WO2023236577A1 - 血小板外泌体富集管 - Google Patents

血小板外泌体富集管 Download PDF

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Publication number
WO2023236577A1
WO2023236577A1 PCT/CN2023/075749 CN2023075749W WO2023236577A1 WO 2023236577 A1 WO2023236577 A1 WO 2023236577A1 CN 2023075749 W CN2023075749 W CN 2023075749W WO 2023236577 A1 WO2023236577 A1 WO 2023236577A1
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platelet
tube
exosome
exosome enrichment
cover
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PCT/CN2023/075749
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English (en)
French (fr)
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郑本冈
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郑本冈
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Publication of WO2023236577A1 publication Critical patent/WO2023236577A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/24—Apparatus for enzymology or microbiology tube or bottle type

Definitions

  • the present invention relates to a blood sampling device, in particular to a platelet exosome enrichment tube.
  • Human blood mainly consists of plasma and blood cells.
  • the main components of blood cells are red blood cells, white blood cells and platelets.
  • Whole blood can be prepared into a variety of ingredients, stored under ideal conditions, and can be effectively used in a variety of treatments.
  • platelets have been found in clinical treatments to stop coagulation in vitro and quickly activate endogenous growth factors, and have good effects on promoting cell growth and repair in the human body.
  • PRP uses exosomes on the surface of platelets to secrete growth factors and anti-inflammatory factors to treat physical discomfort. It is mainly used for osteoarthritis, muscle and ligament injuries, anti-inflammation and tissue repair.
  • PRP can also be used in medical cosmetology and dentistry. For example, its application in medical cosmetology can reduce wrinkles, and its application in dentistry can fill the dental pulp, promote the close contact between the dental implant metal and the pulp cavity, increase growth, and thus increase the success of dental implants. Rate.
  • An object of the present invention is to provide a platelet exosome enrichment tube that utilizes physical
  • the principle is to break the cell membrane of platelets, so that the exosomes of the other three isomers in the cytoplasm of platelets can also be activated and released, thereby obtaining a platelet exosome-enriched solution containing a variety of anti-inflammatory factors and growth factors.
  • a platelet exosome enrichment tube which includes a tube body, a cover body and a plurality of ground glass beads.
  • the pipe body has a closed end, an open end and an accommodating space. The open end is relative to the closed end, and the accommodating space is formed by connecting the closed end to the open end.
  • the cover detachably covers the open end of the pipe body to open and close the accommodation space.
  • the ground glass beads are arranged in the accommodation space, and the surface of each ground glass bead has evenly distributed concave and convex shapes.
  • the closed end of the tube body may be arc-shaped.
  • the tube body may further include a standing portion, which is connected to the closed end, and the end of the standing portion away from the closed end is a flat bottom edge.
  • the material of the tube body can be glass or plastic.
  • the cover may be a spiral tube cap, a plastic cap, a safety cap or a rubber cap.
  • the open end of the tube body is provided with an external thread
  • the inner ring of the cover body is provided with an internal thread.
  • the external thread and internal thread of the tube body and the cover body are tightly coupled.
  • the cover may include an injection port for injecting and/or extracting blood samples in conjunction with the blood collection needle.
  • the cover body may further include a connecting portion protruding from the upper surface of the cover body for connecting to the negative pressure aspirator.
  • the platelet exosome enrichment tube according to the aforementioned embodiment may further include a rubber plug, which is disposed between the tube body and the cover body to seal the accommodation space.
  • the surface roughness (RMS) of each ground glass bead may be 0.01 ⁇ m to 0.5 ⁇ m.
  • the average particle size of each ground glass bead may be 2 mm to 10 mm.
  • Figure 1 is an exploded view of a platelet exosome enrichment tube according to one embodiment of the present invention
  • Figure 2 is an exploded view of a platelet exosome enrichment tube according to another embodiment of the present invention.
  • Figure 3 is a perspective view of a platelet exosome enrichment tube according to another embodiment of the present invention.
  • Figure 4 shows a combined cross-sectional view of the platelet exosome enrichment tube according to Figure 3;
  • Figure 5 is a perspective view of a platelet exosome enrichment tube according to yet another embodiment of the present invention.
  • FIG. 6 shows a combined cross-sectional view of the platelet exosome enrichment tube according to FIG. 5 .
  • FIG. 1 illustrates an exploded view of a platelet exosome enrichment tube 100 according to an embodiment of the present invention.
  • the platelet exosome enrichment tube 100 includes a tube body 110 , a cover 120 and a plurality of ground glass beads 130 .
  • the tube body 110 has a closed end 112, an open end 111 and an accommodating space 113.
  • the open end 111 is relative to the closed end 112, and the accommodating space 113 is formed by the closed end 112 communicating with the open end 111.
  • the closed end 112 of the tube body 110 can be arc-shaped, so that the blood sample can be concentrated at the bottom of the tube when the platelet exosome enrichment tube 100 is in use.
  • the material of the tube body 110 can be glass or plastic, preferably, it can be transparent or translucent plastic or glass, so that the blood stratification can be clearly seen when the platelet exosome enrichment tube 100 is used.
  • the outer wall surface of the tube body 110 may have a plurality of scale marks, so that the internal volume can be directly identified when in use.
  • the inner diameter of the tube body 110 may be 10 mm to 20 mm, the length of the tube body 110 may be 100 mm to 150 mm, and the tube body 110 may accommodate a blood sample of 10 mL to 15 mL, but is not limited thereto.
  • the cover body 120 detachably covers the open end 111 of the tube body 110 to open and close the accommodation space 113 .
  • the cover 120 can be a spiral pipe cap, a plastic cap, a safety cap or a rubber cap.
  • the cover 120 is made of inelastic material such as a spiral pipe cap, a plastic cap or a safety cap, the pipe body 110
  • the open end 111 is provided with an external thread
  • the inner ring of the cover 120 is provided with an internal thread, whereby the external thread and internal thread of the tube 110 and the cover 120 are tightly coupled.
  • the cover 120 is made of an elastic material such as a rubber cap
  • the cover 120 can be directly sleeved on the open end 111 of the tube 110 .
  • the cover 120 may include an injection port 121 for injecting and/or extracting blood samples with a blood collection needle.
  • the blood collection needle may be of a hard-connected type or a soft-connected type.
  • the ground glass beads 130 are disposed in the accommodating space 113 , wherein the surface 131 of each ground glass bead 130 has a uniformly distributed concave and convex shape.
  • the uniformly distributed uneven shape of the surface 131 can be formed by chemical treatment or physical treatment.
  • the chemical treatment method is, for example, a frosting treatment method, in which the glass beads are immersed in a mixed solution of hydrogen fluoride and ammonium fluoride, and the immersed surface is chemically surface treated to obtain the ground glass beads 130 .
  • the physical treatment method includes sand blasting using pressurized air to blow crystalline silica powder and silicon carbide powder onto the surface of the glass beads to obtain frosted glass beads 130;
  • the glass beads are rubbed with a brush of silicon oxide powder, silicon carbide powder, etc. to obtain ground glass beads 130 .
  • the surface roughness (RMS) of each ground glass bead 130 may be 0.01 ⁇ m to 0.5 ⁇ m, and the average particle diameter of each ground glass bead 130 may be 2 mm to 10 mm. More preferably, 20 to 50 ground glass beads 130 can be disposed in the accommodating space 113 of the tube body 110, but it is not limited to this. Please refer to the following Tables 1 to 3.
  • Table 1 shows the properties related to the size of the ground glass beads 130
  • Table 2 shows the chemical properties of the ground glass beads 130
  • Table 3 shows the physical properties of the ground glass beads 130 .
  • the platelet exosome enrichment tube 100 can use the ground glass beads with uniformly distributed concave and convex shapes on the surface to prepare the platelet exosome enrichment solution, using physical principles to break the cell membrane of platelets, so that additional particles can be added to the cytoplasm of platelets.
  • the exosomes of the three isomers can also be activated and released, thereby obtaining a platelet exosome-enriched solution containing multiple anti-inflammatory factors and growth factors, and the obtained platelet exosome-enriched solution contains various types of growth factors.
  • the concentration of factors is several times higher than that of conventional platelet-rich plasma, making the subsequent clinical treatment more effective.
  • FIG. 2 illustrates an exploded view of a platelet exosome enrichment tube 200 according to another embodiment of the present invention.
  • the platelet exosome enrichment tube 200 includes a tube body 210, a cover 220, a plurality of ground glass beads 230 and a rubber stopper 240.
  • the tube body 210 has a closed end 212, an open end 211, and an accommodating space 213.
  • the open end 211 is relative to the closed end 212, and the accommodating space 213 is formed by the closed end 212 communicating with the open end 211.
  • Other technical details of the tube body 210 are the same as those of the tube body 110 and will not be described again.
  • the cover body 220 detachably covers the open end 211 of the tube body 210 to open and close the accommodation space 213.
  • the cover 220 may include an injection port 221 for injecting and/or extracting blood samples in conjunction with the blood collection needle.
  • Other technical details of the cover 220 are the same as those of the cover 120 and will not be described again.
  • ground glass beads 230 are disposed in the accommodating space 213, and the surface (not shown) of each ground glass bead 230 has a uniformly distributed uneven shape. Other technical details of the ground glass beads 230 are the same as those of the ground glass beads 130 and will not be described again here.
  • the platelet exosome enrichment tube 200 further includes a rubber plug 240, which is disposed between the tube body 210 and the cover 220 to seal the accommodation space.
  • the outer diameter of the rubber stopper 240 can be the same as the outer diameter of the tube body 210.
  • the rubber stopper 240 can be provided with a convex portion 241 at one end close to the tube body 210.
  • the outer diameter of the convex portion 241 is the same as the inner diameter of the tube body 210.
  • 240 can be inserted into the tube body 210 through the convex portion 241
  • the open end 211 is used to install the rubber plug 240 on the open end 211 of the tube body 210, and then the cover 220 is wrapped around the rubber plug 240.
  • the rubber stopper 240 can be made of butyl rubber, and a matching blood collection needle can pierce the rubber stopper 240 through the injection port 221 of the cover 220 and enter the accommodation space 213 of the tube body 210 .
  • FIG. 3 is a perspective view of a platelet exosome enrichment tube 300 according to another embodiment of the present invention.
  • FIG. 4 is a combined cross-sectional view of the platelet exosome enrichment tube 300 according to FIG. 3 .
  • the platelet exosome enrichment tube 300 includes a tube body 310, a cover 320, a plurality of ground glass beads 330 and a rubber stopper 340.
  • the tube body 310 has a closed end 312, an open end 311, an accommodating space 313 and a standing part 314.
  • the open end 311 is relative to the closed end 312, and the accommodating space 313 is formed by the closed end 312 communicating with the open end 311.
  • the closed end 312 of the tube body 310 may be arc-shaped, which allows the platelet exosome enrichment tube 300 to concentrate blood samples at the bottom of the tube when in use.
  • the tube body 310 of the platelet exosome enrichment tube 300 further includes a standing portion 314, which is connected to the closed end 312, and the end of the standing portion 314 away from the closed end 312 is a flat bottom.
  • the platelet exosome enrichment tube 300 can be made to stand upright on the desktop, and there is no need to use an additional test tube rack to place or store the platelet exosome enrichment tube. 300, increasing the convenience of use.
  • Other technical details of the tube body 310 are the same as those of the tube body 110 and will not be described again.
  • the cover body 320 detachably covers the open end 311 of the tube body 310 to open and close the accommodation space 313.
  • the cover 320 may include an injection port 321 for injecting and/or extracting blood samples using a blood collection needle.
  • Other technical details of the cover 320 are the same as those of the cover 120 and will not be described again.
  • ground glass beads 330 are disposed in the accommodating space 313 , and the surface (not shown) of each ground glass bead 330 has evenly distributed concave and convex shapes. Other technical details of the ground glass beads 330 are the same as those of the ground glass beads 130 and will not be described again here.
  • the rubber stopper 340 is disposed between the tube body 310 and the cover body 320 to seal the accommodation space. Other technical details of the rubber stopper 340 are the same as those of the rubber stopper 240 and will not be described again here.
  • FIG. 5 shows a perspective view of a platelet exosome enrichment tube 400 according to another embodiment of the present invention.
  • FIG. 6 shows an assembled cross-sectional view of the platelet exosome enrichment tube 400 according to FIG. 5 .
  • the platelet exosome enrichment tube 400 includes a tube body 410 , a cover 420 and a plurality of ground glass beads 430 .
  • the tube body 410 has a closed end 412, an open end 411, an accommodating space 413 and a standing part 414.
  • the open end 411 is relative to the closed end 412, and the accommodating space 413 is connected to the open end 411 by the closed end 412. And formed.
  • the standing part 414 is connected to the closed end 412, and the end of the standing part 414 away from the closed end 412 has a flat bottom edge, so that the platelet exosome enrichment tube 400 can stand upright on a tabletop.
  • Other technical details of the tube body 410 are the same as those of the tube body 110 and will not be described again.
  • the cover 420 detachably covers the open end 411 of the tube 410 to open and close the accommodation space 413 .
  • the cover 420 may include an injection port 421 for injecting and/or extracting a blood sample with a blood collection needle.
  • the cover 420 of the platelet exosome enrichment tube 400 further includes a connecting portion 422 protruding from the upper surface of the cover 420 for connecting with a negative pressure aspirator to increase the capacity of the platelet exosome enrichment tube 400. Scope of application. Other technical details of the cover 420 are the same as those of the cover 120 and will not be described again.
  • ground glass beads 430 are disposed in the accommodating space 413, and the surface (not shown) of each ground glass bead 430 has a uniformly distributed concave and convex shape. Other technical details of the ground glass beads 430 are the same as those of the ground glass beads 130 and will not be described again here.
  • Platelets can secrete a variety of growth factors and anti-inflammatory factors, and have functions such as promoting cell movement, differentiation, division, matrix proliferation, angiogenesis, and balance. They include important growth factors such as platelet-derived growth factor (PDGF). ), transforming growth factor ⁇ (Transforming growth factor ⁇ , TGF ⁇ ), transforming growth factor ⁇ (Transforming growth factor ⁇ , TGF ⁇ ), insulin-like growth factor (Insulin-like growth factor 1, IGF-1), vascular endothelial growth factor (Vascular endothelial growth factor, VEGF) and interleukin 1receptor antagonist IL1-Ra, PDGF can attract stem cells, promote stem cell division, extracellular matrix secretion, stimulate osteogenesis and other functions.
  • PDGF platelet-derived growth factor
  • transforming growth factor ⁇ Transforming growth factor ⁇ , TGF ⁇
  • transforming growth factor ⁇ Transforming growth factor ⁇ , TGF ⁇
  • insulin-like growth factor Insulin-like growth factor 1
  • TGF ⁇ can promote the secretion of extracellular matrix and promote the maturation of mother cells. TGF ⁇ can help improve allergic constitution and regulate the normal development of the immune system.
  • the TGF ⁇ superfamily includes TGF ⁇ 1, TGF ⁇ 2 and TGF ⁇ 3.
  • IGF-1 can promote fracture healing and the growth of skin, muscles, blood vessels, tendons and ligaments.
  • VEGF can promote angiogenesis and inhibit osteoclasts.
  • IL1-Ra can inhibit the inflammatory factor-interleukin 1 and is an important anti-inflammatory factor.
  • a platelet exosome enrichment solution containing multiple anti-inflammatory factors and growth factors can be prepared.
  • blood samples extracted from the subjects were further injected into the tube with a blood collection needle.
  • the invented platelet exosome enrichment tube is placed in a 37°C incubator to stabilize platelet activity. After 1-4 hours, it is taken out from the incubator and placed in the inner sleeve of a flywheel centrifuge, centrifuged at 4000 rpm/min. centrifuge under the conditions for 5 minutes. At this time, the blood sample has been separated into a platelet exosome-enriched liquid layer and a blood cell layer.
  • the platelet exosome enrichment tube used may be the platelet exosome enrichment tube shown in Figure 1, Figure 2, Figure 3 or Figure 5.
  • the platelet exosome-enriched solution (hereinafter referred to as "PRPII”) prepared by the platelet exosome-enriched tube of the present invention was further compared with the platelet-rich plasma (hereinafter referred to as "PRP") prepared by a conventional method.
  • PRPII platelet exosome-enriched solution
  • PRP platelet-rich plasma
  • Table 4 shows the increase in concentration of growth factors required for clinical application of PRPII in various tissue repairs. The values are based on PRP.
  • Table 5 shows the concentrations of PRPII and PRP in each cytokine. The number in parentheses after PRPII indicates the time (hours) of standing in the incubator, and the concentration unit of each cytokine is pg/mL.
  • the results in Table 4 show that the concentration of growth factors required for tissue repair in PRPII prepared from the platelet exosome enrichment tube of the present invention is significantly increased compared to conventional PRP.
  • the results in Table 5 show that the concentration of growth factors is related to the resting time of blood samples at 37°C. The optimal resting time is 3-4 hours to obtain the best results.
  • the anti-inflammatory factors contained in PRPII The concentration of IL1-Ra is 4.15 times that of PRP, and the concentration of PDGF growth factor contained in PRPII is 5.4 times that of PRP.
  • TNF ⁇ tumor necrosis factor ⁇
  • PRPII prepared from the platelet exosome enrichment tube of the present invention was used in clinical trials to further verify the effect of PRPII on reducing the pain index of patients with degenerative arthritis after injection.
  • the subjects in the trial were 30 patients with stage III degenerative arthritis (VAS pain level: 8-10).
  • the above subjects received PRPII treatment once every 2 weeks, for a total of 5 treatments.
  • the trial also includes subjects who received PRP treatment as a control.
  • the strategy of PRP treatment is the same as that of PRPII treatment. Please refer to Table 6 and Table 7.
  • Table 6 shows the pain improvement percentage of the subjects, and Table 7 shows the pain level results of the subjects after the course of treatment.
  • PRPII prepared from the platelet exosome-enriched tube of the present invention can also be used in a variety of clinical treatments, such as dental implant treatment, medical aesthetic treatment, and alopecia. Hair treatment, orthopedic treatment, trauma treatment, ophthalmology treatment, obstetrics and gynecology treatment, etc.
  • dental implant treatment hyaluronic acid is mixed into a semi-solid filling and filled in the dental implant site, which can promote the close connection between the dental implant and the bone and increase the chance of successful dental implantation.
  • medical aesthetic treatment it can be injected under the skin to promote collagen regeneration and improve skin texture.
  • injection into the scalp can regenerate hair.
  • orthopedic treatment it can be directly injected into the fracture defect after surgery to promote bone healing, or injected into bone cavities to promote growth.
  • trauma treatment it can be combined with artificial skin to cover diabetic feet and other chronic ulcerative wounds that are difficult to heal, such as bedsores or burns, to accelerate healing.
  • ophthalmic treatment it can be made into eye drops to treat dry eye syndrome, chronic corneal ulcers or corneal dystrophy.
  • PRPII When used for obstetrics and gynecology treatment, PRPII can be injected into the uterus through the cervix to thicken the endometrium, thereby improving infertility caused by insufficient endometrium hypertrophy and helping the success rate of pregnancy.
  • the platelet exosome enrichment tube of the present invention contains ground glass beads with evenly distributed concave and convex shapes on the surface.
  • physical principles can be used to break the cell membrane of platelets, so that In addition to exosomes on the surface of platelets, exosomes of three other isomers in the cytoplasm can also be activated and released, thereby obtaining a platelet exosome-enriched solution containing multiple anti-inflammatory factors and growth factors, and the obtained
  • concentration of various growth factors in the platelet exosome-enriched solution is several times higher than that of the conventional platelet-rich plasma, which makes subsequent clinical treatment effects better and can be used in a variety of clinical treatments. Great clinical application prospects.

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Abstract

本发明提供一种血小板外泌体富集管，其包含管体、盖体和多个毛玻璃珠，管体具有一封闭端、一开口端及一容置空间，盖体可拆卸地盖合于管体的开口端，毛玻璃珠设置于容置空间内，其中每个毛玻璃珠的表面具有均匀分布的凹凸形状。藉此利用物理原理打破血小板的细胞膜，使在血小板的细胞质中另外3个异构体的外泌体被活化释放，进而得到包含多个抗发炎因子和生长因子的血小板外泌体富集液。

Description

血小板外泌体富集管 技术领域
本发明是有关于一种血液取样装置，特别是一种血小板外泌体富集管。
背景技术
人体血液主要包括血浆和血细胞两部分，血细胞主要成分为红血球、白血球和血小板。全血可以制备成各种成分，在理想条件下保存，可以有效用于各种治疗。其中血小板在临床治疗发现具有体外止凝血和快速启动内源性生长因子的作用，对人体具有良好的促细胞生长和修复作用。
随着现代医学的发展，微量血液成分如富含血小板血浆(platelet rich plasma,PRP)广泛用于临床外科和医学美容，其为从受试者自体抽取血液，收集富含血小板的血浆，及时通过医疗手段重新注入患者皮肤或组织表面，达到自身机体的治疗和恢复医疗作用。可避免利用外体血制品发生的排斥反应。
PRP是利用血小板表面的外泌体分泌生长因子及抗发炎因子来治疗身体的不适，主要用于骨关节炎、肌肉韧带损伤、抗发炎及组织修复。此外，PRP亦可应用于医学美容和牙科，例如在医学美容的应用可减少皱纹，在牙科方面的应用可以填充牙髓，促进植牙金属与牙髓腔密合增加生长，进而增加植牙成功率。
然而血小板除了表面的外泌体能分泌生长因子及抗发炎因子外，另外在血小板的细胞质中另外还有3个异构体(isoforms)的外泌体。习知的PRP只活化了血小板表面的外泌体，因而只有血小板表面的生长因子被活化释放，血小板细胞膜内部另外3个异构体的外泌体没有被利用到。因此发展一种可以打破血小板细胞膜的临床医疗器械，进而利用血小板细胞膜内部3个异构体的外泌体活化释放的生长因子和抗发炎因子，以提高临床应用的疗效和范围至关重要。
发明内容
本发明的一目的是在于提供一种血小板外泌体富集管，其是利用物理 原理打破血小板的细胞膜，使在血小板的细胞质中另外3个异构体的外泌体亦可被活化释放，进而得到包含多种抗发炎因子和生长因子的血小板外泌体富集液。
本发明的一实施方式是在提供一种血小板外泌体富集管，其包含管体、盖体和多个毛玻璃珠。管体具有一封闭端、一开口端及一容置空间，开口端相对于封闭端，容置空间由封闭端连通开口端而形成。盖体可拆卸地盖合于管体的开口端，用以开闭容置空间。毛玻璃珠设置于容置空间内，其中每个毛玻璃珠的表面具有均匀分布的凹凸形状。
依据前述实施方式的血小板外泌体富集管，其中管体的封闭端可为圆弧状。
依据前述实施方式的血小板外泌体富集管，其中管体可更包含站立部，其连接于封闭端，且站立部远离封闭端的一端为平底边。
依据前述实施方式的血小板外泌体富集管，其中管体的材质可为玻璃或塑料。
依据前述实施方式的血小板外泌体富集管，其中盖体可为螺旋管帽、塑料帽盖、安全帽盖或橡胶帽盖。
依据前述实施方式的血小板外泌体富集管，其中管体的开口端设有一外螺纹，且盖体的内圈设有一内螺纹，管体与盖体以外螺纹和内螺纹紧密接合。
依据前述实施方式的血小板外泌体富集管，其中盖体可包含注入口，用以配合采血针注入及/或抽取血液样本。
依据前述实施方式的血小板外泌体富集管，其中盖体可更包含连接部，其凸设于盖体的上表面，用以与负压吸引器连接。
依据前述实施方式的血小板外泌体富集管，可更包含胶塞，所述胶塞设置于管体与盖体之间，用以密闭容置空间。
依据前述实施方式的血小板外泌体富集管，其中每个毛玻璃珠的表面的表面粗度(RMS)可为0.01μm至0.5μm。
依据前述实施方式的血小板外泌体富集管，其中每个毛玻璃珠的平均粒径可为2mm至10mm。
附图说明
为让本发明的上述和其他目的、特征、优点与实施例能更明显易懂，附图的说明如下：
图1绘示本发明一实施方式的血小板外泌体富集管的爆炸图；
图2绘示本发明另一实施方式的血小板外泌体富集管的爆炸图；
图3绘示本发明又一实施方式的血小板外泌体富集管的立体图；
图4绘示依据图3的血小板外泌体富集管的组合剖视图；
图5绘示本发明再一实施方式的血小板外泌体富集管的立体图；以及
图6绘示依据图5的血小板外泌体富集管的组合剖视图。
具体实施方式
以下将参照附图说明本发明的实施例。为明确说明起见，许多实务上的细节将在以下叙述中一并说明。然而，应了解到，这些实务上的细节不应用以限制本发明。也就是说，在本发明部分实施例中，这些实务上的细节是非必要的。此外，为简化附图起见，一些习知惯用的结构与组件在附图中将以简单示意的方式绘示；并且重复的组件将可能使用相同的编号表示。
请参照图1，其绘示本发明一实施方式的血小板外泌体富集管100的爆炸图，血小板外泌体富集管100包含管体110、盖体120和多个毛玻璃珠130。
管体110具有一封闭端112、一开口端111及一容置空间113，开口端111相对于封闭端112，容置空间113由封闭端112连通开口端111而形成。其中管体110的封闭端112可为圆弧状，可以使得血小板外泌体富集管100在使用状态时将血液样本集中于管底。管体110的材质可为玻璃或塑料，较佳地，可以是透明或半透明的塑料或玻璃，使得血小板外泌体富集管100在使用时可以清晰看出血液分层。此外，管体110的外壁面可以具有多个刻度标记，使其在使用状态时，可以直接辨识内部的体积量。更佳地，管体110的内管径直径可为10mm至20mm，管体110的长度可为100mm至150mm，并可容纳10mL至15mL的血液样本，但不以此为限。
盖体120可拆卸地盖合于管体110的开口端111，用以开闭容置空间113。盖体120可为螺旋管帽、塑料帽盖、安全帽盖或橡胶帽盖。当盖体120为螺旋管帽、塑料帽盖或安全帽盖等不具弹性的材质时，前述管体110的 开口端111设有一外螺纹，且盖体120的内圈设有一内螺纹，藉此管体110与盖体120以外螺纹和内螺纹紧密接合。当盖体120为如橡胶帽盖具有弹性的材质时，所述盖体120可直接套接于管体110的开口端111。而盖体120可包含一注入口121，用以配合采血针注入及/或抽取血液样本，采血针可为硬连接式或软连接式。
毛玻璃珠130设置于容置空间113内，其中每个毛玻璃珠130的表面131具有均匀分布的凹凸形状。表面131所具有均匀分布的凹凸形状可经由化学处理方法或物理处理方法所形成。化学处理方法例如施行毛玻璃化处理的方法，可通过将玻璃珠浸渍于氟化氢与氟化铵的混合溶液中，将浸渍面作化学性表面处理而得到毛玻璃珠130。物理处理方法包含利用加压空气将晶质二氧化硅粉末及碳化硅粉末等喷吹于玻璃珠的表面的喷砂处理，以得到毛玻璃珠130；或以经水沾湿的附着有晶质二氧化硅粉末及碳化硅粉末等的刷子磨刷玻璃珠以得到毛玻璃珠130。而每个毛玻璃珠130的表面的表面粗度(RMS)可为0.01μm至0.5μm，每个毛玻璃珠130的平均粒径可为2mm至10mm。更佳地，于管体110的容置空间113中可设置20颗至50颗毛玻璃珠130，但不以此为限。并请参照下表一至表三，其中表一所示为与毛玻璃珠130尺寸相关的性质，表二所示为毛玻璃珠130的化学性质，表三所示为毛玻璃珠130的物理性质。
表一、与毛玻璃珠130尺寸相关的性质

表二、毛玻璃珠130的化学性质
表三、毛玻璃珠130的物理性质

血小板外泌体富集管100可通过其中所包含表面具有均匀分布的凹凸形状的毛玻璃珠，在制备血小板外泌体富集液时，利用物理原理打破血小板的细胞膜，使在血小板的细胞质中另外3个异构体的外泌体亦可被活化释放，进而得到包含多个抗发炎因子和生长因子的血小板外泌体富集液，且所得到的血小板外泌体富集液中各类生长因子的浓度相较于习知的富含血小板血浆增加数倍，使得后续应用于临床的治疗效果更好。
请参照图2，其绘示本发明另一实施方式的血小板外泌体富集管200的爆炸图。血小板外泌体富集管200包含管体210、盖体220、多个毛玻璃珠230和胶塞240。
管体210具有封闭端212、开口端211及容置空间213，开口端211相对于封闭端212，容置空间213由封闭端212连通开口端211而形成。管体210的其他技术细节和管体110相同，在此不再赘述。
盖体220可拆卸地盖合于管体210的开口端211，用以开闭容置空间213。盖体220可包含注入口221，用以配合采血针注入及/或抽取血液样本。盖体220的其他技术细节和盖体120相同，在此不再赘述。
毛玻璃珠230设置于容置空间213内，其中每个毛玻璃珠230的表面(图未绘示)具有均匀分布的凹凸形状。毛玻璃珠230的其他技术细节和毛玻璃珠130相同，在此不再赘述。
血小板外泌体富集管200更包含胶塞240，胶塞240设置于管体210与盖体220之间，用以密闭容置空间。胶塞240的外径可与管体210的外径相同，此外，胶塞240靠近管体210的一端可设有一凸部241，凸部241的外径与管体210的内径相同，胶塞240可通过凸部241塞入管体210的 开口端211，藉此使胶塞240安装于管体210的开口端211，再将盖体220包覆于胶塞240外。而胶塞240的材质可为丁基橡胶，配合的采血针可由盖体220的注入口221穿刺胶塞240而进入管体210的容置空间213。
请参照图3和图4，图3绘示本发明又一实施方式的血小板外泌体富集管300的立体图，图4绘示依据图3的血小板外泌体富集管300的组合剖视图。血小板外泌体富集管300包含管体310、盖体320、多个毛玻璃珠330和胶塞340。
管体310具有封闭端312、开口端311、容置空间313及站立部314，开口端311相对于封闭端312，容置空间313由封闭端312连通开口端311而形成。管体310的封闭端312可为圆弧状，可以使得血小板外泌体富集管300在使用状态时将血液样本集中于管底。而血小板外泌体富集管300的管体310更包含一站立部314，其连接于封闭端312，且站立部314远离封闭端312的一端为平底边。如图4所示，因站立部314的一端为平底边，可使血小板外泌体富集管300直立于使用的桌面，不需再另外使用试管架摆放或收纳血小板外泌体富集管300，增加使用的方便性。管体310的其他技术细节和管体110相同，在此不再赘述。
盖体320可拆卸地盖合于管体310的开口端311，用以开闭容置空间313。盖体320可包含注入口321，用以配合采血针注入及/或抽取血液样本。盖体320的其他技术细节和盖体120相同，在此不再赘述。
毛玻璃珠330设置于容置空间313内，其中每个毛玻璃珠330的表面(图未绘示)具有均匀分布的凹凸形状。毛玻璃珠330的其他技术细节和毛玻璃珠130相同，在此不再赘述。
胶塞340设置于管体310与盖体320之间，用以密闭容置空间，胶塞340的其他技术细节和胶塞240相同，在此不再赘述。
请参照图5和图6，图5绘示本发明再一实施方式的血小板外泌体富集管400的立体图，图6绘示依据图5的血小板外泌体富集管400的组合剖视图。血小板外泌体富集管400包含管体410、盖体420和多个毛玻璃珠430。
管体410具有封闭端412、开口端411、容置空间413及站立部414，开口端411相对于封闭端412，容置空间413由封闭端412连通开口端411 而形成。站立部414连接于封闭端412，且站立部414远离封闭端412的一端为平底边，使血小板外泌体富集管400可直立于使用的桌面。管体410的其他技术细节和管体110相同，在此不再赘述。
盖体420可拆卸地盖合于管体410的开口端411，用以开闭容置空间413。盖体420可包含注入口421，用以配合采血针注入及/或抽取血液样本。血小板外泌体富集管400的盖体420更包含一连接部422，其凸设于盖体420的上表面，用以与负压吸引器连接，以增加血小板外泌体富集管400可应用的范围。盖体420的其他技术细节和盖体120相同，在此不再赘述。
毛玻璃珠430设置于容置空间413内，其中每个毛玻璃珠430的表面(图未绘示)具有均匀分布的凹凸形状。毛玻璃珠430的其他技术细节和毛玻璃珠130相同，在此不再赘述。
试验例
血小板能分泌多种生长因子、抗发炎因子，具有促进细胞移动、分化、分裂、基质增生、血管新生和平衡等功能，其中包含的重要生长因子如血小板衍生生长因子(Platelet-derived growth factor,PDGF)、转化生长因子α(Transforming growth factorα,TGFα)、转化生长因子β(Transforming growth factorβ,TGFβ)、类胰岛素生长因子(Insulin-like growth factor 1,IGF-1)、血管内皮生长因子(Vascular endothelial growth factor,VEGF)和介白素1-受体拮抗剂(Interleukin 1receptor antagonist IL1-Ra)，PDGF能吸引干细胞、促进干细胞分裂细胞外基质分泌、刺激成骨作用等功能。TGFα能促进细胞外基质分泌、促进母细胞成熟。TGFβ能帮助改善过敏体质，调节免疫系统正常发展，TGFβ超家族中包含TGFβ1、TGFβ2和TGFβ3。IGF-1能促进骨折愈合，皮肤、肌肉、血管肌腱及韧带生长。VEGF可以促进血管新生、抑制破骨细胞。IL1-Ra可以抑制发炎因子-介白素1，是重要抗发炎因子。
为测试本发明的血小板外泌体富集管可制备包含多个抗发炎因子和生长因子的血小板外泌体富集液，本试验例进一步将抽取自受试者的血液样本以采血针注入本发明的血小板外泌体富集管，放置于37℃的恒温箱静置以稳定血小板活性，1-4小时后自恒温箱取出并置入飞轮式离心机内套筒中，以4000rpm/min的条件离心5分钟，此时血液样本已分离为血小板外泌体富集液层和血球层。将离心结束后的血小板外泌体富集管取出，再以长针 自盖体的注入口插入管体中，抽取上层的血小板外泌体富集液，即得到制备完成的血小板外泌体富集液，后续可用于注射入受试者所需使用的部位。所使用的血小板外泌体富集管可为图1、图2、图3或图5所示的血小板外泌体富集管。
试验上进一步将以本发明的血小板外泌体富集管所制备的血小板外泌体富集液(以下简称「PRPII」)，与习知方法所制备的富含血小板血浆(以下简称「PRP」)中各类生长因子和细胞因子的浓度进行比较，请参照表四和表五，表四为PRPII于临床应用于各组织修复所需的生长因子的浓度增加倍数，其数值系以PRP为基准。表五为PRPII和PRP于各细胞因子的浓度，其中PRPII后括号的数字表示静置于恒温箱的时间(小时)，而各细胞因子的浓度单位皆为pg/mL。
表四、PRPII于临床应用于各组织修复所需的生长因子的浓度增加倍数
表五、PRPII和PRP于各细胞因子的浓度
表四的结果显示，由本发明的血小板外泌体富集管所制备的PRPII其中所包含各组织修复所需的生长因子的浓度相较于习知的PRP皆有显着地增加。且由表五的结果显示，生长因子浓度与血液样本于37℃的静置时间有关，最佳的静置时间为3-4小时，可以获得最好的结果，其中PRPII所含有的抗发炎因子IL1-Ra浓度为PRP的4.15倍，PRPII所含有的PDGF生长因子浓度是PRP的5.4倍。而PRPII所包含的肿瘤坏死因子α(Tumor necrosis factorα,TNFα)浓度与PRP相比虽稍多，但也不致于因培养时间较长而有大幅增加的情况，显示本发明的血小板外泌体富集管确实可制备含有高浓度各类生长因子的PRPII。
试验上将由本发明的血小板外泌体富集管所制备的PRPII用于临床试验，进一步验证PRPII对于退化性关节炎病人注射后疼痛指数下降的效果。试验上的受试者为30位退化性关节炎第III期的患者(VAS疼痛等级：8-10)，上述受试者每2周接受1次PRPII治疗，总共接受治疗次数为5次，试验上另包含接受PRP治疗的受试者以做为对照，PRP治疗的策略和PRPII治疗相同。请参照表六和表七，表六为受试者的疼痛改善百分比，表七为疗程结束后试者的疼痛等级结果。
表六、受试者的疼痛改善百分比
表七、疗程结束后试者的疼痛等级结果
由表六和表七的结果显示，全部接受PRPII治疗的受试者在疗程结束后疼痛皆获得改善，其中更有80％的受试者的疼痛等级由原本的8-10降至2-4，可证明由本发明的血小板外泌体富集管所制备的PRPII具有显著改善退化性关节炎患者疼痛的效果。
而由本发明的血小板外泌体富集管所制备的PRPII除了退化性关节炎、肌腱损伤的注射治疗的临床运用外，亦可运用于多种临床治疗，例如植牙治疗、医美治疗、秃发治疗、骨科治疗、外伤治疗、眼科治疗和妇产科治疗等。用于植牙治疗时，通过混合玻尿酸变成半固体填塞物填补于植牙处，可以促进植牙体与骨质紧密合，增加植牙成功机率。用于医美治疗时，可注射于皮下促进胶原蛋白再生使肤质改善，也可与脸部注射玻尿酸同时使用，使效果加乘。用于秃发治疗时，注射于头皮可使毛发再生。用于骨科治疗时，术后直接注射骨折缺陷处促进骨愈合，或注射于骨空洞促进生长。用于外伤治疗时，可与人工皮结合覆盖于糖尿病足及其他不易愈合的慢性溃烂伤口，譬如褥疮或烧烫伤，使其加速愈合。用于眼科治疗时，可制成眼药水，治疗干眼症及慢性角膜溃疡或角膜营养不良。用于妇产科治疗时，可由子宫颈注射PRPII入子宫，使子宫内膜肥厚，以改善因为子宫内膜不够肥厚导致的不孕症，帮助怀孕成功比率。
综上所述，本发明的血小板外泌体富集管因包含表面具有均匀分布的凹凸形状的毛玻璃珠，在制备血小板外泌体富集液时，可利用物理原理打破血小板的细胞膜，使在血小板除了表面的外泌体以外，细胞质中另外3个异构体的外泌体亦可被活化释放，进而得到包含多个抗发炎因子和生长因子的血小板外泌体富集液，且所得到的血小板外泌体富集液中各类生长因子的浓度相较于习知的富含血小板血浆增加数倍，使得后续应用于临床的治疗效果更好，并可应用于多种临床治疗，具有极大的临床应用前景。
虽然本发明已以实施方式揭露如上，然其并非用以限定本发明，任何熟习此技艺者，在不脱离本发明的精神和范围内，当可作各种之更动与润饰，因此本发明的保护范围当视后附的权利要求所界定者为准。
符号说明
100,200,300,400:血小板外泌体富集管
110,210,310,410:管体
111,211,311,411:开口端
112,212,312,412:封闭端
113,213,313,413:容置空间
120,220,320,420:盖体
121,221,321,421:注入口
130,230,330,430:毛玻璃珠
131:表面
240,340:胶塞
241:凸部
314,414:站立部
422:连接部

Claims (11)

  1. 一种血小板外泌体富集管，其特征在于，包含：
    一管体，具有一封闭端、一开口端及一容置空间，所述开口端相对于所述封闭端，所述容置空间由所述封闭端连通所述开口端而形成；
    一盖体，其可拆卸地盖合于所述管体的所述开口端，用以开闭所述容置空间；以及
    多个毛玻璃珠，设置于所述容置空间内，其中各所述毛玻璃珠的一表面具有均匀分布的凹凸形状。
  2. 如权利要求1所述的血小板外泌体富集管，其特征在于，所述管体的所述封闭端为圆弧状。
  3. 如权利要求2所述的血小板外泌体富集管，其特征在于，所述管体更包含一站立部，其连接于所述封闭端，且所述站立部远离所述封闭端的一端为平底边。
  4. 如权利要求1所述的血小板外泌体富集管，其特征在于，所述管体的材质为一玻璃或一塑料。
  5. 如权利要求1所述的血小板外泌体富集管，其特征在于，所述盖体为一螺旋管帽、一塑料帽盖、一安全帽盖或一橡胶帽盖。
  6. 如权利要求1所述的血小板外泌体富集管，其特征在于，所述管体的所述开口端设有一外螺纹，且所述盖体的内圈设有一内螺纹，该管体与所述盖体以所述外螺纹和所述内螺纹紧密接合。
  7. 如权利要求1所述的血小板外泌体富集管，其特征在于，所述盖体包含一注入口，用以配合一采血针注入及/或抽取一血液样本。
  8. 如权利要求7所述的血小板外泌体富集管，其特征在于，所述盖体更包含一连接部，其凸设于所述盖体的上表面，用以与一负压吸引器连接。
  9. 如权利要求1所述的血小板外泌体富集管，其特征在于，更包含一胶塞，所述胶塞设置于所述管体与所述盖体之间，用以密闭所述容置空间。
  10. 如权利要求1所述的血小板外泌体富集管，其特征在于，各所述毛玻璃珠的所述表面的表面粗度(RMS)为0.01μm至0.5μm。
  11. 如权利要求1所述的血小板外泌体富集管，其特征在于，各所述 毛玻璃珠的平均粒径为2mm至10mm。
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CN110152747A (zh) * 2019-05-10 2019-08-23 清华大学 微流控芯片以及外泌体的分离方法
TWM589033U (zh) * 2019-01-11 2020-01-11 亞恩生醫股份有限公司 血液分離組件及血液分離管
CN215365724U (zh) * 2021-07-23 2021-12-31 天津市中西医结合医院（天津市南开医院） 一种干细胞外泌体分离提取装置
TWM631631U (zh) * 2022-06-06 2022-09-01 鄭本岡 血小板外泌體富集管
CN217948109U (zh) * 2022-06-06 2022-12-02 郑本冈 血小板外泌体富集管

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CN110152747A (zh) * 2019-05-10 2019-08-23 清华大学 微流控芯片以及外泌体的分离方法
CN215365724U (zh) * 2021-07-23 2021-12-31 天津市中西医结合医院（天津市南开医院） 一种干细胞外泌体分离提取装置
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