WO2018149127A1 - 一种经鼻腔给药用于帕金森病治疗的神经干细胞制剂及其制备方法和应用 - Google Patents

一种经鼻腔给药用于帕金森病治疗的神经干细胞制剂及其制备方法和应用 Download PDF

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WO2018149127A1
WO2018149127A1 PCT/CN2017/101993 CN2017101993W WO2018149127A1 WO 2018149127 A1 WO2018149127 A1 WO 2018149127A1 CN 2017101993 W CN2017101993 W CN 2017101993W WO 2018149127 A1 WO2018149127 A1 WO 2018149127A1
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neural stem
parkinson
disease
cells
cell
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赵雄飞
郑佳威
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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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
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    • C12N2501/23—Interleukins [IL]
    • C12N2501/235—Leukemia inhibitory factor [LIF]

Definitions

  • the invention relates to the technical field of biomedicine, in particular to a neural stem cell preparation for treating Parkinson's disease by nasal administration, a preparation method and application thereof.
  • Parkinson's disease is a clinically common refractory disease.
  • the degeneration of dopaminergic neurons in the substantia nigra and the formation of Lewy bodies are the main pathological features.
  • the clinical manifestations are mainly tremor, bradykinesia, Abnormal muscle rigidity and posture gait, which greatly affects the quality of life of patients.
  • the use of oral levodopa dopamine replacement therapy to alleviate PD motor symptoms has been one of the successes in modern neuroscience transformation studies.
  • levodopa remains the gold standard for the treatment of symptomatic PD.
  • the most effective method for PD treatment at present is to use levodopa replacement therapy, but it is still not effective in preventing or slowing the progression of the disease.
  • only drugs, surgery or electrodes can be used to relieve and control symptoms, and to avoid excessive development.
  • the clinical treatment of Parkinson's disease is based on the oral DA prodrug levodopa, but the drug is effective at first, and the long-term use will result in decreased efficacy and cause a series of complications of dyskinesia.
  • Surgical treatment is mainly based on the destruction of globus pallidus and thalamus, but the recurrence rate is high.
  • the above methods belong to symptomatic treatment, but can not reverse the degeneration of DA neurons. Therefore, it has become an urgent need to find a new technology and a new method that can replace denatured dopaminergic neurons to restore dopamine neurotransmitter function.
  • the object of the present invention is to overcome the deficiencies of the prior art described above and to provide a neural stem cell preparation for the treatment of Parkinson's disease by nasal administration, a preparation method thereof and application thereof.
  • the present invention is different from neurotrophic drugs such as mouse nerve growth factor.
  • the neural stem cell injection is derived from human embryos (through ethical review), is completely 100% human, and uses nasal non-invasive administration to directly act on the central nervous system. It will have a better effect on children with cerebral palsy.
  • the present invention relates to a neural stem cell preparation for the treatment of Parkinson's disease via nasal administration, the method comprising the steps of:
  • S1 Seed cell culture and purification: The primary neural stem cells are cultured and cultured in a serum-free medium to be subcultured to the P5 generation to obtain the seed cells.
  • the primary neural stem cells can be obtained by using a surgical instrument to obtain the ventral midbrain tissue of the discarded fetal brain under a microscope, and then digesting the cells with digestive juice, counting the cells, resuspending them in serum-free complete medium, and inoculation in super
  • the suspension culture was carried out in a 35 ° C, 5% CO 2 incubator, and the liquid was changed every 48 to 72 hours.
  • the cells were cultured for 12 days or more, and continuously cultured to the P5 generation. During the P5 generation, the resulting cells became more and more pure.
  • the obtained P0 to P5 generation cells were placed in a seed cell bank for storage.
  • the neural stem cells of the present invention may be neural stem cells of different animals such as mammals such as primates such as humans, rodents such as mice, and the neural stem cells may be obtained from different sources, such as commercially available neurospheres. a cryopreservation or neural stem cell line, or a neural stem cell induced by mesenchymal stem cells to the nerve; or a neural stem cell induced by other sources, such as iPSC technology, embryonic stem cells (including minimal embryos) Stem cells), sub-total stem cells, human retinal pigment epithelial cells, etc.; or neural stem cells derived from organs and tissues other than nerve tissue organs, such as cord blood and umbilical cord.
  • a cryopreservation or neural stem cell line or a neural stem cell induced by mesenchymal stem cells to the nerve
  • a neural stem cell induced by other sources such as iPSC technology, embryonic stem cells (including minimal embryos) Stem cells), sub-total stem cells, human retinal pigment epithelial cells,
  • P6-P8 generation cell culture The P5 generation neural stem cells obtained in the step S1 were cultured and expanded to a P8 generation by using a serum-free medium.
  • the method of culturing the amplification passage is to coat the cell culture flask with laminin at 37 ° C for 2 hours, and then wash it with DPBS without Ca and Mg for further use; after digesting and counting the P5 generation neural stem cells, no The serum complete medium was resuspended and adjusted to a density of 1 ⁇ 10 5 /ml, inoculated into a coated culture flask, and placed in an adherent culture at 35 ° C in a 5% CO 2 atmosphere; the cell culture was performed at intervals of 48 hours. After changing the medium, the cells can be passaged for 7 days or so until the P8 generation, and the P6-P8 cells are placed in the main cell bank for storage.
  • S3 Working cell bank: Take the P8 cell in step S2, and perform cell characteristics and heterogeneous substance detection. After the test is passed, it is the working cell, which is digested and frozen, and transferred to the working cell bank for use.
  • the serum-free medium in step S2 is composed of DMEM/F12, B27, N2, and neurotrophic factor BFGF, EGF, LIF, without any animal-derived serum component.
  • step S4 Preparation of a therapeutic preparation for Parkinson's disease: the working cells of step S3 are subjected to adherent culture, and the cells are digested and counted when the cells are grown to a logarithmic growth phase; and then work at 0 to 4 ° C.
  • the cell solution and the shaping agent are mixed at a volume ratio of 1:1; the concentration of the working cell solution is 100 ⁇ l of 2.5 ⁇ 10 6 working cells; the plastic agent is Matrigel and 0.4% sodium carboxymethylcellulose solution. Mix in a volume ratio of 1:1.
  • the cryopreserved working cells should be resuscitated first, then cultured in an adherent manner, and the cells are digested and counted when the cells are grown to logarithmic growth phase; then at 0-4 °C 100 ⁇ l of 2.5 ⁇ 10 6 neural stem cells were gently mixed with 100 ⁇ l of the shaping agent, transferred to a pre-cooled container, and stored at a constant temperature of 4 ° C.
  • the present invention has the following beneficial effects:
  • This patent provides a non-invasive treatment of Parkinson's neural stem cells by nasal mucosa adsorption.
  • the preparation can rapidly form a solid membrane under body temperature after being added or injected into the nasal cavity, and adsorbed to the nasal cavity. It absorbs slowly and does not enter the mouth or lungs with the breath, which facilitates the use of patients and doctors, and the test data shows that its efficacy and safety are more than traumatic.
  • the striatum is administered better.
  • the method for culturing neural stem cells in this patent adopts P0 ⁇ P5 as suspension culture and P6 ⁇ P9 as adherent culture.
  • the pre-suspension culture is beneficial to obtain purified neural stem cell clones, which can ensure the purity of neural stem cells obtained.
  • Adherent culture can make neural stem cells proliferate and pass faster, which is conducive to the expansion of cells in the pilot test, and can shorten the incubation time by nearly half.
  • This patent provides a stable, mammalian-derived neural stem cell preparation that can be differentiated into dopamine receptor neurons, oligodendrocytes and stellate cells in the mammalian brain, which can be repaired.
  • the nerves and tissues of the brain can be repaired.
  • the formulation can be used to treat Parkinson's disease without the use of immunosuppressive agents.
  • Figure 1 shows the morphology of neural stem cells, the left picture shows the neural stem cell spheres in suspension culture; the right picture shows the neural stem cells cultured in adherence; the cell proliferation ratio can reach 10 times/generation;
  • Figure 2 shows the identification of neural stem cell characteristics and differentiation ability
  • A-E is the identification of neural stem cells
  • F-M is the identification of neural stem cell differentiation ability
  • Figure 3 shows the purity of neural stem cells by flow identification (>95%)
  • Figure 4 shows the results of immunohistochemical staining of substantia nigra regions in each group of animals after administration of neural stem cells
  • Figure 5 shows the relative content of neurotransmitter DOPAC after administration of neural stem cells
  • Figure 6 shows the relative content of neurotransmitter HVA after administration of neural stem cells
  • Figure 7 is an HPLC detection spectrum of each standard
  • Figure 8 is a HPLC detection map of tissue samples of each group after administration of neural stem cells
  • Figure 9 is a comparison of the results of stride tests in different groups of rats after administration of neural stem cells
  • Figure 10 is a comparison of the results of stride tests in different groups of rats at the 10th week after administration of neural stem cells.
  • the culture vessel is subjected to adherent culture, and the liquid is changed every 2 to 3 days until a distinct neuron colony is formed, and then the colonies formed are collected by cell scraping, and are digested into individual cells using accutase, and fully cultured with neural stem cells.
  • the base is resuspended, suspended and cultured to form a neural stem cell sphere, and the obtained neural stem cell sphere can be digested for passage, and can also be frozen by liquid nitrogen.
  • iPSCs induced pluripotent stem cells
  • the cells were inoculated in a well-coated culture dish for adherent culture, and the solution was changed every 2 to 3 days until a distinct neuron-like colony was formed, and then the colonies formed were collected by cell scraping and digested into individual cells using accutase. Resuspension in a complete medium of neural stem cells, suspension culture, to form a neural stem cell sphere, the obtained neural stem cell sphere can be digested for passage, and can also be frozen by liquid nitrogen.
  • One rat of E14 was intraperitoneally injected with 2% pentobarbital sodium at a dose of 40 mg/kg; the abdomen of rats was immersed in 75% alcohol for 5 min, and the rats were placed in supine position, and the limbs were fixed and cut.
  • the uterus adjacent to the pubic symphysis is free of the intact uterus containing the embryo, and is transferred into a large petri dish containing the neural stem cell preservation solution; the fetal rat with a crown and hip length of about 11 to 12 mm is isolated.
  • the ventral tissue of the midbrain was separated by fine surgery, and placed in a 60 mm sterile Petri dish containing 6 ml of neural stem cell preservation solution.
  • the tissue pieces were divided into small pieces of about 1 mm 3 with forceps, and then transferred to a tube containing 10 ml of neural stem cells. Liquid in a 50 ml centrifuge tube. Then, the cells were digested and counted, resuspended in serum-free complete medium, added to an ultra-low-adsorption T75 flask, and placed in a 35 ° C carbon dioxide incubator for cultivation.
  • the neural stem cells were cultured for 10 to 12 days, and the state of the cells was observed.
  • the neurospheres and culture medium in the low-adsorption T75 flask were pipetted into a 50 ml centrifuge tube and centrifuged at 100 g for 3 minutes to allow the neurospheres to fully precipitate to the bottom of the tube.
  • Aspirate the supernatant add 5 ml of the digested solution to the centrifuge tube, digest it in the incubator for 15 minutes; centrifuge at 4 ° C, 300 g for 5 minutes, discard the supernatant, and add 10 ml of the neural stem cell preservation solution for washing; 4 ° C, 300g, centrifugation for 5 minutes, aspirate the supernatant, add 1 ⁇ 5ml of neural stem cell preservation solution, gently blow to resuspend the cells. The single cell suspension was then counted according to the Standard Procedure for Cell Densitometry. Based on the number of cells counted, the volume of serum-free complete medium to be added was calculated.
  • the resuspended cells were quickly added to a low-adsorption T75 flask and gently shaken from different directions to uniformly distribute the cells in the culture solution, and placed in a carbon dioxide incubator at a culture temperature of 35 ° C and a carbon dioxide concentration of 5%.
  • the cells were serially subcultured until the P5 generation, and the cells were frozen and stored in a seed cell bank for preservation.
  • Culture flask coating Add appropriate amount of laminin solution to the cell culture flask, and put it in a 37 ° C incubator for 2 hours, then wash it with DPBS without Ca and Mg for use;
  • the amplified and purified P5 neural stem cells were digested and counted, resuspended in serum-free complete medium, and adjusted to a density of 1 ⁇ 10 5 /ml, and inoculated into a coated culture flask at 35 ° C.
  • the adherent culture was carried out in a 5% CO 2 incubator; the cell culture was changed every 48 hours, and the cells were continuously cultured for about 7 days to be digested and passaged until the P8 generation, and the cells obtained were placed in the main cell bank for storage.
  • the P8 generation cells were resuspended in serum-free complete medium, adjusted to a density of 1 ⁇ 10 5 /ml, inoculated into a coated culture flask, and placed in a 35 ° C, 5% CO 2 incubator for adherent culture. During the culture process, samples were taken for cell characteristics and heterogeneous substance detection. After the test was passed, the cells were frozen and transferred to a working cell bank for use.
  • the neural stem cells in the working cell bank were resuscitated for adherent culture, and digested according to Example 1.
  • the cells were centrifuged at 4 ° C, 300 g for 5 minutes, the supernatant was aspirated, washed with physiological saline, and the supernatant was aspirated, resuspended in 1-5 ml of physiological saline, centrifuged at 4 ° C, 300 g for 5 minutes, and the supernatant was aspirated.
  • the cells were resuspended by adding 100 ⁇ l of physiological saline, and the prepared cell suspension was mixed 1:1 with the molding agent on ice, and then dispensed into a pre-filled syringe stored at 4 degrees, and placed. Store in a 4 ° C incubator for immediate use and direct injection to the site of administration.
  • the proliferation of neural stem cells in Examples 2 and 3 can be up to 10 times/generation.
  • the P8-derived cells were identified by immunofluorescence.
  • the specific surface markers of the cells expressing neural stem cells can be identified, and the purity can reach more than 95%. At the same time, they can differentiate into neurons and star glue. Qualitocytes, oligodendrocytes, dopamine neurons, etc.
  • the purified P8 cells were identified by flow cytometry, and the purity of neural stem cells was over 95%.
  • Example 6 Pharmacodynamic study of neural stem cells applied to Parkinson's animal model
  • the PD model was induced by microinjection of 6-OHDA into the unilateral MFB. After anesthesia, the rats were placed in a supine position. The nerve stem cell suspension was aspirated 10 ul with a micro-syringe with a silicone hose at the front end. The hose was slowly inserted into the left nasal cavity of the rat, and the cells were slowly injected into the nasal cavity for 15 min. When the neural stem cell fluid completely forms a solid gel, the rat is returned to the cage.
  • Mobile phase 16% aqueous methanol solution containing 40 mM sodium acetate, 15 mM citric acid, 0.25 mM sodium octane sulfonate, 0.2 mM EDTA-2Na, adjusted to pH 4.3 without methanol addition.
  • ECD Electrochemical Detector
  • HPLC detection profile of the tissue samples is shown in Figure 8.
  • Forefoot function was measured before and after treatment in rats using a step test.
  • the experimenter fixed the posterior and hind limbs of the rat's body in one hand, and made it move off the ground.
  • the other hand fixed one side of the forelimb and the other side of the forelimb touched the ground.
  • the rats were moved to the side of the forehand direction and moved to the side at a uniform speed (moving 90 cm within 5 s). ), record the number of steps on the ground side of the ground when moving.
  • the number of steps of the upper limbs on both sides was measured alternately.
  • the forelimb of one rat was repeated 5 times, and the mean was calculated.
  • the staining of TH-positive neurons in the third group of substantia nigra was significantly shallower, indicating that 6-OHDA model induced a large number of neuronal apoptosis in the substantia nigra.
  • the seventh group of substantia nigra TH immunohistochemical staining was slightly deepened, indicating that the administration of Madopar can improve the apoptosis of neurons induced by 6-OHDA to some extent.
  • the staining of the substantia nigra neurons in the first, second, fourth and fifth groups was more obvious than that in the third group, indicating that the different doses of neural stem cells in the nasal cavity can effectively increase the density of neurons in the substantia nigra.
  • the mapping of each standard is shown in Fig. 7.
  • the internal standard DHBA first peaked, the peak time was about 6.5min, the peak time of the neurotransmitter DOPAC was about 9min, the peak time of the neurotransmitter HVA was about 20min, the peak shape was good, and the blank control had no interference.
  • DOPAC and HVA are the main metabolites of dopamine and can be used to reflect changes in dopamine content in the brain. As shown in Figure 5, the other components in the sample map did not interfere with the assay components.
  • the low-dose stem cells after nasal administration can improve the exercise capacity of 6-OHDA-induced Parkinson's model mice, reducing the nigral area nerves.
  • the generation of meta-apoptosis can increase the level of transmitters in the brain of model rats.

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Abstract

一种经鼻腔给药用于帕金森病治疗的神经干细胞制剂及其制备方法及应用，属于生物医药领域。提供的经鼻腔给药用于帕金森病治疗的神经干细胞制剂的制备方法，包括步骤为：种子细胞培养与纯化；P6-P8代细胞培养；工作细胞库；用于帕金森病的治疗制剂的配制。提供的经鼻腔给药用于帕金森病治疗的神经干细胞制剂及其制备方法及应用，该制剂滴加或注射入鼻腔后在体温条件下能迅速形成固态膜状，吸附于鼻腔待细胞进行缓慢吸收，并且不会随呼吸进入口腔或肺部，便捷了病人和医生的使用，且试验数据表明其的疗效和安全性比从创伤性的纹状体给药更好。该制剂可用于治疗帕金森疾病，且不需要使用免疫抑制剂。

Description

一种经鼻腔给药用于帕金森病治疗的神经干细胞制剂及其制备方法和应用
本申请要求于2017年02月16日提交中国专利局、申请号为201710083760.7、发明名称为“一种经鼻腔给药用于帕金森病治疗的神经干细胞制剂”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
本发明涉及生物医药技术领域，尤其涉及一种经鼻腔给药用于帕金森病治疗的神经干细胞制剂及其制备方法和应用。
背景技术
帕金森病(Parkinson’sdisease，PD)是临床上较为常见的难治病症，以黑质多巴胺能神经元变性缺失以及路易小体形成为主要病理特点，临床表现主要为静止性震颤、运动迟缓、肌强直和姿势步态异常，极大影响了患者的生存质量。从19世纪60年代晚期开始，通过口服左旋多巴的多巴胺替代疗法来缓解PD运动症状是现代神经科学转化研究中的成功之一。目前，左旋多巴仍是治疗有症状PD的金标准。PD治疗目前最为有效的方法是采用左旋多巴替代治疗，但仍不能有效阻止或减缓疾病的发展。目前只能采用药物、外科手术或电极的方式进行缓解和控制症状，避免发展速度过快。
自二十世纪八十年代初开展的细胞移植治疗PD以来，人们已尝试过胚胎中脑黑质组织、自体肾上腺髓质，胚胎干细胞、神经干细胞、骨髓干细胞、人畸胎瘤神经元等，并取得了一定的疗效，因此细胞移植治疗PD也越来越多的受到关注。但上述细胞来源均受供体(donors)资源匮乏、伦理问题、成瘤风险大、免疫排斥反应、诱导多巴胺能神经元比例低等问题困扰，限制了细胞移植治疗帕金森病的进程。
目前临床治疗帕金森病的方法以口服DA前体药物左旋多巴为主，但这种药物起初有效，长期服用则会出现疗效下降，并引起一系列运动障碍并发症。外科治疗以苍白球和丘脑的损毁为主要方法，但术后复发率高。 上述方法属于对症治疗，但不能逆转DA能神经元的变性，因此，找到一种能够替代变性的多巴胺能神经细胞以恢复多巴胺神经递质功能的新技术新方法，已成为迫在眉睫的需求。
发明内容
本发明的目的本发明的目的在于克服上述现有技术存在的不足，提供一种经鼻腔给药用于帕金森病治疗的神经干细胞制剂及其制备方法及应用。
本发明与鼠神经生长因子等神经营养药不同，神经干细胞注射液来源自人类胚胎(通过伦理审查)，完全100％人源，并且采用鼻腔无创给药的方式，直接作用于中枢神经系统，预计对小儿脑瘫将有更好的疗效。
本发明的目的是通过以下技术方案来实现的：
本发明涉及一种经鼻腔给药用于帕金森病治疗的神经干细胞制剂，所述方法包括如下步骤：
S1：种子细胞培养与纯化：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传代至P5代即为获得的种子细胞。
原代神经干细胞可以是在显微镜下使用手术器械分离获得废弃胎脑的腹侧中脑组织，然后采用消化液将细胞进行消化，细胞计数后使用无血清完全培养基进行重悬，并接种于超低吸附培养瓶中，35℃，5％CO2培养箱中进行悬浮培养，每间隔48～72小时进行换液，细胞连续培养12天左右即可传代，连续培养至P5代，在P0代到P5代这个过程里，所得的细胞越来越纯。将所得的P0～P5代细胞放入种子细胞库中进行存储。
本发明所述神经干细胞可以是不同动物例如哺乳动物例如灵长类动物例如人、啮齿类动物例如鼠等的神经干细胞，而且所述神经干细胞可以得自不同来源，例如可以商购获得的神经球冻存液或神经干细胞细胞系，或者是间充质干细胞向神经方向诱导获得的神经干细胞；或者是由其他来源经诱导分化而来的神经干细胞，比如iPSC技术，胚胎干细胞(包括极小类胚胎干细胞)，亚全能干细胞，人类视网膜色素上皮细胞等；或者是除神经组织器官以外的其他器官及组织而来的神经干细胞，比如脐带血和脐带。
S2：P6—P8代细胞培养：将步骤S1中得到的P5代神经干细胞用无血清培养基对细胞悬液进行培养扩增传代至P8代。
所述培养扩增传代的方法为将细胞培养瓶采用层粘连蛋白Laminin在37℃包被2小时，然后采用不含Ca、Mg的DPBS洗涤一遍备用；将P5代神经干细胞消化计数后，采用无血清完全培养基进行重悬，并调整密度为1x105个/ml，接种于包被好的培养瓶中，置于35℃，5％CO2环境进行贴壁培养；细胞培养每间隔48小时进行换液，细胞连续培养7天左右即可传代，直至P8代，期间P6-P8细胞放入主细胞库进行存储。
S3：工作细胞库：取步骤S2中P8代细胞，进行细胞特性和异源物质检测，检测合格后，即为工作细胞，将其进行消化冻存，并转移至工作细胞库待用。
优选的，步骤S2中所述无血清培养基是由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。
S4：用于帕金森病的治疗制剂的配制：将步骤S3的工作细胞进行贴壁培养，待细胞生长至对数生长期时将细胞进行消化并计数；然后在0～4℃条件下将工作细胞溶液与塑形剂按体积比1：1进行混合；所述工作细胞溶液的浓度为100微升2.5x106个工作细胞；所述塑性剂为Matrigel与0.4％羧甲基纤维素钠溶液按体积比1:1进行混合。
如果工作细胞是从工作细胞库中的取出的冻存工作细胞先应进行复苏后，再进行贴壁培养，待细胞生长至对数生长期时将细胞进行消化并计数；然后在0～4℃条件下将100微升2.5x106个神经干细胞与100微升塑形剂轻轻混匀，并转移至预冷的容器中，于4℃恒温环境保存。
上述的经鼻腔给药用于帕金森病治疗的神经干细胞制剂的使用方法，将所述的经鼻腔给药用于帕金森病治疗的神经干细胞制剂取出，滴至鼻腔粘膜或注射至皮下。
与现有技术相比，本发明具有如下有益效果：
1.本专利提供了一种无创的经鼻腔粘膜吸附给药的治疗帕金森的神经干细胞制剂，该制剂滴加或注射入鼻腔后在体温条件下能迅速形成固态膜状，吸附于鼻腔待细胞进行缓慢吸收，并且不会随呼吸进入口腔或肺部，便捷了病人和医生的使用，且试验数据表明其的疗效和安全性比从创伤性 的纹状体给药更好。
2.本专利中神经干细胞的培养方法采用P0～P5为悬浮培养、P6～P9为贴壁培养的方式，前期悬浮培养有利于获得纯化的神经干细胞克隆，能够保证获得的神经干细胞的纯度，而贴壁培养能够使神经干细胞更快的增殖传代，有利于细胞的中试扩大培养，并且能缩短接近一半的培养时间。
3.本专利提供了一种性能稳定、哺乳动物来源的神经干细胞制剂，该制剂可在哺乳动物脑内分化成多巴胺受体的神经元，少突胶质细胞和星状胶质细胞，可修复大脑的神经和组织。
4.该制剂可用于治疗帕金森疾病，且不需要使用免疫抑制剂。
附图说明
图1为神经干细胞形态，左图为悬浮培养的神经干细胞球；右图为贴壁培养的神经干细胞；细胞增殖倍数可达10倍/代次；
图2为神经干细胞细胞特性和分化能力的鉴定；A-E为神经干细胞特性鉴定，F-M为神经干细胞分化能力的鉴定；
图3为流式鉴定神经干细胞的纯度(＞95％)；
图4为神经干细胞给药后各组动物黑质区域免疫组化染色结果；
图5为神经干细胞给药后神经递质DOPAC相对含量变化；
图6为神经干细胞给药后神经递质HVA相对含量变化；
图7为各标准品的HPLC检测图谱；
图8为神经干细胞给药后各组组织样品的HPLC检测图谱；
图9为神经干细胞给药后不同组大鼠跨步试验结果比较；
图10为神经干细胞给药后第10周不同组大鼠跨步试验结果比较。
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：神经干细胞种子库细胞的获得
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、通过胚胎组织获取原代细胞
(1)胚胎组织获取
取E14的大鼠1只，以40mg/kg的剂量腹腔注射2％戊巴比妥钠麻醉；75％酒精浸泡消毒大鼠腹部5min，将大鼠仰卧位放置，牵拉固定四肢，剪开后腹正中接近耻骨联合的皮肤游离含有胚胎的完整子宫，移入含神经干细胞保存液的大培养皿内；分离冠臀长11～12mm左右的胎鼠。
(2)原代细胞获取和接种
使用精细手术镊分离中脑腹侧组织，取出放入装有6ml神经干细胞保存液的60mm无菌培养皿中，用镊子将组织块分成约1mm3的小块，然后转移至含有10ml神经干细胞保存液的50ml离心管中。然后进行细胞 消化与计数，采用无血清完全培养基进行重悬，加入超低吸附T75培养瓶中，置于35℃二氧化碳培养箱中进行培养。
4、P1～P5代细胞的扩增传代
神经干细胞培养至10～12天，观察细胞状态，用移液器将低吸附T75培养瓶中的神经球和培养液吸入50ml离心管中，100g离心3分钟，使神经球充分沉淀到管底，吸弃上清，将配好的5ml消化液加入到离心管中，放入培养箱中消化15分钟；4℃，300g，离心5分钟，吸弃上清，加入神经干细胞保存液10ml进行洗涤；4℃，300g，离心5分钟，吸弃上清，加入1～5ml神经干细胞保存液，轻柔吹打使细胞分散重悬。然后按照《细胞密度测定标准操作程序》对单细胞悬液进行计数。根据计数得到的细胞数量，计算需加入的无血清完全培养基的体积。将重悬的细胞迅速加入低吸附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，放入二氧化碳培养箱中，培养温度为35℃，二氧化碳浓度为5％。连续传代培养直至P5代，消化后细胞冻存至种子细胞库进行保存。
实施例2：神经干细胞主细胞库细胞的获得
1、培养瓶包被：将细胞培养瓶中加入适量的层粘连蛋白Laminin溶液，放入37℃培养箱中包被2小时，然后采用不含Ca、Mg的DPBS洗涤一遍备用；
2、将扩增纯化的P5代神经干细胞消化计数后，采用无血清完全培养基进行重悬，并调整密度为1x105个/ml，接种于包被好的培养瓶中，置于35℃，5％CO2培养箱中进行贴壁培养；细胞培养每间隔48小时进行换液，细胞连续培养7天左右即可消化传代，直至P8代，期间所得的细胞放入主细胞库进行存储。
实施例3：神经干细胞工作细胞库细胞的获得
将P8代细胞采用无血清完全培养基进行重悬，并调整密度为1x105个/ml，接种于包被好的培养瓶中，置于35℃，5％CO2培养箱中进行贴壁培养，培养过程中取样进行细胞特性和异源物质检测，检测合格后，将细胞进行冻存，并转移至工作细胞库待用。
实施例4：神经干细胞制剂的制备
将工作细胞库中的神经干细胞复苏进行贴壁培养，参照实施例1消化 细胞，4℃，300g，离心5分钟，吸弃上清，加入生理盐水离心洗涤，吸弃上清，1～5ml生理盐水重悬计数，4℃，300g，离心5分钟，吸弃上清，加入100微升的生理盐水将细胞重悬，在冰上将制备好的细胞悬液与塑形剂进行1:1混匀，然后分装至4度保存的预灌封的注射器中，置于4℃恒温箱中保存备用，使用时直接注射至给药部位。
实施例5：神经干细胞的鉴定
如图1所示实施例2、3里的神经干细胞增殖倍数可达10倍/代次。
如图2所示采用免疫荧光的方法鉴定检测合格后的P8代细胞，能够鉴定细胞表达神经干细胞的特异性表面标志，其纯度可达95％以上；同时也能分化为神经元、星形胶质细胞、少突胶质细胞、多巴胺神经元等。
如图3所示通过流式鉴定鉴定检测合格后的P8代细胞，神经干细胞的纯度可达95％以上。
实施例6：神经干细胞应用于帕金森动物模型的药效学研究
一、帕金森动物模型神经干细胞给药与评价
1.实验分组及免疫组化分析
采用单侧MFB内微量注射6-OHDA诱导PD模型。大鼠麻醉后放置成仰卧位，用前端套有硅胶软管的微量注射器吸取神经干细胞细胞悬液10ul，将软管慢慢伸入大鼠左侧鼻腔中，缓慢将细胞注射入鼻腔，放置15min待神经干细胞液完全形成固态凝胶时，将大鼠放回笼中。
实验分组如下：
Figure PCTCN2017101993-appb-000001
黑质区酪氨酸羟化酶(TH)免疫组化评价
干细胞给药90天后，分别随机取每组大鼠各3只，10％水合氯醛麻醉后，心脏灌注固定。先用生理盐水300ml进行预灌注，将其体内血液冲出，再用中性甲醛固定液400ml进行灌注固定，完整剥取脑组织，继续4℃过夜固定。后进入30％蔗糖溶液4℃脱水至下沉，作连续冰冻冠状切片，片厚为30μm，进行TH染色，见图4所示。
2.高效液相检测神经递质
2.1样品处理方法
干细胞给药90天后，分别随机取每组大鼠各3～6只，即刻处死，解剖取出纹状体，称重并记录。先于液氮中速冻5min后置-80℃保存备用。HPLC检测当天，样品按照100mg加入300μl样品处理液(0.2M高氯酸，0.2mM焦亚硫酸钠，0.01％EDTA-2Na)，同时含有2.5μM DHBA作为内标，超声粉碎(强度60％，时间2min)，14,000rpm离心10min，上清液经0.22μm水相滤膜过滤后用于HPLC检测，以上过程均在4℃下操作。神经递质DOPAC相对含量变化见图5所示；神经递质HVA相对含量变化见图6所示。
2.2色谱条件
色谱柱：DikmaPlatisilODS(5μm，250mm*4.6mm)
流动相：16％甲醇水溶液，含40mM乙酸钠，15mM柠檬酸，0.25mM辛烷磺酸钠，0.2mMEDTA-2Na，未加甲醇前调解pH值至4.3
检测器：电化学检测器(ECD)
流速：1ml/min
电压：500mV
量程：1μA
柱温：25℃
进样体积：50μl
各标准品的HPLC检测图谱见图7所示。
色谱峰结果为：
Figure PCTCN2017101993-appb-000002
Figure PCTCN2017101993-appb-000003
组织样品的HPLC检测图谱见图8所示。
色谱峰结果为：
Figure PCTCN2017101993-appb-000004
3.大鼠行为学评价
采用跨步试验，对大鼠治疗前后分别进行前肢功能测定。实验者一手固定大鼠躯体后半部和后肢，使其离地，另一手固定一侧前肢使另一侧前肢着地，以大鼠正手方向斜向一侧匀速移动大鼠(5s内移动90cm)，记录移动时着地侧前肢步数。交替测量两侧上肢的跨步数。每只大鼠一侧前肢重复5次，计算其均数。
二、实验结果与讨论
1.黑质区酪氨酸羟化酶(TH)免疫组化评价
对各组大鼠黑质区域的免疫组织化学评价结果如图4所示。
第3组黑质区域TH阳性神经元染色明显变浅，说明6-OHDA造模后，会诱导黑质区域神经元的大量凋亡。第7组黑质TH免疫组化染色稍有加深，说明给予美多巴后能一定程度改善由6-OHDA诱导的神经细胞凋亡。第1，2，4，5组黑质神经元染色较为明显，比第3组显著增多，说明鼻腔给予不同剂量的神经干细胞后能有效的提高黑质区域神经元的密度。
2.高效液相检测神经递质
2.1各标准品峰的归属
配制一定浓度的标准品后进样，各标准品的定位图谱如图7所示。内标DHBA最先出峰，出峰时间在6.5min左右，神经递质DOPAC的出峰时间约为9min，神经递质HVA的出峰时间在20min左右，峰形均良好，空白对照无干扰。
2.2纹状体中神经递质含量测定结果
DOPAC和HVA是多巴胺的主要代谢产物，可以反应脑内多巴胺含量变化的指标。如图5所示，样品图谱中其他成分对测定成分无干扰。
DOPAC和HVA的含量相对于植物油处理组的百分比如图5和图6所示。两种递质的变化趋势相似。不同剂量的神经干细胞给药后，递质水平有不同程度的提高。说明干细胞的治疗能提高6-OHDA诱导的帕金森模型大鼠脑内的递质水平。
3.行为学实验结果分析
神经干细胞给药后不同组大鼠跨步试验结果比较见图9所示。
给予6-OHDA进行造模后，模型动物跨步数显著减少；第1～4周给予不同剂量的神经干细胞进行干预，从得到的跨步试验结果看，给药后第10周，给药D组和G组与模型对照组比较有显著改善。神经干细胞给药后第10周不同组大鼠跨步试验结果比较见图10所示。
4.综合讨论
结合行为学、TH神经元染色及神经递质含量检测结果综合分析，中低剂量的干细胞鼻腔给药后对于6-OHDA诱导的帕金森模型鼠的运动能力有一定改善作用，减少黑质区域神经元凋亡的产生，并能提高模型大鼠脑内的递质水平。
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 (11)

  1. 一种经鼻腔给药用于帕金森病治疗的神经干细胞制剂的制备方法，其特征在于，所述方法包括如下步骤：
    S1：种子细胞培养与纯化：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传代至P5代即为获得的种子细胞；
    S2：P6～P8代细胞培养：将步骤S1中得到的P5代神经干细胞用无血清培养基对细胞悬液进行培养扩增传代至P8代；
    S3：工作细胞库：取步骤S2中P8代细胞，进行细胞特性和异源物质检测，检测合格后，即为工作细胞，将其进行消化冻存，并转移至工作细胞库待用；
    S4：用于帕金森病的治疗制剂的配制：将步骤S3的工作细胞进行贴壁培养，待细胞生长至对数生长期时将细胞进行消化并计数；然后在0～4℃条件下将工作细胞溶液与塑形剂按体积比1：1进行混合；所述工作细胞溶液的浓度为100微升2.5x106个工作细胞；所述塑性剂为Matrigel与0.4％羧甲基纤维素钠溶液按体积比1:1进行混合。
  2. 如权利要求1所述的经鼻腔给药用于帕金森病治疗的神经干细胞制剂的制备方法，其特征在于，步骤S1中所述原代神经干细胞来自灵长类动物或啮齿类动物。
  3. 如权利要求1所述的经鼻腔给药用于帕金森病治疗的神经干细胞制剂的制备方法，其特征在于，步骤S1中所述原代神经干细胞来自神经球冻存液或神经干细胞细胞系，间充质干细胞向神经方向诱导获得的神经干细胞、由其他来源经诱导分化而来的神经干细胞和除神经组织器官以外的其他器官及组织而来的神经干细胞中的一种或几种。
  4. 如权利要求3所述的经鼻腔给药用于帕金森病治疗的神经干细胞制剂的制备方法，其特征在于，由其他来源经诱导分化而来的神经干细胞中的其他来源包括胚胎干细胞、亚全能干细胞或人类视网膜色素上皮细胞。
  5. 如权利要求3所述的经鼻腔给药用于帕金森病治疗的神经干细胞制剂的制备方法，其特征在于，除神经组织器官以外的其他器官及组织而来的神经干细胞中的除神经组织器官以外的其他器官及组织包括脐带血或 脐带。
  6. 如权利要求1所述的经鼻腔给药用于帕金森病治疗的神经干细胞制剂的制备方法，其特征在于，步骤S1中培养的温度为35℃；培养的二氧化碳浓度为5％。
  7. 如权利要求1所述的经鼻腔给药用于帕金森病治疗的神经干细胞制剂的制备方法，其特征在于，步骤S2中所述培养扩增传代的方法为将P5代神经干细胞消化计数后，采用无血清完全培养基进行重悬，并调整密度为1x105个/ml，接种，置于35℃，5％CO2环境进行贴壁培养；细胞培养每间隔48小时进行换液，细胞连续培养7天即可传代，直至P8代。
  8. 如权利要求1所述的经鼻腔给药用于帕金森病治疗的神经干细胞制剂的制备方法，其特征在于，步骤S1和S2中所述无血清培养基是由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。
  9. 权利要求1～8任意一项所述的方法制备的经鼻腔给药用于帕金森病治疗的神经干细胞制剂。
  10. 权利要求9中所述的经鼻腔给药用于帕金森病治疗的神经干细胞制剂的应用，其特征在于，将所述用于帕金森病的治疗的神经干细胞制剂滴至鼻腔粘膜或注射至皮下。
  11. 如权利要求10中所述的经鼻腔给药用于帕金森病治疗的神经干细胞制剂的应用，其特征在于，用于帕金森病的治疗的神经干细胞制剂滴至鼻腔粘膜或注射至皮下的体积为10μL。
PCT/CN2017/101993 2017-02-16 2017-09-18 一种经鼻腔给药用于帕金森病治疗的神经干细胞制剂及其制备方法和应用 WO2018149127A1 (zh)

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