WO2022259950A1 - Procédé d'évaluation du vieillissement - Google Patents

Procédé d'évaluation du vieillissement Download PDF

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WO2022259950A1
WO2022259950A1 PCT/JP2022/022466 JP2022022466W WO2022259950A1 WO 2022259950 A1 WO2022259950 A1 WO 2022259950A1 JP 2022022466 W JP2022022466 W JP 2022022466W WO 2022259950 A1 WO2022259950 A1 WO 2022259950A1
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aging
cells
saccharopine
sample
content
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Japanese (ja)
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幸太郎 横手
浩之 江藤
靖夫 大内
尚也 加藤
ひより 金子
善朗 前澤
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国立大学法人千葉大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • 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
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • 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

Definitions

  • the present invention relates to an aging evaluation method, an aging inhibitor screening method, an aging biomarker, and a composition for inhibiting cell aging.
  • Aging is generally thought to be a continuous process characterized by a gradual decline in cellular function.
  • the aging of society is progressing rapidly in Japan and other countries around the world. Among them, a method for simply evaluating the aging state is required.
  • Conventionally, as general aging biomarkers, enhancement of senescence-related beta-galactosidase activity, shortening of telomeres, increased expression of cell cycle inhibitory factors p21, p16, etc. are known, and are widely used in basic research.
  • the analysis of the general aging biomarkers described above requires invasive cell or biological tissue collection, chemical fixation of specimens, purification of nucleic acids or proteins, and analysis using molecular biological techniques, which is complicated. A detailed analysis process is required. In addition, it is necessary to slaughter and destroy the cells and biological tissue specimens collected for analysis. In addition, techniques that use miRNA present in cells, GPNMB gene expression, and changes in DNA methylation as indicators are similarly invasive, requiring complicated analysis processes such as collection of cell specimens and purification and analysis of nucleic acids. . On the other hand, although the technique using 1.5-year-old mouse extracellular vesicle protein is non-invasive, its usefulness as an aging biomarker in humans is unknown. Requires special sample preparation techniques.
  • An object of the present invention is to provide a method for evaluating aging that can be easily performed using a sample that can be collected noninvasively.
  • the inventors of the present invention have found that it is possible to evaluate aging using the L-saccharopine content in a body fluid sample derived from a subject as an index, and that the above problems can be solved, thus completing the present invention.
  • the present invention includes the following aspects. [1] A method for evaluating aging, comprising the step of measuring the L-saccharopine content in a biological sample derived from a subject. [2] The method for evaluating aging according to [1] above, wherein the biological sample is a sample derived from blood or urine. [3] The method for evaluating aging according to [1] or [2] above, wherein the aging to be evaluated is pathological aging.
  • a biomarker of aging which consists of L-saccharopine.
  • a composition for suppressing aging comprising as an active ingredient a substance that suppresses intracellular production of L-saccharopine.
  • the composition of [8] above, wherein the cells are fibroblasts.
  • the composition of [9] above, wherein the fibroblasts are skin-constituting fibroblasts.
  • the present invention provides a method for evaluating aging that can be easily performed using specimens that can be collected noninvasively. Furthermore, according to the present invention, there are provided methods for screening anti-aging means, biomarkers of aging, and compositions for suppressing cell senescence.
  • FIG. 1 shows the ROC curve.
  • FIG. 1A shows the results of healthy subjects vs. WS patients (Healthy/Werner), and FIG. 1B shows the results of ⁇ 70 years old vs. 70 years old or older (Healthy young/old).
  • FIG. 2 shows cell growth curves.
  • FIG. 3 shows the results of senescence-associated ⁇ -galactosidase (SA- ⁇ -gal) staining.
  • SA- ⁇ -gal senescence-associated ⁇ -galactosidase
  • FIG. 4A shows the distribution of L-saccharopine content in plasma specimens from WS patients.
  • FIG. 4B shows the distribution of L-saccharopine content in urine samples from WS patients.
  • the method for evaluating aging of the present invention includes the step of measuring the L-saccharopine content in a body fluid sample derived from a subject. And the aging biomarker of the present invention consists of L-saccharopine.
  • a preferred aspect of the invention is a method for assessing pathological aging.
  • Pathological aging is distinguished from age-associated aging in healthy individuals, where aging already progresses at an early age when it is not observed in the average healthy person.
  • humans include aging due to progeria such as Werner's syndrome (WS), Hutchinson-Gilford-Progeria syndrome (HGPS), Cockayne syndrome, and photoaging due to ultraviolet rays and the like.
  • Subjects include, for example, mammals, including humans.
  • Non-human mammals include monkeys, mice, rats, rabbits, dogs, cats, cows, sheep, horses, pigs, and the like.
  • the actual age of the human being the test subject is preferably 30 years or older, more preferably 40 years or older, and even more preferably 50 years or older, from the viewpoint of aging progression, and suppression of aging progression is expected. From the viewpoint of being able to do so, it is preferably 80 years old or younger, more preferably 75 years old or younger, and even more preferably 70 years old or younger.
  • the actual age of a human being a test subject is preferably 0 years old or older, and preferably 66 years old or younger, 60 years old or younger, 50 years old or younger, or 45 years old. 40 years old or younger, 35 years old or younger, or 30 years old or younger.
  • the human subject is a patient with progeria such as Werner's syndrome (WS), Hutchinson-Gilford-Progeria syndrome (HGPS), Cockayne syndrome, etc.
  • the real age is preferably 0 years old or older.
  • the actual age is the age counted from the date of birth.
  • any biological sample derived from a subject can be used without limitation as long as the L-saccharopine content can be measured.
  • Specific examples include cells, biological tissue, and body fluid samples derived from the subject.
  • Cells and biological tissues include cells of oral mucosa, cells of nasal mucosa, and epidermis, which are easily collected.
  • collected cells or living tissue itself can be used, and cells obtained by culturing the collected cells or living tissue-derived cells (for example, primary cultured cells) can also be used.
  • Body fluid samples include blood-derived samples, lymph fluid, urine, sweat, saliva, nasal discharge, tears, and the like.
  • the biological sample is preferably a body fluid sample, more preferably a blood-derived sample, from the viewpoint that the collection of the biological sample is less burdensome on the test subject and a sufficient amount of the sample can be easily collected for measuring the L-saccharopine content.
  • sample or urine Blood-derived samples include whole blood, serum, and plasma, preferably plasma.
  • a biological sample can be obtained from a subject by methods known to those skilled in the art. For example, urine can be collected by collecting excreted urine. Whole blood can be collected by collecting blood using a syringe or the like. Serum can be obtained, for example, as the supernatant after coagulation of whole blood.
  • Plasma is a portion of whole blood from which blood cells have been removed, and can be obtained, for example, as a supernatant when the whole blood is subjected to centrifugation under conditions such as the presence of sodium citrate that do not cause coagulation of the whole blood.
  • L-saccharopine (systematic name: N-[(S)-5-amino-5-carboxypentyl]-L-glutamic acid, CAS registry number: 997-68-2) is a compound represented by the following chemical formula.
  • L-saccharopine is an intermediate metabolite in L-lysine biosynthesis. It has been reported that L-saccharopine is a toxin that, when accumulated in mitochondria, changes mitochondria into abnormal morphology and impairs their function.
  • L-saccharopine content was higher in fibroblasts and body fluid samples from patients with Werner's syndrome (WS), a progeria disease, than in controls. rice field. They also found that the L-saccharopine content was higher not only in WS patients, but also in body fluid samples from non-WS patients aged 70 years and older than in controls.
  • WS Werner's syndrome
  • the means is preferably mass spectrometry, more preferably tandem mass spectrometry, and even more preferably liquid chromatography/tandem mass spectrometry, from the viewpoint of versatility and convenience.
  • the means for measuring the L-saccharopine content is liquid chromatography/tandem mass spectrometry, the following analysis by liquid chromatography is exemplified.
  • the mass spectrometry method may be either a method of derivatizing a sample using a derivatization reagent or a non-derivatization method without derivatization.
  • Derivatizing reagents include reagents capable of derivatizing amino groups such as 3-aminopyridyl-N-hydroxysuccinimidylcarbamate reagents.
  • the biological sample it is preferable to subject the biological sample to measurement after pretreatment.
  • the biological sample is urine or plasma
  • pretreatment with a sulfosalicylic acid reagent is included.
  • the method of the present invention assesses the aging state of a subject.
  • the aging state can be evaluated by comparing the obtained L-saccharopine content measurement value with a preset reference value.
  • a preset reference value can be set for a predetermined chronological age range.
  • the preset reference value can be set by means known to those skilled in the art, such as ROC analysis.
  • the degree of aging can be determined by fitting the obtained measured value of L-saccharopine content to a preset model curve.
  • a model curve can be generated based on average L-saccharopine content by age.
  • the subject's degree of aging can be assessed by the age determined by fitting the model curve.
  • preventive treatment for suppressing the progression of aging-related diseases positive Rehabilitation treatment etc.
  • Preventive treatments include intake of mitochondria-related supplements such as coenzyme Q10 and nicotinamide mononucleotide (NMN), intake of antioxidant supplements such as ascorbic acid, intake of a lysine-restricted diet, and the like.
  • Active rehabilitation includes exercise training and the like.
  • Another aspect of the present invention is the step of performing the above aging evaluation method, and the progress of aging-related diseases in a subject whose aging state has been evaluated as being equal to or higher than the actual age by the above aging evaluation method. It is a method of treating or inhibiting aging that provides preventative treatment to inhibit it.
  • the present invention is a method for evaluating pathological aging
  • further tests for detecting progeria can be performed.
  • the presence or absence of mutations such as the 3139-1G>C mutation can be detected.
  • the presence or absence of mutations can be detected, for example, by sequencing the human WRN gene on genomic DNA collected from subject-derived somatic cells.
  • the present invention also provides a screening method for anti-aging means.
  • the screening method for the aging inhibitor of the present invention comprises culturing senescent cells in the presence or absence of a test substance; measuring the L-saccharopine content in the obtained cells, culture medium or culture containing cells and culture medium; The step of selecting as a candidate for means is included.
  • Senescent cells used for screening include human-derived senescent cells. Specifically, cells derived from Werner's syndrome (WS) patients, cells in which the WRN gene, which is the causative gene of Werner's syndrome, is mutated or defective, Hutchinson-Gilford-Progeria syndrome (HGPS), premature aging other than WS such as Cockayne syndrome Cells derived from diseased patients, cells derived from elderly people, and the like. Also included are stem cells such as iPS cells obtained by reprogramming these cells, and somatic cells obtained by differentiation-induced stem cells.
  • WS Werner's syndrome
  • HGPS Hutchinson-Gilford-Progeria syndrome
  • stem cells such as iPS cells obtained by reprogramming these cells, and somatic cells obtained by differentiation-induced stem cells.
  • cells artificially induced to age by subculture, drugs, radiation, etc., and genetic mutations that cause aging can be genetically engineered.
  • Cells introduced by techniques are also included.
  • Senescent cells derived from non-human mammals can also be used as senescent cells used for screening.
  • non-human animal models of progeria such as WS and HGPS
  • senescent cells derived from non-human animal models of age-related diseases such as Alzheimer's disease and Klotho gene-deficient animals
  • Non-human cells in which senescence is artificially induced by, for example can be used.
  • ES cells derived from the non-human model animals stem cells such as iPS cells obtained by reprogramming somatic cells derived from the non-human model animals, and somatic cells obtained by differentiation of the stem cells.
  • non-human model animals of progeria such as WS and HGPS; non-human model animals of age-related diseases such as Alzheimer's disease and Klotho gene-deficient animals, etc.
  • Non-human model animals are, for example, non-human mammals such as mice, monkeys and pigs.
  • a test substance is administered to a non-human model animal, and the L-saccharopine content in a body fluid sample of the non-human model animal to which the test substance is administered is measured.
  • In the absence of the test substance means a state in which the test substance is not present and a state in which the test substance is reduced compared to normal culture conditions.
  • Measurement of the L-saccharopine content can be performed on cultures that are cells, culture media, or mixtures containing cells and culture media.
  • composition for suppressing aging also provides compositions for inhibiting aging in subjects to which they are administered.
  • the composition for suppressing aging of the present invention contains, as an active ingredient, a substance that suppresses intracellular production of L-saccharopine.
  • Substances that suppress intracellular production of L-saccharopine are not particularly limited. Examples thereof include candidate substances obtained by the screening method for anti-aging means and substances capable of achieving candidate conditions.
  • One preferred embodiment is an L-lysine restricted diet. As shown in the examples below, the inventors have found that L-lysine restriction reduces L-saccharopine levels, resulting in less mitochondrial damage and improved cell proliferation.
  • the L-lysine-restricted diet can be prepared according to the lysine-restricted diet used for treatment of pyridoxine-dependent epilepsy.
  • Another preferred embodiment is a compound capable of inhibiting the production of L-saccharopine (L-saccharopine production inhibitor). More specifically, preferred embodiments include inhibitors targeting in vivo molecules that act in the lysine degradation metabolic pathway in mitochondria, such as SLC25A29 protein, SLC25A18 protein, GLU1 protein, IDH2 protein, and ⁇ -ketoglutarate. . A preferred embodiment also includes a compound that can enhance the function of NAD+ in the lysine degradation metabolic pathway in mitochondria. L-lysine is mainly used for protein synthesis in cells.
  • L-lysine is transported into the mitochondria by the SLC25A29 protein (solute carrier) and degraded by the lysine-degrading metabolic pathway via L-saccharopine. Specifically, L-saccharopine converts L-lysine and ⁇ -ketoglutarate (2-oxoglutarate) into substrates via the LKR (lysine-ketoglutarate reductase) domain of the AASS protein ( ⁇ -aminoadipate semialdehyde synthase).
  • ⁇ -Ketoglutarate is produced by transamination reaction of L-glutamic acid by GLU1 protein (glutamic acid aminotransferase) and oxidation reaction and decarboxylation by IDH2 protein (isocitrate dehydrogenase) of isocitrate as substrate.
  • GLU1 protein glutamic acid aminotransferase
  • IDH2 protein isocitrate dehydrogenase
  • L-glutamate is transported into mitochondria by the SLC25A18 protein (solute carrier).
  • L-saccharopine is decomposed into glutamic acid and ⁇ -aminoadipic semialdehyde by an oxidation reaction with NAD+ as a coenzyme by the SDH (saccharopine dehydrogenase) domain of the AASS protein.
  • compositions for suppressing aging can be provided in the form of various injections, oral preparations, drip infusions, inhalants, ointments, lotions, sprays, etc., which allow such administration routes.
  • a composition for inhibiting aging can be provided as a pharmaceutical composition.
  • it can be provided as a food composition containing foods with health claims such as foods for special uses, foods for specified health uses, and foods with nutrient function claims; foods with function claims; health supplements; and supplements.
  • a composition for suppressing cell senescence has an effect on a wide range of cells. Specifically, cells of organs such as skin; skeletal muscle; liver, kidney, gastrointestinal tract, lung, and brain are exemplified. Among them, it is particularly effective against fibroblasts, preferably fibroblasts that constitute the skin. Therefore, the composition for suppressing cellular aging is preferably a composition for suppressing aging of cells that constitute the skin.
  • SA- ⁇ -gal Senescence-associated ⁇ -galactosidase staining was also performed using the Cell Senescence Detection Kit (Cell Biolabs) according to the manufacturer's instructions. Cells were washed with PBS and fixed at room temperature using the attached fixing solution. After that, the cells were stained with the attached staining solution overnight at 37°C. Nuclear DNA was stained with DAPI. Stained images were obtained using a BZ-X700 microscope (manufactured by KEYENCE CORPORATION), and the ratio of staining-positive cells was calculated. SA- ⁇ -gal is a marker of cellular senescence. The results are shown in Tables 1 and 2.
  • WS patient-derived fibroblasts had significantly lower cell proliferation potential than the control group.
  • Example 1 Using cells derived from the same patient group and control group as in Reference Example 1, metabolome analysis was performed. Metabolome analysis was entrusted to Human Metabolome Technologies, Inc. (HMT).
  • sample preparation The medium was aspirated from the dish and the cells were washed twice with a 5% mannitol solution. Cells were then treated with 800 ⁇ L of methanol and left for 30 seconds to inactivate the enzyme. Subsequently, the cell extract was treated with 550 ⁇ L of ultrapure water containing an internal standard, and left still for 30 seconds. After obtaining the extract and centrifuging at 2,300 ⁇ g, 4 ° C. for 5 minutes, 800 ⁇ L of the supernatant was passed through a 5 kDa cut-off filter (Ultrafree MC-PLHCC, manufactured by Millipore) at 9,100 ⁇ g, 4 ° C. , 120 minutes to remove macromolecules. The filtrate was concentrated by centrifugation, resuspended in 50 ⁇ L of ultrapure water, and subjected to metabolomic analysis.
  • Ultrafree MC-PLHCC Ultrafree MC-PLHCC, manufactured by Millipore
  • Metabolome analysis was performed by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Specifically, CE-TOFMS analysis was performed using an Agilent CE capillary electrophoresis system (Agilent Technologies) equipped with an Agilent 6210 time-of-flight mass spectrometer. The system is an Agilent G2201AA ChemStation software version B.C.E for CE.
  • Example 2 The L-saccharopine content was quantified by mass spectrometry in plasma samples collected from 29 healthy subjects (22-89 years old) and 10 WS patients (45-66 years old). Mass spectrometry was outsourced to Sumika Chemical Analysis Service, Ltd.
  • L-saccharopine (Toronto Research Chemicals) was dissolved in water/formic acid (100:2, v/v) to prepare a standard stock solution of 10612 ng/mL.
  • the prepared standard stock solution was diluted with water/formic acid (100:2, v/v) to prepare standard solutions of 2, 4, 10, 40, 200, 400, 1000, 2000 and 4000 ng/mL.
  • QC-blank water / formic acid (100: 2, v / v), 40, 400, 2000 ng /mL) was added as a quality check (QC) sample.
  • concentration of each QC sample (corresponding concentration in the plasma sample to be measured) will be 0, 20, 200 and 1000 ng/mL.
  • Pretreatment operation method for plasma sample 100 ⁇ L of plasma sample was placed in a polypropylene tube, and 50 ⁇ L of water/formic acid (100:2, v/v) was added (total 150 ⁇ L). After adding 200 ⁇ L of 5% sulfosalicylic acid aqueous solution to 150 ⁇ L each of the specimen, added calibration curve sample and QC sample, the mixture was stirred for about 30 seconds and centrifuged (setting 4° C., 15000 ⁇ g, 5 minutes). An aliquot of the supernatant was used as an injection sample and 5 ⁇ L was injected into the LC-MS/MS.
  • MS/MS MS/MS used API5000 (AB Sciex Pte. Ltd.).
  • L-saccharopine m/z 277 ⁇ 84 was measured by electrospray ionization (ESI) method, multiple reaction monitoring (MRM) mode, and positive ion detection mode.
  • ESI electrospray ionization
  • MRM multiple reaction monitoring
  • L-saccharopine content in plasma sample The L-saccharopine content in the plasma samples was determined using the measurement results of the plasma samples from healthy subjects and WS patients and the calibration curve prepared above. Table 4 shows the results. Further, FIG. 4A shows a distribution map of the measurement results of plasma specimens of WS patients.
  • Example 3 The L-saccharopine content was quantified by mass spectrometry in urine specimens collected from 29 healthy subjects (22-89 years old) and 10 WS patients (45-66 years old). Mass spectrometry was outsourced to Sumika Chemical Analysis Service, Ltd. Measurement of urinary creatinine content (U-Cre) by an enzymatic method was outsourced to SRL Co., Ltd.
  • L-saccharopine (Toronto Research Chemicals) was dissolved in water/formic acid (100:2, v/v) to prepare a standard stock solution of 10612 ng/mL.
  • the prepared standard stock solution was diluted with water/formic acid (100:2, v/v) to prepare standard solutions of 10, 20, 40, 200, 400, 1000, 2000, 4000, 8000 and 10000 ng/mL.
  • Pretreatment operation method for urine specimen 100 ⁇ L of urine sample was placed in a polypropylene tube, and 50 ⁇ L of water/formic acid (100:2, v/v) was added (150 ⁇ L in total). 20 ⁇ L of APDSTAG Wako Amino Acids Internal Standard Mixture Solution (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) was added to 150 ⁇ L each of the specimen, added calibration curve sample, and QC sample. Furthermore, after adding 200 ⁇ L of a 5% sulfosalicylic acid aqueous solution, the mixture was stirred for about 30 seconds with a mixer and centrifuged (setting 4° C., 15000 ⁇ g, 5 minutes).
  • MS/MS ⁇ Mass spectrometer
  • MS/MS used API5000 (AB Sciex Pte. Ltd.).
  • L-saccharopine content in urine sample The L-saccharopine content in the urine samples was determined using the results of measurement of urine samples from healthy subjects and WS patients and the calibration curve prepared above. Table 4 shows the results. The L-saccharopine content in urine specimens was determined by normalizing with the urinary creatinine (U-Cre) content. Table 4 shows the results. In addition, FIG. 4B shows a distribution map of the measurement results of urine specimens from WS patients.
  • Healthy young group is a group of healthy young people whose real age is 22-67 years old, average: 38.7 ⁇ 2.9 years old
  • Healthy old group is real age 71-89 Age, average: 79.5 ⁇ 2.1 years old healthy elderly group.
  • Age-matched control was 34-67 years old, mean: 47.9 ⁇ 3.3 years old, and the actual age distribution of the WS patient group (36-66 years old, mean: 50.9 ⁇ 3.3 years old).
  • the plasma (Plasma) and urine (Urine) concentrations of L-saccharopine in WS patients were significantly higher than those in the healthy control group. Both plasma and urine L-saccharopine contents were found to be significantly elevated in WS patients when compared with age-matched healthy controls. A linear relationship of L-saccharopine levels with age indicates an increase in L-saccharopine levels in the elderly, and in healthy controls this variable in urine samples showed a significant correlation with age. On the other hand, no such significant relationship was observed in plasma samples from WS patients. Furthermore, when compared between different age groups, L-saccharopine concentrations in both plasma and urine were significantly higher in both the WS group and the healthy elderly group, and significantly higher than the healthy young control group.
  • FIG. 1 shows the ROC curve.
  • the left figure (Fig. 1A) shows the results of healthy subjects vs. WS patients (Healthy/Werner), and the right figure (Fig. 1B) shows the results of under 70 vs. over 70 years (Healthy young/old).
  • L-saccharopine is known to be an intermediate metabolite in the lysine degradation metabolic pathway in mitochondria.
  • the lysine-degrading metabolic pathway is a pathway that degrades excess L-lysine that was not used for protein synthesis, and it was suggested that WS patients have abnormalities in this metabolic pathway. Therefore, in order to suppress the accumulation of L-saccharopine in WS patients, an attempt was made to suppress cell senescence by restricting lysine, an upstream metabolite.
  • WS patient-derived fibroblasts were cultured under control medium (10% FBS, DMEM (146 mg/l for L-lysine containing)) or lysine-restricted medium (10% FBS, DMEM (without L-lysine)) at 5% CO 2 . cultured for 4 months at 37° C. in a humidified incubator. Medium was changed every 2 days. When reaching subconfluence, the cells were passaged at a split ratio of 1: 4 until growth stopped, and the cumulative cell number doubling level per day (Cumulative population doubling level; cPDL ) and senescent cells by ⁇ -galactosidase (SA- ⁇ -gal) staining were calculated.
  • control medium 10% FBS, DMEM (146 mg/l for L-lysine containing)
  • lysine-restricted medium 10% FBS, DMEM (without L-lysine)
  • FIGS. 2 and 3 The results are shown in FIGS. 2 and 3.
  • FIG. 3 *** indicates a p-value ⁇ 0.001. From FIG. 2, it was revealed that the cells cultured in the lysine restriction medium maintained significant cell growth even after 2 months of culture.
  • FIG. 3 revealed that senescence-associated ⁇ -galactosidase (SA- ⁇ -gal) staining significantly reduced the number of positive cells in lysine-limited cells.
  • SA- ⁇ -gal senescence-associated ⁇ -galactosidase

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Abstract

La présente invention a pour objet de fournir un procédé d'évaluation du vieillissement qui peut être facilement mis en oeuvre à l'aide d'un échantillon qui peut être collecté de manière non invasive.　A cet effet, l'invention a pour objet un procédé d'évaluation du vieillissement, le procédé comprenant une étape consistant à déterminer la teneur en L-saccharopine dans un échantillon biologique issu d'un sujet.
PCT/JP2022/022466 2021-06-10 2022-06-02 Procédé d'évaluation du vieillissement WO2022259950A1 (fr)

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