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CANCER CELL DETECTION METHOD USING CELLS OF BIOLOGICAL ORIGIN

外国特許コード F160008804
整理番号 (S2014-1615-N0)
掲載日 2016年8月4日
出願国 世界知的所有権機関(WIPO)
国際出願番号 2015JP076900
国際公開番号 WO 2016047676
国際出願日 平成27年9月24日(2015.9.24)
国際公開日 平成28年3月31日(2016.3.31)
優先権データ
  • 特願2014-193424 (2014.9.24) JP
発明の名称 (英語) CANCER CELL DETECTION METHOD USING CELLS OF BIOLOGICAL ORIGIN
発明の概要(英語) The purpose of the present invention is to provide a novel cancer cell detection method that uses cells of biological origin and that can be used even in cytodiagnosis. In particular, the purpose of the present invention is to provide a novel cancer cell detection method that makes it possible to perform imaging of cells in a living state and a dual detection method for cancer cells in which the aforementioned method is combined with a pre-existing dyeing method for cytodiagnosis. Provided is a cancer cell detection method that uses cells of biological origin and that includes: incubating living cells included in a sample taken from a person together with a fluorescently-labeled L glucose derivative and detecting the fluorescently-labeled L glucose derivative that is taken into the cells; and detecting fluorescence emitted by the L-glucose derivative that is present within the cells while the cells are affixed to a thin glass or plastic plate. Also provided is a dual detection method for cancer cells in which the cancer cell detection method that uses cells of biological origin is combined with a dyeing method using cells that are fixed using an alcohol or the like.
特許請求の範囲(英語) [claim1]
1.  A dual detection method of cancer cells using samples taken from a human, (A) The following living cells cancer detection methods consisting of fluorescence imaging using the process contained in a sample, including:  Possible to detect the cells fluorescent labeling L- glucose derivatives were incubated with fluorescent-labeled L- glucose derivative was incorporated into the cells, wherein the L- glucose derivative, as a fluorescent molecular group 7- nitrobenz-2- oxa group or a derivative having in its molecule an L- glucose derivative, and, In a state where the cells were allowed to adhere to a thin glass or plastic plate, survival detecting the fluorescence emitted by the L- glucose derivative present in the cell, wherein the plate, on its surface, the cells has an area for holding the buffer to maintain a state, and, if the fluorescence which said L- glucose derivative emitted is detected in the cell determines the cell as a cancer cell, A method of detecting the cancer cells, including, as well as (B) a method of detecting cancer consisting of staining method using the following alcohol fixed cells comprises the step,  After the cells were fixed by immersing the plate in ethanol fixative, Papanicolaou staining, Giemsa staining, PAS staining, Gurokotto staining, and were stained by any one of the dyeing method selected from the group consisting of immunocytochemical staining, but a method for detecting the N cells, Double detection method of cancer cells, including.
[2]
[claim2]
2.  It said of cancer consisting of fluorescent imaging detection method a the following steps: (A-1) step live cells contained in a sample collected from a human, and incubated at buffer containing Ki fluorescent labeled L- glucose derivative, incorporating the L- glucose derivative, (A-2) step of buffer solution to replace the buffer solution that does not contain the fluorescent-labeled L- glucose derivative to stop the uptake of the L- glucose derivative, (A-3) wherein the cell a thin glass or be attached to cell attachment areas on the plastic plate is fixed, then holds the buffer provided in the cell adhesion region comprises and is configured to surround plate buffer buffer placed in the holding area for the step of maintaining the cell in a viable state, wherein the buffer solution holding region is designed to surround the plate surface in which the cells are fixed, the buffer holding area It was composed of a buffer holding structure, (A-4) the step of detecting the fluorescence which said L- glucose derivative emits present in the fixed within the cell and, (A-5) the step of detecting cancer cells based on detection of the fluorescence, The method of claim 1 consisting of.
[3]
[claim3]
3.  Wherein step a the following steps: (A-1) a step of fixing by adhering living cells contained in a sample collected from a human, the thin glass or cell attachment region on the plastic plate, (A-2) placed in buffer to buffer holding area for holding the buffer provided in the cell adhesion region comprises and is configured to surround plate, the step of maintaining cells in a viable state, wherein in the buffer storage area is composed of a plate surface the cells are fixed, the buffer holding structure designed to surround the buffer holding region, (A-3) The buffer was exchanged into buffer containing the fluorescent labeled L- glucose derivative, then fixed to the plate cells were incubated, the step of incorporating said L- glucose derivative, (A-4) with a buffer exchanged into buffer without the fluorescent labels L- glucose derivative stop the uptake of the L- glucose derivative, the L- glucose derivative present in said fixed intracellular emitted a step of detecting fluorescence and, (A-5) the step of detecting cancer cells based on detection of the fluorescence, The method of claim 1 consisting of.
[4]
[claim4]
4.  The fluorescent-labeled L- glucose derivative is a 2- [N- (7- nitrobenz-2-oxa-1,3-diazole-4-yl) amino] -2-deoxy -L- glucose (2-NBDLG), sulforhodamine 101 in the 2-position is a mixture of 2-amino-2-deoxy -L- glucose and sulfonamide bond (2-TRLG), of cancer cells according to any one of claims 1 to 3, double detection method.
[5]
[claim5]
5.  In the detection of cancer cells in said step (a-5), further comprising, detecting method according to claim 3 or 4 to determine the cell membrane damage state of the cell fluorescence color of cells fluorescence imaging an index .
[6]
[claim6]
6.  Detection method according to any one of claims 1 to 5, the method (b) is a Papanicolaou stain.
[7]
[claim7]
7.  Detection method according to any one of claims 1 to 6, the thickness of the plate is 0.3mm or less.
[8]
[claim8]
8.  The 衝液 holding structure, the equivalent to the buffer holding region a structure of a plate-shaped or ring-shaped having an opening, having a thickness sufficient to hold the buffer, claims 2 to detection method according to any one of the 7.
[9]
[claim9]
9.  The buffer holding structure is a structure having a thickness of 0.5 ~ 10 mm, made of silicone, the detection method of claim 8.
[10]
[claim10]
10.  Samples taken from the human, cells from a patient of the floating cell solution, scraping peeling cell-derived cell, or an aspiration cell-derived cells, the detection method according to any one of claims 1 to 9.
[11]
[claim11]
11.  Wherein the cell is a cell derived from a patient's sputum, urine, peritoneal fluid, pleural, pericardial, cerebrospinal fluid, bile, pancreatic juice, from the floating cell solution selected from joint fluid or blood, detection method according to claim 10 .
[12]
[claim12]
12.  It said cell is a cell derived from ascites of ovarian cancer patients or endometrial cancer patients, detection method according to claim 11.
[13]
[claim13]
13.  Said plate has markings for indicating the surface and the position information of the cells on the surface opposite to deposit the cells, the detection method according to any one of claims 1 to 12.
[14]
[claim14]
14.  To observe living cells with a laser confocal microscope, a cell observation glass or plastic plate structure comprises a buffer holding structure for holding a cell observation glass or plastic plates with buffer , Cell survival state glass or plastic plate for fixing in, wherein said plate has a buffer holding area for holding the buffer to maintain the cells in a viable state to the surface, the thickness and at 0.3mm or less, and having a marking for indicating the location information of the cells on the surface opposite to the surface for fixing the cell, and, Buffer holding structure for holding a buffer solution in the buffer solution holding region, wherein the buffer retention structure, the silicone plate-shaped or ring-shaped is designed to surround the buffer holding area a buffer holding structure made thick 0.5 ~ 10mm, Cell observation for a glass or plastic plate structure including a.
  • 出願人(英語)
  • ※2012年7月以前掲載分については米国以外のすべての指定国
  • HIROSAKI UNIVERSITY
  • 発明者(英語)
  • YAMADA KATSUYA
  • SASAKI AYAKO
  • ONO KOUKI
  • TONE KIYOSHI
国際特許分類(IPC)
指定国 National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JP KE KG KN KP KR KZ LA LC LK LR LS LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN ST TD TG

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