WO2018227363A1 - 一种防治糖尿病足的干细胞组合物及其应用、干细胞制剂 - Google Patents

一种防治糖尿病足的干细胞组合物及其应用、干细胞制剂 Download PDF

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WO2018227363A1
WO2018227363A1 PCT/CN2017/088012 CN2017088012W WO2018227363A1 WO 2018227363 A1 WO2018227363 A1 WO 2018227363A1 CN 2017088012 W CN2017088012 W CN 2017088012W WO 2018227363 A1 WO2018227363 A1 WO 2018227363A1
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stem cell
cells
preparation
mesenchymal stem
peripheral blood
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PCT/CN2017/088012
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English (en)
French (fr)
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陈海佳
葛啸虎
王一飞
陈婉玲
王小燕
马岩岩
罗二梅
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广州赛莱拉干细胞科技股份有限公司
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Priority to CN201780000488.XA priority Critical patent/CN107454845A/zh
Priority to PCT/CN2017/088012 priority patent/WO2018227363A1/zh
Publication of WO2018227363A1 publication Critical patent/WO2018227363A1/zh

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    • 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
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46433—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule

Definitions

  • the invention relates to the field of stem cell technology, in particular to a stem cell composition for preventing and treating diabetic foot, an application thereof, and a stem cell preparation.
  • Stem cells are a kind of pluripotent cells with self-replication ability and differentiation ability, and have the potential function of regenerating various tissues and organs.
  • the medical community calls them “universal cells.”
  • Stem cell transplantation as a new treatment for angiogenesis, has brought hope to patients with diabetic foot and has become a hot topic in recent years. Its therapeutic mechanism is mainly involved in the formation of neovascularization, initiation and secretion of related factors, Immunoregulation and the ability of stem cells to proliferate and differentiate. After stem cell transplantation, on the one hand, it is integrated into the damaged vascular plexus, and in the ischemic and hypoxic environment, vascular endothelial cells, smooth muscle cells, etc.
  • Stem cells can be induced to differentiate, form new capillaries, and promote angiogenesis; Stem cells can selectively secrete a variety of growth factors such as vascular endothelial growth factor, basic fibroblast growth factor and nerve growth factor through autocrine or paracrine pathways, and bind to corresponding receptors on the endothelial cell membrane to promote Endothelial cells proliferate, migrate, remodel, form new blood vessels, further promote local neovascularization, nerve regeneration and remodeling, etc., and finally improve microcirculation, restore blood flow of affected limbs, improve skin temperature of affected limbs, and promote wound healing. To achieve the purpose of treatment.
  • growth factors such as vascular endothelial growth factor, basic fibroblast growth factor and nerve growth factor through autocrine or paracrine pathways, and bind to corresponding receptors on the endothelial cell membrane to promote Endothelial cells proliferate, migrate, remodel, form new blood vessels, further promote local neovascularization, nerve regeneration and remodeling, etc., and
  • PBMC Peripheral blood mononuclear cell
  • the present invention provides a stem cell composition for preventing and treating diabetic foot, an application thereof, and a stem cell preparation.
  • the stem cell composition can effectively prevent and treat diabetic foot, and the effect is better than that of stem cells or peripheral blood mononuclear cells alone.
  • the present invention provides the following technical solutions:
  • the present invention provides a stem cell composition consisting of mesenchymal stem cells and peripheral blood mononuclear cells.
  • the combination of mesenchymal stem cells and peripheral blood mononuclear cells can effectively treat diabetic foot, and the effect is better than that of stem cells or peripheral blood mononuclear cells alone.
  • the ratio of the number of cells of the stem cell composition mesenchymal stem cells to the peripheral blood mononuclear cells is (1 to 5): (1 to 5).
  • the ratio of the number of cells of the stem cell composition mesenchymal stem cells to peripheral blood mononuclear cells is 1:1.
  • the mesenchymal stem cells are umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells or adipose-derived mesenchymal stem cells.
  • the invention also provides the use of the stem cell composition for the preparation of a medicament for preventing and/or treating diabetic foot.
  • the present invention also provides a stem cell preparation comprising mesenchymal stem cells, peripheral blood mononuclear cells, human serum albumin and a vehicle.
  • the dosage form of the stem cell preparation is an intravenous injection or a subcutaneous injection.
  • the stem cell preparation is an intravenous injection, and the concentration of each component is:
  • Mesenchymal stem cells (1 ⁇ 5) ⁇ 10 5 / mL;
  • Peripheral blood mononuclear cells (1 ⁇ 5) ⁇ 10 5 / mL;
  • Human albumin 5wt%
  • the intravenous infusion intravenously injects the stem cell preparation of the present invention into the patient along the vein at a standard of 5 to 10 ⁇ 10 5 /kg.
  • the stem cell preparation is a subcutaneous injection, and the concentration of each component is:
  • Mesenchymal stem cells (1 ⁇ 5) ⁇ 10 6 / mL;
  • Peripheral blood mononuclear cells (1 ⁇ 5) ⁇ 10 6 / mL;
  • Human albumin 5wt%
  • the stem cell preparation is a subcutaneous injection, and the concentration of each component is:
  • Mesenchymal stem cells 2 ⁇ 10 6 /mL;
  • Peripheral blood mononuclear cells 2 ⁇ 10 6 /mL;
  • Human albumin 5wt%
  • the stem cell preparation containing 2 ⁇ 10 7 mesenchymal stem cells is filled into a 5 mL syringe, and slowly injected into the affected area. After the injection, the needle eye is pressed with sterile gauze for 3 to 5 minutes. Prone for 30 minutes, during the 5min activity 8 to 10 times.
  • the solvent is a compound electrolyte injection, glucose injection or physiological saline.
  • the vehicle is physiological saline.
  • the present invention provides a stem cell composition for preventing and treating diabetic foot, an application thereof, and a stem cell preparation.
  • the stem cell composition consists of mesenchymal stem cells and peripheral blood mononuclear cells.
  • the present invention has at least one of the following advantages:
  • the stem cell composition of the present invention can effectively prevent and treat diabetic foot, and the effect is better than that of stem cells or peripheral blood mononuclear cells alone, and the combined use of mesenchymal stem cells and peripheral blood mononuclear cells has synergistic effect.
  • Mesenchymal stem cells have many advantages such as wide source, abundant content, simple isolation, easy expansion in vitro, no ethical problems, easy autologous/allogene transplantation, low immunogenicity, and small damage to the donor area. . Studies have shown that mesenchymal stem cells can effectively regulate immunity, secrete trophic factors to repair damaged blood vessels, promote neovascularization, improve microenvironment, etc., providing a new way for the treatment of diabetic lower extremity vascular disease.
  • the stem cell composition and the preparation thereof can treat both the symptoms and the root causes, and the treatment effect is obtained; the treatment method is simple and easy, and the patient can accept and use, for those who are unable to perform lower limb bypass, and are old and weak, accompanied by other It is especially appropriate for patients who are unable to undergo surgery.
  • the invention discloses a stem cell composition for preventing and treating diabetic foot, an application thereof and a stem cell preparation, and those skilled in the art can learn from the contents of the paper and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the method and the application of the present invention have been described by the preferred embodiments, and it is obvious that the method and application described herein may be modified or appropriately modified and combined without departing from the scope of the present invention. The technique of the present invention is applied.
  • the stem cell composition for preventing and treating diabetic foot provided by the present invention and the use thereof, and the raw materials or auxiliary materials used in the stem cell preparation are all commercially available.
  • the bone marrow tissue of the patient extracted by bone marrow aspiration was transferred to a 50 ml centrifuge tube, mixed with DMEM-LG medium, and then centrifuged at 500 rpm/min for 10 min. After centrifugation, the upper layer of fat is taken up and the supernatant is discarded;
  • DMEM-LG medium was added to the remaining precipitate to dilute and mix, and added to the tube containing the same volume of Ficoll lymphocyte separation solution as the remaining precipitate, and centrifuged at 2200 rpm/min for 30 min. Collect the mononuclear cells of the aerosol layer after centrifugation, transfer to a new 50 mL centrifuge tube, wash the cells with PBS, centrifuge, and then discard the supernatant. Adjust the cell density to 2 ⁇ 10 5 cell/ml according to the cell count. In a culture dish, placed in an incubator at 37 ° C and a CO 2 concentration of 5%;
  • the cells were changed for the first time at 72h, and the non-adherent cells were removed. After that, the cells were changed once every 2 to 3 days until the cells were grown to a degree of fusion of 80%. The cells were subcultured by trypsin digestion, and after passage to the fourth generation, the cells were observed. The growth state was washed with trypsin and washed with PBS buffer solution, and the cells were counted and collected by centrifugation.
  • Example 3 Culture and collection of adipose-derived mesenchymal stem cells
  • the adipose tissue extracted under aseptic conditions was dispensed into a 50 ml centrifuge tube, 20 ml per tube.
  • An equal volume of 0.5% type I collagenase (final concentration of 0.25%) was added to each tube, thoroughly mixed, sealed, transferred to a constant temperature air shaker, and digested at 37 ° C, 100 rpm for 1 h. Digestion was carried out by adding 4 ml of FBS to each tube and mixing. Centrifuge at 1500 rpm/min for 5 min. Discard the upper two layers of liquid, resuspend the cells in each tube by adding 40 ml of PBS, manually count and centrifuge. The supernatant was discarded, and the cells were seeded in a Petri dish at 1 ⁇ 10 5 /ml, and cultured in an incubator at 37 ° C and a CO 2 concentration of 5%.
  • the plasma, the tunica layer (ie, PBMC layer), the lymphocyte separation solution, and the red blood cell layer were extracted, and the PBMC layer was extracted.
  • the collected PBMC was resuspended in physiological saline, and centrifuged at 500 g. 10 min, repeated washing twice, collecting PBMC;
  • the cell density before rehydration is not more than 3 ⁇ 10 6 /ml
  • the cell density after rehydration is 0.5 ⁇ 1.0 ⁇ 10 6 / ml
  • every 3 days rehydration and additional cell growth Factor until cells were collected on day 14.
  • Example 2 After the umbilical cord mesenchymal stem cells obtained in Example 1 were passed to the fourth passage, the cell growth state was observed, and the cells were digested with trypsin, washed with physiological saline, and collected by centrifugation. Finally, the cell pellet was resuspended in physiological saline and 5% human albumin preparation matrix according to the required reinfusion amount, filtered through a 70 ⁇ m cell sieve, and then injected into a sodium chloride injection bag. After the preparation is made, the freshly prepared preparations are tested within 3 hours, including: stem cell morphology, survival rate, endotoxin, bacteria, fungi, and the like. After the test results are passed, the preparation is placed in a sterile box and transported to the treatment room at a low temperature for treatment.
  • the peripheral blood mononuclear cell pellet of Example 4 was resuspended in physiological saline and 5% human albumin preparation matrix according to the required reinfusion amount, and filtered with a 70 ⁇ m cell sieve to inject sodium chloride injection. In the bag. After the preparation is made, the freshly prepared preparations are tested within 3 hours, including: stem cell morphology, survival rate, endotoxin, bacteria, fungi, and the like. After the test results are passed, the preparation is placed in a sterile box and transported to the treatment room at a low temperature for treatment.
  • umbilical cord mesenchymal stem cell mixed cell preparation The umbilical cord mesenchymal stem cell preparation and the peripheral blood mononuclear cell preparation are thoroughly mixed according to the ratio of the number of cells: 1:1.
  • the concentration of umbilical cord mesenchymal stem cells is (1 ⁇ 5) ⁇ 10 5 / mL (total infusion volume is 100mL, the concentration is determined according to the reinfusion dose), the concentration of peripheral blood mononuclear cells It is (1 ⁇ 5) ⁇ 10 5 / mL (the total infusion volume is 100 mL, the concentration is specifically determined according to the reinfusion dose); human albumin is 5 wt%.
  • the ratio of umbilical cord mesenchymal stem cells to peripheral blood mononuclear cells was 1:1.
  • the concentration of umbilical cord mesenchymal stem cells was 2 ⁇ 10 6 /mL
  • the concentration of peripheral blood mononuclear cells was 2 ⁇ 10 6 /mL
  • human albumin was 5 wt%.
  • the bone marrow mesenchymal stem cell pellet of Example 2 was resuspended in a physiological saline solution and a 5% human albumin preparation base according to the required reinfusion amount, and filtered through a 70 ⁇ m cell sieve to inject a sodium chloride injection. In the bag. After the preparation is made, the freshly prepared preparations are tested within 3 hours, including: stem cell morphology, survival rate, endotoxin, bacteria, fungi, and the like. After the test results are passed, the preparation is placed in a sterile box and transported to the treatment room at a low temperature for treatment.
  • the peripheral blood mononuclear cell pellet of Example 4 was resuspended in physiological saline and 5% human albumin preparation matrix according to the required reinfusion amount, and filtered with a 70 ⁇ m cell sieve to inject sodium chloride injection. In the bag. After the preparation is made, the freshly prepared preparations are tested within 3 hours, including: stem cell morphology, survival rate, endotoxin, bacteria, fungi, and the like. After the test results are passed, the preparation is placed in a sterile box and transported to the treatment room at a low temperature for treatment.
  • Preparation of mixed cell preparation of bone marrow mesenchymal stem cells The bone marrow mesenchymal stem cell preparation and the peripheral blood mononuclear cell preparation are thoroughly mixed according to the ratio of the number of cells in a ratio of 1:1.
  • the concentration of bone marrow mesenchymal stem cells is (1 ⁇ 5) ⁇ 10 5 / mL (total infusion volume is 100mL, the concentration is determined according to the reinfusion dose), the concentration of peripheral blood mononuclear cells It is (1 ⁇ 5) ⁇ 10 5 / mL (the total infusion volume is 100 mL, the concentration is specifically determined according to the reinfusion dose); human albumin is 5 wt%.
  • the ratio of bone marrow mesenchymal stem cells to peripheral blood mononuclear cells was 1:1.
  • the concentration of bone marrow mesenchymal stem cells was 2 ⁇ 10 6 /mL
  • the concentration of peripheral blood mononuclear cells was 2 ⁇ 10 6 /mL
  • the human albumin was 5 wt%.
  • the adipose-derived mesenchymal stem cell pellet of Example 3 was resuspended in physiological saline and 5% human albumin preparation matrix according to the required reinfusion amount, and filtered through a 70 ⁇ m cell sieve to inject sodium chloride injection. In the bag. After the preparation is made, the freshly prepared preparations are tested within 3 hours, including: stem cell morphology, survival rate, endotoxin, bacteria, fungi, and the like. After the test results are passed, the preparation is placed in a sterile box and transported to the treatment room at a low temperature for treatment.
  • the peripheral blood mononuclear cell pellet of Example 4 was resuspended in physiological saline and 5% human albumin preparation matrix according to the required reinfusion amount, and filtered with a 70 ⁇ m cell sieve to inject sodium chloride injection. In the bag. After the preparation is made, the freshly prepared preparations are tested within 3 hours, including: stem cell morphology, survival rate, endotoxin, bacteria, fungi, and the like. After the test results are passed, the preparation is placed in a sterile box and transported to the treatment room at a low temperature for treatment.
  • Preparation of adipose stem cell mixed cell preparation The adipose stem cell preparation and the peripheral blood mononuclear cell preparation were mixed in a ratio of 1:1 of the number of cells.
  • the concentration of adipose-derived mesenchymal stem cells is (1 ⁇ 5) ⁇ 10 5 /mL (the total infusion volume is 100mL, the concentration is determined according to the reinfusion dose), and the concentration of peripheral blood mononuclear cells It is (1 ⁇ 5) ⁇ 10 5 / mL (the total infusion volume is 100 mL, the concentration is specifically determined according to the reinfusion dose); human albumin is 5 wt%.
  • the ratio of adipose-derived mesenchymal stem cells to peripheral blood mononuclear cells was 1:1.
  • the concentration of the adipose-derived mesenchymal stem cells was 2 ⁇ 10 6 /mL
  • the concentration of peripheral blood mononuclear cells was 2 ⁇ 10 6 /mL
  • the human albumin was 5 wt%.
  • Test Example 1 Clinical efficacy of intravenous injection of stem cell injection in the treatment of ischemic diabetic foot
  • the wound shrinkage ranged from 75% to 100% within 4 weeks, most of the new granulation tissue grew, no inflammatory exudate;
  • the wound surface was reduced by ⁇ 75% within 4 weeks, the part of the new granulation tissue grew, and the inflammatory exudate was less;
  • Test Example 2 Clinical efficacy of local injection of stem cell injection preparation for ischemic diabetic foot
  • the subjects were randomly divided into 8 groups, one of which was the control group and the other 7 were the experimental group.
  • the wound surface of the local ulcer was vortex-flushed with a mixture of gentamicin 8 to 160,000 U, 4 to 8 U of common insulin, 10 mg of anisodamine injection and physiological saline, and then dried with a sterile cotton ball. Use sterile gauze once a day.
  • the remaining 7 groups received local injection therapy of umbilical cord MSC, bone marrow MSC, fat MSC, PBMC and their mixed preparations of Examples 5-7, respectively, while applying the conventional treatment regimen.
  • a preparation containing 2 ⁇ 10 7 mesenchymal stem cells (single preparation, mixed preparation) was placed in a 5 mL syringe, and slowly injected into the affected area. After the injection, the needle was pressed with sterile gauze for 3 to 5 minutes, and the patient prone for 30 minutes. During the period of 5 minutes, the affected area was 8 to 10 times.
  • the wound shrinkage ranged from 75% to 100% within 4 weeks, most of the new granulation tissue grew, no inflammatory exudate;
  • the wound surface was reduced by ⁇ 75% within 4 weeks, the part of the new granulation tissue grew, and the inflammatory exudate was less;

Abstract

提供了一种防治糖尿病足的干细胞组合物及其应用、干细胞制剂。该干细胞组合物由间充质干细胞和外周血单个核细胞组成。该干细胞组合物可有效防治糖尿病足。

Description

一种防治糖尿病足的干细胞组合物及其应用、干细胞制剂 技术领域
本发明涉及干细胞技术领域，特别涉及一种防治糖尿病足的干细胞组合物及其应用、干细胞制剂。
背景技术
近年来，随着世界人口老龄化的速度加快，糖尿病患者的数目急剧增加，严重影响了个人的生活质量。糖尿病的危害性很大程度上源于它的多种并发症，其中缺血性糖尿病足是最常见且最严重的并发症之一，且因其较高的病死率、截肢率和感染率，成为糖尿病患者致残和致死的常见原因。糖尿病足的主要临床表现为间歇性跛行、静息痛、足部溃疡和坏疽等，不但给患者造成痛苦，而且给社会和家庭带来了巨大的经济负担。
长期以来对于糖尿病足的治疗处于相对混乱无序的状态，大量患者分散于骨科、普外科、内科、中医科，临床医生多将其归为神经病变和感染，下肢缺血引起的糖尿病足溃疡远未受到应有的重视。目前对缺血性糖尿病足的治疗仍缺乏有效的手段，由于其累及范围广，且有多支及多节段性等特点，传统治疗主要是内科的药物治疗和外科的血流重建,然而对于因远端流出道动脉闭塞性病变导致的足部缺血来说，药物治疗不能从根本上解决问题,外科血流重建是首先应该考虑的方法。但是,此类患者多为年老体弱，经常伴有心脑血管病变，无法接受动脉搭桥或下肢动脉介入等外科治疗而经常面临着截肢的危险。总之，目前临床上一般采用药物、血管搭桥、介入手术等方式治疗，都具有一定局限性，远期效果很不理想。如何促进糖尿病缺血下肢血管再生和有效循环的建立是治疗关键。
干细胞是一类具有自我复制能力和分化能力的多潜能细胞，具有再生各种组织器官的潜在功能，医学界将其称之为“万用细胞”。而干细胞移植作为治疗血管再生的新疗法，为糖尿病足患者带来了希望，并成为近年来研究的热点。其治疗机制主要是参与新生血管形成、启动和分泌相关因子、 免疫调节以及干细胞的增殖和分化能力等有关。干细胞移植后，一方面整合于受损的血管丛，在缺血、缺氧环境下可诱导生成血管内皮细胞、平滑肌细胞等，使其分化、形成新生毛细血管，促进血管再生；另一方面，干细胞可通过自分泌或旁分泌途径等反应性分泌多种生长因子，如血管内皮生长因子、碱性成纤维细胞生长因子和神经生长因子等细胞因子，与内皮细胞膜上的相应受体结合，促进内皮细胞增殖、迁移、重塑，形成新生血管，进一步促进局部新生血管形成、神经再生和重构等，最终可改善微循环，恢复患肢血流，提高患肢皮肤温度，实现促进创面愈合，达到治疗目的。
外周血单个核细胞(Peripheral blood mononuclear cell，PBMC)，即外周血中具有单个核的细胞，包括淋巴细胞和单核细胞。徐世民于2013年发表的《自体外周血干细胞移植治疗糖尿病足的疗效及其与CD34+水平关系的研究》文章中报道了自体外周血干细胞移植后患者有明显的侧支循环建立，局部血运改善，利于溃疡愈合，从而避免了截肢或最大限度降低了截肢平面。
目前还未见将间充质干细胞与外周血单个核细胞共同治疗缺血性糖尿病足的报道。
发明内容
有鉴于此，本发明提供了一种防治糖尿病足的干细胞组合物及其应用、干细胞制剂。该干细胞组合物可有效防治糖尿病足，效果好于干细胞或外周血单个核细胞单独使用的效果。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种干细胞组合物，由间充质干细胞和外周血单个核细胞组成。
在本发明中将间充质干细胞和外周血单个核细胞联合使用后，可有效治疗糖尿病足，效果好于干细胞或外周血单个核细胞单独使用的效果。
作为优选，干细胞组合物中间充质干细胞与外周血单个核细胞的细胞数量比例为(1～5)：(1～5)。
在本发明提供的实施例中，干细胞组合物中间充质干细胞与外周血单个核细胞的细胞数量比例为1：1。
作为优选，间充质干细胞为脐带间充质干细胞、骨髓间充质干细胞或脂肪间充质干细胞。
本发明还提供了该干细胞组合物在制备预防和/或治疗糖尿病足药物中的应用。
本发明还提供了一种干细胞制剂，包括间充质干细胞、外周血单个核细胞、人血白蛋白和溶媒。
在本发明提供的实施例中，干细胞制剂的剂型为静脉注射剂或皮下注射剂。
作为优选，干细胞制剂为静脉注射剂，各组分浓度为：
间充质干细胞：(1～5)×105个/mL；
外周血单个核细胞：(1～5)×105个/mL；
人血白蛋白：5wt％；
溶媒：补足。
在本发明中，静脉输注静脉注射液按照体重以5～10×105个/kg的标准将本发明干细胞制剂沿静脉回输至患者体内。
作为优选，干细胞制剂为皮下注射剂，各组分浓度为：
间充质干细胞：(1～5)×106个/mL；
外周血单个核细胞：(1～5)×106个/mL；
人血白蛋白：5wt％；
溶媒：补足。
优选地，干细胞制剂为皮下注射剂，各组分浓度为：
间充质干细胞：2×106个/mL；
外周血单个核细胞：2×106个/mL；
人血白蛋白：5wt％；
溶媒：补足。
在本发明中，患处局部注射皮下注射剂时，将含2×107个间充质干细胞的干细胞制剂装入5mL注射器，缓慢注入患处，注射完毕后用无菌纱 布按压针眼处3～5min，患者俯卧30min，期间每5min活动患处8～10次。
作为优选，溶媒为复方电解质注射液、葡萄糖注射液或生理盐水。
在本发明提供的实施例中，溶媒为生理盐水。
本发明提供了一种防治糖尿病足的干细胞组合物及其应用、干细胞制剂。该干细胞组合物由间充质干细胞和外周血单个核细胞组成。本发明至少具有如下优势之一：
1、本发明干细胞组合物可有效防治糖尿病足，效果好于干细胞或外周血单个核细胞单独使用的效果，间充质干细胞和外周血单个核细胞联合使用具有协同增效作用。
2、使用安全，没有毒副作用。间充质干细胞具有来源广泛、含量丰富、分离简单、体外扩增稳定容易、不涉及伦理问题、便于自体/异体移植、免疫原性低、对供区损伤小等优势，备受临床医生的重视。研究显示，间充质干细胞能够有效调节免疫，分泌营养因子修复受损血管、促进新生血管生成、改善微环境等，为糖尿病下肢血管病变治疗提供了新途径。
3、本发明干细胞组合物及其制剂能标本兼治，收到治本效果；治疗方法简便易行，患者都能接受和使用，对于那些无法进行下肢搭桥的，及年老体弱、并伴发其他疾病不能接受手术搭桥的患者来说尤为合适。
具体实施方式
本发明公开了一种防治糖尿病足的干细胞组合物及其应用、干细胞制剂，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明提供的一种防治糖尿病足的干细胞组合物及其应用、干细胞制剂中所用原料或辅料均可由市场购得。
下面结合实施例，进一步阐述本发明：
实施例1  脐带间充质干细胞的培养与收集
(1)在无菌条件下，取离开母体时间不超过2h的新鲜脐带，并筛查产妇是否携带传染性疾病病原体，将来源于健康供者(乙肝表面抗原、丙肝抗体、梅毒抗体和艾滋病抗体阴性)的新鲜脐带置于含有双抗的PBS中于4℃保温箱中运回实验室；
(2)将新鲜脐带用无菌的PBS清洗，再用75％乙醇消毒，然后用PBS清洗干净，前后清洗消毒所用溶液体积均为能将脐带浸泡在其中为宜，去除脐带最外层膜及静、动脉后，将脐带剪碎至20mm3大小的块，将其以0.5～1cm的间距均匀接种于培养皿中，缓慢加入50～100μl/cm2完全培养基，置于37℃，CO2浓度为5％的培养箱中静止培养；
(3)细胞贴壁融合后，使用胰酶进行消化，离心，分装至多个培养皿中进行传代培养。
实施例2  骨髓间充质干细胞的培养与收集
(1)在无菌条件下，将通过骨髓穿刺抽取的患者骨髓组织转移至50ml离心管中，加入DMEM-LG培养基等倍稀释混匀后，500rpm/min离心10min。离心后吸取上层的脂肪及上清弃掉；
(2)在剩余沉淀中加入DMEM-LG培养基等倍稀释混匀，并沿管壁加入到含与剩余沉淀等体积的Ficoll淋巴分离液的试管中，2200rpm/min离心30min。收集离心后界面雾状层单个核细胞，转移至新的50mL离心管中，加入PBS洗涤细胞，离心后倒掉上清液，根据细胞计数结果，调整细胞密度为2×105cell/ml接种于培养皿中，置于37℃，CO2浓度为5％的培养箱中静止培养；
(3)细胞培养72h时首次换液，去除未贴壁细胞，此后2～3d换液一次，直到细胞生长至融合度达80％，胰酶消化传代培养，传至第4代后，观察细胞生长状态，用胰酶消化后使用PBS缓冲溶液洗涤，细胞计数后离心收集。
实施例3  脂肪间充质干细胞的培养与收集
(1)将无菌条件下抽取的脂肪组织分装至50ml离心管中，每管20ml。每管中加入等体积的0.5％Ⅰ型胶原酶(终浓度为0.25％)，充分混匀，封口，转移至恒温空气摇床中，37℃、100rpm消化1h。每管加入4ml FBS终止消化，混匀。1500rpm/min离心5min。弃去上两层液体，每管加入40ml PBS重悬细胞，人工计数后离心收集。弃上清，将细胞以1×105个/ml接种于培养皿中，置于37℃，CO2浓度为5％的培养箱中静止培养。
(2)24h后首次换液，弃去悬浮细胞，随后每三天换一次液。当细胞融合度达85％时，胰酶消化传代培养，传至4代后，观察细胞生长状态，用胰酶消化后使用PBS缓冲溶液洗涤，人工计数后离心收集。
实施例4  外周血单个核细胞(Peripheral blood mononuclear cell,PBMC)的制备与收集
(1)采集10～80ml的外周血，将其与生理盐水按体积比1：1的比例混匀，将混匀后的血液混合液与淋巴细胞分离液按体积比2:1的比例缓慢地加至淋巴细胞分离液上层，700g离心20min；
(2)离心后从上到下看，分别为血浆、白膜层(即PBMC层)、淋巴细胞分离液、红细胞层，抽取PBMC层，将收集到的PBMC用生理盐水重悬后，500g离心10min，重复洗涤两次，收集到PBMC；
(3)将收集到的PBMC用RPMI1640基础培养基重悬，按1×106个/ml密度接种于培养瓶中，同时添加细胞生长因子IL-2 500U/ml和IL-1530ng/ml，置于37℃，浓度为5％的CO2培养箱中培养；
(4)诱导5天后，进行补液，补液前细胞密度不大于3×106个/ml，补液后细胞密度在0.5～1.0×106个/ml，然后每隔3天补液和补加细胞生长因子，直至第14天收集细胞。
实施例5  组合制剂的制备
将实施例1获得的脐带间充质干细胞传至第4代后，观察细胞生长状态，用胰酶消化后使用生理盐水洗涤，离心收集。最后把细胞沉淀按照所需回输剂量重悬于生理盐水和5％人血白蛋白制剂基质中，用70μm细胞筛网过滤后注入氯化钠注射液袋中。制剂制成后，3h内对新鲜制得的制剂进行检测，包括：干细胞形态、存活率、内毒素、细菌、真菌等检测。检测结果合格后，将制剂放置在无菌盒中低温运送至诊疗室用于治疗。
根据计数结果，将实施例4的外周血单个核细胞沉淀按照所需回输剂量重悬于生理盐水和5％人血白蛋白制剂基质中，用70μm细胞筛网过滤后注入氯化钠注射液袋中。制剂制成后，3h内对新鲜制得的制剂进行检测，包括：干细胞形态、存活率、内毒素、细菌、真菌等检测。检测结果合格后，将制剂放置在无菌盒中低温运送至诊疗室用于治疗。
脐带间充质干细胞混合细胞制剂的制备：将脐带间充质干细胞制剂和外周血单个核细胞制剂按照细胞数量1:1的比例充分混匀制成。
静脉注射液混合制剂中，脐带间充质干细胞的浓度为(1～5)×105个/mL(总输注体积为100mL，浓度根据回输剂量具体确定)，外周血单个核细胞的浓度为(1～5)×105个/mL(总输注体积为100mL，浓度根据回输剂量具体确定)；人血白蛋白为5wt％。脐带间充质干细胞与外周血单个核细胞比例为1:1。
皮下注射液混合制剂中，脐带间充质干细胞的浓度为2×106个/mL，外周血单个核细胞的浓度为2×106个/mL；人血白蛋白为5wt％。
实施例6  组合制剂的制备
根据计数结果，把实施例2的骨髓间充质干细胞沉淀按照所需回输剂量重悬于生理盐水和5％人血白蛋白制剂基质中，用70μm细胞筛网过滤后注入氯化钠注射液袋中。制剂制成后，3h内对新鲜制得的制剂进行检测，包括：干细胞形态、存活率、内毒素、细菌、真菌等检测。检测结果合格后，将制剂放置在无菌盒中低温运送至诊疗室用于治疗。
根据计数结果，将实施例4的外周血单个核细胞沉淀按照所需回输剂量重悬于生理盐水和5％人血白蛋白制剂基质中，用70μm细胞筛网过滤后注入氯化钠注射液袋中。制剂制成后，3h内对新鲜制得的制剂进行检测，包括：干细胞形态、存活率、内毒素、细菌、真菌等检测。检测结果合格后，将制剂放置在无菌盒中低温运送至诊疗室用于治疗。
骨髓间充质干细胞混合细胞制剂的制备：将骨髓间充质干细胞制剂和外周血单个核细胞制剂按照细胞数量1:1的比例充分混合制成。
静脉注射液混合制剂中，骨髓间充质干细胞的浓度为(1～5)×105个/mL(总输注体积为100mL，浓度根据回输剂量具体确定)，外周血单个核细胞的浓度为(1～5)×105个/mL(总输注体积为100mL，浓度根据回输剂量具体确定)；人血白蛋白为5wt％。骨髓间充质干细胞与外周血单个核细胞比例为1:1。
皮下注射液混合制剂中，骨髓间充质干细胞的浓度为2×106个/mL，外周血单个核细胞的浓度为2×106个/mL；人血白蛋白为5wt％。
实施例7  组合制剂的制备
根据计数结果，把实施例3的脂肪间充质干细胞沉淀按照所需回输剂量重悬于生理盐水和5％人血白蛋白制剂基质中，用70μm细胞筛网过滤后注入氯化钠注射液袋中。制剂制成后，3h内对新鲜制得的制剂进行检测，包括：干细胞形态、存活率、内毒素、细菌、真菌等检测。检测结果合格后，将制剂放置在无菌盒中低温运送至诊疗室用于治疗。
根据计数结果，将实施例4的外周血单个核细胞沉淀按照所需回输剂量重悬于生理盐水和5％人血白蛋白制剂基质中，用70μm细胞筛网过滤后注入氯化钠注射液袋中。制剂制成后，3h内对新鲜制得的制剂进行检测，包括：干细胞形态、存活率、内毒素、细菌、真菌等检测。检测结果合格后，将制剂放置在无菌盒中低温运送至诊疗室用于治疗。
脂肪干细胞混合细胞制剂的制备：将脂肪干细胞制剂和外周血单个核细胞制剂按照细胞数量1:1的比例混合制成。
静脉注射液混合制剂中，脂肪间充质干细胞的浓度为(1～5)×105个/mL(总输注体积为100mL，浓度根据回输剂量具体确定)，外周血单个核细胞的浓度为(1～5)×105个/mL(总输注体积为100mL，浓度根据回输剂量具体确定)；人血白蛋白为5wt％。脂肪间充质干细胞与外周血单个核细胞比例为1:1。
皮下注射液混合制剂中，脂肪间充质干细胞的浓度为2×106个/mL，外周血单个核细胞的浓度为2×106个/mL；人血白蛋白为5wt％。
试验例1  干细胞注射制剂静脉注射治疗缺血性糖尿病足的临床疗效
纳入160例糖尿病足受试者，男女各半，糖尿病病程7～20年。将受试者随机分为8组，其中一组为对照组，其余7组为实验组。对照组局部溃疡创面应用含有庆大霉素8～16万U、普通胰岛素4～8U、山莨菪碱注射液10mg和生理盐水的混合液涡流式冲洗创面，之后用无菌棉球擦干，盖以无菌纱布，每日一次。其余7组在应用常规治疗方案的同时，分别接受脐带MSC、骨髓MSC、脂肪MSC、PBMC及其实施例5-7的混合制剂细胞静脉注射治疗。静脉输注：按照体重均以5×105个/kg的标准将单一间充质干细胞细胞制剂及间充质干细胞与PBMC混合制剂沿静脉回输至患者体内。
具体分组如下：
表1  分组情况及处理措施
Figure PCTCN2017088012-appb-000001
Figure PCTCN2017088012-appb-000002
治疗后，4周后观察疗效，疗效评价标准为：
痊愈：4周内新生肉芽组织全部长全，创面完全愈合；
显效：4周内创面缩小范围于75％～100％之间，新生肉芽组织大部分长出，无炎性渗出液；
好转：4周内创面缩小＜75％，新生肉芽组织部分长出，炎性渗出液较少；
无效：4周内无创面缩小，无新生肉芽组织，炎性渗出液较多。
结果如下表2所示：
表2  各组对缺血性糖尿病足的疗效统计
组别 例数 痊愈 显效 好转 无效 有效率 治愈率
对照组 20 0 5 5 10 50％ 0％
实验组1 20 7 7 6 0 100％ 35％
实验组2 20 14 3 3 0 100％ 70％
实验组3 20 8 7 5 0 100％ 40％
实验组4 20 15 3 2 0 100％ 75％
实验组5 20 6 8 6 0 100％ 30％
实验组6 20 13 5 2 0 100％ 65％
实验组7 20 6 7 7 0 100％ 30％
结果显示：实验组的有效率和治愈率均显著高于对照组，差异具有统计学意义(P＜0.05)，而实验组1、3、5、7间没有统计学差异(P＞0.05)，说明单独使用干细胞制剂或PBMC制剂静脉注射治疗缺血性糖尿病足均能改善患者的症状，部分可达到治愈的疗效；而实验组2、4、6的效果显著好于干细胞与PBMC单独治疗的效果，差异具有统计学意义(P<0.05)，因此结果显示干细胞与PBMC组合使用产生了协同增效作用。
试验例2  干细胞注射制剂局部注射治疗缺血性糖尿病足的临床疗效
纳入160例糖尿病足受试者，男女各半，糖尿病病程7～20年。将受试者随机分为8组，其中一组为对照组，其余7组为实验组。对照组局部溃疡创面应用含有庆大霉素8～16万U、普通胰岛素4～8U、山莨菪碱注射液10mg和生理盐水的混合液涡流式冲洗创面，之后用无菌棉球擦干，盖以无菌纱布，每日一次。其余7组在应用常规治疗方案的同时，分别接受脐带MSC、骨髓MSC、脂肪MSC、PBMC及其实施例5-7的混合制剂细胞局部注射治疗。
局部注射：将含2×107个间充质干细胞的制剂(单一制剂、混合制剂)装入5mL注射器，缓慢注入患处，注射完毕后用无菌纱布按压针眼处3～5min，患者俯卧30min，期间每5min活动患处8～10次。
具体分组如下：
表3  分组情况及处理措施
Figure PCTCN2017088012-appb-000003
治疗后，4周后观察疗效，疗效评价标准为：
痊愈：4周内新生肉芽组织全部长全，创面完全愈合；
显效：4周内创面缩小范围于75％～100％之间，新生肉芽组织大部分长出，无炎性渗出液；
好转：4周内创面缩小＜75％，新生肉芽组织部分长出，炎性渗出液较少；
无效：4周内无创面缩小，无新生肉芽组织，炎性渗出液较多。
结果如下表4所示：
表4  各组对缺血性糖尿病足的疗效统计
组别 例数 痊愈 显效 好转 无效 有效率 治愈率
对照组 20 0 3 7 10 50％ 0％
实验组1 20 8 8 4 0 100％ 40％
实验组2 20 15 2 3 0 100％ 75％
实验组3 20 6 7 7 0 100％ 30％
实验组4 20 13 3 4 0 100％ 65％
实验组5 20 7 8 5 0 100％ 35％
实验组6 20 14 4 2 0 100％ 70％
实验组7 20 6 9 5 0 100％ 30％
结果显示：实验组的有效率和治愈率均显著高于对照组，差异具有统计学意义(P＜0.05)，而实验组1、3、5、7间没有统计学差异(P＞0.05)，说明单独使用干细胞制剂或PBMC制剂局部注射治疗缺血性糖尿病足均能改善患者的症状，部分可达到治愈的疗效；而实验组2、4、6的效果显著好于干细胞与PBMC单独治疗的效果，差异具有统计学意义(P<0.05)，因此结果显示干细胞与PBMC组合使用产生了协同增效作用。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种干细胞组合物，其特征在于，由间充质干细胞和外周血单个核细胞组成。
  2. 根据权利要求1所述的干细胞组合物，其特征在于，所述干细胞组合物中间充质干细胞与外周血单个核细胞的细胞数量比例为(1～5)：(1～5)。
  3. 根据权利要求1或2所述的干细胞组合物，其特征在于，所述干细胞组合物中间充质干细胞与外周血单个核细胞的细胞数量比例为1：1。
  4. 根据权利要求1所述的干细胞组合物，其特征在于，所述间充质干细胞为脐带间充质干细胞、骨髓间充质干细胞或脂肪间充质干细胞。
  5. 如权利要求1至4中任一项所述干细胞组合物在制备预防和/或治疗糖尿病足药物中的应用。
  6. 一种干细胞制剂，其特征在于，包括间充质干细胞、外周血单个核细胞、人血白蛋白和溶媒。
  7. 根据权利要求6所述的干细胞制剂，其特征在于，所述干细胞制剂的剂型为静脉注射剂或皮下注射剂。
  8. 根据权利要求7所述的干细胞制剂，其特征在于，所述干细胞制剂为静脉注射剂，各组分浓度为：
    间充质干细胞：(1～5)×105个/mL；
    外周血单个核细胞：(1～5)×105个/mL；
    人血白蛋白：5wt％；
    溶媒：补足。
  9. 根据权利要求7所述的干细胞制剂，其特征在于，所述干细胞制剂为皮下注射剂，各组分浓度为：
    间充质干细胞：(1～5)×106个/mL；
    外周血单个核细胞：(1～5)×106个/mL；
    人血白蛋白：5wt％；
    溶媒：补足。
  10. 根据权利要求6至9中任一项所述的干细胞制剂，其特征在于，所述溶媒为复方电解质注射液、葡萄糖注射液或生理盐水。
PCT/CN2017/088012 2017-06-13 2017-06-13 一种防治糖尿病足的干细胞组合物及其应用、干细胞制剂 WO2018227363A1 (zh)

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CN106661551A (zh) * 2014-06-06 2017-05-10 福源股份有限公司 由淋巴细胞产生多谱系潜能细胞的方法
CN106074602A (zh) * 2016-06-22 2016-11-09 深圳爱生再生医学科技有限公司 混合细胞制剂及其制备方法与应用

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