WO2018103406A1 - 一种治疗脑部损伤类疾病的神经干细胞注射液及其制备方法和使用方法 - Google Patents

一种治疗脑部损伤类疾病的神经干细胞注射液及其制备方法和使用方法 Download PDF

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WO2018103406A1
WO2018103406A1 PCT/CN2017/101994 CN2017101994W WO2018103406A1 WO 2018103406 A1 WO2018103406 A1 WO 2018103406A1 CN 2017101994 W CN2017101994 W CN 2017101994W WO 2018103406 A1 WO2018103406 A1 WO 2018103406A1
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neural stem
cells
stem cell
cell injection
treating
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French (fr)
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郑佳威
赵雄飞
黄倩莹
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上海安集协康生物技术股份有限公司
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    • 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
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
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    • 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
    • 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
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    • A—HUMAN NECESSITIES
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    • 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
    • A61K9/0085—Brain, e.g. brain implants; Spinal cord
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/23—Interleukins [IL]
    • C12N2501/235—Leukemia inhibitory factor [LIF]

Definitions

  • the invention relates to the field of biomedicine, in particular to a neural stem cell injection solution for treating brain damage diseases, a preparation method thereof and a using method thereof.
  • Pediatric cerebral palsy usually refers to central dyskinesia caused by non-progressive brain injury or brain development abnormalities caused by various causes within one month after birth. Clinically characterized by abnormal posture and muscle tone, muscle weakness, involuntary movement and ataxia, often accompanied by sensation, cognition, communication, behavior and other disorders and secondary skeletal muscle abnormalities, and may have seizures, The disease currently has no ideal treatment, and the disability rate is high, which causes the mental burden and economic burden on the patient and the family.
  • mice nerve growth factor for injection is a biological preparation approved by the SFDA. It is extracted from the submandibular gland of the mouse and has 90% homology with humans. It is administered by intramuscular injection. It is reported that the muscle tension caused by cerebral palsy in children. Symptoms such as motor development, abnormal posture, and abnormal reflection have certain improvement effects.
  • Brain injury mainly includes stroke and traumatic brain injury.
  • NSC neural stem cells
  • a large number of preclinical experiments at home and abroad have shown that neural stem cells (NSC) have certain therapeutic effects on stroke and traumatic brain injury. And some clinical trials have shown that NSC transplantation can treat brain damage.
  • NSCs Neural stem cells
  • NSCs are a group of relatively primitive, self-renewing cells with diverse differentiation potential that can differentiate into neurons, oligodendrocytes, and astrocytes. Under physiological conditions, NSC in the body usually remains at rest; after nerve injury, endogenous NSC can be activated, migrated and differentiated due to changes in the microenvironment to replace damaged cells and reconstruct neural circuits; after genetic modification Exogenous NSC transplantation shows great therapeutic potential and provides a new means for the treatment of brain injury.
  • the object of the present invention is to overcome the deficiencies of the prior art described above, a neural stem cell injection for treating diseases of brain damage, a preparation method thereof and a use method thereof.
  • the invention relates to a method for preparing a neural stem cell injection for treating a brain injury-like disease, the method comprising the following steps:
  • S1 Primary cell culture: the primary neural stem cells are cultured and expanded with a serum-free medium;
  • the source of the primary neural stem cells is one or more of mammalian, primate or rodent neural stem cells; the primary neural stem cells are surgically separated from the cortical tissue of the discarded fetal brain tissue, and then digested The enzyme digests the tissue to obtain a primary neural stem cell suspension; the source method of the primary neural stem cells includes IPSC induction, extraction of embryonic stem cells, extraction of human retinal pigment epithelial cells, or extraction of other organs or tissue stem cells other than nerve tissue organs.
  • the source of primary neural stem cells is neural stem cells of medically discarded human fetal brain cortex tissue.
  • This source is 100% human source. It is a single stem cell product. It is cultured in vitro to P3 generation by self-developed serum-free medium and culture process. The purity of stem cells can reach over 95%, and the activity of neural stem cells is 90. %the above.
  • the primary neural stem cells are surgically separated from the cortical tissue of the discarded fetal brain tissue. After the cells are digested and counted, the cells are resuspended in serum-free complete medium, and the density is adjusted to 1 ⁇ 10 5 /ml, and then inoculated to an ultra low level. The culture flask was incubated in an environment of 35 ° C and 5% CO 2 .
  • the serum-free medium is composed of DMEM/F12, B27, N2, and neurotrophic factors BFGF, EGF, LIF, without any animal source serum components.
  • S2 Purification and expansion of neural stem cells: Digest and pass the primary neural stem cells to the fifth generation, and identify the purity of the cells to 95% or more, as seed cells; P5 to P8 generations As seed cells, seed cells can be frozen for storage.
  • the amplification is completed until the cell diameter exceeds 300 ⁇ m, and then digested and passaged to the P1 generation neural stem cells; the culture method is continuous culture for 10 to 14 days, and observation is performed every 24 hours, and the cell growth state is confirmed to be normal every 48 to 72.
  • the medium was changed in an hour; the morphology of the neural stem cells in suspension culture and adherent culture under the microscope is shown in Fig. 1.
  • the P1 generation neural stem cells were digested and inoculated into an ultra-low adsorption culture flask, and cultured in a 35 ° C, 5% CO 2 environment until passage, and the process of digestion, inoculation, and culture was repeated until passage to P5 generation neural stem cells;
  • the culture method is to change the medium every 48 to 72 hours, and continuously culture for 10 to 14 days.
  • P5 generation neural stem cells were tested for cell characteristics and heterologous substances.
  • Cell characterization included flow characterization of neural stem cell surface markers SOX2 and NESTIN, and their purity was over 95%, as shown in Figure 2.
  • the method of fluorescent immunization identified neural stem cells with a purity and purity of more than 95%, and has various differentiation potentials, which can differentiate into neurons, astrocytes and oligodendrocytes.
  • Heterologous substances are mainly microbiological tests, including sterility test, mycoplasma and endotoxin test, and the results are negative, and the cells are frozen as seed cells after passing the test.
  • step S3 Establishment of a neural stem cell working cell bank: the seed cells in step S2 are counted and suspended in serum medium, and the cells are fused to 80% to 90%, and the cells are digested and passaged, and thus repeated until the cells pass through P9. generation;
  • step S4 Preparation of neural stem cell injection: The P9 generation cells in step S3 were fixed to a microcarrier with physiological saline or loaded into a microcarrier to prepare a clinical neural stem cell injection.
  • the method of suspension culture in step S3 comprises the following steps: the specific method is described in the patent No. 201110173821.1.
  • a nutritional supplement solution comprises 1 ⁇ B27 additive, 1 ⁇ N2 additive, 1.0-3.0 mMol/ L-glutamine of L, sodium pyruvate of 0.5-1.5 mMol/L, NAC of 0.5-1.5 mMol/L, bFGF of 50-150 ng/ml, EGF of 50-150 ng/ml, and 1-15 ng/ml of LIF of DMEM/F12 or DMEM medium;
  • the microcarrier comprises a liposome, a microcapsule or a microsphere carrier.
  • the neural stem cell injection for treating a brain injury type disease prepared by the above preparation method.
  • the above neural stem cell injection contains more than 1 ⁇ 10 6 neural stem cells.
  • the method for using the neural stem cell injection for treating a brain injury-like disease wherein the neural stem cell injection for treating a brain injury-like disease is implanted into a patient; the implantation method includes a fixed-point injection, a vein return, a sheath Internal injection, nasal mucosal adsorption or intramuscular injection.
  • the present invention has the following beneficial effects:
  • the neural stem cell injection of the invention can be derived from the discarded embryonic tissue, completely 100% human origin, and is a single stem cell product, which is cultured in vitro to the P3 generation by a self-developed serum-free medium and culture process.
  • the purity of stem cells can reach over 95%, and the activity of neural stem cells is above 90%.
  • the neural stem cells of the invention can be continuously passaged to the 32nd generation in vitro and maintain good dryness and differentiation potential.
  • the logarithmic cell doubling time can be as fast as 16 to 24 hours, and the viability is over 95%.
  • the value-added multiple (once per generation) can be up to 8 to 10 times.
  • the recovery rate of cells after cryopreservation can reach more than 90%, which is convenient for the preparation and storage of neural stem cell preparations.
  • the ability to differentiate into neurons and oligodendrocytes in vitro is strong.
  • the proportion of neural stem cells differentiated into neurons is 75%-80% (far higher than other similar products, the proportion of spontaneously differentiated neurons in most neural stem cells is about 20%), and differentiates into less gum
  • the ratio of cytoplasmic cells is 4% or more (other neural stem cells spontaneously differentiate into less oligodendrocytes, less than 1%).
  • the neural stem cells of the invention may be derived from IPS, embryonic stem cells, neural stem cells transformed from different mesenchymal stem cells, and neural stem cells derived from cerebral cortex.
  • the injection is used for the treatment of cerebral palsy, stroke, acute and chronic brain injury in children.
  • Figure 1 is a suspension cultured neural stem cell sphere and a single neural stem cell that is undergoing division and proliferation;
  • Figure 2 shows that the purity of neural stem cells is 95% or more by flow detection
  • Figure 3 is a CV staining diagram of a section of brain tissue on day 42 after nasal administration of neural stem cells, with a scale of 2 mm;
  • Figure 4 shows the percentage of cerebral infarct size
  • Figure 5 shows that hNSC can improve sensory, learning, memory and cognitive ability in rats after HI injury.
  • Figure (A) shows the timing of HI modeling, hNSCs administration, and various behavioral tests;
  • Figure (EH) is the performance of the SCT;
  • Figure 6 shows the performance of the Morris water maze.
  • the left picture shows the training time and the right picture shows the time spent in the target quadrant.
  • Figure 7 shows that hNSCs regulate NF- ⁇ B signaling and decrease IL-1 ⁇ expression.
  • A, C, E, G is the corresponding westernblot band;
  • B, D, F, H is the relative intensity of expression of each band;
  • Figure 8 is a behavioral observation chart; Figure A body weight (BWT), Figure B balance beam experiment (BBT), Figure C body elevation swing experiment (EBST), and graph DBederson score experimental data were plotted with GraphPad 5.0;
  • the cell spheres were resuspended in complete medium of neural stem cells, inoculated in a well-coated culture dish for adherent culture, and exchanged every 2-3 days until a distinct neuron-like colony was formed, and then formed by cell scraping.
  • the colonies are digested into individual cells using accutase, resuspended in complete medium of neural stem cells, and cultured in suspension to form neural stem cell spheres.
  • the obtained neural stem cell spheres can be digested for passage or frozen in liquid nitrogen.
  • iPSCs induced pluripotent stem cells
  • the iPSCs cell clones were picked, the cells were gently blown off and resuspended in complete medium without bFGF and EGF, transferred to an ultra-low adsorption culture dish for suspension culture for about 7 days, and half a change every 2 to 3 days. Then, the formed cell spheres were collected by centrifugation.
  • the Laminin-coated petri dish was washed twice with Ca, Mg-free DPBS, and the collected cell pellets were cultured in an adherent dish containing and inoculated in a coated dish, and the solution was changed every 2-3 days until A clear neuron-like colony is formed, and the formed colonies are collected by cell scraping, and are digested into individual cells using an accutase, resuspended in a complete medium of neural stem cells, suspended cultured to form a neural stem cell sphere, and obtained neural stem cells.
  • the ball can be digested for passage, or frozen in liquid nitrogen.
  • the intact embryo/aborent fetus was placed in a hibernationmedium buffer solution in a 100 mm sterile Petri dish and rinsed repeatedly with buffer.
  • one hand holds the ophthalmology
  • the other holds the ophthalmic scissors
  • peels off the skin and bones of the head opens the cranial cavity, exposes the brain tissue, and tears the vascular membrane around the brain tissue with fine surgery.
  • the edge of the cortex was pinched off with a fine surgical procedure, and the cortex was separated and placed in a buffer solution of a 35 mm sterile Petri dish.
  • the cerebral cortex is then divided into tissue pieces of about 1 mm size using a fine surgical procedure.
  • the supernatant was aspirated to about 50 ul with a sterile disposable 3 ml dropper, and 950 ul of the buffer solution was pipetted into the centrifuge tube with a 1 ml tip, and gently sucked for 10 to 15 times to form a single cell suspension.
  • the prepared neural stem cell culture solution was 20 ml and added to a low adsorption T75 flask.
  • the volume of the cell suspension to be inoculated was calculated based on the counted suspension cell density and the final cell seeding density of 200 / ul.
  • the inoculated T75 flask was quickly placed in a carbon dioxide incubator at a culture temperature of 35 ° C and a carbon dioxide concentration of 5%.
  • the T75 culture flask is taken out from the carbon dioxide incubator every 2-3 days according to the "Standard Operation Procedure for Neural Stem Cell Culture Addition Liquid", and placed in the biological safety cabinet. .
  • Using a 2 ml sterile pipette take out 2 ml of the prepared neural stem cell culture supplement solution, and quickly add it to the low-adsorption T75 flask and gently shake it from different directions to evenly distribute the cells.
  • the low-adsorption T75 flask with the rehydration solution was quickly returned to the carbon dioxide incubator at a culture temperature of 35 ° C and a carbon dioxide concentration of 5%.
  • the neural stem cells were cultured for 7-10 days, and the growth state of the cells was observed.
  • the neurospheres and culture medium in the low-adsorption T75 flask were aspirated into a 50 ml centrifuge tube with a sterile pipette, 100 g, 4 ° C, and centrifuged for 10 min to fully precipitate the neurospheres.
  • the supernatant is aspirated as much as possible, and the aspirated supernatant is placed in a sterile container to obtain primary neural stem cells.
  • the prepared 10 ml sterile nerve digest was injected into a centrifuge tube containing primary neural stem cell spheres using a sterile syringe.
  • the centrifuge tube was sealed with a parafilm membrane and allowed to stand for 3 minutes.
  • the cryostat was centrifuged at 4 ° C, 300 g for 10 minutes. Aspirate the supernatant with a sterile disposable pipette, rinse with 1 to 5 ml of LDMEM/F12 with a high-power pipette and pipette, and blow up and down 2 to 3 times to separate the tissue.
  • the cryostat was centrifuged at 4 ° C, 300 g for 10 to 20 minutes. Re-add 1 to 5 mL of hibernationmedium wash and wash a total of three times. Aspirate the supernatant with a sterile disposable pipette. Add 1 ⁇ 5mLDMEM, so that the cell pellet is suspended evenly, taking care not to generate bubbles to make the cell pellet suspend evenly for subsequent counting operations.
  • the volume of the cell suspension to be inoculated was calculated based on the cell density of the suspension obtained by counting. Ten to 30 ml of neural stem cell culture medium was added to three low-adsorption T75 flasks. Mix the cell suspension with a sterile disposable pipette and blist it under the surface with a suitable pipette. Take a calculated volume of single cell suspension (based on the cell suspension density and 50-500/ul) Final detail The cell seeding density) was quickly added to the low-adsorption T75 flask and gently shaken from different directions to evenly distribute the cells in the culture solution. The inoculated low-adsorption T75 flask was quickly placed in a carbon dioxide incubator at a culture temperature of 35 ° C and a carbon dioxide concentration of 5%.
  • the T75 culture flask was taken out from the carbon dioxide incubator every 2 to 3 days according to the "Standard Operation Procedure for Neural Stem Cell Culture and Addition Liquid", and placed in a biological safety cabinet.
  • Using a 2 ml sterile pipette take out 2 ml of the prepared neural stem cell culture supplement solution, and quickly add it to the low-adsorption T75 flask and gently shake it from different directions to evenly distribute the cells.
  • the low-adsorption T75 flask with the rehydration solution was quickly returned to the carbon dioxide incubator at a culture temperature of 35 ° C and a carbon dioxide concentration of 5%. P2 generation cells were obtained.
  • a sterile pipette to inhale the neurospheres and culture medium in the low-absorption T75 flask into a 50ml centrifuge tube, 100g, 4°C, and centrifuge for 10min, so that the neurospheres are fully precipitated to the bottom of the tube, and the supernatant is aspirated as much as possible.
  • the clear liquid is placed in a sterile container and stored in a refrigerator at 4 ° C for storage.
  • the prepared 5-50 ml sterile nerve digest was injected into a centrifuge tube containing P2 neurospheres using a sterile syringe.
  • the centrifuge tube was sealed with a parafilm membrane and allowed to stand for 3 minutes.
  • the cryostat was centrifuged at 4 ° C, 300 g for 10 minutes. Aspirate the supernatant with a sterile disposable pipette, rinse with 5 m LDMEM/F12 with a high-power pipette and pipette, and blow up and down 2 to 3 times to separate the tissue.
  • the cryostat was centrifuged at 4 ° C, 300 g for 5-30 minutes. Wash again by adding 1-10 mL of hibernationmedium and wash a total of three times.
  • the digested P2 cells were inoculated into the T75 flask at a density of 1 ⁇ 10 5 /ml.
  • the culture process and digestion process were the same as those of the P1 cells, and the cells were digested, counted and passaged when the cells were grown to more than 80%. , and so on, until the acquisition of P5 generation cells is obtained.
  • the neurospheres and culture medium in the low-adsorption T75 flask are aspirated into a 50 ml centrifuge tube with a sterile pipette, 100 g, 4 ° C, and centrifuged for 10 min to allow the neurosphere to fully precipitate to the bottom of the tube. Aspirate the supernatant, place the aspirated supernatant in a sterile container, store it in a refrigerator at 4 ° C, and store it.
  • Program cooling using a program desuperheater Place the cells to be frozen in a programmed desuperheater to a temperature below -80 °C according to the standard cryopreservation procedure.
  • the frozen storage box is taken out and transferred to a liquid nitrogen tank for storage.
  • the frozen cells were taken out from the liquid nitrogen tank and placed in a resuscitation rack in a thermostatic water bath (the water temperature was maintained at about 40), and the temperature was re-warmed. When the solid state changes to a liquid state, the recovery is completed.
  • the centrifuge tube was sealed with a parafilm membrane and allowed to stand for 3 minutes. Add the prepared sterilized nerve tissue digestive solution and put it into a carbon dioxide incubator at 35 ° C for 5 to 60 minutes (manually oscillate for 5 seconds every 10 minutes). The cryostat was centrifuged at 4 ° C, 300 g for 10 minutes.
  • 1:3 was inoculated into a low-adsorption T75 flask and 5-50 ml of neural stem cell culture solution was added.
  • Mix the cell suspension with a sterile disposable pipette and pipette again under a liquid level with a suitable range of pipettes to remove the calculated volume of single cell suspension (by counting the cell density of the suspension and 10 to 500 cells/ul The final cell seeding density) was quickly added to the low-adsorption T75 flask and gently shaken from different directions to evenly distribute the cells in the culture.
  • the inoculated low-adsorption T75 flask was quickly placed in a carbon dioxide incubator at a culture temperature of 35 ° C and a carbon dioxide concentration of 5%.
  • the T75 culture flask was taken out from the carbon dioxide incubator every 2 to 3 days according to the growth of the cells and the appearance of the culture solution, and placed in a biological safety cabinet. Using a 2 ml sterile pipette, remove 1-5 ml of the prepared neural stem cell culture supplement solution, quickly add it to the low-absorption T75 flask, and gently shake it from different directions to evenly distribute the cells. The low-adsorption T75 flask with the rehydration solution was quickly returned to the carbon dioxide incubator at a culture temperature of 35 ° C and a carbon dioxide concentration of 5%.
  • the neural stem cells were cultured for 7 to 10 days, and the cell growth state was observed to reach the plateau phase (the cells were observed for 2 consecutive days, and the value added was slow or stagnant) for subculture.
  • the plateau phase the cells were observed for 2 consecutive days, and the value added was slow or stagnant
  • the standard procedure for neurosphere digestion 6 bottles of T75 were used for 1:3 to continue subculture, and the rest were frozen according to the Standard Operating Procedure for Neural Stem Cell Cryopreservation.
  • the cells were blown into individual neural stem cells and 10 ul was counted on a cell automatic counter.
  • the digested individual neural stem cells are inoculated at a concentration of 0.1 to 10 ⁇ 10 5 /mL, cultured in a T75 flask, placed in a carbon dioxide incubator, and the cells are fused to 80% to 90%. %, the cells are digested and passaged, and thus repeated until the cells pass through the P9 generation.
  • the cryopreserved P5-P8 neural stem cells were resuscitated according to the method of Example 3, counted, inoculated, and cultured in a T75 flask, and the carbon dioxide incubator was taken every 2 to 3 days depending on the cell growth and the appearance of the culture solution.
  • Using a 2 ml sterile pipette remove 1-5 ml of the prepared neural stem cell culture supplement solution, quickly add it to the low-absorption T75 flask, and gently shake it from different directions to evenly distribute the cells.
  • the low-adsorption T75 flask with the rehydration solution was quickly returned to the carbon dioxide incubator at a culture temperature of 35 ° C and a carbon dioxide concentration of 5%.
  • the neural stem cells After the neural stem cells are cultured for 10 to 14 days, observe the cell growth. If the cell fusion reaches 85% or more, the cells can be digested, and the neurospheres and culture medium in the low-adsorption T75 flask are inhaled into a 50 ml centrifuge tube with a sterile pipette. 100g, 4 ° C, centrifugation for 10min, so that the neurospheres are fully precipitated to the bottom of the tube, as far as possible to absorb the supernatant, the extracted supernatant is placed in a sterile container for label storage Store in a refrigerator at 4 ° C for later use.
  • the centrifuge tube containing the neural stem cells was taken out, 50 ml of physiological saline was added, the cells were mixed, centrifuged, and centrifuged at 4 ° C for 10 min. After centrifugation, the supernatant was taken, and the above procedure was repeated again for a total of 2 washes, then 1 ml of physiological saline was added for cell counting, and finally, physiological saline was used to make a nerve stem cell injection of 1 ⁇ 10 6 cells/ml.
  • This injection can be used in animal experiments and clinical trials to treat brain injury diseases.
  • Anesthetize with ether fix the animal and expose the left common carotid artery, cross-cut with a coagulator, wait for 1.5 hours, then perform hypoxia for 2 hours (HI) in a 7.8% O 2 /92.2% N 2 hypoxia chamber. .
  • NaH was administered 24 hours after HI modeling.
  • the total number of cells was 3 ⁇ 10 5 /6 ul, 3 ul on each side, and the interval was 5 min.
  • Normal control group 1 normal group
  • ANGE-S001 preparation process fresh P9 generation neural stem cells 3x10 5 were dissolved in 6 ul of physiological saline.
  • hNSCs can significantly reduce the tissue damage caused by HI (5 rats in the model group, nerve) 6 rats in the stem cell group).
  • neural stem cells can significantly reduce the area of cerebral infarction caused by HI.
  • the HI+hNSCs group of mice and sham-operated groups showed consistent preferences in communication and curiosity, while the HI+ saline group was just the opposite. Unlike the HI+saline group, the HI+ hNSCs group and the sham-operated group showed similar nose-point (Fig. G) and ceter-point (Fig. H) indices.
  • HI injury increased the expression of IL-1 ⁇ , phosphorylated I ⁇ B ⁇ and NF- ⁇ BP65 in cells, whereas hNSCs reversed this process.
  • hNSCs significantly enhanced their expression.
  • Example 6 Animal model of brain injury MCAO
  • Animals Adult SD rats (220-250, male and female), a total of 40 rats.
  • Model production success criteria refer to the 5-level scale established by Bederson, the god of the rat Scored by functional impairment. After 2 hours of MCAO rat modeling, the neurological function of the rats was scored. Those with scores of 1 to 3 were determined to be successful mice, and those with grades 0 and 4 were discarded.
  • the intraperitoneal administration of MCAO was performed 24 hours after model establishment, and NSC was administered bilaterally on both sides, 3 ul on each side, and the posture was maintained for 5 min after administration, with an interval of 15 min.
  • the neural stem cell group can significantly repair brain damage caused by cerebral infarction.

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Abstract

一种源于人胎脑皮层来源、用于治疗各种脑损伤的神经干细胞注射液，所述神经干细胞注射液中至少包含1×10 6个神经干细胞。其中所述的神经干细胞为废弃胎脑皮层组织分离培养的神经干细胞，该神经干细胞培养经过组织分离和原代细胞获得培养，神经干细胞纯化及扩大培养，神经干细胞工作细胞库的建立及神经干细胞注射液的制备等步骤。细胞可在无血清培养体系下扩增5000倍。根据细胞使用情况，将种子细胞P5代神经干细胞复苏，计数并无血清培养基悬浮培养，细胞消化传代，如此反复直至细胞传代P9代。此神经干细胞注射液纯度在95％，可用于治疗脑中风，缺血缺氧型脑损伤，小儿脑瘫等多种脑损伤疾病。

Description

一种治疗脑部损伤类疾病的神经干细胞注射液及其制备方法和使用方法
本申请要求于2016年12月05日提交中国专利局、申请号为201611101416.8、发明名称为“一种治疗脑部损伤类疾病的神经干细胞注射液”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及属于生物医药领域，具体涉及一种治疗脑部损伤类疾病的神经干细胞注射液和制备方法及其使用方法。
背景技术
小儿脑瘫通常指出生前到出生后一个月内由各种原因引起的非进行性脑损伤或脑发育异常所导致的中枢性运动障碍。临床上以姿势与肌张力异常、肌无力、不自主运动和共济失调等为特征，常伴有感觉、认知、交流、行为等障碍和继发性骨骼肌肉异常，并可有癫痫发作，该病目前无理想的治疗手段，致残率高，给患者本人及家庭造成的精神负担和经济负担重。
根据卫生部发布的数据计算，我国每年新生儿1600万人，据不同统计，小儿脑瘫发病率在千分之二到千分之五左右，即每年3～8万人；而由于以往人口出生率高、医疗条件落后导致发病率高、治愈率低等原因，有报道称我国已有累积小儿脑瘫患者500万人。因此该病危害十分严重。
小儿脑瘫目前常用的治疗方式有物理治疗、作业治疗、家庭训练、矫形器等。常用的药物有脑神经营养药、肌肉松弛剂等，然而药物治疗只有在必要时才使用，它不能替代功能性训练。注射用鼠神经生长因子是被SFDA批准的一种生物制剂，提取自小鼠的颌下腺，与人的同源性达90％，采用肌肉注射的方式给药，据报道对小儿脑瘫引起的肌张力、运动发育、姿势异常、反射异常等症状有一定改善效果。
目前所有的治疗小儿脑瘫的药物大多为提供营养，缓解症状，不能达到治疗的目的。也就是说，目前市场上并没有特异性治疗小儿脑瘫的有效药物。在临床上通常采取药物和运动康复相结合的办法。最近CFDA批 准的一种注射用鼠神经生长因子生物制剂用于小儿脑瘫的治疗，该药物来源于小鼠的额下腺，与人的同源性达90％，采用肌肉注射方式给药，据报道对小儿脑瘫引起的肌张力，运动发育，姿势异常，反射异常等症状有一定改善效果。
脑损伤主要还包括脑中风和创伤性脑损伤，国内外已有大量临床前实验研究表明神经干细胞(NSC)对于脑中风和创伤性脑损伤有一定的治疗效果。并且有部分临床试验证明NSC移植能够治疗脑损伤。神经干细胞(NSC)是一群较原始的、能自我更新并具有多种分化潜能的细胞，可分化成神经元、少突胶质细胞和星形细胞。在生理条件下，体内的NSC通常保持静息状态；神经损伤后，内源性NSC可因微环境的改变而被激活、迁移和分化，以替代损伤的细胞和重建神经环路；遗传修饰后，外源性NSC移植显示了很大的治疗潜力，为脑损伤的治疗提供了新的手段。
发明内容
本发明的目的在于克服上述现有技术存在的不足，一种治疗脑部损伤类疾病的神经干细胞注射液及其制备方法和使用方法。
本发明的目的是通过以下技术方案来实现的：
本发明涉及一种治疗脑部损伤类疾病的神经干细胞注射液的制备方法，所述方法包括如下步骤：
S1：原代细胞培养：将原代神经干细胞用无血清培养基对细胞悬液进行培养扩增；
其中，原代神经干细胞的来源为哺乳动物、灵长类动物或啮齿类动物的神经干细胞中的一种或几种；所述原代神经干细胞为手术分离废弃胎脑组织的皮层组织，然后消化酶将组织消化离心获得原代神经干细胞悬液；原代神经干细胞的来源方法包括IPSC诱导、提取胚胎干细胞、提取人类视网膜色素上皮细胞或提取神经组织器官以外的其他器官或组织干细胞。
优选的，原代神经干细胞的来源为医疗废弃的人源胎脑皮层组织的神经干细胞。这种来源为完全100％人源，是一种单一的干细胞产品，用自主研发的无血清培养基和培养工艺，在体外培养到P3代，干细胞纯度可达95％以上，神经干细胞活性在90％以上。
优选的，所述原代神经干细胞为手术分离废弃胎脑组织的皮层组织， 细胞消化计数后，采用无血清完全培养基进行重悬，并调整密度为1x105个/ml，然后接种至超低吸附培养瓶中，放入35℃，5％CO2中环境中进行培养。
其中，无血清培养基是由DMEM/F12，B27，N2，以及神经营养因子BFGF,EGF,LIF组成，无任何动物源血清成分。
S2：神经干细胞纯化及扩大培养：将扩增满的原代神经干细胞进行消化、传代，直至传到第5代,并鉴定细胞纯度达95％以上后，作为种子细胞；P5到P8代都可作为种子细胞，种子细胞可以冻存，便于保存。
所述扩增满为至细胞直径超过300μm，然后进行消化传代至P1代神经干细胞；培养方法为连续培养10～14天，期间每24小时进行观察，确认细胞生长状态正常，每隔48～72小时进行换液；悬浮培养和贴壁培养的神经干细胞在显微镜下的形态见图1。
P1代神经干细胞消化后接种至超低吸附培养瓶中，放入35℃，5％CO2的环境中进行培养至传代，重复消化、接种、培养的过程直至传代到P5代神经干细胞；所述培养方法为每间隔48～72小时进行换液，连续培养10～14天。
将P5代神经干细胞进行细胞特性和异源物质检测，细胞特性鉴定包括流式鉴定神经干细胞表面标记物SOX2和NESTIN,检测其纯度达95％以上，见图2。荧光免疫的方法鉴定神经干细胞特性和纯度为95％以上，并具有多种分化潜能，能够分化为神经元，星型胶质细胞和少突胶质细胞。异源物质主要是微生物的检测，包括无菌检测，支原体和内毒素检测，其结果阴性为合格，检测合格后将细胞冻存作为种子细胞。
S3：神经干细胞工作细胞库的建立：将步骤S2中的种子细胞，计数并无血清培养基悬浮培养，待培养瓶细胞融合到80％～90％，将细胞消化传代，如此反复直至细胞传代P9代；
S4：神经干细胞注射液的制备：将步骤S3中的P9代细胞加生理盐水定容或负载于微载体，制成临床用神经干细胞注射液。
优选的，步骤S3的悬浮培养的方法包括以下步骤：具体方法参见申请号为201110173821.1的专利。
(a)将神经干细胞接种在包含bFGF和EGF的DMEM/F12或DMEM 培养基中培养；
(b)2、3或4天后在培养基中添加5～20％体积的营养补充液并继续培养，其中所述营养补充液是包含1×B27添加剂，1×N2添加剂，1.0～3.0mMol/L的L-谷氨酰胺，0.5-1.5mMol/L的丙酮酸钠，0.5～1.5mMol/L的NAC，50～150ng/ml的bFGF，50～150ng/ml的EGF以及1～15ng/ml的LIF的DMEM/F12或DMEM培养基；
(c)监测培养基中形成的神经球直径，当60％以上、优选70％以上神经球直径高于阈值时，分离直径高于所述阈值的神经球，消化后收获，其中所述阈值为200-500μm，例如为250μm、300μm、350μm、400μm或450μm；
(d)未挑中的直径小于所述阈值的剩余细胞保留在原培养基中，将培养基移除1/2-1/4体积，然后添加与移除体积相同体积的(a)中所述的包含bFGF和EGF的培养基继续培养；
(e)在(a)接种神经干细胞10、11、12、13、14或15天后通过合并(c)中收获的细胞和(d)继续培养所得的细胞而收获细胞。
优选的，步骤S4中，所述微载体包括脂质体、微胶囊或微球载体。
上述的制备方法制成的治疗脑部损伤类疾病的神经干细胞注射液。
上述的神经干细胞注射液中包含大于1×106个神经干细胞。
上述的治疗脑部损伤类疾病的神经干细胞注射液的使用方法，将所述治疗脑部损伤类疾病的神经干细胞注射液植入患者体内；所述植入方法包括定点注射，静脉回输、鞘内注射、鼻腔粘膜吸附或肌肉注射。
与现有技术相比，本发明具有如下有益效果：
1、本发明的神经干细胞注射液可来源自认废弃的胚胎组织，完全100％人源，是一种单一的干细胞产品，用自主研发的无血清培养基和培养工艺，在体外培养到P3代，干细胞纯度可达95％以上，神经干细胞活性在90％以上。
2、该发明的神经干细胞在体外可连续传代到第32代并保持良好的干性和分化潜能。对数期细胞倍增时间最快可达16～24小时，活率95％以上。
3、体外扩增能力强，增值倍数(每传代一次)可最高达8～10倍。自主 开发的冻存复苏技术，细胞冻存后复苏率可达90％以上，便于神经干细胞制剂的制备和储存。
4、体外分化为神经元和少突胶质细胞能力较强。在无诱导分化下，此神经干细胞分化为神经元的比例为75％-80％(远高于其他同类产品，大部分神经干细胞自发分化神经元的比例在20％左右)，分化为少突胶质细胞的比例为4％以上(其他神经干细胞自发分化为少突胶质细胞较少，1％以下)。
5、该发明的神经干细胞可以来源于IPS,胚胎干细胞，不同来源间充质干细胞转化得到的神经干细胞，具有大脑皮层来源的神经干细胞。
6、哺乳动物来源的神经干细胞注射液
7、该注射液用于治疗小儿脑瘫，脑卒中，急慢性脑损伤
8、鼻腔粘膜吸附的给药方式和纹状体定点给药治疗脑损伤和小儿脑瘫
9、体外免疫学反应测试显示，此神经干细胞对外周淋巴细胞没有明显的刺激作用，具有较低的免疫原性。
10、药效学实验表明此神经干细胞注射液鼻腔或脑内给予可改善脑损伤大鼠的运动平衡能力和脑损伤面积。移植的神经干细胞可在脑内存活35天以上，并分化为神经元。所有实验动物未出现免疫反应和意外死亡，显示了较好的安全性。
附图说明
图1为悬浮培养的神经干细胞球和正在分裂增殖的单个神经干细胞；
图2为流式检测鉴定神经干细胞纯度为95％以上；
图3为鼻腔给予神经干细胞后第42天脑组织的切片的CV染色图，比例尺为2mm；
图4为脑梗死面积百分比；
图5为hNSC能够改善HI损伤后大鼠的感觉，学习，记忆和认知能力。图(A)为HI造模、hNSCs给药以及各种行为测试的时间安排；图(B-C)为sham+saline(n＝23),sham+hNSCs(n＝24),HI+saline(n＝17)andHI+hNSCs(n＝17)各组大鼠的翻正反射(B)和步态分析(C)的表现；图(D)为各组大鼠的走网试验的表现(N＝12/组)；图(E-H)为SCT的表现；
图6为Morris水迷宫的表现，左图为训练时间，右图为目标象限所花费的时间；
图7为hNSCs能够调节NF-κB信号传导，并降低IL-1β的表达。(A，C，E，G)为对应的westernblot条带；(B，D，F，H)为各条带表达的相对强度；
图8为行为学观察图；图A体重(BWT)、图B平衡木实验(BBT)、图C身体抬高摇摆实验(EBST)、图DBederson评分实验数据分别用GraphPad5.0作图；
图9脑梗死面积结果。
具体实施方式
下面结合实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干调整和改进。这些都属于本发明的保护范围。
实施例1：制得原代神经干细胞
1、从胚胎干细胞(ESCs)诱导获得原代神经干细胞
使用细胞刮挑取ESCs细胞克隆，轻柔吹散，然后使用神经干细胞诱导液重悬，转移至超低吸附培养皿中进行悬浮培养3天左右，收集形成的细胞球，轻柔吹散，再用神经干细胞诱导液进行重悬，超低吸附培养皿中再次悬浮培养3天，收集形成的细胞球；将提前一天使用Laminin包被的培养皿用不含Ca、Mg的DPBS洗涤2次，将收集的细胞球用神经干细胞完全培养基进行重悬，接种于包被好的培养皿中进行贴壁培养，每2-3天进行换液，直至形成明显的神经样集落，然后使用细胞刮收集形成的集落，并使用accutase将其消化成为单个细胞，用神经干细胞完全培养基进行重悬，悬浮培养，使其形成神经干细胞球，获得的神经干细胞球可以消化进行传代，也可以进行液氮冻存。
2、从诱导多能干细胞(iPSCs)诱导获得原代神经干细胞
挑取iPSCs细胞克隆，将细胞轻柔吹散并使用不含bFGF和EGF的完全培养基进行重悬，转移至超低吸附培养皿中进行悬浮培养7天左右，每2～3天进行半量换液，然后离心收集形成的细胞球。将提前一天使用 Laminin包被的培养皿用不含Ca、Mg的DPBS洗涤2次，将收集的细胞球用含有并接种于包被好的培养皿中进行贴壁培养，每2-3天进行换液，直至形成明显的神经样集落，然后使用细胞刮收集形成的集落，并使用accutase将其消化成为单个细胞，用神经干细胞完全培养基进行重悬，悬浮培养，使其形成神经干细胞球，获得的神经干细胞球可以消化进行传代，也可以进行液氮冻存。
3、人源废弃胚胎组织获得原代神经干细胞
1)胚胎组织获取
将完整的胚胎/废弃胎儿置于100mm无菌培养皿的hibernationmedium缓冲溶液中，用缓冲液反复冲洗。在解剖镜下，一手持眼科镊，另一手持眼科剪，剥离头部皮肤和骨骼，打开颅腔，暴露脑组织,用精细手术镊撕去脑组织周围血管膜。用精细手术镊夹断脑皮层的边缘，分离脑皮层，放入35mm无菌培养皿的缓冲溶液中。再用精细手术镊将脑皮层分割成约1mm大小的组织块。
将取材得到的人胚胎/胎儿脑皮层和缓冲溶液用无菌一次性3ml滴管转移进入一个或多个15/50ml离心管，静置5分钟，使组织块充分沉淀到管底，尽量吸弃上清。然后用无菌注射器和0.22um滤膜进行无菌过滤，直接滤入离心管中，每个15ml离心管中需5ml消化液，之后进行轻微晃动。将离心管放入35℃二氧化碳培养箱消化，每5分钟手动振荡一次，消化30分钟后，离心300g，5分钟，20℃。最后用无菌一次性3ml滴管吸弃上清至约50ul处，用1ml枪头吸取950ul缓冲溶液加入离心管，轻柔吹吸10～15次，以致形成单细胞悬液。
2)接种
配制的神经干细胞培养液20ml并加入低吸附T75培养瓶中。根据计数得到的悬液细胞密度和200个/ul的最终细胞接种密度，计算需接种的细胞悬液的体积。用无菌一次性吸管混匀细胞悬液，用合适量程的移液枪在液面下再吹打，取出计算体积的单细胞悬液，迅速加入低吸附T75培养瓶中，并轻轻从不同方向晃动容器，使细胞在培养液中均匀分布。将接种后的T75培养瓶迅速放入二氧化碳培养箱中，培养温度为35℃，二氧化碳浓度为5％。
4)原代细胞培养
原代细胞接种培养后，根据细胞生长情况及培养液外观，按照《神经干细胞培养加液标准操作程序》每过2-3天，从二氧化碳培养箱中取出T75培养瓶，放入生物安全柜中。用2ml的无菌移液管，取出已配置好的神经干细胞培养营养补充液2ml，迅速加入低吸附T75培养瓶中，并轻轻从不同方向晃动，使细胞均匀分布。将加好补液的低吸附T75培养瓶迅速放回二氧化碳培养箱中，培养温度为35℃，二氧化碳浓度为5％。
5)细胞收获
神经干细胞培养至7～10天，观察细胞生长状态，用无菌吸管将低吸附T75培养瓶中的神经球和培养液吸入50ml离心管中，100g，4℃，离心10min，使神经球充分沉淀到管底，尽量吸去上清,将吸取的上清液放入无菌容器中，得到原代神经干细胞。
实施例2：P1～P5代神经干细胞的制备
1、原代神经干细胞传代与培养
将已配好的10ml无菌神经消化液用无菌注射器注入装有原代神经干细胞球的离心管中。将离心管口用parafilm膜密封，静置3分钟。加入准备好的除菌神经组织消化液，放入35℃的二氧化碳培养箱中作用30分钟(每10分钟手动震荡一次5秒)。低温离心机4℃，300g，离心10分钟。用无菌一次性吸管吸弃上清，用大功率移液器和移液管加入1～5mLDMEM/F12冲洗，上下吹打2～3次，使组织分离。低温离心机4℃，300g，离心10～20分钟。重新加入1～5mLhibernationmedium洗涤，共洗涤三次。用无菌一次性吸管吸弃上清。加入1～5mLDMEM，使细胞沉淀悬浮均匀，注意不要产生气泡使细胞沉淀悬浮均匀，用于后续计数操作。
1.1细胞计数
按照《细胞密度测定标准操作程序》对单细胞悬液进行计数。
1.2传代与培养
根据计数得到的悬液细胞密度，计算需接种的细胞悬液的体积。将三个低吸附T75培养瓶中加入10～30ml的神经干细胞培养液。用无菌一次性吸管混匀细胞悬液，用合适量程的移液枪在液面下再吹打，取出计算体积的单细胞悬液(根据计数得到的悬液细胞密度和50～500个/ul的最终细 胞接种密度)，迅速加入低吸附T75培养瓶中，并轻轻从不同方向晃动，使细胞在培养液中均匀分布。将接种后的低吸附T75培养瓶迅速放入二氧化碳培养箱中，培养温度为35℃，二氧化碳浓度为5％。
2、细胞的收获与冻存
P2细胞接种培养后，根据细胞生长情况及培养液外观，按照《神经干细胞培养加液标准操作程序》每过2～3天，从二氧化碳培养箱中取出T75培养瓶，放入生物安全柜中。用2ml的无菌移液管，取出已配置好的神经干细胞培养营养补充液2ml，迅速加入低吸附T75培养瓶中，并轻轻从不同方向晃动，使细胞均匀分布。将加好补液的低吸附T75培养瓶迅速放回二氧化碳培养箱中，培养温度为35℃，二氧化碳浓度为5％。得到P2代细胞。
2.1消化收获
用无菌吸管将低吸附T75培养瓶中的神经球和培养液吸入50ml离心管中，100g，4℃，离心10min，使神经球充分沉淀到管底，尽量吸去上清,将吸取的上清液放入无菌容器中做好标记存放于4℃冰箱中储存备用。
2.2洗涤
将已配好的5-50ml无菌神经消化液用无菌注射器注入装有P2神经球的离心管中。将离心管口用parafilm膜密封，静置3分钟。加入准备好的除菌神经组织消化液，放入35℃的二氧化碳培养箱中作用10～50分钟(每10分钟手动震荡一次5秒)。低温离心机4℃，300g，离心10分钟。用无菌一次性吸管吸弃上清，用大功率移液器和移液管加入5mLDMEM/F12冲洗，上下吹打2～3次，使组织分离。低温离心机4℃，300g，离心5-30分钟。重新加入1-10mLhibernationmedium洗涤，共洗涤三次。
2.3过滤和细胞计数
用无菌一次性吸管吸弃上清。加入2mL DMEM，使细胞沉淀悬浮均匀，按照《细胞密度测定标准操作程序》对单细胞悬液进行计数。
2.4P2代到P5代细胞培养
将消化后的P2代细胞按1×105个/ml的密度接种到T75培养瓶中，培养过程和消化过程与P1代细胞相同，待细胞生长到80％以上的时候进行消化，计数和传代，以此类推，直至获得P5代细胞的获得。
2.4细胞冻存分装
鉴定细胞纯度达95％以上后，用无菌吸管将低吸附T75培养瓶中的神经球和培养液吸入50ml离心管中，100g，4℃，离心10min，使神经球充分沉淀到管底，尽量吸去上清,将吸取的上清液放入无菌容器中做好标记存放于4℃冰箱中储存备用
2.5程序性降温
使用程序降温仪进行程序降温：将需冻存的细胞放置于程序性降温仪中按照标准冻存程序将至-80以下℃。
2.6细胞入库
冻存结束后，将冻存盒取出转移到液氮罐中贮存。
实施例3P6-P9代神经干细胞的制备
1、细胞的复苏
从液氮罐中取出冻存的细胞，放入电热恒温水槽中的复苏架上(水温保持在40左右)，快速晃动复温。由固态变成液态时，复苏即完成。
1.1细胞的洗涤
将离心管口用parafilm膜密封，静置3分钟。加入准备好的除菌神经组织消化液，放入35℃的二氧化碳培养箱中作用5-60分钟(每10分钟手动震荡一次5秒)。低温离心机4℃，300g，离心10分钟。
1.2取样计数和洗涤
用无菌一次性吸管吸弃上清，用大功率移液器和移液管加入1～10mLDMEM/F12冲洗，上下吹打2～3次，使组织分离。低温离心机4℃，300g，离心10分钟。重新加入1～10mLhibernationmedium洗涤，共洗涤三次。用无菌一次性吸管吸弃上清。
1.3传代与培养
将1:3接种于低吸附T75培养瓶中加入5-50ml的神经干细胞培养液。用无菌一次性吸管混匀细胞悬液，用合适量程的移液枪在液面下再吹打，取出计算体积的单细胞悬液(根据计数得到的悬液细胞密度和10～500个/ul的最终细胞接种密度)，迅速加入低吸附T75培养瓶中，并轻轻从不同方向晃动，使细胞在培养液中均匀分布。将接种后的低吸附T75培养瓶迅速放入二氧化碳培养箱中，培养温度为35℃，二氧化碳浓度为5％。
2、种子细胞的传代
P5细胞接种培养后，根据细胞生长情况及培养液外观，每过2～3天，从二氧化碳培养箱中取出T75培养瓶，放入生物安全柜中。用2ml的无菌移液管，取出已配置好的神经干细胞培养营养补充液1-5ml，迅速加入低吸附T75培养瓶中，并轻轻从不同方向晃动，使细胞均匀分布。将加好补液的低吸附T75培养瓶迅速放回二氧化碳培养箱中，培养温度为35℃，二氧化碳浓度为5％。
2.1消化
神经干细胞培养至7～10天，观察细胞生长状态，达到平台期(细胞经连续2日观察，增值缓慢或停滞)进行传代操作。按照《神经球消化标准操作程序》进行。其中6瓶T75用于1:3继续传代培养，其余按照《神经干细胞冻存标准操作程序》冻存。
2.2细胞计数
将细胞吹打成单个神经干细胞，取10ul在细胞自动计数仪上计数。
2.3传代与培养
将消化后的单个神经干细胞，按着0.1～10×105个/mL的浓度接种继续培养，在T75瓶中混匀培养，放入二氧化碳培养箱中,待培养瓶细胞融合到80％～90％，将细胞消化传代，如此反复直至细胞传代P9代。
实施例4:P6～P9代神经干细胞注射液的制备
将冻存的P5～P8神经干细胞按着实施例3的方法进行复苏，计数后接种，在T75培养瓶中培养，根据细胞生长情况及培养液外观，每过2～3天，从二氧化碳培养箱中取出T75培养瓶，放入生物安全柜中。用2ml的无菌移液管，取出已配置好的神经干细胞培养营养补充液1-5ml，迅速加入低吸附T75培养瓶中，并轻轻从不同方向晃动，使细胞均匀分布。将加好补液的低吸附T75培养瓶迅速放回二氧化碳培养箱中，培养温度为35℃，二氧化碳浓度为5％。
待神经干细胞培养10～14天，观察细胞生长情况，如细胞融合达到85％以上，即可消化细胞，用无菌吸管将低吸附T75培养瓶中的神经球和培养液吸入50ml离心管中，100g，4℃，离心10min，使神经球充分沉淀到管底，尽量吸去上清,将吸取的上清液放入无菌容器中做好标记存放 于4℃冰箱中储存备用。
需要时取出装有神经干细胞的离心管，加入50ml生理盐水，细胞混匀后离心，4℃，离心10min。离心后取上清，再次重复以上步骤，共计洗涤2次，然后加入1ml生理盐水进行细胞计数，最后用生理盐水定容成1×106个细胞/ml的神经干细胞注射液。
此注射液可用于动物实验和临床实验治疗脑部损伤类疾病。
实施例5：小儿脑瘫动物模型研究
1、实验动物
动物：SPF级出生后7天的SD大鼠(13-19g，雌雄各半)
2、造模(HI)
乙醚麻醉，动物固定并暴露左侧颈总动脉，用电凝器进行横断，待其恢复1.5小时，然后在7.8％O2/92.2％N2的低氧箱中进行缺氧2小时(HI)。
3、干细胞治疗
HI造模后24小时鼻腔给药，细胞数共3x105/6ul，左右各3ul，两次间隔时间5min。
4、实验分组
正常对照组1(正常组)——给予生理盐水
正常对照组2——ANGE-S001
HI模型对照组(模型组)——给予生理盐水
HI模型给药组(ANGE-S001组)——ANGE-S001给药
ANGE-S001配制过程：将鲜活的P9代神经干细胞3x105溶于6ul生理盐水中。
5、疗效检测和机理探索
(1)鼻腔给予神经干细胞注射液3h后，PKH-26(红)标记的hNSC(3×105细胞/动物)在SD大鼠头部的在体生物发光，SD大鼠头部的在体生物发光成像明显；脑中总的荧光强度明显强于生理盐水给药组；神经干细胞在脑内主要分布在嗅球，皮质，胼胝体和海马区域。
(2)hNSCs对脑损伤的长期影响。见图3。
切片中脑损伤的面积和损伤对侧的脑面积均进行了计算，见图4。可见hNSCs给予后能显著减少HI造成的组织损伤(模型组大鼠5只，神经 干细胞组大鼠6只)。
神经干细胞给予后能明显减少HI引起的脑梗死面积。
(3)hNSC能够改善HI损伤后大鼠的感觉，学习，记忆和认知能力。见图5。
由图(B)可见在给药后第3天，神经干细胞能显著降低翻正实验所需的时间。
由图(C)可见在给药后第5天，神经干细胞能显著降低步态分析的时间。
需要注意的是HI+hNSCs组的老鼠和假手术组在交流和好奇心上表现出一致的偏好，而HI+生理盐水组刚好相反。和HI+saline组大鼠不同，HI+的hNSCs组大鼠与假手术组大鼠表现出相似的nose-point(图G)和ceter-point(图H)指数。
(4)在获得性试验中，HI+saline组大鼠的潜伏期明显比HI+hNSCs组和假手术组大鼠长。在保留试验中，HI+saline组大鼠和sham+saline组和HI+hNSCs组大鼠相比，在目标象限所花费的时间和跨越平台的频率都显著降低，见图6。
(5)鼻腔给药后第3天采用westernblot对IL-1β，磷酸化IκBα，和NF-κBp65的表达，见图7。
如图7所示，HI损伤增加了细胞内IL-1β，磷酸化IκBα和NF-κBP65的表达，而hNSCs能逆转这一过程。对于细胞核NF-κB，hNSCs能显著的增强其表达。
实施例6：脑损伤MCAO动物模型研究
1.实验动物
动物:成年SD大鼠(220-250，雌雄各半)，共40只。
2.造模(MCAO)
术前禁食不禁水12h。动物用10％戊巴比妥钠(32mg/kg)腹腔注射麻醉。参考改进的ZeaLonga方法造模，缺血达到2h后小心抽出栓线，即形成再灌注。假手术对照只是不插入尼龙鱼线，其余步骤同手术组。在手术后保持体温在(37±0.5)℃。
模型制作成功标准：参照Bederson确立的5级评分法，对大鼠的神 经功能缺损进行评分。在MCAO大鼠造模2h后，对大鼠的神经功能进行评分，评分在1～3级者定为造模成功小鼠，0级和4级者弃去。
3、神经干细胞治疗
MCAO造模后24小时鼻腔给药，NSC双侧鼻腔给药，每侧3ul，给药后保持姿势5min，间隔15min。
实验分组
4.行为学观察
造模后第1d、3d、5d、7d、14d、21d、28d、35d、42d、49d、56d分别进行Bederson评分，EBST实验、平衡木实验，每次同步记录体重数据。见图8。
Sham组与MCAO+NS、MCAO+hNSC.、MCAO+rNSC.组比较：*P＜0.05，**P＜0.01；MCAO+NS组与MCAO+hNSC.组比较：#P＜0.05，##P＜0.01；MCAO+NS组与MCAO+rNSC.组比较：无差异；MCAO+hNSC.组与MCAO+rNSC.组比较：^P＜0.05，^^P＜0.01。
5.脑梗死面积结果
由图9可见，脑组织切片结果，神经干细胞组能显著修复脑损伤引起脑梗死。
以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。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，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。对这些实施例的多种修改对本领域的专业技术人员来说是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (15)

  1. 一种治疗脑部损伤类疾病的神经干细胞注射液的制备方法，其特征在于，所述方法包括如下步骤：
    S1：原代细胞培养：将原代神经干细胞用无血清培养基对细胞悬液进行培养扩增；
    S2：神经干细胞纯化及扩大培养：将扩增融合到80％～90％的原代神经干细胞进行消化、传代，直至传到第5代，并鉴定细胞纯度达95％以上后，作为种子细胞；
    S3：神经干细胞工作细胞库的建立：将步骤S2中的种子细胞，计数并无血清培养基悬浮培养，待培养瓶细胞融合到80％～90％，将细胞消化传代，如此反复直至细胞传代P6～P9代；
    S4：神经干细胞注射液的制备：将步骤S3中的P6～P9代细胞加生理盐水定容或负载于微载体，制成临床用神经干细胞注射液。
  2. 如权利要求1所述的治疗脑部损伤类疾病的神经干细胞注射液的制备方法，其特征在于，步骤S1和S3中无血清培养基是由DMEM/F12，B27，N2，以及神经营养因子BFGF，EGF，LIF组成，无任何动物源血清成分。
  3. 如权利要求1所述的治疗脑部损伤类疾病的神经干细胞注射液的制备方法，其特征在于，步骤S1中细胞悬液的细胞浓度为1x105个/ml。
  4. 如权利要求1所述的治疗脑部损伤类疾病的神经干细胞注射液的制备方法，其特征在于，步骤S1中培养扩增的温度为35℃；所述培养扩增的二氧化碳浓度为5％。
  5. 如权利要求1所述的治疗脑部损伤类疾病的神经干细胞注射液的制备方法，其特征在于，步骤S1的原代神经干细胞的来源为哺乳动物、灵长类动物或啮齿类动物的神经干细胞中的一种或几种；所述原代神经干细胞为手术分离废弃胎脑组织的皮层组织，然后消化酶将组织消化离心获得原代神经干细胞悬液。
  6. 如权利要求1所述的治疗脑部损伤类疾病的神经干细胞注射液的制备方法，其特征在于，步骤S1的原代神经干细胞的来源方法包括IPSC 诱导、提取胚胎干细胞、提取人类视网膜色素上皮细胞或提取神经组织器官以外的其他器官或组织干细胞。
  7. 如权利要求1所述的治疗脑部损伤类疾病的神经干细胞注射液的制备方法，其特征在于，步骤S1的原代神经干细胞的来源为医疗废弃的人源胎脑皮层组织的神经干细胞。
  8. 如权利要求1所述的治疗脑部损伤类疾病的神经干细胞注射液的制备方法，其特征在于，步骤S2中扩增融合到80％～90％的原代神经干细胞直径超过300μm。
  9. 如权利要求1所述的治疗脑部损伤类疾病的神经干细胞注射液的制备方法，其特征在于，步骤S2中所述培养方法为每间隔48～72小时进行换液，连续培养10～14天。
  10. 如权利要求1所述的治疗脑部损伤类疾病的神经干细胞注射液的制备方法，其特征在于，步骤S3的悬浮培养的方法包括以下步骤：
    (a)将神经干细胞接种在包含bFGF和EGF的DMEM/F12或DMEM培养基中培养；
    (b)2、3或4天后在培养基中添加5～20％体积的营养补充液并继续培养，其中所述营养补充液是包含1×B27添加剂，1×N2添加剂，1.0～3.0mmol/L的L-谷氨酰胺，0.5～1.5mMol/L的丙酮酸钠，0.5～1.5mMol/L的NAC，50～150ng/ml的bFGF，50～150ng/ml的EGF以及1～15ng/ml的LIF的DMEM/F12或DMEM培养基；
    (c)监测培养基中形成的神经球直径，当60％以上神经球直径高于阈值时，分离直径高于所述阈值的神经球，消化后收获，其中所述阈值为200～500μm，例如为250μm、300μm、350μm、400μm或450μm；
    (d)未挑中的直径小于所述阈值的剩余细胞保留在原培养基中，将培养基移除1/2～1/4体积，然后添加与移除体积相同体积的(a)中所述的包含bFGF和EGF的培养基继续培养；
    (e)在(a)接种神经干细胞10、11、12、13、14或15天后通过合并(c)中收获的细胞和(d)继续培养所得的细胞而收获细胞。
  11. 如权利要求10所述的治疗脑部损伤类疾病的神经干细胞注射液的制备方法，其特征在于，直径高于阈值的神经球达到70％以上。
  12. 如权利要求1所述的治疗脑部损伤类疾病的神经干细胞注射液的制备方法，其特征在于，步骤S4中，所述微载体包括脂质体、微胶囊或微球载体。
  13. 由权利要求1～12中任一项所述的制备方法制成的治疗脑部损伤类疾病的神经干细胞注射液。
  14. 如权利要求13所述的治疗脑部损伤类疾病的神经干细胞注射液，其特征在于所述神经干细胞注射液中包含大于1×106个神经干细胞。
  15. 权利要求14中所述的治疗脑部损伤类疾病的神经干细胞注射液的使用方法，其特征在于，将所述治疗脑部损伤类疾病的神经干细胞注射液植入患者体内；所述植入方法包括定点注射，静脉回输、鞘内注射、鼻腔粘膜吸附或肌肉注射。
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