WO2022021827A1 - 石墨烯的免疫增强新用途 - Google Patents

石墨烯的免疫增强新用途 Download PDF

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WO2022021827A1
WO2022021827A1 PCT/CN2021/074189 CN2021074189W WO2022021827A1 WO 2022021827 A1 WO2022021827 A1 WO 2022021827A1 CN 2021074189 W CN2021074189 W CN 2021074189W WO 2022021827 A1 WO2022021827 A1 WO 2022021827A1
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graphene
cells
irradiation
use according
immune
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French (fr)
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冯晓星
穆祥
冯波
钟金丝
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烯旺新材料科技股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F7/00—Heating or cooling appliances for medical or therapeutic treatment of the human body
    • A61F7/007—Heating or cooling appliances for medical or therapeutic treatment of the human body characterised by electric heating
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61H—PHYSICAL THERAPY APPARATUS, e.g. DEVICES FOR LOCATING OR STIMULATING REFLEX POINTS IN THE BODY; ARTIFICIAL RESPIRATION; MASSAGE; BATHING DEVICES FOR SPECIAL THERAPEUTIC OR HYGIENIC PURPOSES OR SPECIFIC PARTS OF THE BODY
    • A61H39/00—Devices for locating or stimulating specific reflex points of the body for physical therapy, e.g. acupuncture
    • A61H39/06—Devices for heating or cooling such points within cell-life limits
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/44—Elemental carbon, e.g. charcoal, carbon black
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N5/0613—Apparatus adapted for a specific treatment
    • A61N5/0625—Warming the body, e.g. hyperthermia treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61H—PHYSICAL THERAPY APPARATUS, e.g. DEVICES FOR LOCATING OR STIMULATING REFLEX POINTS IN THE BODY; ARTIFICIAL RESPIRATION; MASSAGE; BATHING DEVICES FOR SPECIAL THERAPEUTIC OR HYGIENIC PURPOSES OR SPECIFIC PARTS OF THE BODY
    • A61H2201/00—Characteristics of apparatus not provided for in the preceding codes
    • A61H2201/02—Characteristics of apparatus not provided for in the preceding codes heated or cooled
    • A61H2201/0207—Characteristics of apparatus not provided for in the preceding codes heated or cooled heated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N2005/0635—Radiation therapy using light characterised by the body area to be irradiated
    • A61N2005/0642—Irradiating part of the body at a certain distance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N2005/0635—Radiation therapy using light characterised by the body area to be irradiated
    • A61N2005/0643—Applicators, probes irradiating specific body areas in close proximity
    • A61N2005/0645—Applicators worn by the patient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N2005/0664—Details

Definitions

  • the present disclosure relates to the application of graphene heating products in disease treatment, in particular to the new use of graphene for immune enhancement.
  • Immunity refers to the body's ability to resist foreign invasion and maintain the stability of the internal environment, which reflects the body's ability to distinguish itself from non-self, and to eliminate aliens. Immunity not only refers to the human body's resistance against pathogenic microorganisms, but also includes resistance to non-self antigenic foreign bodies, such as non-pathogenic pollen, drugs and even food, degenerate cells that often appear in normal organisms, and occasional Mutated cancer cells.
  • Immunity is an important physiological function of the human body. People with good immunity will have less pain and stronger physical fitness. Today's medical research shows that many diseases that threaten human health are closely related to the body's immunity. Chronic diseases such as cancer, chronic bronchitis, asthma, allergic rhinitis, chronic hepatitis, and diabetes are all closely related to immunity.
  • Immunity can be divided into innate immunity and acquired immunity according to the way it is acquired.
  • Innate immunity is a non-specific immunity that people have at birth, and can respond quickly to various invading pathogenic microorganisms.
  • Acquired immunity is acquired naturally in the course of life after a person is born, or acquired passively by artificially assisted methods, and is mostly specific immunity.
  • immunoglobulins or immune lymphocytes are formed after stimulation by antigenic substances such as microorganisms.
  • the technical problem to be solved by the present disclosure is to provide a new way to improve immunity.
  • the inventors used the electric heating products made of graphene films on BALB/c mice, beagle dogs and volunteers, respectively. Based on the experiments and observations of the immune response, it is found that the graphene film can enhance immunity by thermal irradiation or sticking to animals or humans.
  • the present disclosure provides new immune-enhancing uses of graphene. Specifically, the present disclosure proposes the following technical solutions:
  • a first aspect of the present disclosure provides the use of graphene in the manufacture of a device for immune enhancement.
  • the present disclosure provides immune-enhancing uses of graphene.
  • the immune enhancement is to increase the thymus index.
  • the immune enhancement is to improve the proliferation ability of lymphocytes.
  • the immune enhancement is to increase the activity of NK cells to kill tumor cells.
  • the immune enhancement includes the treatment of tumors.
  • the tumor is a hematological tumor.
  • the hematological tumor is leukemia or lymphoma; preferably, the leukemia is myeloid leukemia, more preferably, the leukemia is chronic myeloid leukemia; preferably, the lymphoma is T-cell lymphoma tumor.
  • a second aspect of the present disclosure provides the use of graphene in the manufacture of a device for improving the scavenging efficiency of serum urea nitrogen.
  • the present disclosure provides the use of graphene to improve the scavenging efficiency of serum urea nitrogen.
  • the device includes a graphene electrothermal membrane.
  • the graphene electrothermal film includes a graphene film, an electrode and an insulating protective layer, the electrode is arranged on the surface of the graphene film, and the insulating protective layer sandwiches the graphene film and the electrode in the middle .
  • the graphene electric heating film is a transparent graphene electric heating film.
  • the transparent graphene electric heating film uses the transparent graphene film as the heating element, which has higher safety performance than the black film with graphene powder as the heating element; in addition, using a transparent insulating protective layer, the graphene film generates heat Far infrared rays can radiate more to objects.
  • Fig. 1 is a mouse thymus index graph, in the figure, A is the graphene group, B is the isothermal control group, and C is the blank control group;
  • Figure 2 is a graph of the proliferation rate of mouse spleen lymphocytes, in the figure, A is a graphene group, B is an isothermal control group, and C is a blank control group;
  • Figure 3 is a graph showing the activity of NK cells killing YAC-1 tumor cells in mice, in the figure, A is the graphene group, B is the isothermal control group, and C is the blank control group;
  • Fig. 4 is a graph of serum urea nitrogen level in mice, in the figure, A is a graphene group, B is an isothermal control group, and C is a blank control group;
  • Fig. 5 is a Beagle dog lymphocyte proliferation rate graph, in the figure, A is before graphene irradiation, and B is after graphene irradiation;
  • Figure 6 is a graph showing the viability of beagle NK cells killing Yac-1 tumor cells, in the figure, A is a blank group, and B is a graphene irradiation group;
  • Figure 7 is a graph showing the activity of Beagle NK cells killing K562 tumor cells, in the figure, A is a blank group, and B is a graphene irradiation group;
  • Fig. 8 is a graph of human lymphocyte proliferation activity, in the figure, A is before graphene irradiation, and B is after graphene irradiation;
  • Figure 9 is a graph of human lymphocyte proliferation activity, in the figure, A is before graphene is applied to three acupoints, and B is after graphene is applied to three acupoints;
  • Figure 10 is a graph of human lymphocyte proliferation activity, in the figure, A is before graphene is applied to two acupoints, and B is after graphene is applied to two acupoints;
  • Figure 11 is a graph showing the activity of human NK cells in the graphene irradiation group to kill tumor cells
  • Figure 12 is a graph showing the viability of human NK cells to kill tumor cells in three acupoint groups
  • Figure 13 is a graph showing the viability of human NK cells to kill tumor cells in two acupoint groups
  • Figure 14 is a graph of the urea metabolism ability of volunteers, in the figure, A is before graphene irradiation/applied, B is after graphene irradiation/applied, a is before and after irradiation, b is before and after applying three acupoints, c is pasting Apply two acupuncture points;
  • graphene is a two -dimensional carbon nanomaterial composed of carbon atoms in a hexagonal honeycomb lattice with sp hybrid orbitals.
  • Reagent consumables CD4 primary antibody (PE), CD8 primary antibody (FITC), CD3 primary antibody (PerCP), BUN ELISA Kit, ConA, fetal bovine serum, RPMI 1640, DMEM, D-hanks, DMSO, MTT, K562 cells, Yac-1 cells.
  • mice were irradiated at a constant temperature of 37 °C for 1 h, and irradiated continuously for 15 days;
  • Isothermal control group use a heating box to heat the mice at a constant temperature of 37 °C for 1 h, and continue to heat for 15 days;
  • Blank control group normal feeding, no heating irradiation treatment.
  • mice index thymus weight/body weight
  • mice from each group were taken out and sacrificed by decapitation and then immersed in alcohol for disinfection.
  • the spleen was aseptically removed from the above mice, ground and passed through a 200-mesh cell screen, resuspended in RPMI 1640, centrifuged at 1500 r ⁇ min -1 for 10 min, discarded the supernatant, and added to the pellet with an appropriate amount of erythrocyte lysate, and centrifuged at 1500 r ⁇ min -1 for 10 min , RPMI 1640 was washed twice to obtain a single cell suspension, and finally the cell concentration was adjusted to 5X10 6 /mL with RPMI 1640 containing 10% fetal bovine serum.
  • the cells were seeded in a 96-well plate (100 ⁇ L/well), and 10 ⁇ L of Con A was added to each well to stimulate (final concentration of 5 ⁇ g/mL), placed in a 5% CO 2 incubator at 37 °C, and 10 ⁇ L of MTT was added to each well, and continued. After culturing for 4 h, 150 ⁇ L of DMSO was added to each well and shaken for 5 min. After the purple crystals were completely dissolved, the OD value was measured on a microplate reader at 570 nm.
  • NK cell activity [1-(experimental well-effector cell control well)/target cell control well] ⁇ 100%.
  • mice were put into 30 °C water for 60 minutes of load swimming, and immediately after resting for 60 minutes, blood was collected to separate serum, and ELISA kit was used to detect urea in serum. nitrogen content.
  • Thymus is an important immune organ, and its organ index can reflect the strength of the body's immune function to a certain extent.
  • the effect of graphene irradiation on the mouse thymus index is shown in Figure 1.
  • the thymus index of the graphene group was higher. Therefore, graphene irradiation can significantly increase the thymus index in mice.
  • the increase in the thymus index of mice indicates that the graphene environment can promote the development of the thymus gland, a central immune organ, thereby affecting the immune function of mice.
  • NK cells are involved in immunity against viruses, pathogenic microorganisms and tumors in the body. They are the first natural defense line of the body's innate immunity. They are mainly found in peripheral blood and spleen. They do not depend on antibodies and do not require antigen stimulation and sensitization. kill target cells.
  • Graphene irradiation can significantly improve mouse thymus index, lymphocyte proliferation ability, and the ability of NK cells in peripheral blood to kill tumor cells. It is shown that graphene heat treatment can enhance the immunity of BALB/c mice through multiple aspects; at the same time, graphene irradiation can significantly improve the scavenging efficiency of serum urea nitrogen during exercise in mice, thereby enhancing the continuous exercise ability of mice .
  • Blank control group 6 beagle dogs, which were raised normally without heating and irradiation.
  • peripheral blood was drawn from beagle dogs in each group with anticoagulant blood collection tubes.
  • Add the lymphocyte separation solution to the centrifuge tube then draw the diluted blood sample and add it to the liquid surface of the separation solution, centrifuge at 450g for 25 minutes, after centrifugation, suck the T lymphocytes in the centrifuge tube into another centrifuge tube, and add it to the centrifuge tube. 10ml washing solution, mix cells. Centrifuge for 10 min.
  • the cell concentration was adjusted to 5 ⁇ 10 4 /mL with RPMI 1640 containing 10% fetal bovine serum, 100 ⁇ L of cell suspension was aspirated and seeded in a 96-well plate, and 10 ⁇ L of Con A was added to each well to stimulate (the final concentration was 5 ⁇ g/mL), and the cells were placed. After incubating at 37°C in a 5% CO 2 incubator, add 10 ⁇ L of MTT to each well. After continuing to incubate for 4 h, add 150 ⁇ L of DMSO to each well and shake for 5 min. After the purple crystals are completely dissolved, measure the OD value on a microplate reader at 450 nm.
  • NK cell activity [1-(experimental well (effector cell+target cell)-effector cell control well)/target cell control well] ⁇ 100%.
  • peripheral blood was drawn from each group of beagle dogs with a coagulation blood collection tube, and after standing at room temperature for 1 hour, centrifuged at 2500 rpm for 10 minutes, the upper serum was drawn, and the BUN ELISA kit was used to detect the level of urea nitrogen in the serum. Variety.
  • Lymphocyte transformation is the activation of lymphocytes when they are stimulated by antigens or mitotically proliferated, and the cells can be transformed into blast cells.
  • the ability of lymphocyte proliferation determines the number of effector lymphocytes and the strength of the body's immune response, reflecting the body's cellular immune status.
  • NK cells are involved in immunity against viruses, pathogenic microorganisms and tumors in the body. They are the first natural defense line of the body's innate immunity. They are mainly found in peripheral blood and spleen. They do not depend on antibodies and do not require antigen stimulation and sensitization. kill target cells.
  • Example 3 The effect of graphene irradiation on indicators related to the immune response of volunteers
  • Irradiation group 10 volunteers, using a graphene warm moxibustion cover, adjusted to 39 °C for 1 hour, and irradiated continuously for 14 days;
  • Blank control group 10 volunteers, without any irradiation and application.
  • PBMCs were obtained after centrifugation at 1200g for 10 minutes, and CD3+ T cells in PBMCs were isolated with EasySep Human CD3 isolation kit.
  • the cell concentration was adjusted to 5 ⁇ 10 4 /mL with RPMI 1640 containing 10% fetal bovine serum, 100 ⁇ L of cell suspension was aspirated and seeded in a 96-well plate, and 10 ⁇ L of Con A was added to each well to stimulate (the final concentration was 5 ⁇ g/mL), and the cells were placed. After incubating at 37°C in a 5% CO 2 incubator, add 10 ⁇ L of MTT to each well. After continuing to incubate for 4 h, add 150 ⁇ L of DMSO to each well and shake for 5 min. After the purple crystals are completely dissolved, measure the OD value on a microplate reader at 450 nm.
  • NK cell activity [1-(experimental well (effector cell+target cell)-effector cell control well)/target cell control well] ⁇ 100%.
  • peripheral blood was drawn from each group of volunteers with a blood collection tube for promoting coagulation. After standing at room temperature for 1 hour, centrifuged at 2500 rpm for 10 minutes, the upper serum was aspirated, and the BUN ELISA kit was used to detect the levels of urea nitrogen and uric acid in the serum. The change.
  • Lymphocyte transformation is the activation of lymphocytes when they are stimulated by antigens or mitotically proliferated, and the cells can be transformed into blast cells.
  • the ability of lymphocyte proliferation determines the number of effector lymphocytes and the strength of the body's immune response, reflecting the body's cellular immune status.
  • NK cells are involved in immunity against viruses, pathogenic microorganisms and tumors in the body. They are the first natural defense line of the body's innate immunity. They are mainly found in peripheral blood and spleen. They do not depend on antibodies and do not require antigen stimulation and sensitization. kill target cells.
  • Figure 12 shows the effect of graphene sticking on three acupoints of Tanzhong, Zusanli and Quchi on the activity of volunteer NK cells to kill tumor cells.
  • Figure 13 shows the effect of graphene application on Dazhui and Baihui acupoints on the activity of volunteer NK cells to kill tumor cells.
  • Figure 14 shows the metabolism of urea nitrogen in volunteers by graphene irradiation and application. After 14 days of irradiation, the volunteers' urea nitrogen metabolism decreased by an average of 15.25%; after applying three acupoints of Tanzhong, Zusanli and Quchi for 14 days, the volunteers' urea nitrogen metabolism decreased by an average of 19.57%; After 14 days at the two acupoints, the volunteers' urea nitrogen metabolism decreased by an average of 13.3%.

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Abstract

提供了一种石墨烯的免疫增强新用途，可将石墨烯用于制造免疫增强的装置中，通过石墨烯照射或者贴敷的方法，可以增强动物或人的胸腺指数、淋巴细胞增殖能力、NK细胞杀伤肿瘤细胞的活力、血清尿素氮的清除能力。

Description

石墨烯的免疫增强新用途
本申请要求享有在先申请的申请日为2020年7月29日，申请号为202010741791.9，名称为“石墨烯的免疫增强新用途”的中国发明专利申请的优先权。
技术领域
本公开涉及石墨烯发热产品在疾病治疗方面的应用，尤其涉及在石墨烯的免疫增强新用途。
背景技术
免疫力是指机体抵抗外来侵袭，维护体内环境稳定性的能力，体现了机体区分自己和非己，并消灭异己的功能。免疫力不单纯指人体抗致病微生物的抵抗力，还包括对非己的抗原性异物的抵抗力，例如非致病性的花粉、药物甚至食物，正常机体内经常出现的衰残细胞以及偶尔突变出现的癌细胞。
免疫力是人体重要的生理功能，身体免疫力好的人病痛也会少，身体素质更强。当今医学研究表明，威胁人类健康的许多疾病都与人体的免疫力有着密切的关系。癌症、慢性支气管炎、哮喘、过敏性鼻炎、慢性肝炎、糖尿病等慢性疾病，都与免疫力息息相关。
免疫力按其获得方式的不同，可分为先天性免疫力与获得性免疫。先天性免疫，是人一生下来就有的，为非特异性免疫，能对各种入侵的病原微生物作出快速反应。获得性免疫，是人生下来以后在生活过程中自然获得的，或者用人工辅助的方法被动得到，多为特异性免疫。一般是在微生物等抗原物质刺激后才形成的免疫球蛋白或免疫淋巴细胞，能与该抗原起特异性反应，因而作用力强大。
随着社会竞争激烈、人们生活节奏的加快，压力导致人们经常产生免疫力低下等亚健康状态。目前提高人体免疫力采用的方法主要有药物法和食疗法。
发明内容
本公开要解决的技术问题在于，提供一种提高免疫力的新途径。
本发明人为了寻找一种容易实现，对大部分人都有效的免疫力调节的方法，将石墨烯薄膜制成的电热产品分别用于BALB/c小鼠、比格犬以及志愿者身上，进行了免疫反应的实验及观测，发现石墨烯薄膜发热照射或者贴敷动物或人体能够增强免疫力。
本公开提供了石墨烯的免疫增强新用途。具体来说，本公开提出了如下技术方案：
本公开的第一方面提供了石墨烯在制造用于免疫增强的装置中的用途。
另一方面，本公开提供了石墨烯的免疫增强用途。
上述的用途中，所述免疫增强是提高胸腺指数。
上述的用途中，所述免疫增强是提高淋巴细胞增殖能力。
上述的用途中，所述免疫增强是提高NK细胞杀伤肿瘤细胞活力。
上述的用途中，所述免疫增强包括治疗肿瘤。
上述的用途中，所述肿瘤是血液肿瘤。
上述的用途中，所述血液肿瘤是白血病或淋巴瘤；优选地，所述白血病是髓性白血病，更优选地，所述白血病是慢性髓性白血病；优选地，所述淋巴瘤是T细胞淋巴瘤。
本公开的第二方面提供了石墨烯在制造用于提高血清尿素氮的清除效率的装置中的用途。
另一方面，本公开提供了石墨烯的提高血清尿素氮的清除效率的用途。
上述任一用途中，所述装置包括石墨烯电热膜片。
上述用途中，所述石墨烯电热膜片包括石墨烯薄膜、电极和绝缘保护层，所述电极设置在石墨烯薄膜表面，所述绝缘保护层将所述石墨烯薄膜和所述电极夹在中间。
上述用途中，所述石墨烯电热膜片为透明石墨烯电热膜。透明石墨烯电热膜以透明石墨烯薄膜作为发热体，相比以石墨烯粉末作为发热体的黑膜而言，其安全性能更高；另外，采用透明的绝缘保护层，石墨烯薄膜发热产生的远红外线能够更多地辐射到物体。
本公开的有益效果包括：
通过石墨烯照射或者贴敷的方法，可以增强胸腺指数、淋巴细胞增殖能力、NK细胞杀伤肿瘤细胞的活力、血清尿素氮的清除能力。
附图说明
图1为小鼠胸腺指数图，图中，A为石墨烯组，B为等温对照组，C为空白对照组；
图2是小鼠脾脏淋巴细胞增殖率图，图中，A为石墨烯组，B为等温对照组，C为空白对照组；
图3是小鼠NK细胞杀伤YAC-1肿瘤细胞活力图，图中，A为石墨烯组，B为等温对照组，C为空白对照组；
图4是小鼠血清尿素氮水平图，图中，A为石墨烯组，B为等温对照组，C为空白对照组；
图5是比格犬淋巴细胞增殖率图，图中，A为石墨烯照射前，B为石墨烯照射后；
图6是比格犬NK细胞杀伤Yac-1肿瘤细胞的活力图，图中，A为空白组，B为石墨烯照射组；
图7是比格犬NK细胞杀伤K562肿瘤细胞的活力图，图中，A为空白组，B为石墨烯照射组；
图8是人体淋巴细胞增殖活性图，图中，A为石墨烯照射前，B为石墨烯照射后；
图9是人体淋巴细胞增殖活性图，图中，A为石墨烯贴敷三个穴位前，B为石墨烯贴敷三个穴位后；
图10是人体淋巴细胞增殖活性图，图中，A为石墨烯贴敷两个穴位前，B为石墨烯贴敷两个穴位后；
图11是石墨烯照射组人体NK细胞杀伤肿瘤细胞的活力图；
图12是贴敷三个穴位组人体NK细胞杀伤肿瘤细胞的活力图；
图13是贴敷两个穴位组人体NK细胞杀伤肿瘤细胞的活力图；
图14是志愿者尿素代谢能力图，图中，A为石墨烯照射/贴敷前，B为石墨烯照射/贴敷后，a为照射前后，b为贴敷三个穴位前后，c为贴敷两个穴位；
图中：“**”表示P<0.01，“*”表示P<0.05。
具体实施方式
下面将对本公开的技术方案进行清楚、完整地描述。显然，基于本公开中的具体实施方式，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施方式，都属于本公开保护的范围。
需要注意的是，本文所使用的术语仅是为了描述具体实施方式，而非意图限制根据本公开的示例性实施方式。
术语“石墨烯”是一种由碳原子以sp 2杂化轨道组成六角型呈蜂巢晶格的二维碳纳米材料。
下面通过具体实施例来说明本公开的方法。本文未记载的试剂或仪器或操作步骤均是本领域普通技术人员可常规确定的内容：
实施例1 石墨烯照射对小鼠免疫反应相关指标的影响
1试剂和仪器
试剂耗材：CD4一抗(PE)、CD8一抗(FITC)、CD3一抗(PerCP)、BUN ELISA Kit、ConA、胎牛血清、RPMI 1640、DMEM、D-hanks、DMSO、MTT、K562细胞、Yac-1细胞。
仪器：T20全自动细胞计数仪(BioRad)、多功能酶标仪(BIOTEK)、流式细胞仪(Thermo)、超纯水仪(Millipore)、CO 2恒温培养箱(SANYO)、倒置显微镜(OLYMPUS)、水浴锅(山东新华医疗器械有限公司)
2试验方法
(1)BALB/c小鼠分组进行实验处理
实验组：使用石墨烯温灸罩(一种四周贴设石墨烯电热膜的箱子)，对小鼠进行37℃恒温照射1h，连续照射15天；
等温对照组：使用加热箱对小鼠进行37℃恒温加热1h，连续加热15天；
空白对照组：正常饲养，不做加热照射处理。
(2)检测处理
(a)小鼠胸腺指数的测定
实验15d后，每组取出10只BALB/c小鼠，称量体重，脱颈处死后浸泡酒精消毒。将上 述小鼠无菌采集胸腺，计算胸腺指数(胸腺指数＝胸腺重量/体重)。
(b)小鼠脾脏淋巴细胞增殖率的检测
实验15d后，每组取出10只BALB/c小鼠，脱颈处死后浸泡酒精消毒。将上述小鼠无菌摘取脾脏，研磨后过200目细胞筛网，RPMI 1640重悬，1500r·min -1离心10min，弃上清，沉淀加入适量红细胞裂解液，1500r·min -1离心10min，RPMI 1640洗涤2次，制得单细胞悬液，最后用含10％胎牛血清的RPMI 1640调细胞浓度5X10 6/mL。将细胞接种于96孔板(100μL/孔)，并于每孔加入10μLCon A刺激(终浓度为5μg/mL)，置于5％CO 2培养箱37℃培养后，每孔加MTT 10μL，继续培养4h后，每孔加入DMSO 150μL，振荡5min，紫色结晶完全溶解后于酶标仪570nm测定OD值。
(c)NK细胞杀伤肿瘤细胞能力的变化
实验15d后，密度梯度离心法分离脾脏的单个核细胞，随后用NK细胞分离液分离脾脏中的NK细胞。将分离的NK细胞与肿瘤细胞YAC-1按照10:1的比例混合，作用10h后检测各个孔中靶细胞的存活情况，然后计算NK细胞的杀伤率。计算公式：NK细胞活性＝[1-(实验孔-效应细胞对照孔)/靶细胞对照孔]×100％。
(d)血清尿素氮水平的检测
为检测石墨烯照射对小鼠血清中尿素氮水平的影响，在末次照射30min后，将小鼠放入30℃水中负荷游泳60min，休息60min后立即采血分离血清，使用ELISA试剂盒检测血清中尿素氮的含量。
(3)数据分析
(a)石墨烯照射对小鼠胸腺指数的影响
胸腺是重要的免疫器官，其脏器指数可在一定程度上反映机体免疫功能的强弱。石墨烯照射对小鼠胸腺指数的影响如图1所示。由图可见，相对于空白对照组和等温对照组，石墨烯组胸腺指数更高。因此石墨烯照射可以显著提高小鼠胸腺指数。小鼠胸腺指数提高，说明石墨烯环境能促进中枢免疫器官胸腺的发育，从而影响小鼠的免疫功能。
(b)石墨烯照射对小鼠脾脏淋巴细胞增殖的影响
石墨烯照射对小鼠脾脏淋巴细胞增殖的影响如图2所示。可以看出，照射96h时，石墨烯照射组可以显著提高淋巴细胞增殖能力，从而提高其免疫力。
(c)石墨烯照射对NK细胞杀伤肿瘤细胞活力的影响
NK细胞在机体中参与抗病毒、病原微生物和肿瘤的免疫，是机体先天性免疫的第一道天然防线，主要存在于外周血和脾脏中，不依赖抗体也不需要抗原刺激和致敏就能杀伤靶细胞。
石墨烯照射对NK细胞杀伤肿瘤细胞活力的影响如图3所示。可以看出，石墨烯照射组可以显著提高NK细胞杀伤肿瘤细胞活力。
(d)石墨烯照射对血清尿素氮水平的影响
尿素氮清除效率与运动产生的肌肉疲劳效应呈负相关。
石墨烯照射对血清尿素氮水平的影响如图4所示。可以看出，石墨烯照射组可以显著降低血清尿素氮水平，从而可显著降低因运动疲劳造成的尿素氮在体内的积蓄。
结论：
石墨烯照射可以显著提高小鼠胸腺指数、淋巴细胞增殖能力、外周血中NK细胞杀伤肿瘤细胞的能力。表明，石墨烯发热处理可以通过多个方面来增强BALB/c小鼠的免疫力；同时，石墨烯照射可以显著提高小鼠运动过程中血清尿素氮的清除效率，进而增强小鼠的持续运动能力。
实施例2 石墨烯照射对比格犬免疫功能相关指标的影响
1试剂和仪器
同实施例1
2试验方法
(1)比格犬分组进行实验处理
实验组：6只比格犬，使用石墨烯温灸罩，对比格犬进行37℃恒温加热1h，连续照射15天；
空白对照组：6只比格犬，正常饲养，不做加热照射处理。
(2)检测处理
(a)比格犬外周血T淋巴细胞的增殖活性检测
照射结束后，用抗凝采血管抽取各组比格犬外周血5毫升。将淋巴细胞分离液加入离心管中，再吸取稀释后的血液样本加于分离液液面上，450g离心25min，离心后，将离心管中的T淋巴细胞吸取至另一离心管中，并加入10ml清洗液，混匀细胞。离心10min。用含10％胎牛血清的RPMI 1640将细胞浓度调至5×10 4/mL，吸出100μL细胞悬液接种于96孔板，每孔加入10μL Con A刺激(终浓度为5μg/mL)，置于5％CO 2培养箱37℃培养后，每孔加MTT10μL，继续培养4h后，每孔加入DMSO 150μL，振荡5min，紫色结晶完全溶解后于酶标仪450nm测定OD值。
(b)NK细胞杀伤肿瘤细胞能力的变化
照射结束后，用抗凝采血管抽取各组比格犬外周血5毫升。取一只离心管，加入NK细胞分离液，制成梯度界面，将抗凝血加于分离液面上，450g，离心25min。离心后，将NK细胞吸取至另一离心管中，加入10ml清洗液，混匀细胞。250g，离心10min。将分离的NK细胞与K562细胞或YAC-1按照25:1的比例混合，作用5h后检测各个孔中靶细胞的存活情况，然后计算NK细胞的杀伤率。计算公式：NK细胞活性＝[1-(实验孔(效应细胞+靶细胞)-效应细胞对照孔)/靶细胞对照孔]×100％。
(c)血清尿素氮水平的检测
照射结束后，用促凝采血管抽取各组比格犬外周血2毫升，室温下静置1小时后，2500转离心10分钟，吸取上层血清，用BUN ELISA试剂盒检测血清中尿素氮水平的变化。
(3)数据分析
(a)石墨烯照射对比格犬外周血T淋巴细胞增殖活力的影响
淋巴细胞转化是淋巴细胞受到抗原的刺激或有丝分裂增殖时的细胞活化，该细胞可转化为母细胞。淋巴细胞增殖能力决定了效应淋巴细胞的数量和机体免疫应答反应的强度，反映了机体的细胞免疫状态。
石墨烯照射对比格犬外周血淋巴细胞增殖的影响如图5所示。由图可见，石墨烯照射后，比格犬外周血T淋巴细胞的增殖能力明显高于照射之前。石墨烯照射组可以显著提高淋巴细胞增殖能力，从而提高其细胞免疫功能。
(b)石墨烯照射对比格犬外周血NK细胞杀伤肿瘤细胞能力的影响
NK细胞在机体中参与抗病毒、病原微生物和肿瘤的免疫，是机体先天性免疫的第一道天然防线，主要存在于外周血和脾脏中，不依赖抗体也不需要抗原刺激和致敏就能杀伤靶细胞。
石墨烯照射对NK细胞杀伤肿瘤细胞活力的影响如图6和图7所示。可以看出，石墨烯照射组可以显著提高NK细胞杀伤肿瘤细胞K562和Yac-1的杀伤能力。
结论：石墨烯照射可以显著提高比格犬淋巴细胞增殖活力、外周血NK细胞的杀伤肿瘤细胞的活性。表明，石墨烯可多途径改善比格犬的免疫功能。
实施例3 石墨烯照射对志愿者免疫反应相关指标的影响
1试剂和仪器
同实施例1。
2试验方法
(1)分组进行实验处理
照射组：10位志愿者，使用石墨烯温灸罩，调节至39℃照射1小时，连续照射14天；
贴敷三个穴位组：10位志愿者，使用石墨烯贴敷(具体结构可参考专利CN208241915U中提供的理疗贴)，39℃贴敷足三里穴、曲池穴、膻中穴，每次贴敷1小时，连续贴敷14天；
贴敷两个穴位组：10位志愿者，使用石墨烯贴敷，39℃贴敷百会穴和大椎穴，贴敷1小时，连续贴敷14天；
空白对照组：10位志愿者，不做任何的照射和贴敷。
(2)检测处理
(a)志愿者外周血T细胞的增殖活性检测
照射或贴敷试验结束后，用抗凝采血管抽取各组志愿者外周血5毫升。将Lymphoprep分离液加到SepMate-15离心管中，将血液稀释后加到等量的分离液上。1200g离心10分钟后 得到PBMC，用EasySep Human CD3分离试剂盒分离PBMC中的CD3+T细胞。用含10％胎牛血清的RPMI 1640将细胞浓度调至5×10 4/mL，吸出100μL细胞悬液接种于96孔板，每孔加入10μL Con A刺激(终浓度为5μg/mL)，置于5％CO 2培养箱37℃培养后，每孔加MTT 10μL，继续培养4h后，每孔加入DMSO 150μL，振荡5min，紫色结晶完全溶解后于酶标仪450nm测定OD值。
(b)NK细胞杀伤肿瘤细胞能力的变化
照射或贴敷试验结束后，用抗凝采血管抽取各组志愿者外周血5毫升。将Lymphoprep分离液加到SepMate-15离心管中，将血液稀释后加到等量的分离液上。1200g离心10分钟后得到PBMC，用EasySep Human CD56分离试剂盒分离PBMC中的NK细胞。将分离的NK细胞与K562肿瘤细胞细胞或YAC-1肿瘤细胞按照10:1的比例混合，作用5h或10h后检测各个孔中靶细胞的存活情况，然后计算NK细胞的杀伤率。计算公式：NK细胞活性＝[1-(实验孔(效应细胞+靶细胞)-效应细胞对照孔)/靶细胞对照孔]×100％。
(c)血清尿素氮水平的检测
照射结束后，用促凝采血管抽取各组志愿者周血2毫升，室温下静置1小时后，2500转离心10分钟，吸取上层血清，用BUN ELISA试剂盒检测血清中尿素氮和尿酸水平的变化。
(3)数据分析
(a)石墨烯照射、贴敷对志愿者外周血淋巴细胞增殖活力的影响
淋巴细胞转化是淋巴细胞受到抗原的刺激或有丝分裂增殖时的细胞活化，该细胞可转化为母细胞。淋巴细胞增殖能力决定了效应淋巴细胞的数量和机体免疫应答反应的强度，反映了机体的细胞免疫状态。
石墨烯照射对志愿者外周血淋巴细胞增殖的影响如图8所示。由图可见，石墨烯照射后，志愿者外周血T淋巴细胞的增殖能力明显高于照射之前。石墨烯贴敷三个穴位对志愿者外周血淋巴细胞增殖的影响如图9所示。由图可见，石墨烯贴敷膻中、足三里、曲池三个穴位后，志愿者外周血T淋巴细胞的增殖能力显著高于照射之前。石墨烯贴敷两个穴位对志愿者外周血淋巴细胞增殖的影响如图10所示。由图可见，石墨烯贴敷百会和大椎两个穴位后，志愿者外周血T淋巴细胞的增殖能力显著高于照射之前。
可以看出，石墨烯照射和穴位贴敷均可以提高志愿者外周血T淋巴细胞的增殖活性，从而提高其细胞免疫的功能。
(b)石墨烯照射、贴敷对志愿者NK细胞杀伤肿瘤细胞能力的影响
NK细胞在机体中参与抗病毒、病原微生物和肿瘤的免疫，是机体先天性免疫的第一道天然防线，主要存在于外周血和脾脏中，不依赖抗体也不需要抗原刺激和致敏就能杀伤靶细胞。
石墨烯照射对外周血NK细胞杀伤肿瘤细胞活力的影响如图11所示。经过14天照射后，志愿者外周血中NK细胞对K562肿瘤细胞杀伤能力平均提高8.15％(5h)和13.45％(10h)； 对Yac-1肿瘤细胞杀伤能力平均提高16.39％(5h)和23.41％(10h)。
石墨烯贴敷膻中、足三里、曲池三个穴位对志愿者NK细胞杀伤肿瘤细胞活力的影响如图12所示。贴敷膻中、足三里、曲池三个穴位14天后，志愿者外周血中NK细胞对K562肿瘤细胞杀伤能力平均提高4.41％(5h)和5.8％(10h)；对Yac-1肿瘤细胞杀伤能力平均提高7.9％(5h)和9.81％(10h)。
石墨烯贴敷大椎、百会两个穴位对志愿者NK细胞杀伤肿瘤细胞活力的影响如图13所示。贴敷大椎、百会两个穴位14天后，志愿者外周血中NK细胞对K562肿瘤细胞杀伤能力平均提高4.7％(5h)和4.92％(10h)；对Yac-1肿瘤细胞杀伤能力平均提高3.4％(5h)和5.7％(10h)。
可以看出，使用石墨烯温照射14天或者石墨烯贴敷均可提高志愿者外周血NK细胞对K562肿瘤细胞和Yac-1肿瘤细胞的杀伤活力。
(c)石墨烯照射、贴敷对志愿者尿素氮代谢能力的影响
石墨烯照射、贴敷对志愿者尿素氮代谢能力如图14所示。经过14天照射后，志愿者尿素氮代谢能力平均降低15.25％；贴敷膻中、足三里、曲池三个穴位14天后，志愿者尿素氮代谢能力平均降低19.57％；贴敷大椎、百会两个穴位14天后，志愿者尿素氮代谢能力平均降低13.3％。
综上表明，石墨烯照射和穴位贴敷可以提高志愿者对尿素氮的代谢能力。
结论：石墨烯照射和穴位贴敷均可增强志愿者外周血T细胞增殖能力、NK细胞杀伤肿瘤细胞的活性，血清尿素氮的清除能力。表明，石墨烯照射和穴位贴敷可通过多种途径改善人体免疫功能。
以上所述，仅为本公开的具体实施方式，但本公开的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本公开揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本公开的保护范围之内。因此，本公开的保护范围应以所述权利要求的保护范围为准。

Claims (13)

  1. 石墨烯在制造用于免疫增强的装置中的用途。
  2. 石墨烯的免疫增强用途。
  3. 根据权利要求1或2所述的用途，其中，所述免疫增强是提高胸腺指数。
  4. 根据权利要求1或2所述的用途，其中，所述免疫增强是提高淋巴细胞增殖能力。
  5. 根据权利要求1或2所述的用途，其中，所述免疫增强是提高NK细胞杀伤肿瘤细胞活力。
  6. 根据权利要求1或2所述的用途，其中，所述免疫增强包括治疗肿瘤。
  7. 根据权利要求6所述的用途，其中，所述肿瘤是血液肿瘤。
  8. 根据权利要求7所述的用途，其中，所述血液肿瘤是白血病或淋巴瘤；优选地，所述白血病是髓性白血病，更优选地，所述白血病是慢性髓性白血病；优选地，所述淋巴瘤是T细胞淋巴瘤。
  9. 石墨烯在制造用于提高血清尿素氮的清除效率的装置中的用途。
  10. 石墨烯的提高血清尿素氮的清除效率的用途。
  11. 根据权利要求1-10任一项所述的用途，其中，所述装置包括石墨烯电热膜片。
  12. 根据权利要求11所述的用途，其中，所述石墨烯电热膜片包括石墨烯薄膜、电极和绝缘保护层，所述电极设置在石墨烯薄膜表面，所述绝缘保护层将所述石墨烯薄膜和所述电极夹在中间。
  13. 根据权利要求12所述的用途，其中，所述石墨烯电热膜片为透明石墨烯电热膜。
PCT/CN2021/074189 2020-07-29 2021-01-28 石墨烯的免疫增强新用途 WO2022021827A1 (zh)

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