WO2023217112A1 - 间充质干细胞在制备治疗抑郁症的药物中的应用 - Google Patents

间充质干细胞在制备治疗抑郁症的药物中的应用 Download PDF

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WO2023217112A1
WO2023217112A1 PCT/CN2023/092915 CN2023092915W WO2023217112A1 WO 2023217112 A1 WO2023217112 A1 WO 2023217112A1 CN 2023092915 W CN2023092915 W CN 2023092915W WO 2023217112 A1 WO2023217112 A1 WO 2023217112A1
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mesenchymal stem
depression
stem cells
passage
group
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PCT/CN2023/092915
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English (en)
French (fr)
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唐健霞
施松涛
游咏
曾贵荣
李军
曹婧
陈斌
胡胜
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北京中赢谷投资管理有限公司
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Publication of WO2023217112A1 publication Critical patent/WO2023217112A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants

Definitions

  • This application belongs to the field of stem cells, and specifically relates to the application of mesenchymal stem cells in the preparation of drugs for the treatment of depression.
  • MDD Major depressive disorder
  • the diagnostic criteria for MDD are: (A) 5 or more of the following symptoms within 2 weeks Symptoms, exhibiting changes compared to previous functioning. 1) Depressed mood; 2) Loss of interest or pleasure in all or almost all activities; 3) Significant changes in weight or appetite; 4) Insomnia or excessive sleeping; 5) Psychomotor agitation or retardation; 6) Fatigue or energy Inadequacy; 7) Feelings of worthlessness or guilt; 8) Inability to concentrate; 9) Suicidal tendencies; at least one of which is (1) depressed mood or (2) loss of interest or pleasure.
  • Antidepressants currently used clinically include selective serotonin reuptake inhibitors, selective norepinephrine reuptake inhibitors, monoamine oxidase inhibitors, etc. Patients need to continue taking the drugs for 2-4 weeks before they are effective. This will It prolongs the patient's mental and physical suffering and also increases the risk of suicide. On the other hand, antidepressant drugs have serious side effects, including dry mouth, excessive sleep or restlessness, sweating, dizziness, gastrointestinal function reactions, etc. Some may induce epilepsy. In addition, 20–40% of patients are ineffective in first-line antidepressant treatment, and most patients with depression become resistant to treatment in the later stages of treatment.
  • the present application provides an application of mesenchymal stem cells derived from teeth in preparing drugs for treating depression.
  • the present application provides a tooth-derived mesenchymal stem cell for treating depression.
  • the present application provides a drug for treating depression, including mesenchymal stem cells derived from teeth.
  • the present application provides a method of treating depression comprising administering tooth-derived mesenchymal stem cells.
  • the present application provides an application of tooth-derived mesenchymal stem cells in treating depression.
  • the mesenchymal stem cells are mesenchymal stem cells of dental pulp.
  • the mesenchymal stem cells are derived from deciduous teeth, more such as exfoliated deciduous teeth.
  • the mesenchymal stem cells are derived from humans, such as human teeth, such as human deciduous teeth, more such as human exfoliated deciduous teeth.
  • the mesenchymal stem cells are human exfoliated deciduous tooth pulp stem cells.
  • the depressed patient is a human.
  • the depression is depression according to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition.
  • the depression is: mild depression, moderate depression, severe depression, treatment-resistant depression, catatonic depression, melancholic depression, atypical depression, psychotic depression disease, peripartum depression, postpartum depression, bipolar I depression, or bipolar II depression.
  • the depressive disorder is major depressive disorder (MDD).
  • the mesenchymal stem cells are passage 50 or less, such as passage 40 or less, such as passage 30 or less, such as passage 20 or less, such as passage 15 or less. , for example, it is less than or equal to the 10th generation, for example, it is less than or equal to the 5th generation, for example, it is the 3rd to 5th generation. In some embodiments, the mesenchymal stem cells are passage 4.
  • the mesenchymal stem cells are administered once every 1-30 days, such as once every 1-2 weeks (i.e., every 7-14 days), more such as once every week (i.e., every 7-14 days). Dosing once per day).
  • the dosage of mesenchymal stem cells is 10 4 -10 8 cells/20g, for example, 10 5 -10 7 cells/20g, more such as 10 6 ⁇ 10% cells/20g, more For example, it is 10 6 pieces/20g.
  • the mesenchymal stem cells are administered by injection, such as intravenously.
  • the drug is a drug for administration by injection, for example, a drug for administration by intravenous injection.
  • the drug is a long-acting drug. In some embodiments, the treatment is long-acting treatment.
  • the drug for treating depression prepared from mesenchymal stem cells of the present application can still have the effect of treating depression after stopping administration for a long period of time (for example, up to 6 weeks) without the need for
  • the effect of treating depression can be maintained only if administered continuously or in short intervals (for example, one week).
  • the fluoxetine drug group developed drug resistance after long-term administration, and its antidepressant effect was significantly reduced, while the mesenchymal stem cell group could still continue to exert its antidepressant effect after 6 weeks of drug withdrawal.
  • Figure 1 is an experimental flow chart in some embodiments of the present application.
  • Figure 2 shows the body weight measurement results of each group of mice after the end of treatment in some embodiments of the present application.
  • Figure 3 shows the body temperature measurement results of each group of mice after the treatment in some embodiments of the present application.
  • Figures 4 and 5 show the sugar water preference of each group of mice in the first and second sugar water preference tests in some embodiments of the present application.
  • Figures 6A-6C and Figures 7A-7C show the total distance traveled by mice in each group of autonomous activities, the distance traveled in the central zone, and the residence time in the central zone during the first and second open-field testing of some embodiments of the present application.
  • Figures 8A and 8B show the tail suspension immobility time of each group of mice in the first and second tail suspension tests in some embodiments of the present application.
  • Figure 9 shows the immobility time of each group of mice in the forced swimming test according to some embodiments of the present application.
  • Figure 10 shows the use of flow cytometry to detect changes in the ratio of CD4 + and CD8 + in mouse lymphocytes in some embodiments of the present application.
  • Figure 11 shows the use of flow cytometry to analyze immune cell changes in some embodiments of the present application.
  • Figure 12 shows HE sections of heart, liver, spleen, lung and kidney in some embodiments of the present application.
  • Figure 13 shows the fluorescence signal intensity of SHED enriched in the liver in some embodiments of the present application.
  • Figure 14 shows changes in LPC content detected by plasma lipid metabolomics in some embodiments of the present application.
  • MSCs mesenchymal stem cells
  • SHED human exfoliated deciduous teeth
  • SHED can still exert long-lasting antidepressant properties.
  • the study by Dutta et al. (S. Dutta, P. Sengupta, Life Sciences 2016, 152: 244-248) calculated that 9 days in mice is approximately equivalent to 1 year in humans. Based on this, it can be inferred that SHED is equivalent to the effect of SHED on humans. It can still exert its antidepressant properties at least about 4 years and 8 months after discontinuation of administration.
  • SHED transplantation may be effective in the treatment of MDD and has great clinical application value.
  • SHED may be achieved by regulating the immune imbalance of MDD, regulating neurotransmitters, secreting neurotrophic factors, activating and repairing the mature damaged nerve cells in the brain.
  • Example 1 Isolation and culture of dental pulp stem cells (SHED) from human exfoliated deciduous teeth
  • Collect retained deciduous teeth from children aged 6 to 12 years old who are in good health isolate the pulp of the deciduous teeth aseptically, and use a solution containing type I collagenase (Collagenase I, 2 mg/ml) and neutral enzyme (Neutral protease II, 4 mg/ml)
  • type I collagenase Collagenase I, 2 mg/ml
  • neutral enzyme Neutral protease II, 4 mg/ml
  • Terminate digestion with a-MEM medium containing 15% fetal bovine serum centrifuge at 1500 rpm for 5 minutes to remove the supernatant, collect the cell pellet at the bottom, resuspend in a-MEM medium containing 15% fetal bovine serum, and inoculate T25 cell culture flask, culture at 37°C, 5% CO2 .
  • the medium was changed for the first time after 3 days, and floating impurities in the supernatant were aspirated.
  • culture medium was added to continue culturing. Dense P0 colonies will be visible after about 7 to 10 days, and the cells will be passaged to P1 and P3 generations and frozen in liquid nitrogen for subsequent experiments.
  • mice Forty 8-week-old C57BL6 male mice weighing 22–24 g were used. Except for the 10 mice in the normal control group, the remaining 30 mice were subjected to chronic unpredictable mild stress (CUMS).
  • the stress factors included fasting, water deprivation, forced swimming, strobe, and noise. , restraint, tilting cage, wet cage, day and night reversal, etc. During the restraint period, the animals were fasted and water-free, and were treated continuously for 28 days.
  • the modeling methods are detailed in Table 1.
  • mice After 28 days of continuous modeling, the mice were tested for their preference for sugar water. Based on the results of the sugar water preference test and taking into account their body weight, the mice were randomly divided into equal groups. There were 10 mice in each of the model control group, fluoxetine group, and SHED group (normal control group, model control group). , fluoxetine group and SHED group are abbreviated as N, C, F, S respectively in the attached figures). The normal control group and the model control group were given 400 ⁇ L/20g of distilled water by gavage every day; the mice in the SHED group were administered via the tail vein, and the dosage concentration was that each mouse was injected with P4 generation exfoliated deciduous tooth pulp stem cells (SHED) once a week.
  • SHED deciduous tooth pulp stem cells
  • the dose was 1 ⁇ 10 6 pieces/20g, resuspended in 200 ⁇ L PBS; the fluoxetine group was given the corresponding drug solution by gavage, and the dosage volume was 20 mL/kg (fluoxetine dosage concentration was 5.2 mg/kg), continuously every day Administer until collection. After administration in each group, the mice continued to undergo chronic unpredictable stress.
  • the specific grouping and treatment methods are shown in Figure 1. After treatment, the model group had the lowest weight, while both the SHED group and the fluoxetine group returned to normal weight ( Figure 2).
  • the body temperature of the mice in this group was measured before and after each administration. The body temperature of the mice fluctuated around 35.5–37.5°C, and no obvious persistence of body temperature was found after administration. Increase or decrease ( Figure 3).
  • mice were tested 2 weeks after the 5th dose (named the "first behavioral test") and 6 weeks after the 6th dose (named the “second behavioral test”).
  • Sugar water preference test empty field, tail suspension, forced swimming and other behavioral tests. After the test is completed, the tissue is taken for relevant tests.
  • (1) Sugar water preference test The mice were tested for sugar water preference.
  • the sugar water preference test was divided into a training period and a test period. The two days before the test were used as a training period to allow the animals to fully adapt to drinking sucrose water. In the first 24 hours, the animals were given two bottles of 1% sucrose water. The second 24 hours is also the training period. The mice are given one bottle of 1% sucrose water and one bottle of pure water. No fasting and no water for 8 hours before the test.
  • mice were given 1 bottle of 1% sucrose water and 1 bottle of pure water at the same time, and the amount of sugar water and pure water that the animals drank within 15 hours was tested.
  • the positions of the two bottles were swapped to avoid the influence of positional preference.
  • sugar water preference index sugar water consumption weight (g)/total liquid consumption weight (g) ⁇ 100%).
  • the results of the first sugar water preference test showed that the model group consumed less sugar water, while the sugar water intake of the SHED group and the fluoxetine group had no significant difference compared with the normal control group, indicating the effectiveness of SHED treatment ( Figure 4).
  • the results of the second sugar water preference test showed that the model group consumed less sugar water, while the sugar water intake of the SHED group and the fluoxetine group had no significant difference compared with the normal control group, indicating the effectiveness of SHED treatment (Figure 5).
  • the first empty field test found that the total distance traveled by the mice in the model group increased, but the distance and residence time in the central area were significantly reduced compared with the normal control group, indicating that the mice in the model group had obvious depressive behavior, and the SHED group and fluoxetine
  • the SHED group reduced the total journey time and prolonged the distance and residence time of mice in the central zone, and compared with the fluoxetine group, the SHED group was better than the fluoxetine group in extending the distance and residence time in the central zone ( Figure 6).
  • the open-field test in the second behavioral test found that compared with fluoxetine, SHED injection treatment was still better than the fluoxetine group in improving the autonomous activity behavior of mice 6 weeks after drug withdrawal (Figure 7).
  • Tail suspension A fully automatic mouse tail suspension instrument (TST-100 tail suspension video analysis system, Chengdu Taimeng Technology Co., Ltd.) was used to detect the immobility time of mice within 5 minutes.
  • the model group showed the least immobility time in the first tail suspension test. It may be due to the anxiety state at this time that it struggled, so it showed a decrease in tail suspension immobility time. Both the SHED and fluoxetine groups were effective.
  • mice were tested in a transparent plexiglass bottle (20 cm in diameter, 50 cm in depth, filled with 23-25°C water, 25 cm in depth). The mice were forced to swim alone for 5 minutes. Detect the immobility time of mice within 5 minutes. Because forced swimming will affect subsequent results, the forced swimming experiment was only conducted during the second behavioral test. The results showed that the model group had the longest immobile time, indicating that the mice in the model group were in a state of despair and gave up struggling. The immobility time of the SHED group was significantly less than that of the model group, and there was almost no difference from the normal control group.
  • mice were anesthetized by inhalation with isoflurane, the eyeballs were removed to collect blood, and the whole blood of the mice was collected and then all mice were killed by cervical dislocation and then the materials were collected.
  • Mouse peripheral blood mononuclear cells were extracted using a mouse peripheral blood mononuclear cell isolation kit, and CD4 + and CD8 were detected by flow cytometry. + T cell ratio.
  • the specific steps are as follows: Use CD3, CD4 and CD8 antibodies to stain the cells, incubate on ice for 30 minutes, stop staining, wash away the antibodies, resuspend in PBS buffer and put on the machine. The results are shown in Figure 10.
  • the liver, part of the spleen, heart, lungs, and kidneys were fixed in 4% paraformaldehyde, sliced and stained with HE. The remaining spleen was ground into a single cell suspension, and flow cytometry was used to detect the ratio of CD4 + and CD8 + T cells.
  • the specific steps are as follows: Use CD3, CD4 and CD8 antibodies to stain the cells, incubate on ice for 30 minutes, stop staining, wash away the antibodies, resuspend in PBS buffer and put on the machine. The results are shown in Figure 11.
  • the ratio of lymphocytes in plasma and spleen proves that SHED is safe for the body in treating depression and does not cause significant changes in the body's immune cells.
  • HE sections of the heart, liver, spleen, lungs and kidneys showed that SHED treatment did not cause obvious pathological changes in the organs of mice ( Figure 12), confirming the safety of SHED treatment.
  • SHED treatment may completely improve the pathological changes caused by chronic unpredictable stress in mice, rather than simply improving depressive-like behavior.
  • Mesenchymal stem cells have homing properties and tend to flock to damaged organs.
  • mice from each group were taken and injected into the tail vein with PKH-26 fluorescently labeled SHED.
  • the injection dose was 1 ⁇ 10 6 /20g and resuspended in 200 ⁇ L PBS.
  • the tail vein was collected 24 hours later, and each organ was scanned for fluorescence signals. It was found that SHED was significantly enriched in the liver of the model group and fluoxetine administration group, while SHED was not significantly enriched in the liver of the normal control group and SHED group ( Figure 13), which suggests that the SHED treatment group may have improved liver damage caused by chronic unpredictable stress.

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Abstract

间充质干细胞在制备治疗抑郁症的药物中的应用。所述间充质干细胞是牙髓的间充质干细胞，例如是人脱落乳牙牙髓干细胞。

Description

间充质干细胞在制备治疗抑郁症的药物中的应用 技术领域
本申请属于干细胞领域，具体涉及间充质干细胞在制备治疗抑郁症的药物中的应用。
背景技术
重性抑郁障碍(Major depressive disorder，MDD)是一种严重类型的精神疾病，根据精神障碍诊断与统计手册第五版，MDD的诊断标准为：(A)2周内出现5个或以上的下列症状，表现出与先前功能相比不同的变化。1)心境抑郁；2)对所有或几乎所有活动丧失兴趣或愉悦感；3)体重或食欲的明显改变；4)失眠或睡眠过多；5)精神运动性激越或迟滞；6)疲劳或精力不足；7)感到自己毫无价值或内疚；8)注意力不集中；9)自杀倾向；其中至少1项是(1)心境抑郁或(2)丧失兴趣或愉悦感。(B)这些症状引起有临床意义的痛苦，或导致社交、职业或其他重要功能方面的损害。(C)这些症状不能归因于某种物质的生理效应或其他躯体疾病。诊断标准A–C构成了重性抑郁发作。MDD发病率可高达18％。MDD中50％的人群有自杀倾向且其中有10％实施过自杀，因此致死率非常高。
目前临床使用的抗抑郁药，包括选择性5-羟色胺再摄取抑制剂，选择性去甲肾上腺素再摄取抑制剂，单胺氧化酶抑制剂等，患者需要持续进药2–4周后才能显效，这将延长患者的精神与身体痛苦，同时也增加了自杀风险。另一方面，抗抑郁药物副作用较大，包括口干、睡眠过多或坐立不安、出汗、头晕、胃肠功能反应等，有些可能诱发癫痫。此外还有20–40％的患者对一线抗抑郁药物治疗无效，同时治疗后期大部分抑郁症患者对治疗产生抗性。
因此，需要一种更为优异，避免现有抗抑郁药的副作用、治疗无效、使患者产生抗性等不利因素的抗抑郁药物。
发明内容
一方面，本申请提供了一种来源于牙的间充质干细胞在制备治疗抑郁症的药物中的应用。
一方面，本申请提供了一种用于治疗抑郁症的来源于牙的间充质干细胞。
一方面，本申请提供了一种治疗抑郁症的药物，包括来源于牙的间充质干细胞。
一方面，本申请提供了一种治疗抑郁症的方法，包括施用来源于牙的间充质干细胞。
一方面，本申请提供了一种来源于牙的间充质干细胞在治疗抑郁症中的应用。
在一些实施方案中，所述间充质干细胞是牙髓的间充质干细胞。在一些实施方案中，所述间充质干细胞来源于乳牙，更例如是脱落乳牙。在一些实施方案中，所述间充质干细胞来源于人，例如是人的牙，例如是人的乳牙，更例如是人脱落乳牙。在一些实施方案中，所述间充质干细胞是人脱落乳牙牙髓干细胞。
在一些实施方案中，所述抑郁症的患者是人。在一些实施方案中，所述抑郁症是根据精神障碍诊断与统计手册第五版判断的抑郁症。在一些实施方案中，所述抑郁症为：轻度抑郁症、中度抑郁症、重度抑郁症、难治性抑郁症、紧张性抑郁症、忧郁性抑郁症、非典型抑郁症、精神病性抑郁症、产期抑郁症、产后抑郁症、I型双相抑郁症、或II型双相抑郁症。在一些实施方案中，所述抑郁症是重性抑郁障碍(MDD)。
在一些实施方案中，所述间充质干细胞为小于等于第50代，例如是小于等于第40代，例如是小于等于第30代，例如是小于等于第20代，例如是小于等于第15代，例如是小于等于第10代，例如是小于等于第5代，例如是第3代至第5代。在一些实施方案中，所述间充质干细胞为第4代。
在一些实施方案中，所述间充质干细胞为每1–30日给药一次，例如是每1–2周(即每7–14日)给药一次，更例如是每周(即每7日)给药一次。在一些实施方案中，所述间充质干细胞的给药量为104-108个/20g，例如是105-107个/20g，更例如是106±10％个/20g，更例如是106个/20g。在一些实施方案中，所述间充质干细胞为注射给药，例如是静脉注射给药。在一些实施方案中，所述药物为用于注射给药的药物，例如是用于静脉注射给药的药物。
在一些实施方案中，所述的药物是长效药物。在一些实施方案中，所述的治疗是长效治疗。
在一些实施方案中，本申请的间充质干细胞制备的治疗抑郁症的药物，可以达到在较长时间内停止给药后(例如长达6周)仍然能够起到治疗抑郁效果，而不需要如现有技术的治疗抑郁症的药物一般，不间断地、或者短间断地(例如1周)给药才能够持续维持治疗抑郁效果。并且氟西汀药物组在长期给药后出现了药物抵抗作用，抗抑郁行为的作用明显下降，而间充质干细胞组停药6周后依然可以持续发挥抗抑郁症状的作用。
附图说明
图1为本申请的一些实施例中的实验流程图。
图2为本申请的一些实施例中，治疗结束后各组小鼠的体重测量结果。
图3为本申请的一些实施例中，治疗结束后各组小鼠的体温测量结果。
图4和图5为本申请的一些实施例的第一次和第二次糖水偏爱检测中，各组小鼠的糖水偏爱度。
图6A–6C和图7A–7C为本申请的一些实施例的第一次和第二次空场检测中，各组小鼠的自主活动总路程、中央区路程以及中央区停留时间。
图8A和图8B为本申请的一些实施例的第一次和第二次悬尾检测中，各组小鼠的悬尾不动时间。
图9为本申请的一些实施例的强迫游泳检测中，各组小鼠的不动时间。
图10为本申请的一些实施例中，利用流式细胞术检测小鼠淋巴细胞CD4+及CD8+比例变化。
图11为本申请的一些实施例中，利用流式细胞术分析免疫细胞变化。
图12为本申请的一些实施例中，心、肝、脾、肺和肾的HE切片。
图13为本申请的一些实施例中，SHED在肝脏中富集的荧光信号强弱。
图14为本申请的一些实施例中，血浆脂质代谢组学检测LPC的含量变化。
具体实施方式
与其他组织来源的间充质干细胞(mesenchymal stem cells，MSCs)相比，由发明人首次分离鉴定的口腔颌面组织来源的MSCs——人脱落乳牙牙髓干细胞(stem cells from human exfoliated deciduous teeth，SHED)，发育于早期外胚叶间充质头部的神经嵴细胞。SHED不仅具备MSCs的一般特征，还易于获取和扩增数量，符合伦理，同时SHED具有更高的自我增殖能力、神经向分化和免疫调节能力。结合SHED在治疗抑郁症的实验中呈现出的优异的实验结果、尤其是在停止给药较长时间后仍能持久发挥抗抑郁作用的性能(这区别于大多数抗抑郁治疗药物)，发明人推测，这也可能是SHED相较于其他MSCs，在治疗抑郁症相关的疾病中获得更理想的治疗效果的原因之一。
持久发挥抗抑郁作用的性能，从实施例中SHED在治疗抑郁症的实验中呈现出的实验结果可以看到：在停止给药较长时间(6周)后，相较于大多数的抗抑郁治疗药物，SHED仍能持久发挥抗抑郁作用的性能。Dutta等人的研究(S.Dutta,P.Sengupta,Life Sciences 2016,152:244-248)推算出小鼠的9天约相当于人类的1年时间，据此可以推测相当于SHED对于人类在停止给药至少约4年8个月后，仍能发挥抗抑郁作用的性能。
发明人认为，SHED移植可能对MDD治疗有效，且具有极大的临床应用价值。SHED可能通过调节MDD的免疫失衡、神经递质调节、分泌神经营养因子、激活和修复脑内已经受损的神经细胞成熟来实现。
以下通过具体的实施例进一步说明本申请的技术方案，具体实施例不代表对本申请保护范围的限制。其他人根据本申请理念所做出的一些非本质的修改和调整仍属于本申请的保护范围。
实施例1人脱落乳牙牙髓干细胞(SHED)的分离和培养
收集身体健康状态良好的6至12岁儿童滞留乳牙，无菌分离乳牙牙髓，使用含I型胶原酶(Collagenase I，2mg/ml)和中性酶(Neutral protease II，4mg/ml)的溶液进行消化，消化条件为37℃摇床180rpm转速45分钟。用含15％胎牛血清的a-MEM培养基终止消化，1500rpm 5分钟离心去除上清液后收集底部的细胞沉淀，用含15％胎牛血清的a-MEM培养基重悬后，接种于T25细胞培养瓶，37℃、5％CO2培养。3天后第一次换液，吸除上清液漂浮杂质，PBS清洗后，加入培养液继续培养。约7至10天后可见密集的P0集落，传代至P1，直至传至P3代细胞冻存于液氮中用于后续实验。
实施例2慢性不可预知性温和应激(CUMS)小鼠模型的建立
使用8周龄的C57BL6雄性小鼠40只，体重22–24g之间。除正常对照组的10只外，将其余30只小鼠进行慢性不可预知性温和应激(chronic unpredictable mild stress，CUMS)处理，应激因素包括禁食、禁水、强迫游泳、频闪、噪声、束缚、倾笼、湿笼、昼夜颠倒等，束缚期间动物禁食禁水，连续处理28天，造模方式详见表1。
表1慢性不可预知性应激(CUMS)造模时间表

实施例3分组及给药
对连续造模28天后的小鼠进行糖水偏爱检测，根据糖水偏爱检测结果兼顾体重对小鼠随机平均分组，模型对照组、氟西汀组、SHED组各10只(正常对照组、模型对照组、氟西汀组、SHED组在附图中分别简记为N、C、F、S)。正常对照组和模型对照组每天灌胃给予蒸馏水400μL/20g；SHED组小鼠经尾静脉给药，给药浓度为每只小鼠每周一次注射P4代脱落乳牙牙髓干细胞(SHED)，注射剂量为1×106个/20g，200μL PBS重悬；氟西汀组分别给予相应药液灌胃，给药体积为20mL/kg(氟西汀给药浓度为5.2mg/kg)，连续每天给药直至取材。各组给药后小鼠继续进行慢性不可预知性应激，具体分组及处理方式见图1。治疗结束后，模型组的体重最低，而SHED组与氟西汀组均恢复了正常体重(图2)。为了评估SHED注射对于CUMS小鼠机体的安全性，每次给药前后均对该组小鼠进行体温测量，小鼠体温围绕35.5–37.5℃上下波动，均未见给药后体温持续性的明显升高或降低(图3)。
实施例4行为学检测
分别于治疗过程中第5次给药后2周(命名为“第一次行为学检测”)和第6次给药后6周(命名为“第二次行为学检测”)对小鼠进行糖水偏爱检测、空场、悬尾、强迫游泳等行为学检测。检测结束后取组织进行相关检测。
(1)糖水偏爱检测：小鼠进行糖水偏爱检测，糖水偏爱测试分为训练期和测试期。测试前2天作为训练期，使动物充分适应蔗糖饮水，第1个24小时给予动物两瓶1％蔗糖水。第2个24小时也是训练期，给予小鼠1瓶1％蔗糖水，1瓶纯水。测试前不禁食，禁水8小时。测试期，同时给予小鼠1瓶1％蔗糖水，1瓶纯水，测试15小时内动物分别饮用糖水和纯水水量。测试中间，将两瓶位置互换以避免位置偏爱影响因素。测试结束，计算糖水偏爱指数(糖水偏爱指数＝糖水消耗重量(g)/总液体消耗重量(g)×100％)。第一次糖水偏爱检测结果显示，模型组对糖水消耗减少，而SHED组和氟西汀组糖水摄入与正常对照组相比无显著差异，说明SHED治疗的有效性(图4)。第二次糖水偏爱检测结果显示，模型组对糖水消耗减少，而SHED组和氟西汀组糖水摄入与正常对照组相比无显著差异，说明SHED治疗的有效性(图5)。
(2)空场检测：糖水偏爱测试后进行空场检测，将动物放入测试箱(Behav动物自主活动行为分析系统，上海吉量科技有限公司产品)中，适应3分钟后开始检测，检测10分钟内小鼠自发活动。空场检测是观察研究实验动物神经精神变化、进入开阔环境后的各种行为，是评价实验动物在新异环境中自主行为、探究行为与紧张度的一种方法。第一次空场检测发现模型组小鼠总路程增加，但在中央区的路程和停留时间与正常对照组相比明显减少，说明模型组小鼠有明显的抑郁行为，SHED组和氟西汀组减少了总路程时间，延长了小鼠在中央区的路程和停留时间，而且与氟西汀组相比，SHED组延长中央区路程和中央区停留时间的效果好于氟西汀组(图6)。第二次行为学检测中的空场检测发现与氟西汀相比，SHED注射治疗停药6周后改善小鼠自主活动的行为仍然优于氟西汀组(图7)。
(3)悬尾：采用小鼠全自动悬尾仪(TST-100悬尾视频分析系统，成都泰盟科技有限公司)检测小鼠5分钟内不动时间。模型组在第一次悬尾试验中表现为最少不动时间，可能是由于此时的焦虑不安状态，导致其挣扎故表现为悬尾不动时间的减少，SHED和氟西汀组均能有效缓解小鼠的挣扎时间(图8A)；第二次悬尾试验，模型组表现为悬尾不动时间增加，处于一种“行为绝望状态”，显示为典型的抑郁样行为，而SHED治疗组和氟西汀组悬尾不动时间均比模型组少，且SHED组改善效果更佳(图8B)。
(4)强迫游泳：小鼠在透明的有机玻璃瓶中测试(直径20厘米，深度50厘米，装满23-25℃的水，深度25厘米)。老鼠被强迫单独游泳5分钟。检测小鼠5分钟内不动时间。因强迫游泳后会影响后续结果，仅在第二次行为学检测时行强迫游泳实验。结果表明，模型组不动时间最长，说明模型组小鼠处于绝望状态，放弃挣扎。SHED组不动时间比模型组明显少，并且与正常对照组几乎无差异，而氟西汀给药组与模型组并无明显差异，并且SHED组与氟西汀给药组相比有明显统计学差异，在一定程度上说明了传统药物氟西汀对于重性抑郁障碍治疗的局限性和药物抵抗性。强迫游泳的结果表明了SHED治疗小鼠重性抑郁障碍(MDD)的优越性和治疗作用的持续性(图9)。
实施例5小鼠的取材
行为学检测结束后取组织进行相关检测，取材前禁食15小时。异氟烷行小鼠吸入麻醉，摘除眼球取血，收集小鼠全血后脱颈处死所有小鼠后取材。
血浆用于流式细胞术检测小鼠淋巴细胞CD4+及CD8+比例变化，使用小鼠外周血单个核细胞分离试剂盒提取小鼠外周血单个核细胞，通过流式细胞术检测CD4+及CD8+T细胞比例。具体步骤如下：使用CD3、CD4和CD8抗体对细胞进行染色，冰上孵育30分钟后中止染色，洗去抗体，PBS缓冲液重悬上机。结果如图10所示。
4％多聚甲醛中固定肝脏、部分脾脏、心脏、肺脏、肾脏，切片行HE染色剩余脾脏研磨成单细胞悬液，利用流式细胞术检测CD4+及CD8+T细胞比例。具体步骤如下：使用CD3、CD4和CD8抗体对细胞进行染色，冰上孵育30分钟后中止染色，洗去抗体，PBS缓冲液重悬上机。结果如图11所示。
血浆和脾中淋巴细胞的比例证明了SHED治疗抑郁症对于机体的安全性，并不会引起机体免疫细胞的明显变化。心、肝、脾、肺和肾的HE切片显示SHED治疗不会引起小鼠明显的脏器病理性变化(图12)，证实了SHED治疗的安全性。
由于SHED治疗作用的持续性，推测SHED治疗可能彻底改善了小鼠由于慢性不可预知应激导致的病理性改变，而不仅仅是单纯的改善抑郁样行为。间充质干细胞具备归巢特性，倾向于向有病损的器官趋集。
小鼠取材前每组各取3只小鼠，均尾静脉注射PKH-26荧光标记的SHED，注射剂量为1×106个/20g，200μL PBS重悬。尾静脉24小时后取材，取各脏器行荧光信号扫描，发现SHED在模型组和氟西汀给药组的肝脏富集明显，而SHED在正常对照组和SHED组的肝脏富集不明显(图13)，这提示SHED治疗组可能改善了慢性不可预知应激引起的肝损伤。
因此对剩余每组的7个血浆样品共28个样品送迈维生物公司进行脂质代谢组学分析，发现溶血磷脂酰胆碱(LPC)：LPC(16:1)，LPC(18:4)，LPC(0:0/22:4)，LPC(20:5)在模型组明显减少，而SHED组这四类LPC含量比模型组明显增加，并且与正常对照组几乎无差异，而氟西汀给药组与模型组并无明显差异，并且SHED组与氟西汀给药组相比有明显统计学差异(图14)，该部分数据说明SHED治疗能发挥其长效性是有其独特性的，可能通过彻底改善肝脏受损的功能来实现。

Claims (10)

  1. 来源于牙的间充质干细胞在制备治疗抑郁症的药物中的应用。
  2. 如权利要求1所述的应用，其特征在于，所述间充质干细胞是牙髓的间充质干细胞。
  3. 如权利要求1所述的应用，其特征在于，所述间充质干细胞来源于乳牙。
  4. 如权利要求1所述的应用，其特征在于，所述间充质干细胞来源于人。
  5. 如权利要求1所述的应用，其特征在于，所述间充质干细胞是人脱落乳牙牙髓干细胞。
  6. 如权利要求1所述的应用，其特征在于，所述抑郁症为：轻度抑郁症、中度抑郁症、重度抑郁症、难治性抑郁症、紧张性抑郁症、忧郁性抑郁症、非典型抑郁症、精神病性抑郁症、产期抑郁症、产后抑郁症、I型双相抑郁症、或II型双相抑郁症。
  7. 如权利要求1所述的应用，其特征在于，所述抑郁症是重性抑郁障碍。
  8. 如权利要求1所述的应用，其特征在于，所述间充质干细胞为小于等于第50代；
    优选地，所述间充质干细胞为小于等于第40代；
    优选地，所述间充质干细胞为小于等于第30代；
    优选地，所述间充质干细胞为小于等于第20代；
    优选地，所述间充质干细胞为小于等于第15代；
    优选地，所述间充质干细胞为小于等于第10代；
    优选地，所述间充质干细胞为小于等于第5代；
    优选地，所述间充质干细胞为第3代至第5代；
    优选地，所述间充质干细胞为第4代。
  9. 如权利要求1所述的应用，其特征在于，所述药物为用于注射给药的药物。
  10. 如权利要求1所述的应用，其特征在于，所述药物为长效药物。
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