CN115192609A

CN115192609A - Application of mesenchymal stem cells in preparation of medicine for treating depression

Info

Publication number: CN115192609A
Application number: CN202210509140.6A
Authority: CN
Inventors: 唐健霞; 施松涛; 游咏; 曾贵荣; 李军; 曹婧; 陈斌; 胡胜
Original assignee: XIANGYA STOMATOLOGICAL HOSPITAL CENTRAL SOUTH UNIVERSITY
Current assignee: Beijing Youwei Technology Co ltd
Priority date: 2022-05-10
Filing date: 2022-05-10
Publication date: 2022-10-18
Also published as: WO2023217112A1

Abstract

The application belongs to the field of stem cells, and particularly relates to application of mesenchymal stem cells in preparation of a medicament for treating depression. The mesenchymal stem cell is, for example, a mesenchymal stem cell of dental pulp, for example, a human exfoliated deciduous tooth dental pulp stem cell.

Description

Application of mesenchymal stem cells in preparation of medicine for treating depression

Technical Field

The application belongs to the field of stem cells, and particularly relates to application of mesenchymal stem cells in preparation of a medicament for treating depression.

Background

Major Depressive Disorder (MDD) is a serious type of mental illness, and according to the fifth edition of the diagnostic and statistical manual for mental disorders, the diagnostic criteria for MDD are: (A) Within 2 weeks, 5 or more of the following symptoms appeared, showing different changes compared to the previous function. 1) Mood depression; 2) Loss of interest or pleasure in all or almost all activities; 3) Significant changes in body weight or appetite; 4) Insomnia or hypersomnia; 5) Psychomotor agitation or retardation; 6) Fatigue or lack of energy; 7) Feel worthless or guilt by oneself; 8) Inattention; 9) A tendency to suicide; at least 1 of which is (1) depression of mood or (2) loss of interest or pleasure. (B) These symptoms cause clinically significant distress or result in impairment of social, occupational, or other important functions. (C) These symptoms cannot be attributed to the physiological effects of a substance or other physical disorder. Diagnostic criteria a-C constitute major depressive episodes. MDD incidence can be as high as 18%. The mortality rate is very high in MDD because 50% of the population is suicidal and 10% of them have suicide.

The existing clinical antidepressant drugs, including selective 5-hydroxytryptamine reuptake inhibitor, selective norepinephrine reuptake inhibitor, monoamine oxidase inhibitor, etc., need to be taken for 2-4 weeks for the patients to take effect, which prolongs the mental and physical pains of the patients and increases the suicide risk. On the other hand, antidepressant drugs have large side effects, including dry mouth, excessive sleep or restlessness, sweating, dizziness, gastrointestinal functional reaction and the like, and some epilepsy may be induced. In addition, 20-40% of patients are not effective for first-line antidepressant drug therapy, while most patients with depression in the later stages of treatment develop resistance to the treatment.

Therefore, there is a need for an antidepressant drug which is superior, avoids the side effects of the existing antidepressants, is ineffective in treatment, and causes patients to develop resistance.

Disclosure of Invention

In one aspect, the present application provides a use of mesenchymal stem cells derived from teeth for the preparation of a medicament for the treatment of depression.

In one aspect, the present application provides a dental-derived mesenchymal stem cell for use in the treatment of depression.

In one aspect, the present application provides a medicament for treating depression, comprising mesenchymal stem cells derived from teeth.

In one aspect, the present application provides a method of treating depression comprising administering mesenchymal stem cells derived from teeth.

In one aspect, the present application provides a use of a tooth-derived mesenchymal stem cell for treating depression.

In some embodiments, the mesenchymal stem cell is a mesenchymal stem cell of dental pulp. In some embodiments, the mesenchymal stem cells are derived from deciduous teeth, more e.g., exfoliated deciduous teeth. In some embodiments, the mesenchymal stem cells are derived from a human, such as a human tooth, such as a human deciduous tooth, more such as a human deciduous tooth. In some embodiments, the mesenchymal stem cell is a human exfoliated deciduous tooth dental pulp stem cell.

In some embodiments, the patient with depression is a human. In some embodiments, the depression is a depression as judged according to the fifth edition of the manual for mental disorder diagnosis and statistics. In some embodiments, the depression is: mild depression, moderate depression, major depression, treatment-resistant depression, stress depression, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, type I bipolar depression, or type II bipolar depression. In some embodiments, the depression is Major Depressive Disorder (MDD).

In some embodiments, the mesenchymal stem cell is from 50 passages or less, such as from 40 passages or less, such as from 30 passages or less, such as from 20 passages or less, such as from 15 passages or less, such as from 10 passages or less, such as from 5 passages or less, such as from 3 passages to 5 passages. In some embodiments, the mesenchymal stem cell is passage 4.

In some implementationsIn a regimen, the mesenchymal stem cells are administered once every 1-30 days, for example once every 1-2 weeks (i.e., every 7-14 days), more for example once a week (i.e., every 7 days). In some embodiments, the mesenchymal stem cells are administered in an amount of 10 ⁴ -10 ⁸ Per 20g, e.g. 10 ⁵ -10 ⁷ Per 20g, more for example 10 ⁶ . + -. 10% per 20g, more preferably 10 ⁶ Per 20g. In some embodiments, the mesenchymal stem cells are administered by injection, for example, intravenously. In some embodiments, the drug is a drug for administration by injection, for example, a drug for intravenous administration.

In some embodiments, the drug is a long acting drug. In some embodiments, the treatment is a long-acting treatment.

In some embodiments, the medicament for treating depression prepared from mesenchymal stem cells of the present application can achieve the effect of treating depression after stopping administration for a longer time (for example, for up to 6 weeks), and can maintain the effect of treating depression continuously without administration for a short time (for example, for 1 week) as in the prior art medicaments for treating depression. And the fluoxetine drug group has drug resistance after long-term administration, the effect of the antidepressant action is obviously reduced, and the mesenchymal stem cell group can still continuously play the role of the antidepressant symptom after stopping taking the drug for 6 weeks.

Drawings

Fig. 1 is a flow chart of an experiment in some embodiments of the present application.

Figure 2 is a body weight measurement of each group of mice after treatment is completed, in some embodiments of the present application.

Figure 3 is a body temperature measurement of groups of mice after treatment is completed, in some embodiments of the present application.

Fig. 4 and 5 are sugar water preference levels for each group of mice in the first and second sugar water preference tests of some embodiments of the present application.

Fig. 6A-6C and 7A-7C are graphs of total distance of autonomous activity, central zone distance, and central zone residence time for each group of mice in the first and second null field tests according to some embodiments of the present application.

Fig. 8A and 8B are graphs of tail suspension immobility times for each group of mice in the first and second tail suspension tests according to some embodiments of the present disclosure.

FIG. 9 is a graph of the immobility time of each group of mice in forced swim test according to some embodiments of the present application.

FIG. 10 is a graph showing flow cytometry detection of mouse lymphocyte CD4 in some embodiments of the present application ⁺ And CD8 ⁺ The ratio is changed.

FIG. 11 is a graph of flow cytometry analysis of immune cell changes, in some embodiments of the present application.

Fig. 12 is a HE section of heart, liver, spleen, lung, and kidney in some embodiments of the present application.

Fig. 13 is a graph of the intensity of fluorescence signals enriched in liver by SHED, in some embodiments of the present application.

Figure 14 is a graph of plasma lipid metabolomics to detect changes in LPC content in some embodiments of the present application.

Detailed Description

In contrast to Mesenchymal Stem Cells (MSCs) derived from other tissues, MSCs derived from oromaxillofacial tissues, human exfoliated deciduous dental pulp stem cells (SHED), which were first isolated and identified by the inventors, developed neural crest cells of the mesenchymal head of early ectodermal leaves. The SHED not only has the general characteristics of MSCs, but also is easy to obtain and amplify, accords with ethics, and has higher self-proliferation capacity, neural differentiation capacity and immunoregulation capacity. In combination with the excellent experimental results presented by SHED in experiments on depression treatment, especially the ability to exert a sustained antidepressant effect over a prolonged period of time after discontinuation of drug administration (which is different from most antidepressant therapeutic agents), the inventors speculate that it may also be one of the reasons why SHED achieves a more desirable therapeutic effect in the treatment of depression-related diseases than other MSCs.

The property of exerting an antidepressant effect lastingly, as can be seen from the experimental results presented in the examples for SHED in experiments for the treatment of depression: after a longer period of time (6 weeks) after discontinuation of the administration, the SHED still exerts its antidepressant effect longer than most antidepressant therapeutic drugs. The studies by Dutta et al (s. Dutta, p. Segupta, life Sciences 2016, 152.

The inventors believe that SHED transplantation may be effective in MDD treatment and have great clinical utility. SHED may be achieved by modulating the immune imbalance of MDD, neurotransmitter regulation, secretion of neurotrophic factors, activation and repair of already damaged neuronal cell maturation in the brain.

The technical solutions of the present application are further illustrated by the following specific examples, which do not represent a limitation to the scope of the present application. Insubstantial modifications and adaptations of the concepts taught herein by others are intended to be covered by the present disclosure.

Example 1 isolation and culture of human exfoliated deciduous tooth pulp Stem cells (SHED)

The retained deciduous teeth of children 6 to 12 years old, which are in good health, were collected, the pulp of the deciduous teeth was aseptically separated, and digestion was performed using a solution containing Collagenase I (collagen I,2 mg/ml) and Neutral enzyme (Neutral protease II,4 mg/ml) under conditions of 45 minutes at 37 ℃ with 180rpm of a shaker. Terminating the digestion with a-MEM medium containing 15% fetal calf serum, centrifuging at 1500rpm for 5 minutes to remove the supernatant, collecting the bottom cell pellet, resuspending in a-MEM medium containing 15% fetal calf serum, inoculating to T25 cell culture flask, and culturing at 37 deg.C and 5% CO ₂ And (5) culturing. Changing the culture solution for the first time after 3 days, sucking floating impurities in the supernatant, washing with PBS, and adding the culture solution for continuous culture. After about 7 to 10 days, dense P0 colonies were visible, passaged to P1, until P3 passage cells were frozen in liquid nitrogen for subsequent experiments.

Example 2 establishment of a Chronic Unpredictable Mild Stress (CUMS) mouse model

40C 57BL6 male mice, 8 weeks old, were used, weighing between 22-24 g. In addition to 10 of the normal control groups, 30 mice were treated with Chronic Unpredictable Mild Stress (CUMS) including fasting, water deprivation, forced swimming, stroboflash, noise, restraint, cage tilt, wet cage, reversal of day and night, etc., during which the animals were fasted and water deprived, for 28 consecutive days, in a molding regimen as detailed in table 1.

TABLE 1 Chronic unpredictable stress (CUMS) modeling schedule

Example 3 grouping and administration

And (3) carrying out sugar water preference detection on the mice after 28 days of continuous molding, and randomly and averagely grouping the mice according to the sugar water preference detection result and considering the weight, wherein 10 mice respectively comprise a model control group, a Fluoxetine group and a SHED group (the Normal control group, the model control group, the Fluoxetine group and the SHED group are respectively marked as Normal (N), CUMS (C), fluoxetine (F) and SHED (S) groups in the attached drawing). The normal control group and the model control group are administrated with distilled water of 400 mu L/20g by intragastric administration every day; the mice in SHED group were administered via tail vein at a concentration of 1 × 10P 4-generation exfoliated deciduous dental pulp stem cells (SHED) per mouse per week ⁶ Resuspend each 2 g of PBS at 200. Mu.L; the fluoxetine groups are respectively administered with corresponding liquid medicines for intragastric administration, the administration volume is 20mL/kg (the fluoxetine administration concentration is 5.2 mg/kg), and the continuous daily administration is carried out until the materials are obtained. Mice continued to develop chronic unpredictable stress after each group was dosed, and the specific grouping and treatment was shown in figure 1. After the treatment was over, the model group had the lowest body weight, while both the SHED and fluoxetine groups recovered normal body weight (FIG. 2). To assess the safety of the SHED injection against the CUMS mouse body, body temperature measurements were taken before and after each dose of the mice in this group, and the body temperature of the mice fluctuated around 35.5-37.5 ℃ without any significant increase or decrease in body temperature persistence after the dose administration (FIG. 3).

Example 4 behavioural testing

The mice were tested for glucose preference, empty field, tail suspension, forced swimming, etc. 2 weeks after the 5 th dose (designated "first behavioural test") and 6 weeks after the 6 th dose (designated "second behavioural test") during the treatment period, respectively. And taking the tissue after the detection is finished to carry out related detection.

(1) And sugar water preference detection: the mice are subjected to a sugar water preference test, which is divided into a training period and a testing period. The 2 days before testing was used as a training period to allow the animals to acclimatize to sucrose drinking, and two bottles of 1% sucrose water were given to the animals 24 hours prior to the 1 st hour. The 2 nd 24 hours is also the training period, and the mice were given 1 bottle of 1% sucrose solution, 1 bottle of pure water. No food was fasted and water was withheld for 8 hours before testing. During the test period, 1 bottle of 1% sucrose water and 1 bottle of pure water are given to the mice at the same time, and the animals respectively drink the amounts of the sucrose water and the pure water within 15 hours of the test. In the middle of the test, the two bottles were interchanged in position to avoid positional preference. At the end of the test, the sugar water preference index (sugar water preference index = sugar water consumption weight (g)/total liquid consumption weight (g) × 100%) was calculated. The results of the first sugar water preference test showed that the model group had reduced sugar water consumption, whereas the sugar water intake was not significantly different between the SHED group and fluoxetine group compared to the normal control group, indicating the effectiveness of SHED treatment (FIG. 4). The results of the second sugar water preference test showed that the model group had reduced sugar water consumption, whereas the sugar water intake was not significantly different between the SHED group and fluoxetine group compared to the normal control group, indicating the effectiveness of SHED treatment (FIG. 5).

(2) And (3) detecting an empty field: the method comprises the following steps of carrying out empty field detection after sweet water preference test, putting animals into a test box (Behav animal autonomous activity behavior analysis system, product of Shanghai Ji Mass science and technology Co., ltd.), starting detection after adapting to 3 minutes, and detecting spontaneous activity of mice within 10 minutes. The open field detection is a method for observing and researching the neuropsychiatric changes of experimental animals and various behaviors after the experimental animals enter an open environment, and is used for evaluating the autonomous behaviors of the experimental animals in a new and different environment and researching the behaviors and the tensity. The first empty field test shows that the total course of the mice in the model group is increased, but the course and the residence time in the central area are obviously reduced compared with those in the normal control group, which indicates that the mice in the model group have obvious depression behaviors, the total course time is reduced by the SHED group and the fluoxetine group, the course and the residence time of the mice in the central area are prolonged, and the effect of prolonging the course and the residence time in the central area by the SHED group is better than that of the fluoxetine group compared with the fluoxetine group (figure 6). The empty field test in the second behavioural test found that the behaviour towards improving locomotor activity in mice after 6 weeks of withdrawal of the SHED injection therapy was still superior to that of the fluoxetine group compared to fluoxetine (figure 7).

(3) Suspending the tail: the immobility time of the mouse within 5 minutes is detected by adopting a full-automatic tail suspension instrument (TST-100 tail suspension video analysis system, chengdotai Union science and technology Co., ltd.). The model group showed minimal immobility time in the first tail suspension test, probably due to the anxious state at this time, struggled and thus showed reduction in tail suspension immobility time, both the SHED and fluoxetine groups effectively alleviated struggling time of mice (fig. 8A); in the second tail suspension test, the model group showed an increase in the time of tail suspension immobility, in a "behavior despair state", which was shown to be typical depressive-like behavior, whereas the tail immobility time was less in both the SHED-treated group and the fluoxetine group than in the model group, and the SHED group improved better (FIG. 8B).

(4) Forced swimming: the mice were tested in clear plexiglass bottles (20 cm diameter, 50 cm depth, 25 cm depth filled with 23-25 ℃ water). The mice were forced to swim alone for 5 minutes. Mice were tested for immobility over 5 minutes. Because the subsequent results are influenced after forced swimming, the forced swimming experiment is only carried out during the second ethological detection. The result shows that the immobility time of the model group is longest, which indicates that the mice of the model group are in a despair state and abandon struggling. The immobility time of the SHED group is obviously shorter than that of the model group and almost has no difference with the normal control group, the fluoxetine administration group has no obvious difference with the model group, and the SHED group has obvious statistical difference with the fluoxetine administration group, thereby explaining the limitation and drug resistance of the traditional drug fluoxetine to the treatment of the major depressive disorder to a certain extent. The results of forced swimming indicated the superiority of SHED in the treatment of Major Depressive Disorder (MDD) in mice and the persistence of the therapeutic effect (FIG. 9).

EXAMPLE 5 mouse selection

After the behavioral test is finished, taking tissues for relevant test, and fasting for 15 hours before taking materials. Isoflurane-induced mice are subjected to inhalation anesthesia, eyeballs are removed, blood is taken, and after whole blood of the mice is collected, the neck is removed, all the mice are killed, and then the materials are taken.

Method for detecting mouse lymphocyte CD4 by using blood plasma through flow cytometry ⁺ And CD8 ⁺ Changing the proportion, extracting mouse peripheral blood mononuclear cells by using a mouse peripheral blood mononuclear cell separation kit, and detecting CD4 by flow cytometry ⁺ And CD8 ⁺ T cell ratio. The method comprises the following specific steps: cells were stained with CD3, CD4 and CD8 antibodies, incubated on ice for 30 minutes and then the staining was stopped, the antibodies washed off and resuspended in PBS buffer on the machine. The results are shown in FIG. 10.

Fixing liver, partial spleen, heart, lung and kidney in 4% paraformaldehyde, slicing, HE-staining, grinding the rest spleen into single cell suspension, and detecting CD4 by flow cytometry ⁺ And CD8 ⁺ T cell ratio. The method comprises the following specific steps: cells were stained with CD3, CD4 and CD8 antibodies, incubated on ice for 30 minutes and then the staining was stopped, the antibodies washed off and resuspended in PBS buffer on the machine. The results are shown in FIG. 11.

The ratio of plasma and lymphocytes in the spleen demonstrates the safety of SHED in treating depression in the body, and does not cause significant changes in the body's immune cells. HE sections of heart, liver, spleen, lung and kidney showed that the SHED treatment did not cause significant pathological changes in the organs of the mice (fig. 12), confirming the safety of SHED treatment.

Due to the persistence of the therapeutic effect of SHED, it is speculated that SHED treatment may completely ameliorate pathological changes in mice due to chronic unpredictable stress, rather than merely solely ameliorating depressive-like behavior. Mesenchymal stem cells have homing properties and tend to recruit to diseased organs.

Before the mice are taken, 3 mice are taken in each group, and PKH-26 fluorescence labeled SHED is injected into the tail vein of each group, with the injection dose of 1 multiplied by 10 ⁶ Each aliquot was resuspended in 20g, 200. Mu.L of PBS. The tail vein is taken after 24 hours, and fluorescent signal scanning is carried out on each organ, so that the liver enrichment of the SHED in the model group and the fluoxetine administration group is obvious, and the liver enrichment of the SHED in the normal control group and the SHED group is not obvious (figure 13), which indicates that the SHED treatment group can possibly improve the liver injury caused by chronic unpredictable stress.

Thus a total of 28 samples from the remaining 7 plasma samples of each group were sent to vitamin, inc for lipid metabolomics analysis, finding Lysophosphatidylcholine (LPC): LPC (16.

Claims

1. Application of mesenchymal stem cells derived from teeth in preparation of medicines for treating depression.

2. The use of claim 1, wherein the mesenchymal stem cell is a dental pulp mesenchymal stem cell.

3. The use of claim 1, wherein the mesenchymal stem cells are derived from deciduous teeth.

4. The use of claim 1, wherein the mesenchymal stem cells are of human origin.

5. The use of claim 1, wherein the mesenchymal stem cell is a human exfoliated deciduous tooth dental pulp stem cell.

6. The use of claim 1, wherein the depression is: mild depression, moderate depression, major depression, treatment resistant depression, dysthymia, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, type I bipolar depression, or type II bipolar depression.

7. The use of claim 1, wherein the depression is major depressive disorder.

8. The use of claim 1, wherein the mesenchymal stem cells are passage no more than 50;

preferably, the mesenchymal stem cells are passage 40 or less;

preferably, the mesenchymal stem cells are passage 30 or less;

preferably, the mesenchymal stem cells are passage 20 or less;

preferably, the mesenchymal stem cells are passage 15 or less;

preferably, the mesenchymal stem cells are passage 10 or less;

preferably, the mesenchymal stem cells are passage 5 or less;

preferably, the mesenchymal stem cell is passage 3 to passage 5;

preferably, the mesenchymal stem cell is passage 4.

9. The use of claim 1, wherein the medicament is a medicament for administration by injection.

10. The use of claim 1, wherein the medicament is a long acting medicament.