restrictions on the thickness of the extracted tissue also depend on the density and tissue properties of the cell gaps in the tissue, but even if the thickness of the extracted tissue is thick, as described below, it is cooled immediately after the extraction. If the tissue temperature is lowered, the metabolism of the removed tissue is suppressed, and an appropriate tissue recovery process is performed, at least about 200 to 300 microns from the surface layer can be observed while maintaining physiological activity.
  • the collected tissue is preferably immediately taken out into a Krebs-Ringer solution (KRB, pH 7.4) at about 0 ° C. and cooled.
  • the coolant may be an appropriately selected organ preservation solution or the like.
  • the subsequent steps can be performed using a Krebs-Ringer solution or a buffer solution such as physiological saline having a function equivalent to that of the Krebs-Ringer solution.
  • the biological tissue-derived tissue piece can be trimmed as necessary before applying the method of the present invention.
  • the shape can be adjusted by performing appropriate trimming using a cutting blade or other cutting means so that the tissue can be easily observed while cooling the tissue under a stereomicroscope.
  • trimming is performed so that the direction can be confirmed. May be.
  • Treating a tissue piece collected from a living body (processed by trimming or the like as necessary) at a temperature of 29 to 33 ° C. restores damaged tissue.
  • the treatment temperature in the recovery treatment is possible at 25 ° C. or higher, but in order to obtain sufficient recovery, it is preferably carried out between 29-33 ° C., and the treatment time is at least 30 minutes, preferably at least 40 minutes. More preferably, the treatment temperature and treatment time are at least 60 minutes or longer, depending on the organ or organ from which the tissue is derived, or on the degree of tissue damage, and further on the intended measurement. Can be changed as appropriate. Thereafter, the temperature is returned to 35.5 to 37.5 ° C.
  • the temperature is 35.5 to 37.5 ° C, preferably 36.5 to 37.5 ° C, more preferably about 37 ° C.
  • the tissue is initially damaged at about 32 ° C. for 40 minutes or more and damaged by biopsy or trimming. After recovering the temperature, a method of reheating to 37 ° C. can be mentioned.
  • the tissue piece placed at a temperature of 35.5 to 37.5 ° C.
  • the collected tissue piece is the side facing the gastrointestinal lumen ( Epithelial cells) facing the lens side of the confocal microscope and sandwiched between nylon meshes placed on a temperature-controlled perfusion chamber placed on a confocal microscope or other device capable of fluorescence observation Can be fixed.
  • Application of the fluorescently labeled L-glucose derivative can be performed, for example, as follows.
  • the perfusate is converted from the KRB solution to the fluorescent L-glucose derivatives 2-NBDLG (100 ⁇ M) and 2-TRLG using a general method of replacing the dosing solution using a solenoid valve or a drop due to hydrostatic pressure. It is possible to administer the fluorescent L-glucose mixed solution to the living tissue by a method of contacting the tissue by switching to a (20 ⁇ M) mixed solution for a certain period of time and then returning the perfusate to the KRB solution. At that time, a hemichannel inhibitor Carbenoxolone may be added for the purpose of suppressing non-specific uptake.
  • the time for applying the fluorescently labeled L-glucose derivative to the tissue is not limited to this, but it is sufficient to be within several tens of minutes. For example, even when the application time is within 20 minutes, within 10 minutes, or about 5 minutes, sufficient uptake of 2-NBDLG into cancer cells in the tissue can be achieved.
  • Fluorescence can be detected according to a known method (for example, methods described in Patent Document 1 and Patent Document 2).
  • the time required for washing off the excess fluorescent solution after the administration of the fluorescently labeled L-glucose derivative solution is not limited to the removal of the fluorescent solution in the chamber, but the fluorescent solution that has not entered the cell by entering the tissue gap.
  • the time required for the removal depends on the thickness and shape of the tissue. In general, the evaluation is usually possible in about 10 minutes and is maintained for about 30 minutes. If you want to prevent changes in fluorescence fading, decomposition, outflow, etc. over time, keep the tissue at 0 ° C to maintain the state immediately after the end of administration, and then increase the tissue activity around 0 ° C. You may observe it while holding it down.
  • the time for holding the tissue at 0 ° C. is not particularly limited as long as the tissue can be maintained alive and the activity of the tissue can be suppressed. Specifically, for example, within 24 hours, preferably Within 4 hours, more preferably within 1 hour. Thereby, it becomes possible to perform the process b and the process c in another place.
  • the living tissue-derived tissue piece can be maintained alive. Therefore, the tissue piece can be used for further examination as necessary.
  • a tissue piece in which cancer is detected using the method of the present invention is observed as a normal pathological tissue specimen.
  • the tissue is fixed with formalin or the like according to a conventional method, and a histopathological specimen is prepared for pathological diagnosis. At that time, it is important to pay attention so that the surface observed by fluorescence coincides with the sliced surface of the pathological specimen and the orientation of the tissue matches.
  • the tissue is clamped with nylon mesh or the like from the top and fixed, and after observation, formalin is fixed while maintaining the same arrangement.
  • the correspondence between the living tissue and the pathological specimen may be determined from the shape of the reconstructed tissue.
  • a living tissue is placed in a commercially available low-melting agarose, and the orientation of the tissue is fixed by lowering to 0 ° C., followed by observation with a fluorescence microscope through agar at low temperature, and then commercially available.
  • the tissue temperature is lowered to, for example, minus 20 ° C., and then the tissue is sliced into a thickness of 100-200 microns in parallel with the observation surface.
  • Ordinary paraffin pathological sections may be prepared after the cut sections are fixed in formalin. The latter has an advantage that it is easy to match the direction of fluorescence observation with the direction of slicing a pathological section. Also, since the fluorescence image changes depending on the depth in the tissue, a tomographic photograph is taken by appropriately using a confocal microscope, a deconvolution microscope, or other methods that enable three-dimensional observation, and a pathological tissue specimen It is also possible to correspond.
  • the above processes are: a) collection, b) cooling, c) tissue trimming as needed, d) repair of tissue damage under mild low temperature, e) administration and uptake process of fluorescent glucose at body temperature, f) Flushing process of fluorescent glucose, g) Tissue orientation determination method corresponding to the fixed histopathological specimen image, h) Fluorescence observation method, and i) Tissue fixation method after observation and rough thin as needed
  • a method of handling with a histopathological specimen such as a cutting operation, and a double examination is possible, a rapid examination using a living tissue and an examination using a histopathological specimen. Not only is this possible, but histopathology specimen examination is facilitated.
  • the method is not limited to the above-described method as long as each purpose is satisfied, and various changes and improvements can be made using a known technique.
  • the cancer tissue targeted by the cancer detection method of the present invention is not particularly limited.
  • cancer in which squamous epithelium and glandular tissue are seen such as stomach cancer, esophageal cancer, cervical cancer, and biliary tract cancer.
  • a wide range of cancer tissues such as absorptive epithelial cancer such as large intestine and transitional epithelial cancer such as bladder cancer can be mentioned.
  • the biological tissue from which the tissue piece is derived in the method for maintaining cells in the living tissue piece of the present invention in a living state is not particularly limited, and can include various organs and organs, such as the digestive tract, trachea, bladder Vagina, other various lumens, oral cavity, uterus, cervix, prostate, and other organs.
  • the present inventors from a patient suspected of early gastric cancer or gastric cancer, immediately before performing mucosal resection (Endoscopic omucosal resection, EMR) or submucosal dissection (endoscopic submucosal dissection, ESD), Tissue excised from the area estimated to be the lesion and the area surrounding the margin for EMR / ESD and surrounding the lesion, and fluorescently labeled in vitro (ex vivo) L-glucose derivatives (2-NBDLG and 2-TRLG) were applied to detect cancer.
  • EMR Endoscopic omucosal resection
  • ESD endoscopic submucosal dissection
  • Example 1 Preparation of living tissue-derived tissue piece
  • EMR endoscopic mucosal resection
  • B) the tissue was immediately cooled.
  • the biopsy tissue was fixed by sandwiching it with a nylon mesh placed on the top and bottom.
  • Example 2 For each of the tissues (A and B) to which fluorescently labeled L-glucose was applied, a transmitted light image (differential interference contrast image, hereinafter referred to as DIC) and green before administration of the fluorescently labeled glucose derivative while perfusing the KRB solution An autofluorescence image of the channel was observed.
  • the superposition is shown in FIG.
  • the dark areas in the left and right figures are from the biopsy tissue collected from the site determined by the endoscopist as a tumor lesion at the time of endoscopic biopsy, and from the site determined to be a non-tumor tissue surrounding the lesion, respectively. It is the collected biopsy tissue.
  • the vertical and horizontal lines are nylon grids that keep the tissue from moving.
  • the distance between the center of one grid and the center of the next grid is 400 microns.
  • the perfusate was switched from the KRB solution to a mixed solution containing 2-NBDLG (100 ⁇ M) and 2-TRLG (20 ⁇ M), which are fluorescent L-glucoses, using a solenoid valve.
  • the mixed solution was administered to the biopsy tissue for 5 minutes, and washing was started after completion of the administration.
  • FIG. 3 shows the superposition of the DIC image and the confocal fluorescence image observed in the green channel reflecting the uptake of 2-NBDLG when 18 minutes have passed after the start of washing after the end of the administration.
  • a strong fluorescent signal due to the uptake of 2-NBDLG into the cells was observed, suggesting the possibility of the presence of a tumor in this region.
  • FIG. 4 shows the superposition of the fluorescent images thus obtained.
  • a significant uptake of red 2-TRLG was observed in a considerable area around the tumor on the right side, suggesting the possibility of cells that non-specifically take up the fluorescently labeled L-glucose derivative, such as damaged cells or inflammatory cells. It was.
  • the region showing 2-TRLG uptake was limited. Therefore, it was suggested that the uptake of 2-NBDLG seen in the region of the suspected tumor on the left side of FIG. 3 includes specific uptake.
  • FIG. 5 shows the superposition of. If there is uptake of 2-TRLG that emits red fluorescence in addition to uptake of 2-NBDLG that emits green fluorescence, the synthesized color becomes yellow. Looking at the lower region of the suspected tumor area on the left, it is presumed that the red 2-TRLG has not been taken in, so it is presumed that the cell membrane has been kept healthy. It was suggested that there was a tumor in the area.
  • FIG. 6 shows a superposition of the fluorescence image observed in the green channel reflecting 2-NBDLG uptake and the red channel reflecting 2-TRLG uptake when 18 minutes have passed since the start of washing after completion of the administration. Shown in As estimated in FIG. 3, strong uptake of green 2-NBDLG was observed in the lower region of the suspected tumor region on the left side.
  • Example 3 Observation by histopathological image
  • the biopsy tissue used in Example 2 was fixed in formalin and then subjected to H & E staining to obtain a histopathological image.
  • the results are shown in FIG.
  • a tumor was actually present in the region under the tissue biopsied from the region where the left tumor was suspected and 2-NBDLG was strongly taken up.
  • the region where 2-NBDLG in the upper left tissue was not taken up showed a normal tissue image. Metaplasia was observed in the middle region between the two.
  • tissue degeneration was observed in the region around the tumor on the right side, and in FIGS. 3 and 6, a completely normal tissue image was observed when 2-NBDLG on the right side of the tissue was not taken up.
  • FIG. 8 shows the correspondence image obtained from pathological diagnosis added to the fluorescence image of the biopsy tissue obtained in Example 2.
  • the L-glucose derivative 2-NBDLG showing green fluorescence is strongly taken up by cells in the tissue collected from the lesion, and the normal nuclear state around the lesion (size and location of the nucleus in)
  • the uptake in tissue regions showing the cell and cytoarchitecture was clearly different.
  • the state of uptake of 2-TRLG showing red fluorescence, which is different from 2-NBDLG, into cells can visualize the uptake of cells damaged in the cell membrane and in the inflamed part. It was found to be distinguishable from the selective uptake of 2-NBDLG shown.
  • the method of the present invention provides a new method for detecting cancer using a living tissue-derived tissue fragment.

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Abstract

　La présente invention vise à procurer un procédé pour réaliser une image d'un cancer dans un échantillon de tissu frais, en remplacement, ou en complément, d'un spécimen coloré à l'hématoxyline et à l'éosine, pendant le diagnostic du cancer. La présente invention vise également à procurer un procédé pour maintenir des fragments de tissu biologique à l'état vivant ex vivo. À cet effet, l'invention porte sur un procédé, dans lequel procédé un fragment de tissu collecté à partir d'un corps vivant est traité pendant au moins 30 minutes ou plus à une température de 29 à 33° C, puis est ensuite amené à 35,5 à 37,5° C, ce par quoi les fonctions biologiques du tissu peuvent être maintenues à l'état normal. L'invention porte également sur un procédé pour détecter un cancer dans un fragment de tissu biologique par imagerie, par traitement du tissu en présence de 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-L-glucose (2-NBDLG).
PCT/JP2015/052350 2014-01-30 2015-01-28 Procédé de détection de cancer employant un fragment de tissu biologique, et procédé pour maintenir des cellules dans un fragment de tissu biologique ex vivo WO2015115478A1 (fr)

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