WO2020224197A1 - 治疗缺血性疾病的脐带动脉旁干细胞注射液及其制备方法 - Google Patents

治疗缺血性疾病的脐带动脉旁干细胞注射液及其制备方法 Download PDF

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WO2020224197A1
WO2020224197A1 PCT/CN2019/113020 CN2019113020W WO2020224197A1 WO 2020224197 A1 WO2020224197 A1 WO 2020224197A1 CN 2019113020 W CN2019113020 W CN 2019113020W WO 2020224197 A1 WO2020224197 A1 WO 2020224197A1
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artery
umbilical cord
para
stem cell
umbilical
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French (fr)
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丁利军
孙海翔
徐璐
李一凡
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南京鼓楼医院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
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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
    • A61K35/14—Blood; Artificial blood
    • A61K35/19—Platelets; Megacaryocytes
    • 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
    • A61K35/48—Reproductive organs
    • A61K35/51—Umbilical cord; Umbilical cord blood; Umbilical stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/16—Blood plasma; Blood serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

Definitions

  • the invention belongs to the field of medicine, and relates to an injection, in particular to an umbilical artery stem cell injection for treating ischemic diseases and a preparation method thereof.
  • Lower extremity ischemic disease is a common peripheral arterial disease, which refers to the stenosis or occlusion of lower extremity arteries and insufficient blood perfusion caused by various reasons, which leads to intermittent claudication, ulcers, gangrene and other ischemic manifestations of the lower extremities.
  • Class disease At present, there is no effective treatment and control method for the treatment of ischemic diseases of the lower extremities, and it is not enough to induce the formation of sufficient new blood vessels in a wide range of ischemic and hypoxic areas. Finding high-quality stem cell transplantation to improve the blood perfusion of ischemic lower limbs is an important issue to be solved urgently.
  • the present invention provides a paraumbilical artery stem cell injection for treating ischemic diseases and a preparation method thereof, so as to overcome the defects of the prior art.
  • the present invention provides a para-umbilical cord artery stem cell injection for the treatment of ischemic diseases, comprising suspended para-umbilical cord artery stem cells and a solvent.
  • the present invention provides a para-umbilical cord artery stem cell injection for the treatment of ischemic diseases, which may also have the characteristic that the solvent is platelet-rich cord plasma.
  • the present invention provides a para-umbilical cord artery stem cell injection for the treatment of ischemic diseases, which may also have the characteristic that the content of para-umbilical cord artery stem cells is 5 ⁇ 10 6 /mL.
  • 5 ⁇ 10 6 /mL means that 5 ⁇ 10 6 para-umbilical artery stem cells are suspended in 1 mL of solvent.
  • the present invention provides a paraumbilical artery stem cell injection for the treatment of ischemic diseases, which may also have the feature that the solvent is PBS for injection containing human serum albumin.
  • the present invention provides a para-umbilical cord artery stem cell injection for the treatment of ischemic diseases, which may also have the following characteristics: wherein the content of para-umbilical cord artery stem cells is 5 ⁇ 10 6 /mL, and the content of human serum albumin is 5% of the volume of PBS.
  • the present invention also provides a preparation method of the above-mentioned para-umbilical cord artery stem cell injection for the treatment of ischemic diseases.
  • the para-arterial stem cells are derived after culturing the umbilical cord artery, which is digested by enzymatic digestion to obtain a single para-umbilical cord artery stem cell. After washing twice with PBS, Gently mix with platelet-rich umbilical cord plasma to obtain para-umbilical cord artery stem cell injection.
  • the present invention provides a preparation method of para-umbilical cord artery stem cell injection for the treatment of ischemic diseases, which may also have the following characteristics: wherein the preparation process of para-umbilical cord artery stem cells is: taking fresh human umbilical cord after full-term birth.
  • the present invention provides a preparation method of paraumbilical cord artery stem cell injection for the treatment of ischemic diseases, which may also have the following characteristics: wherein the preparation method of platelet-rich umbilical cord plasma is: adding 750IU to a 50mL centrifuge tube Collect 40 mL of cord blood with heparin sodium; centrifuge at 200 g at low speed for 10-15 minutes at room temperature to collect the upper liquid; centrifuge the upper liquid again at high speed at 2000 g for 10 min, platelet sedimentation at the bottom of the centrifuge tube, discard part of the supernatant plasma, leave 200uL plasma and bottom sediment Platelets, gently resuspend to obtain platelet-rich umbilical cord plasma (PRP).
  • PRP platelet-rich umbilical cord plasma
  • the present invention provides a preparation method of para-umbilical cord artery stem cell injection for the treatment of ischemic diseases, which may also have the feature that the obtained single-cell suspension of para-umbilical cord artery stem cells does not contain calcium or magnesium. Wash 2 times with PBS, then resuspend the cell pellet with platelet-rich umbilical cord plasma and mix gently to prepare para-umbilical cord artery stem cell injection; store the para-umbilical cord artery stem cell injection at 4°C for a short time and use within 12 hours. Shake gently before use.
  • the present invention also provides a preparation method of the above-mentioned para-umbilical cord artery stem cell injection for the treatment of ischemic diseases.
  • the para-arterial stem cells are derived from umbilical cord artery tissue culture, and a single para-umbilical cord artery stem cell is obtained by enzymatic digestion, using PBS without calcium and magnesium Wash twice, then resuspend the cell pellet in PBS containing 5% human serum albumin and mix gently to prepare the para-umbilical cord artery stem cell injection; store the para-umbilical cord artery stem cell injection at 4°C for a short period of time and use within 12 hours , Shake gently before use.
  • the present invention provides a para-umbilical cord artery stem cell injection for the treatment of ischemic diseases and a preparation method thereof.
  • the para-umbilical cord artery stem cells can spontaneously form blood vessels or vasculature in vivo without the help of growth factors.
  • the bud-like or tube-like structure can also promote the formation of vascular endothelial cells in the body.
  • PRP platelet-rich umbilical cord plasma
  • Umbilical cord artery stem cells and platelet-rich umbilical cord plasma have a synergistic effect, and the effect is significant.
  • Transplantation of human para-umbilical artery stem cell injection resuspended in platelet-rich umbilical cord plasma for treatment of lower limb ischemia model mice significantly improved the blood supply of the ischemic lower limb.
  • Figure 1a shows the immunofluorescence staining of vWF, mCD31, and hCD31 of matrigel plugs of human para-arterial stem cells of human umbilical cord and Wharton’s gel-derived mesenchymal stem cells;
  • Figure 1b shows the quantitative statistics of vWF, mCD31, and hCD31 immunofluorescence staining of human umbilical artery para-arterial stem cells and matrigel plugs of the Wharton’s gel-derived mesenchymal stem cell group;
  • Figure 1c shows the mCD31 immunohistochemical staining of matrigel plugs of human umbilical artery stem cells and Wharton’s gel mesenchymal stem cells;
  • Figure 1d is a quantitative statistical diagram of mCD31 immunohistochemical staining of matrigel plugs of human umbilical artery stem cells and Wharton’s gel mesenchymal stem cell groups;
  • Figure 2a shows the mCD31 immunohistochemical staining of ischemic gastrocnemius in the human umbilical cord Wharton's jelly mesenchymal stem cell injection, para-arterial stem cell injection, and PBS transplantation group;
  • Figure 2b is a quantitative statistical diagram of mCD31 immunohistochemical staining of ischemic gastrocnemius in the human umbilical cord Wharton's glue-derived mesenchymal stem cell injection, para-arterial stem cell injection, and PBS transplantation group;
  • Figure 3a is a Doppler ultrasound blood flow chart of ischemic lower limbs of mice in the transplantation group of human umbilical cord Wharton's jelly mesenchymal stem cell injection and para-arterial stem cell injection;
  • Figure 3b shows the blood flow in the ischemic lower limbs of mice in the transplantation group of human umbilical cord Wharton's jelly mesenchymal stem cell injection and para-arterial stem cell injection.
  • An umbilical cord artery stem cell injection for the treatment of ischemic diseases comprising suspended umbilical cord artery stem cells and platelet-rich umbilical cord plasma.
  • the content of stem cells adjacent to the umbilical cord artery was 5 ⁇ 10 6 /mL.
  • para-arterial stem cells are derived from umbilical cord artery tissue culture, and a single para-umbilical cord artery stem cell is obtained by enzymatic digestion, and washed twice with calcium and magnesium-free PBS. Then the cell pellet is resuspended with platelet-rich umbilical cord plasma, and gently mixed to prepare the para-umbilical cord artery stem cell injection.
  • the para-umbilical artery stem cell injection is stored at 4°C for a short time, and used within 12 hours. Shake gently before use.
  • the preparation process of umbilical cord artery stem cells is: take fresh human umbilical cord 7-15 cm after full-term birth, and transport it to the laboratory on ice with PBS containing 10% blue chain double antibody and 1% heparin within 4 hours; Take out the umbilical cord from the clean bench and squeeze out the blood in the umbilical cord. After repeated washing with PBS until there is no blood and blood clots, scissors will trim the two sections, cut the umbilical cord along the long axis of the blood vessel in the umbilical cord, and bluntly separate the umbilical cord artery with forceps; The umbilical cord artery was cut into pieces of 1-2mm 3 in the direction perpendicular to the umbilical cord artery.
  • the tissue pieces were evenly spread on the bottom of a 100mm cell culture dish and placed in an incubator with 5% CO 2 and saturated humidity at 37°C for 3 hours; then slowly added Put 5ml of DMEM-LG medium into a 100mm dish and place it in an incubator lightly; add 3ml and 2ml of DMEM-LG medium on the 3rd and 7th day of the first week, and then change the medium twice in the second week. After that, the full amount of fluid was changed twice in the third week, during which the adherent cells crawling out of the tissue mass were observed; after 3 weeks, the umbilical artery tissue mass was removed when the cells grew to 80% fusion, 0.05% trypsin digestion, and subculture; P3- P5 generation cells are used for injection suspension preparation.
  • the preparation method of platelet-rich umbilical cord plasma is: add 750 IU heparin sodium to a 50 mL centrifuge tube to collect 40 mL of cord blood; centrifuge at 200 g at low speed for 10-15 min at room temperature to collect the upper liquid; centrifuge the upper liquid again at high speed at 2000 g for 10 min , Platelet deposition at the bottom of the centrifuge tube, discard part of the supernatant plasma, leave 200uL plasma and platelets deposited at the bottom, gently resuspend to obtain platelet-rich umbilical cord plasma (PRP).
  • PRP platelet-rich umbilical cord plasma
  • the application method of the paraumbilical artery stem cell injection for the treatment of ischemic diseases is: injection. Specifically, multiple injections in the gastrocnemius muscle.
  • mice are randomly grouped together.
  • mice in each litter are sorted according to their initial body weight.
  • the computer randomly generates 20 two-digit random numbers.
  • the size is sorted corresponding to the body weight.
  • 5 groups are randomly grouped, 4 mice in each group, and the weight of each group is calculated after grouping. There was no statistical difference between the means.
  • each group of 4 animals are kept in cages. Mark the group number, cage number and mark the animal serial number of each cage with picric acid. After the mice were grouped and adapted to the environment, they were reared for 1 week, and the living conditions and weight changes of the mice were observed.
  • mice in each group had gained satisfactory weight, appeared healthy and active, docile and non-irritating, and weighed an average of 20-30g, reaching 7-9 Weekly age, the average weight of each group has no significant difference. Meet the criteria for the selected experiment and enter the experiment.
  • mice were injected with a cell suspension subcutaneously along the groin on the ventral side, and divided into 4 groups: 1 Para-umbilical artery stem cell group: 200 ⁇ l Matrigel Matrigel+50 ⁇ l PBS containing 7.5 ⁇ 10 5 para-umbilical cord artery stem cells; 2Umbilical cord Wharton’s glue stem cell group: 200 ⁇ l Matrigel Matrigel+50 ⁇ l PBS containing 7.5 ⁇ 10 5 umbilical cord Wharton’s glue stem cells; 3Negative control group: 200 ⁇ l Matrigel Matrigel+50 ⁇ l PBS; 4Positive control group: 200 ⁇ l Matrigel Matrigel+50 ⁇ l PBS contains 37.5ng of bFGF.
  • 1 Para-umbilical artery stem cell group 200 ⁇ l Matrigel Matrigel+50 ⁇ l PBS containing 7.5 ⁇ 10 5 para-umbilical cord artery stem cells
  • 2Umbilical cord Wharton’s glue stem cell group 200 ⁇ l Matrigel Matrigel
  • mice were sacrificed, and the subcutaneous matrigel plugs of each group were taken out.
  • the frozen block After the frozen block is made, it can be placed in a cryostat to freeze section.
  • the thermostat microtome (Leica) knife chamber is set to -25°C, and the machine chamber is set to -20°C.
  • the position and angle of the anti-roll plate should be placed in an appropriate place.
  • the slide is attached to the tissue section, and it should not be moved up and down.
  • the sections are placed at room temperature for 30 minutes, they are placed at 4°C and fixed in acetone for 5-10 minutes. Wash with PBS 5min ⁇ 3 times.
  • the frozen sections were dried at room temperature for 15 minutes, and the sections could be sealed at room temperature with PBS containing 10% normal goat serum for 1 hour.
  • Drop the primary antibody or primary antibody working solution mCD31, Abcam; hCD31, BD; vWF, Abcam
  • DAPI working solution was added dropwise to stain the nucleus at room temperature for 10-20 minutes (working concentration 0.1% staining for 15 minutes).
  • Recover DAPI add 5-10 ⁇ l of anti-fluorescence attenuation mounting tablets or neutral gum, and mount the coverslips with cleaned covers, then you can take pictures under a fluorescence microscope or a confocal microscope.
  • the prepared slices are placed in a slicing box, placed in a refrigerator at 4°C, and can be stored for about a week.
  • Figure 1a shows the vWF, mCD31, and hCD31 immunofluorescence staining images of the matrigel plugs of the human umbilical artery para-arterial stem cells and Wharton’s gel-derived mesenchymal stem cell group.
  • Figure 1c shows the mCD31 immunohistochemical staining of matrigel plugs of human umbilical artery stem cells and Wharton’s gel mesenchymal stem cell group
  • Figure 1d shows human umbilical artery stem cells and Wharton’s gel Quantitative statistics of mCD31 immunohistochemical staining of matrigel plugs in the mesenchymal stem cell group.
  • Umbilical cord Wharton's gel mesenchymal stem cell group It is suggested that the ability of human umbilical artery stem cells to vascularize in vivo and to promote vascularization of mouse endothelial cells is significantly better than that of Warton's glue-derived mesenchymal stem cells.
  • mice The lower limb ischemia model experiment in mice is performed on the paraumbilical artery stem cell injection for the treatment of ischemic diseases:
  • mice are 6-8 weeks old and are kept in the IVC animal room of the Animal Experiment Center, Gulou Hospital, Nanjing University School of Medicine, at a room temperature of 22°C, with a 12-hour light/dark light cycle (06: 00-18: 00) , Free drinking and eating.
  • mice After weighing the mice with a tray scale, they were anesthetized by intraperitoneal injection of 0.3 ml/kg of 1% chloral hydrate. After the mice were anesthetized, they were placed in the supine position, and the mice were fixed on the surgical board with rubber bands. Routine skin preparation, iodophor disinfection, and sterile hole towels. Use ophthalmic forceps to gently lift the skin, and cut a longitudinal incision with ophthalmic scissors from the groin to the inner thigh along the direction of the blood vessel, about 5mm in length.
  • mice Four weeks after modeling, the nude mice were randomly divided into three groups: PBS group, PRP group containing umbilical cord Wharton’s gel stem cells (umbilical cord Wharton’s gel stem cell injection), and PRP group containing umbilical cord arterial stem cells (by umbilical cord artery) Stem cell injection), the sham operation group served as a control.
  • the ischemic lower limb gastrocnemius muscle tissue of nude mice was fixed with 4% paraformaldehyde.
  • the ischemic lower limbs of nude mice were sliced continuously with a thickness of 5 ⁇ m, and one of every 5 slices was selected for HE staining. One section was stained by immunohistochemistry. Each slice was randomly selected from 5 high-powered fields (100 ⁇ ) to count the number of blood vessels stained brown by CD31 antibody. Any vascular endothelial cells or clusters of vascular endothelial cells stained brown are counted as one blood vessel. Lumen>8 red blood cells, blood vessels with thicker muscle layers were not counted.
  • the laser Doppler detects the blood flow index.
  • Figure 2a shows the mCD31 immunohistochemical staining of ischemic gastrocnemius in the human umbilical cord Wharton's jelly mesenchymal stem cell injection, paraarterial stem cell injection, and PBS transplantation group.
  • Figure 2b is a quantitative statistical graph of mCD31 immunohistochemical staining of ischemic gastrocnemius muscle in the human umbilical cord Wharton's jelly mesenchymal stem cell injection, para-arterial stem cell injection, and PBS transplantation group (the number of vascular rings per high-power field of view & each high-powered field of view Number of lower blood vessel rings/number of muscle fibers).
  • the mCD31 immunohistochemical results showed the deficiency of the human umbilical artery stem cell injection group
  • the number of new blood vessels in the lower limbs was more than that in the umbilical cord Watong's glue injection group and the PBS group.
  • the data quantification results show the number of blood vessel rings per high power field and the number of blood vessel rings/muscle fiber per high power field. It was significantly higher than that in the umbilical cord Wharton’s gel-derived mesenchymal stem cell injection group, suggesting that the PRP injection of human umbilical cord artery stem cells significantly improved the angiogenesis of ischemic lower limbs in mice.
  • Figure 3a is a Doppler ultrasound blood flow chart of ischemic lower limbs of mice in the transplantation group of human umbilical cord Wharton's jelly mesenchymal stem cell injection and para-arterial stem cell injection.
  • Figure 3b shows the blood flow in the ischemic lower limbs of mice in the transplantation group of human umbilical cord Wharton's jelly mesenchymal stem cell injection and para-arterial stem cell injection.
  • An umbilical cord artery stem cell injection for treating ischemic diseases comprising suspended umbilical cord artery stem cells and PBS for injection containing human serum albumin.
  • the content of stem cells adjacent to the umbilical cord artery is 5 ⁇ 10 6 /mL
  • the content of human serum albumin is 5% of the volume of PBS.
  • para-arterial stem cells are derived from umbilical cord artery tissue culture, and a single para-umbilical cord artery stem cell is obtained by enzymatic digestion, and washed twice with calcium and magnesium-free PBS. Then the cell pellet was resuspended in PBS containing 5% human serum albumin and mixed gently to prepare the para-umbilical cord artery stem cell injection; the para-umbilical cord artery stem cell injection was stored at 4°C for a short period of time and used within 12 hours. Shake gently.

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Abstract

一种治疗缺血性疾病的脐带动脉旁干细胞注射液及其制备方法，包括悬浮的脐带动脉旁干细胞和溶剂。溶剂可以为富含血小板的脐带血浆或含有5%人血清白蛋白的注射用PBS。其制备方法为脐带动脉组织培养后衍生出动脉旁干细胞，酶解法消化获得单个所过脐带动脉旁干细胞，经PBS洗涤两次后，再与溶剂轻轻混匀，获得脐带动脉旁干细胞注射液。

Description

治疗缺血性疾病的脐带动脉旁干细胞注射液及其制备方法 技术领域
本发明属于药物领域，涉及一种注射液，尤其涉及一种治疗缺血性疾病的脐带动脉旁干细胞注射液及其制备方法。
背景技术
下肢缺血性疾病是一种常见的外周动脉疾病，是指由各种原因导致的下肢动脉狭窄或闭塞、血流灌注不足，从而导致下肢出现间歇性跛行、溃疡、坏疽等缺血表现的一类疾病。目前下肢缺血性疾病的治疗尚未有有效的治疗和控制方法，不足以在广泛的缺血缺氧区域内诱导足够的新生血管的构建。寻找优质的干细胞移植改善缺血下肢的血流灌注是亟待解决的重要问题。
目前，人脐带间充质干细胞的研究方面尚有缺陷。主要包括：(1)目前临床应用的主要是脐带华通氏胶间充质干细胞/脐带干细胞混合物，特性不明确，细胞表面CD146表达量低，其血管生成能力较弱。(2)下肢缺血性疾病患者的康复有赖于充分的血供重建，人脐带华通氏胶间充质干细胞在下肢缺血性疾病中的应用疗效不佳。
发明内容
本发明提供一种治疗缺血性疾病的脐带动脉旁干细胞注射液及其制备方法，以克服现有技术的缺陷。
为实现上述目的，本发明提供一种治疗缺血性疾病的脐带动脉旁干细胞注射液，包括悬浮的脐带动脉旁干细胞和溶剂。
进一步，本发明提供一种治疗缺血性疾病的脐带动脉旁干细胞注射液，还可以具有这样的特征：其中，溶剂为富含血小板的脐带血浆。
进一步，本发明提供一种治疗缺血性疾病的脐带动脉旁干细胞注射液，还可以具有这样的特征：其中，脐带动脉旁干细胞的含量为5×10 6/mL。其中，5×10 6/mL指，1mL溶剂中悬浮5×10 6个脐带动脉旁干细胞。
进一步，本发明提供一种治疗缺血性疾病的脐带动脉旁干细胞注射液，还可以具有这样的特征：其中，溶剂为含人血清白蛋白的注射用PBS。
进一步，本发明提供一种治疗缺血性疾病的脐带动脉旁干细胞注射液，还可以具有这样 的特征：其中，脐带动脉旁干细胞的含量为5×10 6/mL，人血清白蛋白的含量为PBS体积的5％。
本发明还提供上述治疗缺血性疾病的脐带动脉旁干细胞注射液的制备方法，脐带动脉组织培养后衍生出动脉旁干细胞，酶解法消化获得单个脐带动脉旁干细胞，经PBS洗涤两次后，再与富含血小板的脐带血浆轻轻混匀，获得脐带动脉旁干细胞注射液。
进一步，本发明提供一种治疗缺血性疾病的脐带动脉旁干细胞注射液的制备方法，还可以具有这样的特征：其中，脐带动脉旁干细胞的制备过程为：足月生产后取新鲜人脐带7-15cm，4h内用含有10％青链双抗和1％肝素的PBS冰上4℃运至实验室；在超净工作台内取出脐带，挤出脐带中的血，PBS反复冲洗直至无血液和血块后，剪刀修齐两断面，沿脐带内血管长轴方向剪开脐带，用镊子钝性分离脐带动脉；将脐带动脉沿与脐带动脉垂直方向剪碎至1-2mm 3大小组织块，组织块均匀铺于100mm细胞培养皿底，放置于5％CO 2、37℃饱和湿度的培养箱内3h；然后缓慢加入5ml DMEM-LG培养基至100mm皿中，轻放置于培养箱中培养；第一周第3天和第7天各加DMEM-LG培养基3ml、2ml，之后第二周半量换液2次，之后第三周全量换液2次，期间观察组织块周围贴壁细胞爬出情况；3周后细胞长到80％融合时去除脐带动脉组织块，0.05％胰酶消化，传代培养；取P3-P5代细胞用于注射悬液制备。
进一步，本发明提供一种治疗缺血性疾病的脐带动脉旁干细胞注射液的制备方法，还可以具有这样的特征：其中，富含血小板的脐带血浆的制备方法为：在50mL离心管中加入750IU肝素钠收集脐带血40mL；在室温下200g低速离心10-15min，收集上层液体；对该上层液体再次2000g高速离心10min，血小板沉积离心管底部，弃去部分上清血浆，留200uL血浆及底部沉积的血小板，轻轻重悬获得富含血小板的脐带血浆(PRP)。
进一步，本发明提供一种治疗缺血性疾病的脐带动脉旁干细胞注射液的制备方法，还可以具有这样的特征：其中，获得的脐带动脉旁干细胞的单细胞悬液，使用不含钙、镁的PBS洗涤2次，然后细胞沉淀采用富含血小板的脐带血浆加以重悬，轻轻混匀制成脐带动脉旁干细胞注射液；脐带动脉旁干细胞注射液放于4℃短暂储存，12h内使用，使用前轻轻摇匀。
本发明还提供上述治疗缺血性疾病的脐带动脉旁干细胞注射液的制备方法，脐带动脉组织培养后衍生出动脉旁干细胞，酶解法消化获得单个脐带动脉旁干细胞，使用不含钙、镁的PBS洗涤两次，然后细胞沉淀采用含有5％人血清白蛋白的PBS加以重悬，轻轻混匀制成脐带动脉旁干细胞注射液；脐带动脉旁干细胞注射液放于4℃短暂储存，12h内使用，使用前轻轻摇匀。
本发明的有益效果在于：本发明提供一种治疗缺血性疾病的脐带动脉旁干细胞注射液及 其制备方法，脐带动脉旁干细胞不需要借助生长因子，能够体内自发形成类似于血管或脉管系统的芽样或管样结构，同时可以促进血管内皮细胞在体内形成血管样结构。此外，富含血小板脐带血浆(PRP)中存在多种血管生成因子，参与新生血管的生成；PRP中还含有众多能促进干细胞增殖、分化、迁移、黏附的细胞因子和蛋白质。脐带动脉旁干细胞与富含血小板脐带血浆协同作用，效果显著。重悬于富含血小板的脐带血浆的人脐带动脉旁干细胞注射液移植治疗下肢缺血模型小鼠，其缺血下肢血流供应显著改善。
附图说明
图1a为人脐带动脉旁干细胞与华通氏胶间充质干细胞组matrigel plugs的vWF、mCD31、hCD31免疫荧光染色图；
图1b为人脐带动脉旁干细胞与华通氏胶间充质干细胞组matrigel plugs的vWF、mCD31、hCD31免疫荧光染色量化统计图；
图1c为人脐带动脉旁干细胞与华通氏胶间充质干细胞组matrigel plugs的mCD31免疫组化染色；
图1d为人脐带动脉旁干细胞与华通氏胶间充质干细胞组matrigel plugs的mCD31免疫组化染色量化统计图；
图2a为人脐带华通氏胶间充质干细胞注射液、动脉旁干细胞注射液、PBS移植组缺血腓肠肌的mCD31免疫组化染色；
图2b为人脐带华通氏胶间充质干细胞注射液、动脉旁干细胞注射液、PBS移植组缺血腓肠肌的mCD31免疫组化染色量化统计图；
图3a为人脐带华通氏胶间充质干细胞注射液、动脉旁干细胞注射液移植组小鼠缺血下肢的多普勒超声血流图；
图3b为人脐带华通氏胶间充质干细胞注射液、动脉旁干细胞注射液移植组小鼠缺血下肢的血流量化。
具体实施方式
以下结合具体实施例对本发明作进一步说明。
实施例1
一种治疗缺血性疾病的脐带动脉旁干细胞注射液，包括悬浮的脐带动脉旁干细胞和富含血小板的脐带血浆。其中，脐带动脉旁干细胞的含量为5×10 6/mL。
治疗缺血性疾病的脐带动脉旁干细胞注射液的制备方法为：脐带动脉组织培养后衍生出动脉旁干细胞，酶解法消化获得单个脐带动脉旁干细胞，使用不含钙、镁的PBS洗涤2次，然后细胞沉淀采用富含血小板的脐带血浆加以重悬，轻轻混匀制成脐带动脉旁干细胞注射液。脐带动脉旁干细胞注射液放于4℃短暂储存，12h内使用，使用前轻轻摇匀。
其中，脐带动脉旁干细胞的制备过程为：足月生产后取新鲜人脐带7-15cm，4h内用含有10％青链双抗和1％肝素的PBS冰上4℃运至实验室；在超净工作台内取出脐带，挤出脐带中的血，PBS反复冲洗直至无血液和血块后，剪刀修齐两断面，沿脐带内血管长轴方向剪开脐带，用镊子钝性分离脐带动脉；将脐带动脉沿与脐带动脉垂直方向剪碎至1-2mm 3大小组织块，组织块均匀铺于100mm细胞培养皿底，放置于5％CO 2、37℃饱和湿度的培养箱内3h；然后缓慢加入5ml DMEM-LG培养基至100mm皿中，轻放置于培养箱中培养；第一周第3天和第7天各加DMEM-LG培养基3ml、2ml，之后第二周半量换液2次，之后第三周全量换液2次，期间观察组织块周围贴壁细胞爬出情况；3周后细胞长到80％融合时去除脐带动脉组织块，0.05％胰酶消化，传代培养；取P3-P5代细胞用于注射悬液制备。
其中，富含血小板的脐带血浆的制备方法为：在50mL离心管中加入750IU肝素钠收集脐带血40mL；在室温下200g低速离心10-15min，收集上层液体；对该上层液体再次2000g高速离心10min，血小板沉积离心管底部，弃去部分上清血浆，留200uL血浆及底部沉积的血小板，轻轻重悬获得富含血小板的脐带血浆(PRP)。
治疗缺血性疾病的脐带动脉旁干细胞注射液的应用方法为：注射用。具体的，于腓肠肌内多点注射。
对脐带动脉旁干细胞进行体内基质胶塞实验：
1、BALB/C免疫缺陷鼠随机分组
各窝小鼠共20只按初始体重大小排序，由计算机随机产生20个两位数随机数字，大小排序与体重相对应，随机分组、产生5组，每组4只，分组后统计各组体重均数间无统计学差异。按饲养密度要求每组4只分笼饲养。标记组号、笼号并用苦昧酸标记每笼动物序号。将小鼠分组后适应环境饲养1周，观察小鼠生活状态，体重变化；1周后各组小鼠体重增长满意，外观健康活跃，温顺无激惹，体重平均20-30g，达7-9周龄，每组平均体重无显著性差异。符合入选实验标准，进入实验。
2、体内成血管模型的建立
小鼠麻醉后于腹侧沿腹股沟下方皮下注入细胞混悬液，分成4组，分别为：①脐带动脉旁干细胞组：200μl Matrigel基质胶+50μl PBS内含7.5×10 5个脐带动脉旁干细胞；②脐 带华通氏胶干细胞组：200μl Matrigel基质胶+50μl PBS内含7.5×10 5个脐带华通氏胶干细胞；③阴性对照组：200μl Matrigel基质胶+50μl PBS；④阳性对照组：200μl Matrigel基质胶+50μl PBS内含bFGF 37.5ng。
3、冰冻切片免疫荧光染色
14d后处死小鼠，取出各组皮下的matrigel plugs。将组织块平放于软塑小盒内(直径约2cm)，可适量加OCT包埋剂浸没组织，然后将特制小盒缓缓平放入盛有液氮的小杯内，当盒底部接触液氮时保持小盒原位。切勿进入液氮中，约10-20s组织冰结成块。制成冻块后即可置入恒冷箱切片机冰冻切片。恒温切片机(Leica)刀室设置为-25℃、机室设置为-20℃。抗卷板的位置及角度放置适当位置，载玻片附贴组织切片，切勿上下移动。
切片室温放置30min后，放至4℃丙酮固定5-10min。PBS洗5min×3次。冰冻切片室温晾干15min，可用含10％正常山羊血清的PBS室温封闭切片1小时。滴加适当比例稀释的一抗或一抗工作液(mCD31，Abcam；hCD31，BD；vWF，Abcam)，均匀覆盖组织面，且保证整个过程中不会使组织干涸。将切片放在加了PBS的免疫组化湿盒，室温孵育2小时或4℃过夜。第二天先将湿盒放到37℃回温1h，然后吸取片上的一抗进行回收，将切片插入到小染缸PBS冲洗。滴加用PBS稀释好的二抗Alexa Fluor 488-conjugated goatanti-rabbit IgG(1∶200，Invitrogen)/Alexa Fluor 555-conjugated goat anti-rabbit IgG(1∶200，Invitrogen)(避光)置于烘箱中37℃孵育1小时。切片置于染缸内，PBS洗5min×3次。滴加DAPI工作液染核，室温10-20min(工作浓度0.1％染色15min)。回收DAPI，滴加5-10μl抗荧光衰减封片剂或中性树胶，采用处理干净的盖玻片封片，即可到荧光显微镜或者共聚焦显微镜下观察拍照。做好的片放在切片盒内，置于4℃冰箱，可保存一周左右。
图1a为人脐带动脉旁干细胞与华通氏胶间充质干细胞组matrigel plugs的vWF、mCD31、hCD31免疫荧光染色图，图1b为人脐带动脉旁干细胞与华通氏胶间充质干细胞组matrigel plugs的vWF、mCD31、hCD31免疫荧光染色量化统计图，图1c为人脐带动脉旁干细胞与华通氏胶间充质干细胞组matrigel plugs的mCD31免疫组化染色，图1d为人脐带动脉旁干细胞与华通氏胶间充质干细胞组matrigel plugs的mCD31免疫组化染色量化统计图。
关于人脐带动脉旁干细胞、脐带华通氏胶干细胞体内自成环及促成环能力的比较，如图1a、1b和1c所示，vWF、mCD31、hCD31免疫荧光结果显示人脐带动脉旁干细胞复合Matrigel基质胶移植至免疫缺陷小鼠腹股沟皮下后，胶块内新生血管数多于华通氏胶间充质干细胞，如图1d所示，数据量化结果显示其每高倍镜荧光强度亦显著高于人脐带华通氏胶间充质干细胞组。提示人脐带动脉旁干细胞体内自成血管及促小鼠内皮细胞成血管能力显著优于华通氏 胶间充质干细胞。
对治疗缺血性疾病的脐带动脉旁干细胞注射液进行小鼠的下肢缺血模型实验：
1、实验动物
BALB/C裸鼠雄鼠为6-8周，饲养于南京大学医学院附属鼓楼医院动物实验中心IVC动物房，室温22℃，采用12小时明/暗光照周期(06：00-18：00)，自由饮水和进食。
2、模型构建
用托盘称对小鼠称重以后，按照0.3ml/kg的1％水合氯醛腹腔注射麻醉。小鼠被麻醉后，置仰卧位，用橡皮筋将小鼠固定于手术板上。常规备皮，碘伏消毒，铺无菌洞巾。使用眼科镊轻轻提起皮肤，从腹股沟到大腿内侧沿着血管走向用眼科剪剪开一纵形切口，长约5mm。在20倍的解剖显微镜视野下使用尖镊轻轻地刺破膜状血管鞘，暴露股动脉、静脉及其分支。用7号线结扎股总动脉远端及其各分支。
3、实验分组
裸鼠建模完毕后四周，随机分为三组：PBS组，含有脐带华通氏胶干细胞的PRP组(脐带华通氏胶干细胞注射液)，含有脐带动脉旁干细胞的PRP组(脐带动脉旁干细胞注射液)，假手术组作为对照。随机选取缺血下肢腓肠肌5个点，PBS组注射PBS 100μl；含有脐带华通氏胶干细胞的PRP组注射混有5×106脐带华通氏胶干细胞的PRP悬液100μl；含有脐带动脉旁干细胞的PRP组注射混有5×106脐带动脉旁干细胞的PRP悬液100μl。裸鼠手术完毕后饲养在IVC动物房中。
4、免疫组化微血管密度计数
裸鼠缺血下肢腓肠肌肌肉组织，4％多聚甲醛固定。裸鼠缺血下肢以5μm厚度连续切片，每5张切片选1张行HE染色。1张切片行免疫组化染色。每张切片随机选取5个高倍镜视野(100×)计数，被CD31抗体染成棕色的血管数目，任何染成棕色的血管内皮细胞或血管内皮细胞簇均为一个血管计数。管腔＞8个红细胞，具有较厚肌层的血管不计数。
5、激光多普勒检测血流变化
小鼠麻醉成功后，俯卧位于激光多普勒仪专用灰色软垫上，使用双面胶固定其双足使其足底面朝上且左右对称，在小鼠正上方调整扫描探头的位置，距小鼠大约15cm，激光多普勒仪检测血流指数。
图2a为人脐带华通氏胶间充质干细胞注射液、动脉旁干细胞注射液、PBS移植组缺血腓肠肌的mCD31免疫组化染色。图2b为人脐带华通氏胶间充质干细胞注射液、动脉旁干细胞注射液、PBS移植组缺血腓肠肌的mCD31免疫组化染色量化统计图(每高倍镜视野下血管环数& 每高倍镜视野下血管环数/肌纤维数)。
关于人脐带动脉旁干细胞注射液、脐带华通氏胶干细胞注射液移植后小鼠腓肠肌新生血管数量的比较，如图2a所示，mCD31免疫组化结果显示人脐带动脉旁干细胞注射液组的缺血下肢新生血管数目多于脐带华通氏胶注射液组和PBS组，如图2b所示，数据量化结果显示其每高倍镜视野下血管环数和每高倍镜视野下血管环数/肌纤维数显著高于脐带华通氏胶间充质干细胞注射液组，提示人脐带动脉旁干细胞PRP注射液显著改善小鼠缺血下肢的血管新生。
图3a为人脐带华通氏胶间充质干细胞注射液、动脉旁干细胞注射液移植组小鼠缺血下肢的多普勒超声血流图。图3b为人脐带华通氏胶间充质干细胞注射液、动脉旁干细胞注射液移植组小鼠缺血下肢的血流量化。
关于人脐带动脉旁干细胞注射液、脐带华通氏胶干细胞注射液移植后小鼠腓肠肌血流量的比较，如图3a和3b所示，超声多普勒血流图及数据量化结果显示人脐带动脉旁干细胞注射液组的缺血下肢血流丰度显著高于脐带华通氏胶注射液组和PBS组，提示人脐带动脉旁干细胞注射液改善小鼠缺血下肢血供能力显著优于华通氏胶间充质干细胞注射液。
实施例2
一种治疗缺血性疾病的脐带动脉旁干细胞注射液，包括悬浮的脐带动脉旁干细胞、含人血清白蛋白的注射用PBS。其中，所述脐带动脉旁干细胞的含量为5×10 6/mL，人血清白蛋白的含量为PBS体积的5％。
治疗缺血性疾病的脐带动脉旁干细胞注射液的制备方法为：脐带动脉组织培养后衍生出动脉旁干细胞，酶解法消化获得单个脐带动脉旁干细胞，使用不含钙、镁的PBS洗涤两次，然后细胞沉淀采用含有5％人血清白蛋白的PBS加以重悬，轻轻混匀制成脐带动脉旁干细胞注射液；脐带动脉旁干细胞注射液放于4℃短暂储存，12h内使用，使用前轻轻摇匀。

Claims (10)

  1. 一种治疗缺血性疾病的脐带动脉旁干细胞注射液，其特征在于：
    包括悬浮的脐带动脉旁干细胞和溶剂。
  2. 根据权利要求1所述的治疗缺血性疾病的脐带动脉旁干细胞注射液，其特征在于：
    其中，所述溶剂为富含血小板的脐带血浆。
  3. 根据权利要求2所述的治疗缺血性疾病的脐带动脉旁干细胞注射液，其特征在于：
    其中，所述脐带动脉旁干细胞的含量为5×10 6/mL。
  4. 根据权利要求1所述的治疗缺血性疾病的脐带动脉旁干细胞注射液，其特征在于：
    其中，所述溶剂为含有人血清白蛋白的注射用PBS。
  5. 根据权利要求4所述的治疗缺血性疾病的脐带动脉旁干细胞注射液，其特征在于：
    其中，所述脐带动脉旁干细胞的含量为5×10 6/mL；
    人血清白蛋白的含量为PBS体积的5％。
  6. 如权利要求2所述的治疗缺血性疾病的脐带动脉旁干细胞注射液的制备方法，其特征在于：
    脐带动脉组织培养后衍生出动脉旁干细胞，酶解法消化获得单个所述脐带动脉旁干细胞，经PBS洗涤两次后，再与所述富含血小板的脐带血浆轻轻混匀，获得脐带动脉旁干细胞注射液。
  7. 根据权利要求6所述的治疗缺血性疾病的脐带动脉旁干细胞注射液，其特征在于：
    其中，所述脐带动脉旁干细胞的制备过程为：
    足月生产后取新鲜人脐带7-15cm，4h内用含有10％青链双抗和1％肝素的PBS冰上4℃运至实验室；
    在超净工作台内取出脐带，挤出脐带中的血，PBS反复冲洗直至无血液和血块后，剪刀修齐两断面，沿脐带内血管长轴方向剪开脐带，用镊子钝性分离脐带动脉；
    将脐带动脉沿与脐带动脉垂直方向剪碎至1-2mm 3大小组织块，组织块均匀铺于100mm细胞培养皿底，放置于5％CO 2、37℃饱和湿度的培养箱内3h；
    然后缓慢加入5ml DMEM-LG培养基至100mm皿中，轻放置于培养箱中培养；
    第一周第3天和第7天各加DMEM-LG培养基3ml、2ml，之后第二周半量换液2次，之后第三周全量换液2次，期间观察组织块周围贴壁细胞爬出情况；
    3周后细胞长到80％融合时去除脐带动脉组织块，0.05％胰酶消化，传代培养；
    取P3-P5代细胞用于注射悬液制备。
  8. 根据权利要求6所述的治疗缺血性疾病的脐带动脉旁干细胞注射液，其特征在于：
    其中，所述富含血小板的脐带血浆的制备方法为：
    在50mL离心管中加入750IU肝素钠收集脐带血40mL；
    在室温下200g低速离心10-15min，收集上层液体；
    对所述上层液体再次2000g高速离心10min，血小板沉积离心管底部，弃去部分上清血浆，留200uL血浆及底部沉积的血小板，轻轻重悬获得富含血小板的脐带血浆。
  9. 根据权利要求6所述的治疗缺血性疾病的脐带动脉旁干细胞注射液，其特征在于：
    其中，获得的脐带动脉旁干细胞的单细胞悬液，使用不含钙、镁的PBS洗涤2次，然后细胞沉淀采用所述富含血小板的脐带血浆加以重悬，轻轻混匀制成脐带动脉旁干细胞注射液；
    所述脐带动脉旁干细胞注射液放于4℃短暂储存，12h内使用，使用前轻轻摇匀。
  10. 如权利要求4所述的治疗缺血性疾病的脐带动脉旁干细胞注射液的制备方法，其特征在于：
    脐带动脉组织培养后衍生出动脉旁干细胞，酶解法消化获得单个所述脐带动脉旁干细胞，使用不含钙、镁的PBS洗涤两次，然后细胞沉淀采用含有5％人血清白蛋白的PBS加以重悬，轻轻混匀制成脐带动脉旁干细胞注射液；
    所述脐带动脉旁干细胞注射液放于4℃短暂储存，12h内使用，使用前轻轻摇匀。
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