[go: up one dir, main page]

WO2025026131A1 - Procédé de préparation de lymphocytes tcd8 + naïfs et utilisation correspondante dans la préparation de médicaments destinés à retarder la sénescence - Google Patents

Procédé de préparation de lymphocytes tcd8 + naïfs et utilisation correspondante dans la préparation de médicaments destinés à retarder la sénescence Download PDF

Info

Publication number
WO2025026131A1
WO2025026131A1 PCT/CN2024/106994 CN2024106994W WO2025026131A1 WO 2025026131 A1 WO2025026131 A1 WO 2025026131A1 CN 2024106994 W CN2024106994 W CN 2024106994W WO 2025026131 A1 WO2025026131 A1 WO 2025026131A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
primitive
preparation
aging
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2024/106994
Other languages
English (en)
Chinese (zh)
Inventor
王宏林
吴玥
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai First Peoples Hospital
Original Assignee
Shanghai First Peoples Hospital
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai First Peoples Hospital filed Critical Shanghai First Peoples Hospital
Publication of WO2025026131A1 publication Critical patent/WO2025026131A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • 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

Definitions

  • the present invention relates to the field of medical technology, and in particular to a method for preparing CD8 + primitive T cells and an application thereof in preparing anti-aging drugs.
  • CD8 + T cell subsets are the initial state of CD8 + T cells before they exert their effects. They can rapidly clone and expand to form phenotypic and functional heterogeneous effector cells, and then migrate to all corners of the body to exert antiviral and anti-tumor functions.
  • CD8 + naive T cells interact with antigen-presenting cells in the first phase, driving the process of CD8 + naive T cells to differentiate into effector cells and activate them rapidly. This interaction continues at the site of infection to regulate the immune response and kill target cells.
  • the purpose of the present invention is to address the deficiencies in the prior art and provide a preparation and application of CD8 + primitive T cells for delaying aging, thereby providing a new intervention strategy for delaying aging.
  • the present invention provides the use of CD8 + primitive T cells in the preparation of anti-aging drugs, wherein the CD8 + primitive T cells are young CD8 + primitive T cells.
  • the above-mentioned young CD8 + primitive T cells are derived from mammals with the same genetic background.
  • "same genetic background” means that the donor mouse and the recipient mouse are of the same mouse strain to avoid cell rejection reaction in mice.
  • the above-mentioned young CD8 + primitive T cells can delay the aging of the subject and improve the subject's athletic ability.
  • the present invention also provides a method for preparing a cell preparation for delaying aging, the method comprising the following steps:
  • Step S1 obtaining tissue cells, adding red blood cell lysis solution and standing for 4-6 minutes, and then adding buffer to dilute and terminate the reaction;
  • the above tissues may be one or a combination of peripheral blood, lymph, spleen or bone marrow.
  • the above buffer solution is a PBS solution containing 10% bovine serum albumin.
  • Step S2 centrifuge the cell solution in which the reaction has been terminated in step S1 at 300-500 ⁇ g for 5 minutes, discard the supernatant, add cell separation solution and resuspend the cells until the cell concentration reaches 0.5 ⁇ 10 5 / ⁇ L-2 ⁇ 10 5 / ⁇ L;
  • Step S3 separating and collecting CD8 + primitive T cells to obtain the cell preparation for delaying aging.
  • the separation and collection method includes magnetic bead sorting.
  • the present invention also provides a cell preparation for delaying aging prepared according to the above preparation method, wherein the cell preparation comprises young CD8 + primitive T cells.
  • the cell concentration in the above cell preparation is 1.2 ⁇ 10 4 / ⁇ L - 2.4 ⁇ 10 4 / ⁇ L.
  • the present invention has the following beneficial effects:
  • the present invention discovered for the first time that CD8 + primitive T cells are related to delaying individual aging, and successfully verified this result through a mouse model experiment.
  • CD8 + primitive T cells from young mice were implanted into old mice, the aging of the old mice was delayed and their motor ability was enhanced, confirming that the CD8 + primitive T cells of young individuals can indeed delay the aging of old individuals, providing a new research and development idea for the exploration of anti-aging drugs.
  • FIG1 is a cluster comparison diagram of various cell subsets in the CD8 + naive T cells of the juvenile group and the elderly group in Example 1 of the present invention.
  • FIG. 2 is a schematic diagram of the mouse adoptive transfer model of Example 2 of the present invention.
  • A Flow chart of the construction of the adoptive transfer model of CD8 + naive T cells in aged mice according to Example 2 of the present invention.
  • FIG3 is a mouse adoptive transfer model for evaluating the regulatory effect of young CD8 + naive T cells on aging in Example 2 of the present invention.
  • D Statistical graph of the total moving distance, total moving time, non-moving time, and frequency of entering the central area of 10-week-old positive control group mice, 30-month-old control group mice, and 30-month-old treatment group mice within 5 minutes in the open field experiment of Example 2 of the present invention.
  • the inventors of the present application found for the first time through in-depth research that individual CD8 + naive T cells are associated with delayed aging. Furthermore, the inventors of the present application successfully verified this result through a mouse model experiment. After the CD8 + naive T cells from young mice were implanted into old mice, the aging of the old mice was delayed and their motor ability was enhanced.
  • Example 1 CD8 + naive T cells undergo phenotypic changes during aging, suggesting the potential feasibility of targeting CD8 + naive T cells to delay aging.
  • the inventors previously collaborated with the Department of Orthopedics of Shanghai Sixth People's Hospital to collect bone marrow samples from 8 volunteers during orthopedic surgery, and performed single-cell transcriptome sequencing analysis on all CD45 + immune cells in the bone marrow. Inclusion criteria: 1 All age groups; 2 Healthy people without basic metabolic diseases in physical examinations or normal clinical tests.
  • the above 8 volunteers were divided into a young group (3-15 years old) and an elderly group (70-95 years old). Cells were extracted from fresh tissues using a syringe until the tissue block turned from red to white. Red blood cells were further lysed using ACK lysis buffer to prepare a single cell suspension. Each single cell suspension was labeled with an anti-human CD45 staining antibody, and CD45 + immune cells, including T cells, B cells, NK cells, DC cells, etc., were sorted out using a flow cytometer BD FACSAriacell sorter, with a viability of more than 90%.
  • Chromium Single Cell 3' v3 (10x Genomics) library preparation was performed by Shanghai Xuran Biotechnology Co., Ltd. according to the manufacturer's instructions.
  • the resulting libraries were sequenced using the Illumina HiSeq 4000 platform. Trimmed data were processed using CellRanger (10x Genomics, version 3.0), and cells were further screened, processed, and analyzed using the Seurat package (version 3.1.2) in R (version 3.6.3) software.
  • Seurat package version 3.1.2
  • R version 3.6.3
  • the individual's CD8 + primitive T cell phenotype is related to aging and changes during the aging process. This can be used as a starting point to intervene in the aging process, which is verified by the following mouse model experiment.
  • CD45.1 4-6 week-old mice (equivalent to 5-year-old humans) with CD45.1 strain background were used as CD8 + naive T cell donor mice
  • 16-month-old mice (equivalent to 50-year-old humans) with CD45.2 strain background were used as recipient mice. Both CD45.1 and CD45.2 markers can be detected by flow cytometry.
  • the purpose of selecting different strains for donor mice and recipient mice is to facilitate the distinction between donor and recipient cells and to subsequently detect whether the CD8 + naive T cells of young mice are successfully colonized in the recipient mice.
  • CD45.1 mice of 4-6 weeks old were used as donor mice, and spleen tissue of donor mice was used as the tissue source of CD8 + naive T cells.
  • Mouse spleen tissue was selected because mouse spleen is easy to obtain and contains more CD8 + naive T cells.
  • the CD8 + naive T cells in a single donor mouse can be used by multiple recipient mice.
  • CD8 + naive T cells were sorted using MagniSort TM Mouse CD8 Na ⁇ ve T cell Enrichment Kit (Invitrogen, Thermo Fisher Scientific, USA) to obtain CD8 + naive T cell suspension.
  • the CD8 + primitive T cells extracted above were dissolved in a sterile PBS solution, and the cells were resuspended at a concentration of 1.2 ⁇ 10 6 /100 ⁇ L-2.4 ⁇ 10 6 /100 ⁇ L.
  • mice with CD45.2 strain background 16-month-old mice with CD45.2 strain background were selected as recipient mice, and the recipient mice were randomly divided into two groups, control group and treatment group.
  • the control group mice were injected with an equal amount of normal saline, and the treatment group mice were injected with CD8 + original T cell suspension from the above donor mice through the tail vein every month, with 125 ⁇ L injected per mouse. After the injection, the mice were observed for 1 hour, and continued to be raised after confirmation.
  • the overall flow chart is shown in Figure 2 A.
  • mice from the treatment group were randomly selected to verify whether the CD8 + naive T cells from the donor mice had colonized in the recipient mice as follows:
  • blood was collected from the eye sockets of the mice in the treatment group. 100 ⁇ L of blood was collected from each mouse and placed in a 1.5 ml EP tube containing anticoagulant sodium heparin to prevent blood clotting. In another 15 ml centrifuge tube, 8 ml of double distilled water and 1 ml of fetal bovine serum (FBS) were added and mixed evenly. Then, the blood of the mice collected above was added and mixed upside down for 1 minute to lyse the red blood cells. Then, 1 ml of 10 ⁇ PBS was added to terminate the lysis reaction. Centrifuge at 300 ⁇ g for 5 minutes, discard the supernatant, and resuspend and wash the cells with PBS for flow cytometry staining.
  • FBS fetal bovine serum
  • mice After orbital blood sampling, the mice were euthanized, and the spleen and lymph nodes were removed and ground after full-body alcohol disinfection.
  • the femur was taken and the cells in the bone marrow were flushed out using a syringe.
  • the cells from the three parts were prepared as single cell suspensions and then stained with flow cytometry antibodies together with the cells lysed from the above blood.
  • the fluorescent labeled antibodies added to the flow cytometry included anti-mouse CD45.1 and anti-mouse CD45.2.
  • Figure 2B shows that the CD45.1-positive cells from the donor mice have been successfully colonized in the recipient mice, and the mouse adoptive transfer model has been completed, and the subsequent steps can be continued.
  • Open field test In this experiment, 10-week-old mice (equivalent to 18-year-old humans) were selected as the positive control group and the open field test was performed together. In this experiment, 10 mice were tested in each group, and each mouse was tested 3 times, and the results were averaged. The specific operation steps are as follows:
  • Pole climbing test In this experiment, 10-week-old mice (equivalent to 18-year-old humans) were selected as the positive control group and the pole climbing test was performed together. In this experiment, 6 mice were tested in each group, and each mouse was tested 3 times, and the results were averaged. The specific operation steps are as follows:
  • the present invention has completed the experimental study of the effect of delaying aging by identifying a unique immune cell subset with anti-aging potential in young individuals and successfully constructing a corresponding mouse cell therapy model.
  • the present invention proposes for the first time that the key component of the immune system, CD8 + na ⁇ ve T cells from young individuals, has the effect of delaying systemic aging of the body, and has completed verification in mouse experiments based on the independently constructed cell therapy model.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Wood Science & Technology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Genetics & Genomics (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Biochemistry (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Toxicology (AREA)
  • Virology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Microbiology (AREA)
  • Hematology (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de préparation de lymphocytes T CD8+ naïfs et une utilisation correspondante dans la préparation de médicaments destinés à retarder la sénescence. Les lymphocytes T CD8+ naïfs sont des lymphocytes T CD8+ naïfs jeunes. Il est découvert pour la première fois que les lymphocytes T CD8+ naïfs sont liés à un retardement de la sénescence individuelle et le résultat est vérifié avec succès au moyen d'expériences de modèle de souris. Après l'implantation de lymphocytes T CD8+ naïfs provenant d'un corps de jeune souris dans un corps de souris âgée, la sénescence de la souris âgée est retardée et la capacité d'exercice est améliorée. Il est démontré que les lymphocytes T CD8+ naïfs de jeunes individus peuvent vraiment retarder la sénescence d'individus âgés, et une nouvelle idée de recherche et de développement est fournie pour la recherche de médicaments destinés à retarder la sénescence.
PCT/CN2024/106994 2023-07-28 2024-07-23 Procédé de préparation de lymphocytes tcd8 + naïfs et utilisation correspondante dans la préparation de médicaments destinés à retarder la sénescence Pending WO2025026131A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202310940841.X 2023-07-28
CN202310940841.XA CN117771272A (zh) 2023-07-28 2023-07-28 Cd8+原始t细胞的制备方法及其在制备延缓衰老药物中的应用

Publications (1)

Publication Number Publication Date
WO2025026131A1 true WO2025026131A1 (fr) 2025-02-06

Family

ID=90378866

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2024/106994 Pending WO2025026131A1 (fr) 2023-07-28 2024-07-23 Procédé de préparation de lymphocytes tcd8 + naïfs et utilisation correspondante dans la préparation de médicaments destinés à retarder la sénescence

Country Status (2)

Country Link
CN (1) CN117771272A (fr)
WO (1) WO2025026131A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117771272A (zh) * 2023-07-28 2024-03-29 上海市第一人民医院 Cd8+原始t细胞的制备方法及其在制备延缓衰老药物中的应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013014535A1 (fr) * 2011-07-22 2013-01-31 Evrogen Joint Stock Company Procédés et compositions pour l'augmentation de la diversité des lymphocytes t
KR20130139576A (ko) * 2012-06-13 2013-12-23 가톨릭대학교 산학협력단 Ｉｌ-2 및 ｉｌ-2 항체를 유효성분으로 포함하는 노화 및 노인면역질환의 예방 또는 치료용 조성물
CN115047190A (zh) * 2022-07-25 2022-09-13 深圳丹伦基因科技有限公司 一种生物标志物及其应用
CN116355846A (zh) * 2021-12-28 2023-06-30 北京永泰生物制品有限公司 一种质量稳定可控的扩增活化淋巴细胞的方法及其用于防治神经科疾病中的用途
CN117771272A (zh) * 2023-07-28 2024-03-29 上海市第一人民医院 Cd8+原始t细胞的制备方法及其在制备延缓衰老药物中的应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013014535A1 (fr) * 2011-07-22 2013-01-31 Evrogen Joint Stock Company Procédés et compositions pour l'augmentation de la diversité des lymphocytes t
KR20130139576A (ko) * 2012-06-13 2013-12-23 가톨릭대학교 산학협력단 Ｉｌ-2 및 ｉｌ-2 항체를 유효성분으로 포함하는 노화 및 노인면역질환의 예방 또는 치료용 조성물
CN116355846A (zh) * 2021-12-28 2023-06-30 北京永泰生物制品有限公司 一种质量稳定可控的扩增活化淋巴细胞的方法及其用于防治神经科疾病中的用途
CN115047190A (zh) * 2022-07-25 2022-09-13 深圳丹伦基因科技有限公司 一种生物标志物及其应用
CN117771272A (zh) * 2023-07-28 2024-03-29 上海市第一人民医院 Cd8+原始t细胞的制备方法及其在制备延缓衰老药物中的应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
FAGNONI FRANCESCO F, VESCOVINI ROSANNA, PASSERI GIOVANNI, BOLOGNA GIOVANNI, PEDRAZZONI MARIO, LAVAGETTO GIAMPAOLO, CASTI AMOS, FRA: "Shortage of circulating naive CD8 ϩ T cells provides new insights on immunodeficiency in aging", IMMUNOBIOLOGY, vol. 95, no. 9, 1 May 2000 (2000-05-01), pages 2860 - 2868, XP093273021 *

Also Published As

Publication number Publication date
CN117771272A (zh) 2024-03-29

Similar Documents

Publication Publication Date Title
JP7580202B2 (ja) 精製済み間葉系幹細胞組成物および間葉系幹細胞組成物を精製する方法
Greish et al. Human umbilical cord mesenchymal stem cells as treatment of adjuvant rheumatoid arthritis in a rat model
US11828757B2 (en) Methods of measuring potential for therapeutic potency and defining dosages for autologous cell therapies
CN110675914A (zh) 一种筛选肿瘤特异性t细胞及tcr的方法
US20230051925A1 (en) Vesicle and use thereof
CN113136362A (zh) 一种囊泡及其应用
JP2024511052A (ja) 濃縮された制御性ｔ細胞の集団及びその産生方法
WO2025026131A1 (fr) Procédé de préparation de lymphocytes tcd8 + naïfs et utilisation correspondante dans la préparation de médicaments destinés à retarder la sénescence
Fazazi et al. Myelin-reactive B cells exacerbate CD4+ T cell-driven CNS autoimmunity in an IL-23-dependent manner
Wei et al. Umbilical Cord‐Derived Mesenchymal Stem Cells Attenuate S100‐Induced Autoimmune Hepatitis via Modulating Th1 and Th17 Cell Responses in Mice
CN116421629B (zh) 干细胞外泌体在预防或治疗衰老的药物中的应用
Hoffman et al. Comparative computational RNA analysis of cardiac-derived progenitor cells and their extracellular vesicles
Fenton et al. Single-cell characterisation of mononuclear phagocytes in the human intestinal mucosa
JP2025530540A (ja) 細胞の品質属性の検出方法、検出装置及びそれらの使用
CN110731970A (zh) 一种治疗过敏性鼻炎的细胞制剂
CN119039403A (zh) 乙型肝炎病毒抗原肽及其应用
CN101506353A (zh) 外周造血干细胞的处理方法
CN111773382A (zh) 一种Ad-NK4修饰的DC疫苗构建方法
HK40086175A (zh) 改进的人源化模型小鼠的构建方法
CN114990061B (zh) 一种诱导扩增中央记忆性t细胞的培养方法
CN113502271B (zh) 一种用于治疗炎症性肠病的间充质干细胞及其应用
Saleh Understanding the Impact of Duchenne Muscular Dystrophy Disease Severity on Human Skeletal Muscle Progenitor Cell Delivery
CN115747158A (zh) 一种体外高效扩增人黏膜相关恒定t细胞的方法
Oberholster et al. Comprehensive proteomic analysis of JC polyomavirus-infected human astrocytes and their excreted vesicles
CN117660330A (zh) 密度梯度离心分离细胞的方法及car-t细胞的制备方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24848089

Country of ref document: EP

Kind code of ref document: A1

DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)