WO2021132576A1 - アポトーシスにより細分化されたヌクレオソームを標的とする自家蛍光によるリキッド・バイオプシィ法 - Google Patents
アポトーシスにより細分化されたヌクレオソームを標的とする自家蛍光によるリキッド・バイオプシィ法 Download PDFInfo
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Definitions
- the present invention targets subdivided nucleosomes released into the blood by cell apoptosis, excites and detects their autofluorescence by surface plasmon enhancement, and obtains epigenetic information for cancer-centered disease information. Regarding the underlying liquid biopsy method.
- CAD caspase-activated DNase
- the activated CAD cleaves DNA in nucleosome units, so it is said to be fragmented as DNA that is a multiple of approximately 200 bp (Non-Patent Document 5).
- This fragmented DNA contains both genetic information and epigenetic information carried by genomic DNA. That is, although the genetic information carried by genomic DNA is in the base sequence, it may show different information even if the base sequence is the same. That is, the DNA in our cells exists in the form of being wrapped around the histone protein, and when the histone protein undergoes chemical modification such as acetylation, the state of the DNA changes.
- the genetic information that shows such changes is called epigenetics, but it is the cells that make up one individual exert different functions even though they have the same genomic DNA received from the fertilized egg.
- epigenetics of Epigenetics information acts as a switch, which changes the structure of DNA by adding or eliminating molecules and turns on and off the expression of genes in genomic DNA.
- the epigenetics of each cell is important (Professor of Tohoku University: Takahiro Arima, "Epigenetics and Evolution Knowing from Humans"). Therefore, it is said that most naturally occurring cancer cells have DNA methylation and chromatin abnormalities.
- a definitive diagnosis of cancer is generally made by a so-called biopsy method, in which a part of cancer tissue is collected by puncture or endoscopic treatment and a histopathological examination of the tissue piece is performed.
- Many biopsies are invasive procedures with risks such as bleeding and complications of bacterial infections, and biopsies can be difficult at some major sites.
- a liquid biopsy an attempt to diagnose a disease with an accuracy comparable to that of a pathological diagnosis using easily collectable samples such as blood and urine is called a liquid biopsy. It is attracting a great deal of attention as being suitable for clinical application due to its rapidity and simplicity.
- the targets of this liquid biopsy include exosomes and peripheral blood circulation microRNA (microRNA:) in addition to circulating tumor cells (CTC) and peripheral blood circulating tumor DNA (circulating tumor DNA: ctDNA). miRNA) and so on.
- microRNA peripheral blood circulation microRNA
- CTC circulating tumor cells
- ctDNA peripheral blood circulating tumor DNA
- CTC blood circulating tumor cells
- EMT epithelial mesenchymal conversion
- CTC circulates in the blood and invades other tissues to form metastatic tumors (metastasis). Since this CTC is present in the peripheral blood of cancer patients, it is expected that detection of this CTC can be used to determine the process of metastasis and to predict the prognosis of treatment.
- CTC is present in a very small amount, and there is a drawback that it is very difficult to detect.
- peripheral blood circulation microRNA and exosomes containing it are expected to be particularly useful for early diagnosis.
- microRNA complementarily binds to messenger RNA (mRNA) before it is translated into protein in cells and inhibits the translation of that mRNA, which is important in various life phenomena as a so-called fine tuner for gene expression.
- mRNA messenger RNA
- fine tuner for gene expression This is because it plays a role, and it is known that the expression control mechanism of microRNA is disrupted in cancer cells, and for example, microRNA that promotes cell proliferation is highly expressed.
- exosomes almost all cells secrete extracellular vesicles (exosomes), and it is said that early cancer can be diagnosed by detecting exosomes secreted by early-stage cancer cells in body fluids. Because.
- this type of liquid biopsy method including the diagnostic technique using the above-mentioned circulating tumor cells (CTCs, Circulating Tumor Cells), uses CD45 antibody beads after lysing red blood cells from a cancer patient sample (4 mL of peripheral blood). It is necessary to use to remove blood cells and sort cytokeratin-positive and CD45-negative cell fractions, and there is a limit to the provision of a liquid biopsy method in clinical applications that can be analyzed in a short time ( Non-Patent Documents 1, 2 and 3).
- the present inventors have conducted diligent research, and as a result, in apoptotic cells, the inhibitory factor of CAD (caspase-activated DNase) is decomposed, and activated CAD is obtained. Since the DNA is cleaved in nucleosome units, it is fragmented as DNA that is a multiple of approximately 200 bp. This fragmented DNA (fragmented nucleosome) after apoptosis contains both genetic information and epigenetic information carried by genomic DNA, and when histone protein undergoes chemical modification such as acetylation, the appearance of DNA changes.
- CAD caspase-activated DNase
- fragmented DNA fragmented nucleosome
- histone protein fragmented nucleosome
- autofluorescence having a brightness equal to or higher than a predetermined value that can be confirmed with a fluorescence microscope is generated. It was found that it emits light and is observable (Fig. 1).
- tumor suppressor genes including p53 are methylated by epigenetics, and although detection of methylated nucleosomes is indispensable for cancer disease information, methylated nucleosomes are selectively collected. It was difficult to do. Moreover, the fragmented DNA captured from the blood after apoptosis, that is, the fragmented nucleosomes aggregate somewhat on the biochip, and the autofluorescent colonies emitted by them are observed as multiple colonies like the constellations of the night sky. It was observed as a spread of about 25 ⁇ m in the small one and a spread of about 150 ⁇ m in the large one.
- Non-Patent Document 6 Non-Patent Document 6
- Circulating Tumor Cells Circulating tumor cells isolation and diagnostics: toward routine clinical use.
- Cancer Res 2011; 71: 5955-60 GorgesTM, Pantel K :. Circulating tumor cells as therapy-related biomarkers in cancer patients.
- Permuth-Wey Jet al. A Genome-Wide Investigation of MicroRNA Expression Identification Biologically-Meaningful MicroRNAs That Distinguish between High-Risk and Low-Risk Intraductal Papillary.Mucinous Neoplasmsof Ellen Heitzer, Peter Ulz and Jochen B.
- fragmented DNA (subdivided nucleosomes) released by pathological cell death represented by apoptosis in blood or other body fluids due to the occurrence of a disease is the subject of examination.
- carcinogenesis is a genetic disease caused by abnormalities in cancer genes and tumor suppressor genes, but to bases represented by methylation. It has become clear that carcinogenesis is caused by epigenetic genetic abnormalities due to modification, and not only qualitative abnormalities of tumor suppressor genes but also quantitative abnormalities due to hypermethylation are important for the carcinogenic mechanism.
- the problem to be solved by the present invention is to target fragmented DNA (fragmented nucleosomes) released into the blood by apoptosis, and to perform histone modification analysis and chromatin structure analysis using tissue samples to perform various diseases, especially.
- a detection method for detecting and diagnosing a tumor leading to the onset of cancer as a disease-related substance quickly and easily by a liquid biopsy method using autofluorescence of fragmented DNA (fragmented nucleosome). It is in.
- a sample prepared by directly or diluting a culture solution containing body fluid or cells is brought into contact with a measurement substrate having a plasmon metal mesocrystal region in the sample, and fragmented DNA (fragmented nucleosome) in the sample.
- the captured fragmented DNA (subdivided nucleosome) on the plasmon metal mesocrystal is exposed to a single wavelength excitation light by a laser light source or an LED light source.
- the fluorescence image of the fluorescent colony of fragmented DNA is constant by irradiating the excitation light with a certain wavelength width to obtain the excitation light from the filter through a filter and enhancing its autofluorescence by the surface plasmon enhancing effect.
- the excitation light a single wavelength excitation light from a laser light source or an excitation light having a constant wavelength width that acquires the excitation light from an LED light source or the like through a filter is used.
- ROI is adopted in adopting a region of predetermined brightness from the observed fluorescent colonies. Then, the reason why the pixels having the brightness equal to or higher than the predetermined threshold value in the fluorescent colony are adopted is to improve the measurement accuracy.
- the pixels of the fluorescent colonies having a brightness equal to or higher than the predetermined value adopted by binarization are adopted and the total area thereof is calculated. Alternatively, the ratio (ratio value) of the total area of two different wavelength regions is calculated.
- the adopted RGB wavelength range is preferably the G or B region, and it is preferable to calculate the G / B or B / G ratio. This is because when the spectroscopic spectrum of the fluorescent colony is observed, a peak peculiar to a disease, particularly cancer, is observed in the B or G region.
- the target to be captured as a detection target is fragmented DNA (subdivided nucleosome) that is a multiple of about 200 bp in which activated CAD (caspase-activated DNase) cleaves DNA in nucleosome units. Therefore, it contains both the genetic information carried by the genomic DNA and the epigenetics information, and it is preferable to perform the ratio determination in order to make it easy to extract only the epigenetics information.
- the captured fragmented DNA fragmented nucleosome
- the captured fragmented DNA is excited at the same or different two-wavelength excitation wavelengths in the B or G region to obtain fluorescent colonies, and the fluorescent colonies are filtered through different two-wavelength filters.
- This is a liquid biopsy method in which the ratio of each total area value of the pixels collected from the cells is the B region / G region or the G region / B region.
- the body fluid as a sample is plasma or serum separated from lymph or blood, and fragmented DNA (fragmented nucleosome) binds to histone protein and shows a positive charge, so that it can be selectively captured. ..
- fragmented DNA fragment nucleosome
- the target is to identify the canceration of the iPS cell.
- a disease-related substance activated CAD (caspase-activated DNase), cleaves DNA in nucleosome units, and a fragmented DNA (subdivided nucleosome) in a multiple of about 200 bp is selected.
- Autofluorescence which usually becomes noise in fluorescence diagnosis, is manifested by the surface plasmon enhancing effect, and fluorescent colonies having a brightness equal to or higher than a predetermined threshold are adopted, and the total area value of fluorescent colonies above a predetermined threshold, or
- bioproteins have autofluorescence, but this autofluorescence provides a signal (desirably fluorescently labeled) background (such as autofluorescence, which is not desirable to be fluorescently labeled) in the fluorescence observation of the target. It forms and becomes noise.
- the autofluorescence of this bioprotein reflects the structure of the bioprotein itself, the structural analysis by autofluorescence is particularly effective in the following points. That is, in recent years, based on the analysis results performed so far, not only genomic changes but also epigenome changes have accumulated in the process of cancer cell development and progression, and epigenome modification is used for chemical control and physical control. It is roughly divided. A representative of chemical control is the modification of various histone tails called "histone codes".
- the gene expression state correlates well with the chromatin state of the promoter region where the transcription initiation site is located, but the chromatin state of the enhancer is also deeply involved in the gene expression regulation peculiar to the cell lineage. Recently, it has been shown that the enhancer region using H3K27ac as an index is also important in the three-dimensional structure morphology (genome topology) of genes. Therefore, observation of autofluorescence using clinical tissue specimens suggests the possibility of early diagnosis of cancer and identification of cancer sites by histone modification analysis and chromatin structural analysis. That is, the protein conjugate to which the cell-free DNA is bound stabilizes the DNA in the nucleosome when the DNA wraps around the histone to form a nucleosome and the histone protein is methylated.
- Non-Patent Document 5 Non-Patent Document 5
- the autofluorescence of disease-related substances including free DNA (cfDNA) and fragmented DNA (subdivided nucleosomes) is manifested by the surface plasmon enhancing effect, and the total area is based on the fluorescent colonies having a brightness of a predetermined threshold or higher. It has been found that by calculating the ratio value by the value or the two wavelength ratios that correlate with RGB and / or it, it is possible to diagnose the disease, in particular, to identify cancer patients, benign tumor patients, and healthy subjects.
- fragment DNA fragment nucleosomes
- cfDNA cell-free DNA
- the DNA wrapped around histones binds to histone proteins and exists stably by methylation (various tumor suppressor genes hypermethylated from cancer cells by the cancer and epigenetics: hypermethylated RB genes).
- P14 and p53 are included in addition to p16). Therefore, by providing a chip showing a positive charge, these can be selectively adsorbed or captured.
- the substance captured on such a chip manifests autofluorescence by the surface plasmon effect due to specific excitation light, and when these are analyzed with a fluorescence microscope, the fluorescence wavelength indicates that the healthy subject, cancer patient, and other diseased patients. I found that I could identify. That is, cell-free DNA is also released into the blood from healthy cells via apoptosis, but tumor cell-free DNA is released from cancer cells, which are due to their own methylation and histone methylation. It binds tightly and releases disease-specific fragmented DNA (fragmented nucleosomes) from other diseased cells.
- the DNA released from the cells of these healthy subjects, cancer patients, and other diseased patients has not only genetic genetic abnormalities but also epigenetic genetic abnormalities due to modification to bases, and is released from each cell.
- fragmented DNA fragmented nucleosomes
- epigenetic modification is roughly classified into its chemical control and physical control, and is affected.
- the fluorescence wavelength spectrum or spectral peak differs depending on the site (according to the spectroscopic spectrum, a slight peak was observed around 515 nm in the fragmented DNA (subdivided nucleosome) of the cancer patient). Therefore, calculating the ratio value based on the total area value of the fluorescent colonies above the adopted predetermined threshold value or the ratio of the total area value of the fluorescent colonies above the adopted predetermined threshold value in the two wavelength region includes cancer. , Extremely effective in evaluating various diseases.
- protein conjugates are formed. Autofluorescence can be manifested by plasma enhancement and observed with a fluorescence microscope, which can be useful for early diagnosis of diseases. This is because post-translational modification abnormalities of proteins are associated with the development of various diseases, but proteomics analysis can be easily and quickly performed on specimens of patients with various diseases such as cancer, lifestyle-related diseases, and infectious diseases.
- these fragmented DNAs are released into blood and other body fluids by pathological cell death typified by apoptosis due to the occurrence of the disease, and thus have a relationship with the disease. deep.
- the autofluorescence of the nucleosome itself after apoptosis is characterized by strong autofluorescence in the blue (B) region in malignant tumors and strong autofluorescence in the green (G) region in benign tumors (Fig. 9).
- B blue
- G green
- Fig. 9 shows at the high-brightness area values of the region and the green (G) region by the Ratio value (B / G or G / B)
- test level Proteo test level
- FIG. 1 shows the obtained fluorescence image using a DM (dichroic mirror) 405-445 / 514 manufactured by Olympus, and shows a method of adopting 10 points having high brightness for each sample.
- FIG. 2 shows images of 470-490 nm and 600-620 nm obtained using DM405-445 / 514 manufactured by Olympus.
- FIG. 3 is a graph showing a spectroscopic spectrum acquired using BS10 / 90.
- FIG. 4 shows a silica gel drying container, in which (a) shows a dropping state of crystals obtained by centrifuging blood, and (b) shows a state in which plasma is dried in the container with silica gel.
- First step A of the first method of the present invention Explanatory drawing of making a measuring chip (proteo chip), Second step B: Explanatory drawing for determining the analysis range of the proteochip, Third step C: It is explanatory drawing of quantification of the area of (adopted fluorescent colony) of a proteo chip.
- Image acquisition step (1) ⁇ analysis range determination step (2) ⁇ fluorescent colony adoption step (3) ⁇ RGB calculation step of total area value of adopted colonies in RGB fluorescent image (4) ⁇ obtained RGB
- the conceptual diagram of the Ratio calculation method of this invention is shown.
- the liquid biopsy method it is preferable to adopt the following steps in the first method.
- the first method fragmented DNA (subdivided nucleosomes) captured by single-wavelength excitation light from a laser or excitation light of a certain wavelength width adopted by a filter is excited, and the total area of pixels above a predetermined threshold of the fluorescent colony is calculated.
- Equipment used KEYENCE, fluorescence microscope BZ-X710
- Light source Metal halide lamp 80W
- Fluorescence filter BZ-X filter DAPI (460 ⁇ 25nm)
- Analysis software BZ-X Analyzer a) Selective capture step of cancer-related substances: Fig.
- Measurement substrate having a plasmon metal mesocrystal region showing a negative charge on the surface of the sample A sample prepared by directly or diluting a culture solution containing body fluid or cells is brought into contact with a proteochip (Fig. 5A (1)) (Fig. 5A). (2)), The positively charged protein conjugate in the sample is charged by the plasmon metal mesocrystal as a disease-related substance (Fig. 5A (3)) Step b) Fluorescent image acquisition step: (1) Set the proteo chip with cancer-related substances on the fluorescence microscope, determine the measurement position (X, Y axis) of the chip while looking at the bright field image, and click the autofocus button to focus the chip (1) Z-axis, focus).
- a substance with strong fluorescence blue deposits, a binarization threshold value of 13 or more based on the brightness value under the following measurement conditions
- the area value thereof is calculated (unit: ⁇ m 2 ). From the area value, 0 to 19999 is classified into three stages of low cancer risk A, 20000 to 29999 is classified into observation required B, and 30,000 to 3 stages with cancer risk.
- FIG. 6 is a second method of the present invention, which is a method of adopting a fluorescence colony having a certain threshold value or more from a fluorescence image and calculating a ratio value with RGB and / or a two wavelength ratio that correlates with it.
- FIG. 6 (1) three RGB fluorescent images are acquired for each sample.
- the analysis range is determined and surrounded by ROI (FIG. 6 (2)).
- “circularity and brightness of fluorescent colonies” are used as threshold values, analysis conditions are determined, and dust and the like are excluded (FIG. 6 (3)).
- the total area value of the fluorescent colonies adopted from the RGB fluorescence image is calculated (FIG. 6 (4)). The details are as follows.
- An LED light source was used as the light source, and a fluorescence wavelength filter was attached to the Olympus upright microscope BX-63 to limit the fluorescence wavelengths to be measured and acquire fluorescence images.
- the excitation light of B is 375 to 400 nm and the fluorescence wavelength is in the range of 460 ⁇ 25 nm.
- the excitation light of G is 470 to 495 nm, and the fluorescence wavelength is in the long wavelength range of 510 nm or more.
- the excitation light of R is 530 to 550 nm, and the fluorescence wavelength is in the long wavelength range of 575 nm or more.
- B is the excitation light and fluorescence wavelength that strongly emits autofluorescence of cancer-related substances
- G is the excitation light and fluorescence wavelength that strongly emits autofluorescence of benign diseases.
- B deposits with a brightness value in the range of 28000 to 50000 were selected, and deposits with a circularity of 0.3 or less were set to be removed.
- G is a setting that selects deposits with a brightness value in the range of 27,000 to 50,000 and removes deposits with a circularity of 0.3 or less.
- R is a setting that selects deposits with a brightness value in the range of 21000 to 50000 and removes deposits with a circularity of 0.3 or less.
- This brightness value setting range was set according to the fluorescence intensity of the autofluorescence of the adhering substance and the light intensity of the LED light source.
- the analysis software is set to automatically remove foreign substances with irregular shapes and improve the analysis accuracy.
- the range of the brightness value is in the range of 10000 to 70,000, preferably about 20000 to 50,000, and the range of circularity is in the range of 0.9 to 0, preferably about 0.3. This circularity is adjusted according to the situation of the fluorescent colonies. From the RGB area value calculated by this measurement and analysis setting, the ratio value of the two wavelength ratios such as B / G and G / R is calculated, and for the large intestine, the ratio value of B / G is 1.9 to 1.0 for malignant tumors. The range was around 0.1 for benign tumors and 0.2-0.8 for healthy subjects. By measuring the fluorescence by dividing the excitation light in this way, it is possible to obtain a fluorescence image of stronger autofluorescence in each excitation light. By measuring and analyzing the same cancer-related substance at the same position on the biochip with different settings of excitation light and fluorescence wavelength, it is possible to extract more autofluorescence peculiar to the substance and improve the accuracy of the data. I was able to improve.
- FIG. 7 shows an image of a microscope stage when four proteochips (measurement boards) are set in a holder to automate measurement.
- the measurement positions (X-axis and Y-axis) of the chips (1) to (4) are registered in advance, and the focus (z-axis) of the chips (1) to (4) is adjusted by the initiative.
- the X, Y, and Z axes are determined, three types of RGB measurements on the four chips are automatically performed, and four RGB fluorescent images are obtained.
- the sample body fluid is not only plasma or serum separated from lymph or blood, but also contains urine, saliva, etc., and cell free DNA (cfDNA) in which a protein conjugate binds to histone protein and exhibits a positive charge.
- the cell-free DNA comprises circulating tumor DNA (ctDNA) released from the cell.
- the excitation light having a wavelength of 405 nm used for exciting the tumor-affinitive fluorescent substance, and in that case, the fluorescence wavelength is observed around 630 nm.
- the protein conjugate captured in the present invention contains circulating tumor DNA (ctDNA), which is a nucleosome bound to histone protein or a higher-order chromatin, and when histone is modified for methylation, it is a liquid by autofluorescence.
- ctDNA circulating tumor DNA
- the substrate having the plasmon metal mesocrystal region used in the method of the present invention is called a proteochip.
- the manufacturing method is as follows (see Patent Document 1). 1) Aqueous metal complex is chemically reduced by an electrode potential difference on a metal substrate having an electrode potential (high ionization tendency) lower than that of the metal forming the complex to aggregate quantum crystals (nano-sized metal complex crystals).
- a quantum crystal of the silver complex is formed by aggregating an aqueous solution of silver thiosulfate on a copper or a copper alloy having an electrode potential (high ionization tendency) lower than that of silver by using a chemical reduction method. ..
- the concentration of the metal complex in the aqueous solution should be determined mainly by considering the size of the quantum crystal to be formed, and when using a dispersant, the concentration should also be considered, and usually 100 ppm to 5000 ppm. However, a concentration of 500 to 2000 ppm is preferable for preparing a nanosize called a nanocluster depending on the function of the ligand.
- E ° represents the standard electrode potential
- R represents the gas constant
- T represents the absolute temperature
- Z represents the ionic value
- F represents the Faraday constant.
- the metal complex is a plasmon metal complex selected from Au, Ag, Pt or Pd, it has a localized surface plasmon resonance enhancing effect on excitation light.
- the metal complex when it is a silver complex, it is preferably formed by the reaction of silver halide with a silver complexing agent having a stability constant (production constant) (log ⁇ i) of 8 or more, and as silver halide.
- a silver complexing agent having a stability constant (production constant) (log ⁇ i) of 8 or more, and as silver halide.
- Silver chloride is preferable, and the complexing agent is preferably one selected from thiosulfate, thiocyanate, sulfite, thiourea, potassium iodide, thiosalicylate, and thiocyanurate.
- the silver complex has quantum dots composed of nanoclusters having an average diameter of 5 to 20 nm, and the size of the quantum crystal is 100 to 200 nm.
- the plasmon metal mesocrystal in the proteochip used in the present invention is an oxide of the quantum crystal of the plasmon metal complex, and is a negative charge required to supplement the methylated nucleosome that is positively charged in the blood with an electric charge.
- the acicular nanocrystals of silver oxide containing silver peroxide show a surface plasmon enhancing effect by irradiation with excitation light represented by laser light, and autofluorescence of cancer-related substances represented by adsorbed histone. Has been found to be preferable for detecting.
- a silver ion aqueous solution can be formed by constant potential electrodeposition using an Ag / AgCl electrode, or by oxidizing a silver quantum crystal by an alkali treatment, which is a silver complex quantum. It can be easily formed by subjecting a crystal, for example, a silver thiosulfate quantum crystal, to an alkali treatment (treatment with an aqueous sodium hypochlorite solution).
- Patent Document 2 Localized plasmon fluorescence consisting of silver nanoparticles with a uniform grain system by dispersing chips (Patent Document 2) and metal nanoparticles in an organic solvent, volatilizing the organic solvent, and self-assembling the metal nanoparticles in the two-dimensional direction.
- An reinforcing sheet Patent Document 3 may be used.
- sample used in the present invention Specimens are prepared from body fluids containing blood. Since erythrocytes show strong autofluorescence, it is better to centrifuge and remove only plasma. In the case of cancer disease as a disease-related substance, the dilution ratio is determined by diluting 10 to 50 times so as to facilitate the measurement of autofluorescence of fragmented DNA (fragmented nucleosome). A 20 to 30-fold dilution is desirable for a silver oxide mesocrystal formed by alkali-treating a silver thiosulfate complex quantum crystal prepared by dropping an aqueous solution of a silver thiosulfate complex of about 1000 ppm onto phosphor bronze.
- fragmented DNA fragmented nucleosomes
- protein conjugates Protein-bound DNA fragments: nucleosome or chromatin
- vacuum drying or desiccant vacuum drying or desiccant because it is selectively captured by plasmon metal mesocrystals showing a negative charge and showing a surface plasmon enhancing effect by excitation light
- fragmented DNA fragmented nucleosomes
- iPS cells may contain cancerous gene DNA, they can be used as a sample, and the canceration of iPS cells can be identified and removed by the following autofluorescence.
- Fluorescent image acquisition process Here, the fragmented DNA (subdivided nucleosome) captured on the plasmon metal mesocrystal substrate is irradiated with excitation light to enhance the autofluorescence of the captured fragmented DNA (subdivided nucleosome) by the surface plasmon enhancing effect. , Fluorescent colonies are acquired as fluorescent images.
- excitation light a laser light source of excitation light having a diameter of 405 nm, which is said to be suitable for exciting a hematoporphyrin derivative (tumor-affinitive fluorescent substance) having different accumulation / excretion characteristics between normal tissue and lesion tissue, was used.
- the ratio value is calculated by the total area value of the pixels of the fluorescent colony above the adopted predetermined threshold value, or the total area ratio of the pixels in RGB and the two wavelength regions correlating with it. As a result, healthy subjects, benign tumors, and malignant tumors can be identified by the total area value and the ratio value of the total area in different wavelength regions.
- the brightness at each wavelength is displayed at a ratio of the brightness of 470 nm as 100%, and a graph is created with the average value of 10 points as data for each sample. Further, as shown in FIG. 3, it was confirmed that the peak of the cancer-related substance exists at a wavelength of around 610 nm.
- Target object and its autofluorescent image of the captured target object observed with a fluorescence microscope In the present invention, fragmented DNA (fragmented nucleosomes) abnormally generated in cancer diseases and other diseases is targeted for detection. Protein conjugates associated with this type of disease are selectively captured by plasmon metal mesocrystals due to their positive charge, and are enhanced by the surface plasmon enhancing effect of plasmon metal mesocrystals by irradiation with excitation light, and are confirmed by fluorescence microscopy. It has been found that autofluorescence having a brightness equal to or higher than a predetermined value can be emitted (Fig. 1).
- cell free DNA (cfDNA) in human plasma is released into the blood as a protein bond associated with histone or TF, preferentially survives, and in healthy individuals is mainly derived from the cell line of the myeloid lymphatic system. Contributions from one or more additional tissues are considered for certain medical conditions, including nucleosome footprints that infer cell types from cfDNA in pathological conditions such as cancer, and the origin of that tissue.
- Non-Patent Document 5 Cell, 2016 January 14; 164: Cell-free DNA is an in vivo nucleosome footprint that informs its tissues-of-origin).
- Non-Patent Document 4 Circulating Tumor DNA as a Liquid Biopsy for Cancer; Climinal Chemistry 2015; 61: 112-123).
- Fragmented DNA fragment nucleosomes
- histones nucleosomes
- chromatin fibers
- Globulins are also positively charged, but their increase is up to 2 times or less compared to other cancer-related substances, whereas the substance detected in the present invention increases 100 times or more with cancer progression. Therefore, the increase other than globulin is said to have detected cancer-related substances, indicating that the total area of pixels above a certain threshold of the adopted fluorescent colonies is associated with the stage of cancer.
- Example 1 A 1000 ppm aqueous solution of silver thiosulfate was prepared, one drop thereof was dropped onto a phosphor bronze plate, the solution was left to stand for about 3 minutes, and the solution was blown off.
- the photograph showing various SEM images of the nanoparticle aggregate (quantum crystal) produced in Example 1 (see FIG. 1 of Patent Document 1), it is a thin hexagonal columnar crystal of about 100 nm, and the surface has irregularities on the order of several nm. Is expressed. No facets peculiar to metal nanocrystals could be confirmed.
- FIG. 1 of Patent Document 1 In the photograph showing the relationship between the standing time after dropping on the phosphorus bronze slope and the quantum crystal shape (Fig.
- Quantum crystal construction In the case of a 1000 ppm silver thiosulfate complex aqueous solution, when the quantum crystal is dropped on a phosphorus bronze plate and left for 3 minutes, it is formed into a hexagonal columnar shape of about 100 nm, and each hexagonal columnar quantum crystal may have irregularities on the order of several nm. Although it was confirmed from the SEM image, the facet peculiar to the metal nanocrystal could not be confirmed, and the element derived from silver and the complex ligand was detected by the EDS element analysis. It is presumed that the irregularities appearing on the surface are spread by forming quantum dots as clusters of silver in the complex.
- the equilibrium potential of the silver thiosulfate complex is 0.33 and that of copper. Since it is equivalent to the electrode potential (0.34), only silver (0.80) is deposited on the copper substrate, and in the case of phosphorus bronze, the electrode potential is 0.22, which is slightly low, and thus the silver complex.
- Patent Document 1 9 and 10 of Patent Document 1 are estimated from the results of EDS (FIG. 8 of Patent Document 1), and the peak near 529 eV is the O peak derived from silver peroxide (AgO), which is 530 eV.
- the nearby peak is recognized as an O peak derived from silver oxide (Ag2O).
- the O peak derived from silver peroxide (AgO) at the peak near 529 eV is larger than the O peak derived from silver oxide (Ag2O) at the peak near 530 eV. It can be said that it shows that silver peroxide is formed in the vicinity of the substrate. It is presumed that this is due to the catalytic action during mesocrystal formation and the electrode potential of the substrate.
- the EDS measurement was performed using the above-mentioned recrystallized substrate model: JEOL Ltd./JSM-7001F (field emission analysis scanning electron microscope). Further, the same result was not obtained even when the quantum crystal of silver thiosulfate was treated with an aqueous solution of hypochlorous acid, an aqueous solution of 0.01 specified caustic soda, an aqueous solution of 0.01 specified hydrochloric acid, and an aqueous solution of 0.1 molar sodium carbonate. Therefore, it is considered that the formation of the acicular crystals is caused by the above reaction in the presence of silver ions and thiosulfate ions. Silver oxide is negatively charged in an aqueous solution and is reduced by light to precipitate metallic silver. Since silver peroxide has a remarkable tendency, it is considered that a positively charged cancer-related substance is adsorbed and a surface plasmon enhancing effect between the adsorbed cancer-related substance and silver particles can be obtained.
- Nucleosomes are the basic building blocks of chromatin and have a structure in which DNA is wrapped around a histone octamer consisting of four types of histones (H2A, H2B, H3, H4). Histones play a role in packaging DNA. In addition, it plays an important role in regulating the accessibility of DNA and in gene regulation. Post-translational modifications of histones regulate their interaction with DNA and other nucleoproteins, affecting reversible gene expression. As the types of histone modifications, methylation, acetylation, phosphorylation, ubiquitination, SUMOylation, citrullination, and ADP liposylation are mainly known.
- Peripheral gene expression is activated or suppressed depending on which site in the histone sequence undergoes these modifications.
- various histone code-related antibodies Anti-histone antibodies manufactured by Genetex
- the combination of these post-translational modification sites of histones and their effects on gene expression can be used to obtain the modified state of the histone code captured by the plasmonic chip of the present invention.
- the histone code hypothesis can be verified.
- fragmented DNA fragmented nucleosome
- fluorescence imaging based on the amount of captured crystals.
- a peak was observed around 515 nm in a sample collected from the blood of a patient, and the peak could not be observed in a sample collected from the blood of a healthy person. It is considered that the determination can be made in consideration of the total area of the pixels equal to or larger than the predetermined threshold value or the ratio of the total area of the pixels having the predetermined threshold value or more in the two wavelength regions in the RGB region G / G, G / B and the like. And information about how cancer develops and progresses through this chromatin remodeling event tells clinicians how such cancers are likely to respond to specific chemotherapeutic agents. It allows for more accurate predictions, and in this way, chemotherapy based on knowledge of tumor chemosensitivity can be reasonably designed.
- the Rio value of the total area ratio of pixels equal to or larger than a predetermined threshold in the two wavelength region is calculated from the fluorescence image obtained by RGB, but the fluorescence image obtained by G tends to increase the number of benign tumors. Yes, the fluorescence image obtained in B tends to increase malignant tumors. Therefore, in the examples, when the Radio value was analyzed by G / R for the prostate, the results ranged from about 2.0 for benign tumors, 1.7 for healthy subjects, and 1.76 to 1.86 for malignant tumors. On the other hand, when the Radio value of the large intestine was analyzed by B / G, it had a range of 1.9 to 2.0 for malignant tumors, 0.2 to 0.8 for healthy subjects, and around 0.1 for benign tumors. It became.
- BP 470 to 495 nm was used as a filter for excitation light on the molecular side, and BA 510 to 550 nm was used as a fluorescence filter.
- the total pixel area is calculated as a molecule of Ratio by adopting pixels having a threshold value equal to or higher than a predetermined value in the fluorescence image obtained by using the mirror unit.
- BP 460 to 480 nm was used as the excitation light filter on the denominator side
- BA 495 to 540 nm was used as the fluorescence filter.
- the total pixel area is calculated as the denominator of Ratio by adopting pixels having a threshold value equal to or higher than a predetermined value in the fluorescence image obtained by using the mirror unit.
- the ratio of the above G-region numerator / G-region denominator was calculated and compared with the stage separately determined by the inspection, as shown in Table 1 below. From this, it was found that the results obtained by the method of the present invention are closely related to the malignant / benign and stage determined by the actual examination.
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Abstract
Description
標的とすべき断片化DNA(細分化ヌクレオソーム)は、ctDNAを含む、細胞遊離DNA(cfDNA)がヒストンタンパクと結合して最小単位のヌクレオソームまたはそれが高次化したクロマチンとして血中に存在してくる。しかも、ヒストンに巻き付いたDNAはヒストンタンパクと結合し、メチル化することで安定に存在する(前記癌とエピジェネチックスにより、ガン細胞から高メチル化した各種ガン抑制遺伝子:高メチル化したRB遺伝子、p16以外にp14並びにp53が含まれる)。したがって、プラス電荷を示すチップを提供することで、これらを選択的に吸着または捕捉することができる。かかるチップ上に捕捉される物質は特定の励起光により自家蛍光を表面プラズモン効果で顕在化して現れ、これらを蛍光顕微鏡で分析すると、その蛍光波長により、健常者、がん患者、その他の疾患患者を識別できることを見出した。すなわち、健常者の細胞からも細胞遊離DNAがアポトーシスを介して血中に放出されるが、がん細胞からは腫瘍細胞遊離DNAが放出され、これらはそれ自身のメチル化及びヒストンのメチル化で強固に結合し、他の疾患細胞からは疾患特有の断片化DNA(細分化ヌクレオソーム)を放出する。これら健常者、がん患者、その他の疾患患者の細胞から放出されるDNAはgeneticな遺伝子異常だけでなく、塩基への修飾によるepigeneticな遺伝子異常が発生しており、各細胞からの放出される断片化DNA(細分化ヌクレオソーム)には化学的又は物理的に相違が見られる。その結果、健常者、がん患者、その他の疾患患者の細胞から放出される断片化DNA(細分化ヌクレオソーム)に自家蛍光の波長に相違がみられることを見出した。また、がん患者から採取したがん関連物質の蛍光波長をより詳細に分類すると、エピゲノム修飾が、その化学的制御と物理的制御に大別され、影響を受けるため、がん原発部位、転移部位により蛍光波長スペクトルまたはスペクトルピークが異なってくる(分光スペクトルによると、がん患者の断片化DNA(細分化ヌクレオソーム)には515nm付近にわずかなピークが観測された)。したがって、採択された所定の閾値以上の蛍光コロニーの総面積値、又は2波長域での、採択された所定の閾値以上の蛍光コロニーの総面積値比でratio値を演算することはガンを含め、各種疾病の評価に極めて有効である。
本発明によれば、具体的に、これら断片化DNA(細分化ヌクレオソーム)は疾患の発生により血液その他体液中にアポトーシスに代表される病理的な細胞死によって放出されるため、疾患との関係が深い。特に、アポトーシス後のヌクレオソーム自体の自家蛍光の特徴が悪性腫瘍ではブルー(B)領域の自家蛍光が強く、良性腫瘍ではグリーン(G)領域の自家蛍光が強く現れるため(図9)、ブルー(B)領域とグリーン(G)領域の輝度の高い面積値をRatio値(B/G又はG/B)で見ると、健常者、良性腫瘍、悪性腫瘍の識別をすることに成功し、ガンの超早期診断、再発判定、転移判定、治療効果のモニタリングが可能となることが分かった(図10)。アポトーシス後の細分化されたヌクレオソームは遺伝子異常により発生するメチル化ヌクレオソームの検出を可能とするため、図11に示す多段階発がん仮説モデルに示すように、本発明の検査レベル(Proteo検査レベル)は現在の画像診断レベル(PET-CT,CT,MRI)以前のガン関連物質の検出に基づき、超早期発見が可能となることがわかった。
使用機器:キーエンス社、蛍光顕微鏡BZ-X710
光源:メタルハライドランプ80W
蛍光フィルタ:BZ-XフィルタDAPI(460±25nm)
解析ソフト:BZ-X Analyzer
a)がん関連物質の選択的捕捉工程:図5A
試料中表面マイナス電荷を示すプラズモン金属メソ結晶領域を有する測定基板:プロテオチップ(図5A(1))に、体液または細胞を含む培養液をそのまま又は希釈して作成した検体を接触させ(図5A(2))、検体中のプラス電荷を示すタンパク結合体を疾病関連物質としてプラズモン金属メソ結晶に電荷捕捉させる(図5A(3))工程
b)蛍光画像取得工程:
(1) がん関連物質が付着したプロテオチップを蛍光顕微鏡にセットし、明視野画像を見ながらチップの測定位置(X,Y軸)を決めて、オートフォーカスボタンをクリックしてチップのフォーカス(Z軸、ピント)を合わせる。測定設定をBZ-XフィルタDAPIに切り替えて蛍光画像の測定を開始する。
このプラズモン金属メソ結晶(直径約8mm)上に捕捉されたタンパク結合体に励起光を照射して、捕捉タンパク結合体の自家蛍光を表面プラズモン増強効果により増強し、蛍光コロニーを蛍光画像(図5B)として取得する工程
c)蛍光コロニーの採択工程:
解析範囲(直径5mm)内にある蛍光コロニーの輝度を二値化して所定の閾値以上の輝度の蛍光コロニーを採択する工程
d)演算工程:
採択された所定の閾値以上の蛍光コロニーの総面積値(図5C)を算出する。ここでは、蛍光の強い物質(青色の付着物、以下の測定条件では輝度値に基づいて二値化閾値13以上とする)を採択し、その面積値を計算する(単位μm2)。
面積値から0~19999をガンリスクの低い場合A、20000~29999を要観察B、30000~ガンリスクありの3段階に分類して判定する。
一般的な蛍光測定は蛍光色素をつけてその色素特有の蛍光波長で観察するのが一般的である。励起波長や蛍光波長は色素の種類により決まっている波長を設定し測定する。色素を用いた蛍光測定は色素の蛍光を見ていて、物質そのものの自家蛍光を見る測定法ではない。
一方、自家蛍光は物質そのものの化学組成や化学構造、官能基、修飾化合物、生物学的構造などで発すると考えられる。つまり、自家蛍光はそれぞれ物質固有の蛍光波長を持っていると考えられる。本発明ではがん関連物質固有の自家蛍光を見る。一般的な色素の波長ではなくがん関連物質固有の自家蛍光の波長を複数の励起光から測定する方法である。がん関連物質固有の自家蛍光を直接測定するための最適な励起光と蛍光波長を特定した。がん関連物質の自家蛍光を分光しその蛍光波長を分析した結果から、強い自家蛍光を発するRGBの励起光とRGBの自家蛍光の測定する波長を決めた。光源にはLED光源を使用し、オリンパス正立顕微鏡BX-63に蛍光波長フィルタをつけて測定する蛍光波長を限定し蛍光画像を取得した。
具体的には
Bの励起光は375~400nmで蛍光波長は460±25nmの範囲
Gの励起光は470~495nmで蛍光波長は510nm以上の長波長の範囲
Rの励起光は530~550nmで蛍光波長は575nm以上の長波長の範囲である。
Bはがん関連物質の自家蛍光が強く発する励起光と蛍光波長で、Gは良性疾患の自家蛍光が
強く発する励起光と蛍光波長である。がん関連物質が付着したバイオチップをオリンパス正立顕微鏡BX-63にセットし、LED光源と蛍光波長フィルタを上記Bの設定にする。バイオチップのリアルタイム蛍光画像を見ながらフォーカス(Z軸)を合わす。フォーカス(Z軸)と測定位置(X,Y軸)が決まれば、Bの設定の蛍光画像を測定する。次に、LED光源と蛍光波長フィルタを上記Gの設定にし、フォーカス(Z軸)を合わせ先ほどのB設定と同じ測定位置(X,Y軸)に合わし、蛍光画像を測定する。測定位置(X,Y軸)を合わすことにより、チップの同じ位置の同じがん関連物質のそれぞれの蛍光画像を取得することが出来る。同じようにR設定のLED光源と蛍光波長フィルタにして、同じ測定位置(X,Y軸)の蛍光画像を測定する。
解析法
イメージングソフトウェア「cellSens」(日本オリンパス光学社製)を使用して解析を行った。
それぞれの励起光で測定した3種類の蛍光画像のバイオチップに付着した同じ位置の同じがん関連物質をROIで囲って解析範囲を決め、下記RGBの設定範囲の物質の面積値を算出する。Bは輝度値28000~50000の範囲の付着物を選択し、また円形度0.3以下の付着物を除去する設定とした。
Gは輝度値27000~50000の範囲の付着物を選択し、また円形度0.3以下の付着物を除去する設定。
Rは輝度値21000~50000の範囲の付着物を選択し、また円形度0.3以下の付着物を除去する設定。
この輝度値設定範囲は付着物質の自家蛍光の蛍光強度やLED光源の光の強度により範囲を設定した。また円形度0.3で解析することにより、解析ソフトが自動でいびつな形状の異物を除去し解析精度を上げる設定にした。輝度値の範囲は10000~70000の範囲で好ましくは20000~50000程度の範囲で、円形度の範囲は0.9~0の範囲で、好ましくは0.3程度である。蛍光コロニーの状況に応じてこの円形度は調整される。この測定および解析設定により算出されたRGBの面積値からB/G、G/Rなどの2波長比のRatio値を算出し、大腸に関してはB/GのRatio値で悪性腫瘍が1.9~1.0の幅を持ち、良性腫瘍で0.1前後、健常者で0.2~0.8となった。
このように励起光を分けて蛍光測定をすることにより、各励起光におけるより強い自家蛍光の蛍光画像を得ることが出来る。この手法により、バイオチップ上の同じ位置の同じがん関連物質を違う設定の励起光、蛍光波長で測定し解析することより、物質固有の自家蛍光をより多く引き出すことが出来、データの精度を向上することができた。
本発明方法で用いるプラズモン金属メソ結晶領域を有する基板をプロテオチップという。その製造方法は、以下の通りである(特許文献1参照)。
1)金属錯体水溶液を錯体を形成する金属より卑なる電極電位(イオン化傾向の大きい)金属基板上で電極電位差により化学還元して量子結晶(ナノサイズの金属錯体結晶)を凝集させている。銀錯体の場合、チオ硫酸銀水溶液を銀より卑なる電極電位(イオン化傾向の大きい)の銅または銅合金上で凝集させることにより銀錯体の量子結晶を化学還元法を採用して形成している。詳しくは、金属錯体の水溶液中の濃度は主として形成する量子結晶のサイズを考慮して決定すべきであり、分散剤を使用するときはその濃度をも考慮するのがよく、通常、100ppmから5000ppmの範囲で使用できるが、配位子の機能にも依存してナノクラスタというべきナノサイズを調製するには500から2000ppmの濃度が好ましい。
2)量子結晶を形成する金属錯体は担持金属の電極電位Eと相関する式(I)で示される錯体安定度定数(logβ)以上を有するように選択される。
式(I):E゜=(RT/|Z|F)ln(βi)
(ここでE゜は、標準電極電位、Rは、気体定数、Tは、絶対温度、Zは、イオン価、Fは、ファラデー定数を表す。)
ここで、金属錯体が、Au、Ag、PtまたはPdから選ばれるプラズモン金属の錯体である場合は、励起光に対して局在表面プラズモン共鳴増強効果を有する。特に、金属錯体が銀錯体であるときは、安定度定数(生成定数)(logβi)が8以上の銀錯化剤とハロゲン化銀との反応により形成されるのがよく、ハロゲン化銀としては塩化銀が好ましく、錯化剤としてはチオ硫酸塩、チオシアン酸塩、亜硫酸塩、チオ尿素、ヨウ化カリ、チオサリチル酸塩、チオシアヌル酸塩から選ばれる1種であるのが好ましい。銀錯体は平均直径が5~20nmであるナノクラスタからなる量子ドットを有し、量子結晶のサイズが100~200nmとなる。
3)本発明で用いるプロテオチップにおける、プラズモン金属メソ結晶とは、プラズモン金属錯体の量子結晶の酸化物であり、血中でプラスに帯電するメチル化ヌクレオソームを電荷で補足するに必要なマイナス帯電で、励起光の照射により表面プラズモン増強効果を発揮し、補足したメチル化ヌクレオソームの自家蛍光を増強する効果を有するものをいう。銀錯体量子結晶の場合、アルカリ処理(次亜塩素酸ナトリウム水溶液で処理)すると、以下の反応により銀ハロゲン化物を核として過酸化銀を含み、銀酸化物の複合物の針状ナノ結晶群が形成されるものと思われ(特許文献1の図5)、しかも水中で(-)荷電を帯びる一方、DNAが巻き付いたヒストンが(+)荷電を帯びるため(特許文献1の図7(a))、この遊離ヌクレオソームに代表される正電荷を帯びたがん関連物質を選択的に吸着することが見出された。しかも過酸化銀を含む銀酸化物の針状ナノ結晶群は、レーザ光に代表される励起光の照射により表面プラズモン増強効果を示し、吸着されたヒストンに代表されるがん関連物質の自家蛍光を検出するのに好ましいことが見出されている。
Na2S2O3+4NaClO+H2O→Na2SO4+H2SO4(2NaHSO4)+4NaCl
Ag++NaCl→ AgCl + Na+
Ag++3NaOCl→ 2AgCl + NaClO3 + 2Na+
Ag++OH- → AgOH
2Ag++ 2OH- → Ag2O+H2O
4)本発明の銀酸化物の複合針状ナノ結晶群は、過酸化銀を含む銀酸化物が自己組織化してニューロン状の三次元超構造体(メソ結晶)を形成するもので(特許文献1の図8及び9)、銀イオン水溶液をAg/AgCl電極を用いて定電位電析を行って、又は銀の量子結晶をアルカリ処理で酸化することにより形成することができるが、銀錯体量子結晶、例えばチオ硫酸銀量子結晶をアルカリ処理(次亜塩素酸ナトリウム水溶液で処理)することによって容易に形成することができる。
5)疾病関連物質を吸着することができる限り、蛍光標識マーカーの高度検出と感度と迅速性を実現するピッチ350nmの周期構造をもつ基板に銀と酸化亜鉛の薄膜を成膜してなるプラズモニックチップ(特許文献2)や金属ナノ粒子を有機溶媒中に分散させ、有機溶媒を揮発させて金属ナノ粒子を二次元方向に自己組織化して粒系の揃った銀ナノ微粒子からなる局在プラズモン蛍光増強シート(特許文献3)を用いてもよい。
血液を含む体液から検体を作成する。赤血球は強い自家蛍光を示すので、遠心分離して血漿をのみを取り出すのがよい。疾患関連物質としてガン疾患の場合は、10~50倍希釈して断片化DNA(細分化ヌクレオソーム)の自家蛍光を測定しやすくするように希釈率を決定する。リン青銅上におよそ1000ppmのチオ硫酸銀錯体水溶液を滴下して作成したチオ硫酸銀錯体量子結晶をアルカリ処理して酸化形成した銀酸化物メソ結晶に対しては20~30倍希釈が望ましい。細胞の場合は機械的破砕がその物性を変化させないので好ましい。そして、ここで都合のいいことには、断片化DNA(細分化ヌクレオソーム)は安定なタンパク結合体(Protein-bound DNA fragments: nucleosome or chromatin)を形成し、比較的強い正電荷を示す。それ故、負電荷を示し、かつ励起光により表面プラズモン増強効果を示すプラズモン金属メソ結晶に選択的に捕捉され、しかも断片化DNA(細分化ヌクレオソーム)は安定であるため、真空乾燥又は乾燥剤(シリカゲル)乾燥後保存して蒸留水等に再溶解しても乾燥前のタンパク結合体の自家蛍光の特徴を再現することができる。乾燥剤(シリカゲル)乾燥ではこれは現場での採血、遠心分離での血漿の採取だけでなく、図4に示すように、容器内で乾燥させ、郵送での検査依頼を可能とするものとなる。また、全ての疾患には異常たんぱく質の蓄積を原因とする場合が多く、例えば、アルツハイマー病、パーキンソン病、筋萎縮性側索硬化症、ハンチントン病などの神経変性疾患に共通する特徴は、神経細胞内に凝集した異常タンパク質の蓄積によるものである。異常タンパク質は細胞毒性を持つため、神経細胞変性や細胞死を引き起こす。ほとんどの異常タンパク質は、ユビキチン化によって目印がつけられプロテアソームで分解されるが、プロテアソームは凝集した異常タンパク質を壊すことができず、神経変性疾患における神経細胞では、ユビキチン化された異常タンパク質の凝集体が蓄積していることから、異常タンパク質の検出ががん以外の疾病関連物質の検出として、リキッド・バイオプシィ法を用いて可能であることが示唆されるところである。さらに、iPS細胞はガン化遺伝子DNAを含む場合があるので、検体とし、以下の自家蛍光で、iPS細胞のがん化の識別を行い、除去することもできる。
ここでは、上記プラズモン金属メソ結晶基板上に捕捉された断片化DNA(細分化ヌクレオソーム)に励起光を照射して、捕捉断片化DNA(細分化ヌクレオソーム)の自家蛍光を表面プラズモン増強効果により増強し、蛍光コロニーを蛍光画像として取得する。
励起光としては正常組織と病変組織でその集積・排出特性が異なるヘマトポルフィリン誘導体(腫瘍親和性蛍光物質)を励起するに適するとされる405nmの励起光のレーザ光源を用いた。タンパク結合体は血液を採取して遠心分離にかけ、得られる血漿を蒸留水で30倍希釈して用いた。
c)蛍光コロニーの採択工程:
オリンパスDM(ダイクロイックミラー)405-445/514を用い、図1の結果が得られたので、各サンプル毎の輝度の高いポイントを例えば10点(疾病に応じ、採取ポイント数は決められる)抽出し、中心付近に円形のROIを作成し、スペクトルデータを算出した。蛍光コロニーの10点採択は専用ソフト「cellSens」で二値化し、所定の閾値以上の輝度の蛍光コロニーを採択する。
その他の画像取得条件は次の通りである。
レーザ: 405nm 50%
対物レンズ: 10倍(MPLFLN10×)
ピンホール径:500nm
取得波長:460-504nm
スリット幅:4nm
ステップ:2nm
解像度:1024×1024
平均化:4回
d)演算工程:
採択された所定の閾値以上の蛍光コロニーのピクセルの総面積値、又はRGB及びそれと相関する2波長域でのピクセルの総面積比でratio値を演算する。
その結果、総面積値ならびに異なる波長域の総面積のRatio値により健常者、良性腫瘍、悪性腫瘍を識別することができる。また、R、G、Bの2波長の比率及びそれと相関する2波長の比率(ratio)により健常者、良性腫瘍、悪性腫瘍を識別する。
オリンパス製DM405-445/514を使用して470-490nm及び600-620nmの画像を取得し、Ratio値に変化があるかどうか確認した結果、図2の結果を得た。前立腺に関し、G/Rで良性腫瘍の場合2.0前後、健常者で1.70前後、悪性腫瘍で1.76を超え、1.86までの幅を持ち、ステージとの関連を示すことがわかった。この結果はサンプル数の増加によりデータ精度を向上させることができる。
また、ポイントごとの輝度が異なるため、各波長での輝度を470nmの輝度を100%とした割合にて表示し、10ポイントの平均値をサンプル毎のデータとしてグラフを作成した。また、図3のように610nm前後の波長にがん関連物質のピークが存在することを確認できた。
本発明ではガン疾患及びその他の疾患で異常に発生する断片化DNA(細分化ヌクレオソーム)を検出対象とする。この種疾患に関連するタンパク結合体は、正電荷を有することよりプラズモン金属メソ結晶に選択的に捕捉され、励起光の照射によりプラズモン金属メソ結晶の表面プラズモン増強効果により増強され、蛍光顕微鏡で確認できる所定以上の輝度を有する自家蛍光を発光することが見出されている(図1)。これらのタンパク結合体は疾患の発生により血液その他体液中にアポトーシスに代表される病理的な細胞死によって放出され、図1に示す夜空の星座のように複数のコロニーが観測され、小さいものでは、25μm程度の広がり、大きいものでは150μm程度の広がりとして観測される。アポトーシス細胞ではCAD(カスパーゼ活性化DNase)の阻害因子が分解され、活性化したCADがDNAをヌクレオソーム単位で切断するため、およそ200bpの倍数の断片化DNA(細分化ヌクレオソーム)として捕捉されるためである。詳しくは、ヒト血漿中の細胞遊離DNA(cfDNA)はヒストンまたはTFと関連するタンパク質結合として血中に放出され、優先的に生き延び、健常者では主に骨髄系リンパ系の細胞系に由来するが、特定の病状では1つまたは複数のさらなる組織からの寄与が考えられており、がんなどの病理学的状態におけるcfDNAから細胞タイプを推測するヌクレオソームの足跡(footprint)を含み、その組織の起源を知らせる(非特許文献5:Cell,2016January14;164:Cell-free DNA comprises an in vivo nucleosome footprint that informs its tissues-of-origin)が明らかにされている。因みに,腫瘍の悪性度がより高度な懐死に結び付き、循環血液中に腫瘍DNA(ctDNA)が増加することが報告されており、血漿DNAがヌクレアーゼで切断されたヌクレオソームに類似した予測可能な断片化パターンを示し、健常者とガン患者でcfDNAのサイズ分布を評価でき、血漿中のcfDNAが腫瘍形成や転移の進行に関与することも報告されており、リキッド・バイオプシィの診断バイオマーカ―としてcfDNAの重要性が明らかにされている(非特許文献4:Circulating Tumor DNA as a Liquid Biopsy for Cancer; Climinal Chemistry 2015;61:112-123)。
がん患者を区別することができるだけでなく、がん発生部位の識別性が示唆される。
血清中のがん関連物質として断片化DNA(細分化ヌクレオソーム)は、DNAがひと巻きされたヒストン(ヌクレオソーム)、それらが集まりひも状になった構造のクロマチン(線維)を含む。グロブリンも正電荷を帯びるが、その増加は他のがん関連物質に比べて最大2倍以下であるのに対し、本発明で検知される物質はがん進行に伴う増加が100倍以上に達するので、グロブリン以外の増加はがん関連物質が検知されているとされ、採択された蛍光コロニーの一定閾値以上のピクセルの総面積はガンのステージと関連することを物語る。
(実施例1)
チオ硫酸銀1000ppm水溶液を調製し、その1滴をりん青銅板上に滴下し、約3分間放置し、溶液を吹き飛ばすと、SEM像でみると、量子結晶が作成されていた。実施例1で製造したナノ粒子凝集体(量子結晶)の各種SEM像を示す写真(特許文献1の図1参照)では、100nm前後の薄い六角柱状結晶であって、表面に数nmオーダの凹凸が発現している。金属ナノ結晶に特有のファセットは確認できなかった。りん青銅坂上に滴下後の放置時間と量子結晶形状の関係を示す写真(特許文献1の図6)では、まず、六角形の量子結晶が生成し、形状を維持しつつ成長するのが認められ、量子結晶のEDSスペクトル(元素分析)の結果を示すグラフ(特許文献1の図4)では、りん青銅板上に形成された結晶は銀及び錯体配位子由来の元素を検出したが、銅板上にチオ硫酸銀1000ppm水溶液を調製し、その1滴を滴下し、約3分間放置し、溶液を吹き飛ばした場合は、銀のみを検出したに過ぎなかった。
量子結晶は1000ppmチオ硫酸銀錯体水溶液の場合、りん青銅板上に滴下して3分間放置すると、100nm前後の六角柱状に形成され、各六角柱状の量子結晶は数nmオーダの凹凸を持つことがSEM像から確認されたが、金属ナノ結晶に特有のファセットは確認できず、EDS元素分析で銀及び錯体配位子由来の元素を検出されたため、全体は銀錯体のナノ結晶であって、その表面に現れる凹凸は錯体中の銀がクラスタとして量子ドットを形成して広がっていると推測される。本発明の銀錯体量子結晶がりん青銅板上に形成される一方、銅基板上には銀のみのナノ粒子が析出する現象を見ると、チオ硫酸銀錯体の平衡電位が0.33で銅の電極電位(0.34)と同等であるため、銅基板上には銀(0.80)のみが析出し、りん青銅の場合は0.22と電極電位がわずかに卑であるため、銀錯体の結晶が析出したものと思われる。したがって、量子結晶を作成するためには1)錯体水溶液が500~2000ppmという希薄な領域であること、2)金属錯体水溶液の平衡電位に対し担持金属の電極電位がわずかに卑であること、3)電極電位差で金属錯体が凝集させることが重要であると思われる。また、1000ppmチオ尿素銀錯体水溶液を使用した場合も同様であった。
上記量子結晶基板に5%次亜塩素酸ソーダ水溶液を滴下して2分間処理して除去すると特許文献1の図11に示す結晶構造が見られ、針状の結晶とラクビーボール状の塊と大きい塊が見られたので、それぞれの組成をEDSスペクトル(元素分析)で分析すると、以下の反応式から針状の結晶はともに塩化銀と酸化銀の複合結晶からなるものと考えられるが、特許文献1の図7の結果は塩素は確認できず、銀と酸素が支配的であることがわかる。Na2S2O3+4NaClO+H2O→Na2SO4+H2SO4+4NaCl (1)
Ag++NaCl→ AgCl + Na+(2)
Ag++3NaOCl→ 2AgCl + NaClO3 + 2Na+ (3)
Ag++OH- → AgOH (4)2Ag++ 2OH- → Ag2O +H2O(5)
したがって、本発明に係るメソ結晶の形成には銀イオンとチオ硫酸イオンが塩素イオンの存在下にアルカリ酸化反応により生ずるものと思われるが、通常の水溶液中では酸化銀が形成されるに過ぎないが、以下のXPS測定から過酸化銀が支配的に形成されていると推測される。
XPS測定:
上記量子結晶基板に次亜塩素酸ナトリウム水溶液25μlを2分間滴下し、
再結晶基板を作り、エッチングせずそのまま(使用機種:アルバック・ファイ(株)/PHI5000VersaProbeII(走査型X線光電子分光分析装置))でAgとOとをXPS測定した。また、比較対象のため、酸化銀の粉と塩化銀の粉のAgを測定した。他方、再結晶基板をアルゴンガスクラスターイオン銃で5分間エッチングしてAgとOをXPS測定した。XPS測定結果(特許文献1の図9及び図10)をEDSの結果(特許文献1の図8)から推測して、529eV付近のピークは過酸化銀(AgO)に由来するOピークで、530eV付近のピークは酸化銀(Ag2O)に由来するOピークであると認められる。エッチングした場合に、酸素量は減少するが、529eV付近のピークの過酸化銀(AgO)に由来するOピークが、530eV付近のピークは酸化銀(Ag2O)に由来するOピークよりも大きいことは基板近傍に過酸化銀が形成されているのを物語るものといえる。これは、メソ結晶形成時の触媒作用と基板の電極電位が影響しているものと推測される。 なお、EDS測定は上記再結晶基板を使用機種:日本電子株式会社/JSM-7001F(電界放出形分析走査電子顕微鏡)を用いて行った。
また、チオ硫酸銀の量子結晶を次亜塩素酸水溶液、0.01規定苛性ソーダ水溶液、0.01規定塩酸水溶液、0.1モル炭酸ナトリウム水溶液で処理しても同様の結果は得られなかった。よって、この針状結晶の形成には銀イオンとチオ硫酸イオンの存在下に上記反応により生ずるものと思われる。酸化銀は水溶液中で負電荷を帯び、光により還元されて金属銀を析出させる。過酸化銀はその傾向が顕著なので、正電荷のがん関連物質を吸着し、しかも吸着したがん関連物質と銀粒子との間の表面プラズモン増強効果が得られるものと思われる。
ヌクレオソームはクロマチンの基本的構成単位で、4種のヒストン(H2A、H2B,H3、H4)からなるヒストン8量体にDNAが巻きついた構造をしているが、ヒストンはDNAをパケージングするという役割に加え、DNAのアクセシビリティを調節すること及び、遺伝子制御にも重要な役割を果たしている。ヒストンの翻訳後修飾により、DNAやその他の核蛋白質との相互作用が制御され、可逆的な遺伝子発現に影響を及ぼす。ヒストン修飾の種類として、メチル化、アセチル化、リン酸化、ユビキチン化、SUMO化、シトルリン化、ADPリポシル化、が主として知られている。ヒストンの配列中、どの部位がこれらの修飾を受けるかによって、周囲の遺伝子発現は活性化または抑制される。こうしたヒストンの翻訳後修飾部位の組み合わせと遺伝子発現への影響を各種ヒストンコード関連抗体(Genetex社抗ヒストン抗体)を使用すると、本発明のプラズモニックチップで捕捉されたヒストンコードの修飾された状態を、蛍光画像で観測すると、ヒストンコード仮説の検証を行うことができる。
光源にはLED光源(XYLIS製波長360~770nm)を使用し、オリンパス正立顕微鏡BX-63に以下の蛍光波長フィルタをつけて測定する蛍光波長を限定し蛍光画像を取得した。各波長域の所定の閾値以上のピクセルを採択し、その総面積を採択するのは上記と同様である。
9名の大腸検査患者の試料(血液から遠心分離して採取した血漿)を上記バイオチップ上に滴下して検体を作成し、分子側のG領域および分母側G領域の所定閾値以上のピクセルの総面積を求めた。分子側の励起光のフィルタとしてBP470~495nmを用い、蛍光フィルタとしてBA510~550nmを用いた。上記ミラーユニットを用いて得られる蛍光画像の所定以上の閾値のピクセルを採択してRatioの分子としてピクセル総面積を算出する。
他方、分母側の励起光のフィルタとしてBP460~480nmを用い、蛍光フィルタとしてBA495~540nmを用いた。上記ミラーユニットを用いて得られる蛍光画像の所定以上の閾値のピクセルを採択してRatioの分母としてピクセル総面積を算出する。以上のG領域分子/G領域分母のRatioを算出し、別途検査で決定したステージと比較すると、以下の表1の通りであった。これにより、本発明方法で得られる結果は実際の検査で決められる悪性/良性及びステージと密接な関係が見られることが分かった。
Claims (5)
- a)試料中プラズモン金属メソ結晶領域を有する測定基板を用い、体液または細胞を含む培養液をそのまま又は希釈して作成した検体を接触させ、検体中の断片化DNA(細分化ヌクレオソーム)を疾病関連物質としてプラズモン金属メソ結晶に電荷捕捉させる工程と、b)このプラズモン金属メソ結晶上の捕捉された断片化DNA(細分化ヌクレオソーム)に励起光を照射して、その自家蛍光を表面プラズモン増強効果により増強し、断片化DNA(細分化ヌクレオソーム)の蛍光コロニーの蛍光画像を一定の測定領域(ROI)を決め、RGBのいずれかの領域の蛍光コロニー画像を取得する工程と、c)該蛍光コロニー画像の所定の閾値以上の輝度を示すピクセルを採択する工程と、d)採択された測定領域の所定の波長域での所定の閾値以上のピクセルの総面積値、又は採択された測定領域の異なる2波長領域の、所定の閾値以上のピクセルの総面積値のratioを演算する工程を含むことを特徴とする自家蛍光によるリキッド・バイオプシィ法。
- プラズモン金属メソ結晶が過酸化銀メソ結晶を含む、プラズモン金属錯体量子結晶の酸化物であって、プラス電荷を有し、かつ表面プラズモン増強効果を有する請求項1記載のリキッド・バイオプシィ法。
- 捕捉した断片化DNA(細分化ヌクレオソーム)がB又はG領域の同一又は異なる二波長域の励起波長で励起されて蛍光コロニーを得、該蛍光コロニーを異なる二波長域のフィルタを介して採取し、B領域の所定の閾値以上のピクセルの総面積値を悪性腫瘍とする一方、G領域の所定の閾値以上のピクセルの総面積値を良性腫瘍とし、そこから採取したピクセルの各総面積値のratio値がB領域/G領域又はG領域/B領域のRatio値である請求項1記載のリッキド・バイオプシィ法。
- 所定の閾値以上のピクセルの総面積値のB領域/G領域のRatio値で、悪性腫瘍で1.9から2.0の幅を持ち、健常者で0.2から0.8、良性腫瘍で0.1前後である請求項2記載のリッキド・バイオプシィ法。
- 捕捉した断片化DNA(細分化ヌクレオソーム)がメチル化されたガン抑制遺伝子、高メチル化したRB遺伝子、p16以外にp14、p53等を含む請求項1記載のリッキド・バイオプシィ法。
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