Application of human amniotic epithelial stem cells in preparation of medicine for treating depression diseases

Abstract

The invention discloses application of human amniotic epithelial stem cells in preparation of drugs for treating depression diseases. The human amniotic epithelial stem cells are harvested after being subjected to pancreatin digestion, centrifugation and resuspension in sequence after being cleaned by mechanically separating an amnion on the inner surface of a discarded in-vitro placenta tissue; and a human amniotic epithelial stem cell regimen for the treatment of depression; the cell is wide in source, free of ethical dispute, low in immunogenicity and free of tumorigenicity, and a novel safe and effective way is provided for depression treatment. The cell preparation can be administered through intravenous injection, intrathecal injection or nasal delivery, and the action mechanism of the cell preparation is that the cell preparation can inhibit excessive activation of glial cells and relieve depression-related neuroinflammatory response, so that the neuron microenvironment is improved. The traditional Chinese medicine composition can effectively promote hippocampal neurogenesis (DCX positive cell increase and the like), regulate the activity of 5-hydroxytryptamine neurons and glutamic acid neurons to restore the steady state of a neural network, and remarkably improve the depression-like behavior of a model mouse.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular, the application of human amniotic epithelial stem cells in the preparation of a drug for treating depressive disorders. BACKGROUND

[0002] Major Depressive Disorder (MDD) is a mental illness characterized by persistent and significant emotional depression, decreased interest, and lack of energy. It has high incidence, high disability rate, and high suicide risk. The incidence of depression has gradually increased in recent years, and the course of the disease is chronic and recurrent. The etiology and pathogenesis of depression are complex, involving genetics, neuroendocrine, neuroimmunity, and neuroplasticity disorders. Currently, the commonly used antidepressants in clinical practice mainly act on monoamine neurotransmitters (such as 5-hydroxytryptamine, 5-HT) to increase the concentration of inter-synaptic monoamine substances to alleviate symptoms. However, this type of drug has a slow onset of action, and about 30%-50% of patients have poor efficacy, and progress to refractory depression, which urgently needs the development of new treatment strategies.

[0003] Previous studies have confirmed that patients with chronic depression have reduced hippocampal volume and insufficient dentate gyrus neurogenesis, which impairs the flexibility of learning and cognition, hinders normal cognitive processes, and ultimately leads to severe damage to memory and emotional function. Inflammation plays an important role in the pathophysiological mechanism of depression. Microglia are immune cells in the central nervous system that play a key role in maintaining brain homeostasis and responding to inflammatory signals. Studies have shown that microglia in patients with depression are abnormally activated and release a series of inflammatory mediators such as cytokines, chemokines, and prostaglandins, which can directly affect the function of neurons, impair neuroplasticity and neurogenesis, and then lead to the occurrence of depressive symptoms. Therefore, regulating neuroinflammation, promoting neurogenesis, and restoring neural network homeostasis are important targets for the treatment of depression. SUMMARY

[0004] To solve the problems in the background art, the present application provides a new strategy and scheme based on human amniotic epithelial stem cells (hAESCs) to address the limitations of existing antidepressants and the pathogenic mechanisms of depression. The aim is to safely and effectively improve depressive symptoms by inhibiting neuroinflammation, promoting neurogenesis, and regulating neurotransmitter balance.

[0005] In order to achieve the above-mentioned purpose, the application provides application of human amniotic epithelial stem cells (hAESCs) in preparation of a medicament for treating depression, and a specific treatment mechanism is explored.

[0006] I. Application of human amniotic epithelial stem cells in preparation of a medicament for treating depression.

[0007] The human amniotic epithelial stem cells are prepared in the following manner: (1) The amnion is separated from the inner surface of discarded ex vivo human placental tissue by mechanical means; (2) After the obtained amnion is carefully washed, it is subjected to 0.125% trypsin digestion, centrifugation and resuspension in sequence, and the human amniotic epithelial stem cells are harvested.

[0008] In the specific implementation, the amnion can be collected from the placental tissue discarded after cesarean section of healthy puerperae (syphilis spirochetes, HIV, hepatitis A and hepatitis B are all negative). The written informed consent of the donor is obtained for all collections.

[0009] The human amniotic epithelial stem cells are passage 1-2 cells.

[0010] II. Application of a pharmaceutical composition of human amniotic epithelial stem cells, the application of the pharmaceutical composition in preparation of a medicament for treating depression, the pharmaceutical composition of human amniotic epithelial stem cells comprising human amniotic epithelial stem cells.

[0011] III. The pharmaceutical composition of human amniotic epithelial stem cells comprises human amniotic epithelial stem cells and pharmaceutically acceptable carriers.

[0012] The pharmaceutically acceptable carriers are at least one selected from microcapsules, microspheres, liposomes, micelles or sustained-release materials and other pharmaceutically acceptable carriers.

[0013] The human amniotic epithelial stem cells are passage 1-2 cells.

[0014] The surface markers of the human amniotic epithelial stem cells satisfy: expression of EpCAM (CD326), E-cadherin (CD324), ZO-1 and Pan-CK; no expression of CD73, CD90 and CD105; no expression of HLA-II class antigens (HLA-DR and HLA-DQ), but expression of HLA-G.

[0015] This invention utilizes multiple biological effects of human amniotic epithelial stem cells to improve depressive symptoms, including increasing the number of new neurons in the hippocampus of depressed mice, increasing the concentration of anti-depression-related proteins brain-derived neurotrophic factor (BDNF) and serotonin (5-HT), and reducing the inflammatory microenvironment in the brain.

[0016] This invention application also provides a cell therapy method for treating depression, wherein the human amniotic epithelial stem cells are resuspended in physiological saline to obtain a cell injection solution, and the cell injection solution is administered within 6 hours after preparation.

[0017] IV. A cell preparation for treating depression, comprising an effective therapeutic dose of human amniotic epithelial stem cells and a pharmaceutically acceptable carrier, wherein the dosage concentration of the cell preparation is 1 × 10⁻⁶. 6 cells / mouse (based on mouse model).

[0018] The cell preparation is in the form of an injection, and the route of administration includes intravenous injection, intrathecal injection or nasal delivery, and can be made into a cell-biomaterial composite preparation.

[0019] The human amniotic epithelial stem cells described therein promote neuronal survival, improve the inflammatory microenvironment, effectively promote hippocampal neurogenesis (such as an increase in DCX-positive cells), regulate the activity of serotonergic and glutamatergic neurons to restore neural network homeostasis, and effectively and significantly improve depressive-like behavior in model mice.

[0020] Human amniotic epithelial cells (hAESCs) are a type of perinatal stem cell, possessing the dual characteristics of embryonic and adult stem cells. They are not only pluripotent but also possess immunomodulatory capabilities, helping to reduce neuroinflammatory responses and thus decrease neuronal damage. Furthermore, hAESCs can secrete various neurotrophic factors that help protect and promote neuronal survival and function. In animal models and in vitro experiments of neurological diseases such as stroke, spinal cord injury, Parkinson's disease, and Alzheimer's disease, hAESCs have shown the potential to promote nerve regeneration and repair. Based on these capabilities of nerve regeneration and protection, release of neurotrophic factors, and immunomodulation, hAESC transplantation could serve as a novel treatment for neuropsychiatric disorders.

[0021] This invention provides the use of human amniotic epithelial cells as a therapeutic agent for depression. Experimental verification has shown that the human amniotic epithelial stem cells have a significant therapeutic effect on depression. Compared with traditional antidepressants, this cell therapy agent has the advantages of good safety, low incidence of side effects, and significant antidepressant efficacy.

[0022] The present invention employs neuroscience methods to intervene in depressive-like mice using amniotic epithelial stem cells, demonstrating the effectiveness of hAESCs in treating depressive-like behaviors.

[0023] This invention demonstrates that treatment with human amniotic epithelial stem cells (hAESCs) reversed depressive-like behavior in mice with chronic unpredictable stress (CUMS). hAESCs can cross the blood-brain barrier and migrate directionally to the hippocampus in mice, promoting the secretion of hippocampal brain-derived neurotrophic factor (BDNF). Under behaviorally induced stress, hAESCs enhance the activity of hippocampal serotonergic (5-HT) neurons and reduce the neurotoxicity caused by excessive excitation of glutamatergic (GLU) neurons. Simultaneously, by inhibiting excessive activation of glial cells, they block depression-related inflammatory responses, improve the neuronal microenvironment, reduce neuronal damage and apoptosis, and promote the transformation of newborn neurons into mature neurons (neurogenesis), significantly improving depressive-like behavior in mice.

[0024] The core advantages of this invention lie in utilizing the multiple biological functions of hAESCs: 1. Targeting and Penetration: Intravenously infused hAESCs can cross the blood-brain barrier and migrate directionally to the hippocampus, reaching peak distribution 14-21 days post-transplantation. 2. Neuroinflammatory Regulation: hAESCs significantly inhibit microglial overactivation, reduce the levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α in the hippocampus, and improve the neuronal microenvironment. 3. Neurotrophic Support: Significantly enhances the expression of brain-derived neurotrophic factor (BDNF) in the hippocampus, promoting neuronal survival and functional recovery. 4. Neurotransmitter Balance: Enhances the activity of 5-HTergic neurons, inhibits excessive excitation of glutamatergic neurons, and restores neural network homeostasis. 5. Neural Development Promotion: Increases the number of DCX (a marker of newborn neurons) and NeuN (a marker of mature neurons) positive cells in the hippocampus, promoting neurogenesis. 6. Behavioral improvement: In a mouse model of chronic unpredictable stress (CUMS), hAESCs treatment significantly reduced immobility time in the forced swimming test (FST) and tail suspension test (TST) (P<0.01) and restored sucrose preference (P<0.01), confirming its antidepressant efficacy.

[0025] Experimental results showed that administration of hAESCs significantly improved depressive-like behaviors, specifically by reducing immobility time in the forced swimming test (FST) and tail suspension test (TST), increasing sucrose consumption, enhancing serotonergic (5-HT) neurons, and reducing the overexcitation activity of glutamatergic (GLU) neurons.

[0026] Mechanism studies have shown that hAESCs improve the neuronal microenvironment by inhibiting excessive activation of glial cells, reducing depression-related inflammatory responses, and increasing the content of neurotrophic factors in the hippocampus, ultimately reducing neuronal apoptosis and promoting neurogenesis, thereby reversing depressive behavior.

[0027] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit the scope of this application.

[0028] This invention utilizes human amniotic epithelial cells for the treatment of depression, fully leveraging the advantages of human amniotic epithelial cells, which mainly possess the following advantages: (1) It can maintain its pluripotency for a long time and has the potential to differentiate into the three germ layer tissues unique to embryonic stem cells; (2) The cell surface hardly expresses MHC II molecules, so it will not cause inflammation, allergy and immune response, and the transplant matching requirements are correspondingly reduced; (3) It has the ability to regulate immune responses in vivo and in vitro, and can secrete a variety of immunomodulatory factors, anti-angiogenic proteins or anti-inflammatory factor-related proteins when cultured in vitro; (4) It has low immunogenicity and can be regarded as immune pardon cells. It has no antigen presentation function. After transplantation, it can reduce the source of immune cells and avoid the occurrence of immune rejection. (5) It does not express telomerase reverse transcriptase and has no tumorigenicity (including benign tumors, sarcomas and carcinomas); (6) It has a strong ability to proliferate and can maintain a strong proliferative capacity in previous generations (about 5 generations); (7) It has a wide range of sources, is easy to obtain, has no application restrictions, and does not involve ethical issues.

[0029] Beneficial technical effects of the present invention: The invention utilizes human amniotic epithelial stem cells, which is an ideal treatment for depression and has great potential in clinical treatment.

[0030] As cells isolated from discarded placentas, human amniotic epithelial stem cells (HMSCs) have the advantages of being readily available and free from ethical concerns. HMSCs are non-tumorigenic and have low immunogenicity. They can synthesize and secrete various anti-inflammatory and neurotrophic factors, and contain multiple active substances that can inhibit the inflammatory activation of microglia, thereby reversing the pathological changes in brain regions, primarily the hippocampus, in the brain of those with depression, restoring brain health, and reversing depression. Attached Figure Description

[0031] To make the objectives, technical solutions, and test results of this invention clearer, the following figures are provided for illustration.

[0032] Figure 1 These are the basic properties of human amniotic epithelial stem cells.

[0033] Figure 2 A diagram showing the behavioral results of treating depression-like mice with human amniotic epithelial stem cells.

[0034] Figure 3This image shows the tracing results of human amniotic epithelial stem cells in the mouse brain.

[0035] Figure 4 The figure shows the effect of human amniotic epithelial cell therapy on the activity of 5-HT neurons and GLU neurons in the brain.

[0036] Figure 5 Figure showing the effect of human amniotic epithelial cell therapy on the expression of neurotrophic factor BNDF in the brain.

[0037] Figure 6 This diagram illustrates the regulatory effects of human amniotic epithelial stem cells on the inflammatory microenvironment in the brain.

[0038] Figure 7 This is a schematic diagram illustrating the inhibitory effect of human amniotic epithelial stem cells on the secretion of inflammatory factors by microglia (BV2).

[0039] Figure 8 This diagram shows the results of human amniotic epithelial stem cells in influencing the survival and transformation of neurons in the hippocampus of the brain.

[0040] Figure 9 Transcriptome results of the hippocampus in mice with depression after treatment with human amniotic epithelial stem cells. Detailed Implementation

[0041] The present invention will be described in detail below with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention. The present invention is further illustrated below by way of embodiments, but this does not limit the present invention to the scope of the embodiments described. Experimental methods in the following embodiments that do not specify specific conditions are performed according to conventional methods and conditions, or according to the product instructions. All reagents, cells, and animals used in the examples of the present invention are commercially available. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0042] The embodiments of the present invention are as follows: 1. Extraction, separation and identification of human amniotic epithelial stem cells.

[0043] The amniotic epithelial cells described in this invention are derived from humans. The amnion can be isolated from an isolated human placenta, washed with physiological buffer to remove blood cells, and mechanically removed any remaining chorionic villi and blood vessels. Isolation refers to the removal of cells from a tissue sample and separation from other tissues. Single cells can be isolated from intact human amniotic epithelial tissue using any conventional techniques or methods, including mechanical forces (shredding or shearing forces) and enzymatic digestion with a combination of proteases such as collagenase, trypsin, lipase, releasing enzyme, and pepsin.

[0044] 1.1 In a preferred embodiment of the invention, a method for isolating amniotic epithelial cells from amniotic tissue is provided, the method comprising the following steps: The amnion was obtained from placental tissue through mechanical separation. After cleaning, the amnion is digested with digestive enzymes, and the digested liquid is centrifuged to obtain human amniotic epithelial cells.

[0045] In a preferred embodiment of the present invention, the human amnion is obtained with the authorization and consent of the parturient. Placental tissue is taken from a healthy parturient after cesarean section, the placenta is cut with a cross-shaped scalpel, and the whole amnion is obtained through mechanical separation.

[0046] In another preferred embodiment of the present invention, the human amniotic epithelial cells obtained in step 2 can be further cultured, with the preferred culture conditions being: 1 × 10⁻⁶ cells / mL. 6 -1×10 8 Cells were seeded into culture dishes at a density of 1 cell / plate and placed in a CO2 incubator. After the human amniotic epithelial cells adhered, the culture medium was changed. Once 80% confluence was achieved, the dishes were placed in a 3% oxygen environment and cultured for 24 hours. After the cells had contiguously grown onto the plates, they were digested and cryopreserved.

[0047] Those skilled in the art can concentrate viable cell populations using other known methods. These post-processing washing / concentration steps can be performed individually or simultaneously. In addition to the methods described above, viable cell populations can be further purified or enriched after cell washing or culture to reduce contaminating and dead cells. Cells can be separated from suspensions using techniques such as buoyancy density sedimentation centrifugation, differential adhesion to and elution from a solid phase, immunomagnetic beads, fluorescence laser cell sorting (FACS), or other techniques. Examples of these different techniques and apparatus for performing them can be found in the prior art and commercially available products.

[0048] There are no restrictions on the type of basal culture medium used in this invention, as long as it is suitable for cell culture. Preferred media include DMEM and NPBM. There are no restrictions on the types of other components that may be contained in the basal culture medium mentioned above, but preferred components include F-12, FCS, and neural survival factors, etc.

[0049] The amniotic epithelial cell preparation of the present invention comprises the active ingredient amniotic epithelial cells and a pharmaceutically acceptable carrier.

[0050] Considering the type of disease to be treated, those skilled in the art can appropriately select the appropriate state of the amniotic epithelial cells: untreated collected cells (crude extract); partially purified cells; or purified cells that have then been cultured and expanded.

[0051] The pharmaceutically acceptable carriers described in this invention refer to substances with a suitable benefit / risk ratio that are suitable for use in humans and / or animals, without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), such as pharmaceutically acceptable solvents, suspending agents, or excipients that promote cell survival and deliver formulated cells to humans or animals. The carrier is selected according to a suitable planned route of administration. The carriers of this invention include, but are not limited to, various physiological buffers, such as physiological saline, phosphate buffer, artificial cerebrospinal fluid; or whole serum, umbilical cord serum; and may also include various artificial scaffolds, including but not limited to gelatin sponges, demineralized bone, polyglycolic acid (PGA), polylactic acid (PLA), and copolymers thereof.

[0052] Amniotic epithelial cells can be administered to patients using any suitable method, such as intravenous injection or intraspinal injection. These cells are typically contained in pharmaceutically acceptable liquid culture media. Cell administration can be repeated or continuous (e.g., via continuous infusion into cerebrospinal fluid). Generally, multiple administration routes should be administered at intervals of at least 7-10 days. Another approach is to combine cells with biological materials for administration. Combining these two methods can yield better therapeutic effects.

[0053] The appropriate dosage of amniotic epithelial cells will vary depending on the patient's age, sex, weight, health condition, and other factors. Typically, the dosage range for each administration is approximately 10... 3 -10 9 Cells, typically about 10 6 -10 7 cell.

[0054] 1. Flow cytometry analysis: (1) Prepare washing buffer: sterile PBS solution containing 2% (v / v) FBS; (2) Add 0.25% (w / v) trypsin digestion solution to the cell culture dish, place it in a 37℃ incubator for digestion, and after stopping digestion, collect the cells in a centrifuge tube and centrifuge at 4℃, 500×g for 5min. (3) Discard the supernatant, add 1 ml of washing buffer to resuspend the cells, transfer the cell suspension to a 1.5 ml EP tube, and centrifuge at 4°C, 500 × g for 3 min; (4) Discard the supernatant, add 1 ml of washing buffer to resuspend the cells, and centrifuge at 4°C, 500×g for 3 min; (5) Discard the supernatant, add 50 μl of washing buffer to each tube to resuspend the cells, then add an appropriate amount of antibody with a fluorescent label, resuspend again, and incubate at 4°C in the dark for 30 min; (6) Centrifuge at 4°C, 500×g for 3 min, discard the supernatant, add 1 ml of washing buffer to each tube to resuspend the cells, centrifuge again, and discard the supernatant; (7) Repeat step 6 twice; (8) Add 500 μl of washing buffer to each tube to resuspend the cells, then transfer the cell suspension to a flow cytometry tube for machine detection.

[0055] (9) Process the data using FlowJo analysis software.

[0056] The experimental results are as Figure 1 shown. The experimental results show that the cell morphology of human amniotic epithelial stem cells observed by optical microscopy and the detection of important cell surface markers. It can be observed that human amniotic epithelial stem cells are oval-shaped, with extremely low expression of CD31, CD34, CD45, and CD144, that is, they are not mesenchymal stem cells, not hematopoietic stem cells, not endothelial progenitor cells; with extremely low expression of HLA-DR and HLA-DQ, and high expression of HLA-G, that is, hAESCs have biological safety for allogeneic use.

[0057] 2. Construction of a mouse model of chronic unpredictable stress-induced depression Sixty clean-grade C57BL / 6 healthy adult male mice, 8 weeks old, weighing 23 - 28 g, were provided by Jiangsu Jicui Yakang Biotechnology Co., Ltd. [Experimental Animal License No. SYXK (Su) 2022 - 0004]. After the start of the study, they were housed in a standard animal experiment center throughout the process. All mice were housed separately in cages, with the same mouse strain in each cage. Together with other mice of the same strain in parallel experiments, there were 4 - 5 mice in each cage. The environmental temperature was 22 - 25°C, and the humidity was 65 - 70%. During the whole experiment, except for the operations required for model preparation, the mice had free access to drinking water and food, natural light, and good ventilation conditions, while maintaining a relatively quiet breeding environment.

[0058] After one week of acclimatization, all mice were randomly divided into three groups: a blank control group (Control group), a model group (CUMS group), and a cell group (hAESCs group), with 20 mice in each group. Mice in the model group and cell group received CUMS to establish a mouse model of depression. Nine specific stressors were used in this experiment: 24 hours of wet cage, 12 hours of reversed day-night light, 24 hours of fasting, 24 hours of water deprivation, 8 hours of cage tilting at 45°, 3 minutes of tail clamping (one clamp every 10 minutes for 1 minute, for a total of 3 times), 4 hours of restraint, 5 minutes of swimming in ice water, and 24 hours of co-caging (15 mice per cage). The model establishment process lasted for 4 weeks, with one stressor applied daily, and different stimuli used for two consecutive days. The order of stimuli was randomly determined as follows: (1) Wet cage: For 24 hours, sterile water is added to the mouse bedding to keep the bedding moist. The water level should cover the surface of the bedding to maintain its wet state. (2) Reversed day and night lighting: Lights are turned on at night for illumination and turned off during the day to keep it dark for 12 hours. (3) Fasting: Depriving mice of the opportunity to eat for 24 hours; (4) Water deprivation: Mice were deprived of the opportunity to drink water for 24 hours. (5) 45° tilted cage: For 8 hours, the mouse cage was tilted at a 45-degree angle; (6) Tail clamping: For 3 minutes, use colored tail clamps to clamp the mouse's tail 1 cm from the tip of the tail. Clamp the tail once every 10 minutes, each time for 1 minute, for a total of 3 times; (7) Restraint: For 4 hours, the mice were placed in a restraint tube with a diameter of 3 cm and a length of 12 cm; (8) Ice water swimming: For 5 minutes, the test animal was placed in water at a temperature of 4°C, with its toes just touching the bottom of the tub. The animal was removed after 5 minutes. (9) Co-cage rearing: 15 mice were kept together in the same cage for 24 hours.

[0059] 3. For example Figure 2 As shown, behavioral experiments observed the effect of human amniotic epithelial stem cells on improving the depressive state of CUMS mice. 3.1 The forced swimming test was used to assess the depressive-like behavior in mice.

[0060] On the first day of the experiment, a transparent cylindrical acrylic water tank with a diameter of 25 cm and a height of 60 cm was used. Warm water (25±2℃) was poured into the tank to a depth of approximately 30 cm, and mice were forced to swim in it for 15 minutes as a pre-stimulation. After swimming, the mice were gently wiped clean and returned to their cages, and the warm water in the cylinder was replaced. On the second day of the experiment, under the same conditions, mice were forced to swim for 6 minutes. Each mouse was video-recorded for 6 minutes, with the first 2 minutes considered an adaptation period and their performance during this period not included in the experimental results. The time the mice remained still during the last 4 minutes was also recorded.

[0061] 3.2 Tail suspension test (TST) The tail suspension test was used to assess the degree of despair and helplessness in mice. In a quiet, dark environment, the mice were suspended from a horizontal bar 30 cm above the ground by tape, rope, and clips at a distance of 1-2 cm from the tail. A cylindrical plastic tube was placed over the base of the tail, and the mice were kept upright. The tail suspension test was conducted for 6 minutes, and each mouse was filmed for 6 minutes. The first 2 minutes were considered an adaptation period for the mice, and their behavior during this period was not included in the experimental results. The time the mice remained still during the last 4 minutes was filmed and recorded.

[0062] The results are as follows Figure 2 As shown in the leftmost sub-image.

[0063] 3.3 Sucrose preference test (SPT) A sucrose preference test was used to assess anhedonia in mice. Mice were housed individually. On the first day of the experiment, each cage contained two bottles of 1% sucrose solution. On the second day, each cage contained one bottle of 1% sucrose solution and one bottle of purified water, with the bottles placed randomly. On the third day, all mice were fasted and deprived of water for 24 hours. The formal testing began on the fourth day. Each cage was randomly placed with two water bottles, one containing weighed 1% sucrose solution and the other containing purified water. After 12 hours, the bottles were swapped. The two bottles were weighed again after 12 hours. The amount of sucrose solution consumed and the amount of purified water consumed by each mouse were recorded. Sucrose preference was calculated as a percentage of total water consumption, specifically: Sucrose preference = (Sucrose solution consumption / (Sucrose solution consumption + Purified water consumption)) x 100%.

[0064] The experimental results showed that in the tail suspension test, after modeling, the immobility time of mice in the model group was significantly longer than that of mice in the blank control group (P<0.001), indicating their state of depression and despair. After hAESC transplantation, the immobility time of mice was significantly reduced compared to the model group (P<0.01), while there was no statistically significant difference in immobility compared to the normal group. This indicates that hAESC transplantation can increase the struggle time in a despair environment in CUMS model mice, reduce immobility time, and improve their state of depression and despair.

[0065] After CUMS modeling, the model group mice exhibited a longer immobility time during the forced swimming test compared to the control group (P<0.05), indicating their depressed and hopeless state. The immobility time of the cell group mice after hAESC transplantation was significantly reduced, showing a statistically significant difference compared to the model group (P<0.05) and the normal group, indicating that hAESC transplantation can improve the depressive mood and increase the struggling time of the mice in the depression model environment.

[0066] In the sucrose preference experiment, after CUMS modeling, the model mice showed a significant decrease in water preference compared to the blank control group (P<0.001), indicating that the mice had lost pleasure after depression modeling. The sucrose preference index of the mice after hAESCs transplantation treatment increased significantly, with a significant difference compared to the model group (P<0.01). However, compared with the normal group, the sucrose preference result of the cell group mice was slightly lower, but there was no statistical difference. This suggests that hAESCs treatment can promote sucrose preference behavior in depressed mice and improve anhedonia.

[0067] The results are as follows Figure 2 The middle subgraph and the right subgraph are shown.

[0068] 4. Raman imaging to trace the distribution of human amniotic epithelial stem cells in the mouse brain after intravenous infusion. To investigate whether transplanted hAESCs can cross the blood-brain barrier and enter the brain, and their spatial and temporal distribution, this invention uses gold nanoparticle-labeled hAESCs, which are then injected into mice via tail vein. On days 1, 3, 7, 14, and 21 post-transplantation, the mice are anesthetized, and their brains are sectioned. Raman imaging is used to detect gold nanoparticle signals in the prefrontal cortex, anterior cingulate cortex, hippocampus, and posterior cingulate cortex, indicating the spatiotemporal distribution of hAESCs. The preparation of the human amniotic epithelial stem cell suspension is described in Specific Implementation Scheme 1. This invention provides a gold nanoparticle labeling method to track stem cells, allowing for the tracking of human amniotic epithelial stem cells entering the brain after tail vein injection in mice and analyzing their distribution within the brain.

[0069] The method for labeling human amniotic epithelial stem cells with gold nanoparticles is as follows: 200 μL of Au-UrDTTC nanoparticles (2 mg / mL in PBS) are mixed with 15 × 10⁻⁶ ppm of PBS. 6 After incubating with complete culture medium for 6 hours, the nano-gold-hAESCs were centrifuged at 300g for 10 minutes. The supernatant was washed away, and the nano-gold-hAESCs were collected in PBS. Viable cells were counted by trypan blue staining, and the viable cell density was adjusted to 1 million cells / 100μL. The nano-gold-hAESCs were then injected into mice via the tail vein. On days 1, 3, 7, 14, and 21 post-transplantation, mice (n=3) were anesthetized with isoflurane (2.3%), and their brains were sectioned (100μm thick). Slices from the prefrontal cortex, anterior cingulate cortex, hippocampus, and posterior cingulate cortex were collected. Raman scanning was performed using a 785 nm laser with StreamLine acquisition mode. Multiple spectra were acquired under continuous laser illumination, with continuous linear movement during the microscopic phase. The spatial resolution along the scan line was set to 14.2 μm in length and 200 μm in width. All Raman mapping images were scanned using a 150mW laser with a 5x objective and an integration time of 0.2s. The total imaging time depends on the scanned area.

[0070] After the mouse depression model was established and behavioral assessments were completed, depressed mice were selected and treated with stem cell therapy via tail vein administration. The specific procedure was as follows: a prepared suspension of human amniotic epithelial stem cells was injected into the mice via tail vein at a concentration of 1 million cells / 100 μL.

[0071] Experimental results are as follows Figure 3 As shown, the results indicated that the fluorescence signal in the mouse brain gradually increased over time. On day 14, gold nanoparticle signals were distributed in the prefrontal cortex, anterior cingulate cortex, hippocampus, and posterior cingulate cortex. On day 21, the fluorescence signal in the hippocampus was stronger than in other brain regions. This suggests that hAESCs can cross the blood-brain barrier and enter the mouse brain, primarily distributing in the hippocampus.

[0072] 5. Optical fiber records the activation status of specific neurons under different behaviors.

[0073] To investigate the effects of hAESCs intervention on neuronal activity in the mouse hippocampus, the dynamic changes in 5-HT release during different behaviors in each group of mice were detected using fiber optic recording. Stereotactic injection was used to co-express 5-HT2.0 and iGluSnFR in the CA1 region of the mouse hippocampus.

[0074] 5.1 Stereoscopic Injection of Customized Viruses into the Brain The viral vector was designed and manufactured by Schmidt Technology (Wuhan, China). Mice were anesthetized using a 2-5% isoflurane inhalation anesthesia machine. After shaving and disinfecting the head, the mice were fixed in a three-dimensional frame using incisor and ear strips. A midline incision was made from above the forehead to the ear line using ophthalmic scissors to expose the skull, and the periosteum was removed with 3% hydrogen peroxide to expose the meninges. To verify that the skull was straight in all planes, a hole was drilled in the skull at the target injection site, and a 1ml Hamilton microinjector was inserted into the injection site, with the needle left in place for 10 minutes before and after injection. First, 200 nL of 5-HT probe (AAV-hSyn-r5-HT2.0, AAV2 / 9, Wuhan Shumi Brain Science Technology Co., Ltd.) and 200 nL of GLU probe (rAAV-hSyn-iGluSnFR(A184S)-WPRE-hGH PA, AAV2 / 9, Wuhan Shumi Brain Science Technology Co., Ltd.) were injected into the left hippocampal CA1 region (BregmaAP: -3.16 mm; ML: -3.2 mm; DV: 3.5 mm) at a rate of 40 nL / min and left in place for 10 minutes. Then, a 250 mm optical fiber was inserted into a ceramic sleeve and implanted 0.2 mm above the injection site. After implantation and injection, the wound was sutured and disinfected. Mice were returned to their cages to monitor viral expression, and further procedures were performed at least 3 weeks post-surgery.

[0075] 5.2 Fiber Optic Recording of Neuronal Activity The activity of serotonergic (5-HT) neurons and glutamatergic (GLU) neurons in the CA1 region of the left hippocampus was assessed using a fiber optic photometry system. Matched optical fibers were connected to the implanted fiber using a sleeve. A 2-meter-long optical fiber connected the commutator and the implanted fiber. 473nm and 580nm laser beams with wavelengths of 10–20 μW were focused by a 20x objective lens and reflected from a dichroic mirror to the optical commutator to record calcium ion signals. The photomultiplier tube current was converted into an analog voltage signal by an amplifier and then recorded as a digital signal (100 Hz) by the fiber optic photometry system. This reflected the fluorescence signal intensity. Finally, normalized AF values ​​and trajectories were visualized using custom-written MATLAB code.

[0076] Experimental results are as follows Figure 4As shown, the results indicated that in the CUMS depression model, compared with the blank control group, the activity of 5-HTergic neurons in the hippocampus was significantly inhibited while the activity of glutamatergic neurons was excessive. The sucrose consumption in the sucrose preference test was reduced (P<0.01), and the immobility time in the forced swimming / tail suspension test was prolonged (P<0.01), with behavioral manifestations consistent with the degree of neural activity. In the tail suspension test, the 5-HT fluorescence signal in the hAESCs treatment group was significantly increased compared with the model group (P<0.001), but the fluorescence intensity was still lower than that in the blank control group (P<0.05). This indicates that hAESCs intervention can partially reverse the reduction of 5-HT in the CA1 region of the hippocampus. The hAESCs treatment group showed a significant effect in reversing the excessive activation of glutamatergic neurons in depressed mice, with no significant difference compared with the blank group. In the forced swimming test, the 5-HT fluorescence signal continued to increase regardless of whether the mice struggled, and only gradually began to decrease after the mice left the water. The peak 5-HT fluorescence signal in the blank control group and the cell therapy group was significantly higher than that in the model group. The activity of GLU neurons in the treatment group was significantly reduced compared to CUMS mice, showing a difference from the blank control group. When mice drank sugar water, this invention observed a significant increase in 5-HT fluorescence and a significant reduction in GLU signal overactivation in the treatment group. There was no statistically significant difference in 5-HT and GLU fluorescence peaks between the blank control group and the cell therapy group. These data indicate that after hAESCs treatment, mice showed increased 5-HT secretion in the hippocampal CA1 region and inhibited GLU neuronal overexcitation during behavioral tests, demonstrating the recovery of neural networks after hAESCs treatment.

[0077] 6. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of neurotrophic factors and inflammatory factors in the brains of mice after stem cell therapy. The concentrations of inflammatory cytokines IL-1, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and brain-derived neurotrophic factor (BDNF) in mouse hippocampal tissue homogenates were detected using an ELISA kit. The prepared mouse hippocampal tissue homogenate was diluted 5-fold and added to a 96-well plate according to the manufacturer's instructions. The specific steps are as follows: (1) Prepare a coating solution by diluting the capture antibody with coating buffer; (2) Coat each well of the plate with 100 μL of coating solution. Cover the plate and incubate overnight at 2-8°C. (3) Aspirate the liquid from each well and wash three times with 200 mL of washing buffer per well. After washing, invert the plate and gently tap it on absorbent paper to remove any remaining liquid; (4) At room temperature, add 200 μL of blocking buffer to each well and block for 1 hour; (5) Suction, invert the orifice plate and gently tap it on absorbent paper to remove residual liquid; (6) Prepare standard and sample dilution solutions in blocking buffer; (7) Add 100 mL of standard (two copies) and the sample to the designated wells. Gently shake continuously (500 rpm) at room temperature for 1 hour; (8) Aspirate the liquid from each well and wash each well 5 times with >200L of washing buffer. After washing, invert the plate and gently tap it on absorbent paper to remove any remaining liquid; (9) Prepare the detection antibody solution by diluting the detection antibody in a blocking buffer; (10) Add 100 μL of detection antibody solution to each well. Gently shake continuously at room temperature (approximately 500 rpm, for 2 hours); (11) Aspirate the liquid from each well and wash 5 times with >200 μL of washing buffer in each well. After washing, invert the plate and gently tap it on absorbent paper to remove any residual liquid; (12) Prepare streptavidin-horseradish peroxidase working solution by diluting with blocking buffer at a ratio of 1:5000; (13) Add 100 μL of streptavidin-horseradish peroxidase working solution to each well. Gently shake continuously (approximately 500 rpm) at room temperature for 30 minutes; (12) Prepare streptavidin-horseradish peroxidase working solution by diluting with blocking buffer at a ratio of 1:5000; (13) Add 100 μL of streptavidin-horseradish peroxidase working solution to each well. Gently shake continuously (about 500 rpm) at room temperature for 30 minutes; (14) Aspirate the liquid from each well and wash each well 5 times with >200 mL of washing buffer. After washing, invert the plate and gently tap it on absorbent paper to remove any remaining liquid; (15) Add 100 mL of substrate solution to each well. Incubate the plate at room temperature for 30 minutes; (16) Add 100 μL of stop solution to each well; (17) Measure the absorbance at 450 nm within 30 minutes after adding the stop solution.

[0078] Experimental results are as follows Figure 5As shown in the results, statistical analysis revealed that after cell therapy, brain-derived neurotrophic factor (BNDF) in the brains of depressed mice was significantly upregulated (P<0.001), indicating that human amniotic epithelial stem cell therapy has a promoting effect on the recovery of damaged brains.

[0079] Furthermore, because inflammation plays a crucial role in the progression of depression, neuroinflammation and neuroplasticity are interconnected: inflammation is a key node in the pathological changes of neuroplasticity. ELISA detection methods showed that, compared to the blank control group, as... Figure 6 As shown, the levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in the hippocampus of mice induced by CUMS were significantly increased (IL-1β: P < 0.0001, IL-6: P < 0.01, TNF-α: P < 0.05). Compared with the model group, the expression of IL-1β, IL-6, and TNF-α was significantly inhibited 4 weeks after hAESC transplantation (IL-1β: P < 0.001, IL-6: P < 0.05, TNF-α: P < 0.05), with no statistically significant difference compared with the normal group. These results indicate that hAESC transplantation can alleviate CUMS-induced neuroinflammation in the hippocampus of mice.

[0080] 7. Immunofluorescence 7.1 Cellular Immunofluorescence An inflammatory stimulation model was established using the microglia cell line BV2 and lipopolysaccharide (LPS) (100 μM). The treatment group consisted of co-cultured BV2 cells and human amniotic epithelial stem cells. The inflammatory marker iNOS was detected by immunofluorescence.

[0081] BV2 cell treatment: Remove cells from the cell culture incubator, observe the cells, discard the culture medium, wash cells 2-3 times with 1×PBS, add 500-800µL of 4% paraformaldehyde to each well, and fix at room temperature for 15 min; discard the paraformaldehyde solution, wash 3 times with 1×PBS, 5 min each time; cell permeabilization: permeabilize with 1×PBS containing 0.25% Triton X-100 at room temperature for 1-10 min; discard the permeabilization solution, wash 3 times with 1×PBS, 5 min each time; Blocking: Add 500-800µL of blocking buffer (900µL PBS + 100µL horse serum) to each well and block at room temperature for 1 hour; Primary antibody incubation: Dilute the antibody with 1×PBS at a ratio of 1:50-1:200, incubate at room temperature for 2 hours, then recover the primary antibody dilution solution, and wash 3 times with 1×PBS for 5 minutes each time; Secondary antibody incubation: Dilute the fluorescent secondary antibody with 1×PBS at a ratio of 1:200-1:500, incubate at room temperature in the dark for 1 hour, then discard the fluorescent secondary antibody dilution solution, and wash 3 times with 1×PBS for 5 minutes each time; DAPI staining: DAPI was diluted with 1×PBS at a ratio of 1:1000 and the cells were stained at room temperature for 1 min. Cell washing: Discard the DAPI diluent and wash three times with 1×PBS for 5 minutes each time; Observe and photograph the positive areas under an inverted fluorescence microscope.

[0082] Experimental results are as follows Figure 7 As shown, the results indicated that lipopolysaccharide (LPS) stimulation significantly activated microglia, causing morphological changes. Most microglia differentiated into M1 inflammatory microglia and secreted a large number of inflammatory factors, with high iNOS expression. After co-culturing with human amniotic epithelial stem cells, the activation of microglia was greatly reduced, and the expression of inflammatory factors decreased significantly. Data analysis showed that this was statistically significant, indicating that hAESC treatment reversed the inflammatory activation of microglia.

[0083] 7.2 Tissue Immunofluorescence The number and density of neurons in the hippocampus of mice in the blank control group, the depression group, and the human amniotic epithelial stem cell therapy group were determined by using the neuronal labeling antibody NEUN.

[0084] Mouse brain tissue collection: Mice were anesthetized with isoflurane, and pre-cooled saline was perfused into the left ventricle until the liver turned white. Then, 4% paraformaldehyde (PFA, pH=7.4) fixative was perfused for 15 minutes. The brain tissue was completely removed and fixed in 4% PFA at 4°C for 24 hours. The fixed brain tissue was then transferred to 20% sucrose-PFA solution (4°C) for dehydration until it settled (approximately 48 hours), followed by further dehydration in 30% sucrose-PBS solution until it settled completely (approximately 15 hours) to remove ice crystals and maintain tissue morphology.

[0085] OCT embedding and frozen section preparation: After dehydration, the brain tissue was blotted dry with filter paper, placed flat in an aluminum foil mold with the ventral side down, and covered with OCT embedding agent. The tissue was then rapidly frozen to -80°C for fixation. The cryostat was pre-cooled to -20°C (box temperature) and -22°C (sample head temperature). The section thickness was adjusted to 20-35 μm, and an anti-roll plate was used to prevent tissue curling. After sectioning, the sections were attached to poly-L-lysine slides and stored at -40°C for long-term preservation.

[0086] Immunofluorescence staining procedure: Equilibrate sections to room temperature for 15 minutes, wash three times with PBS (5 minutes each time) to remove OCT. Incubate with 0.3% Triton X-100 (prepared in PBS) at room temperature for 20 minutes. Add blocking buffer containing 10% normal goat serum, 0.3% glycine, and 0.2% Triton X-100, and incubate at 37°C for 2 hours. Dilute primary antibody according to the manufacturer's instructions (e.g., anti-NeuN antibody, 1:500; anti-DCX antibody, 1:200), incubate overnight in a humidified chamber at 4°C, and wash three times with PBST (5 minutes each time). Under light-protected conditions, add fluorescently labeled secondary antibody (e.g., Alexa Fluor 594-labeled anti-rabbit IgG, 1:200), and incubate at 37°C for 1 hour. Stain nuclei with DAPI (2 μg / ml) for 5 minutes, wash with PBS, and dry in the dark. Mount the slide and acquire images.

[0087] Experimental results are as follows Figure 8 As shown, the results indicated that, compared to healthy mice, CUMS mice exhibited decreased cell density in the hippocampus, a lower DCX+ / NEUN+ positivity rate, and a significantly reduced immunofluorescence rate from newborn neurons to mature neurons, indicating decreased neurogenesis. However, after hAESC treatment, hippocampal neurons showed some recovery.

[0088] 8. RNA-seq cDNA library construction: Mice were euthanized by cervical dislocation, and RNA was extracted from hippocampal brain tissue. Concentration was determined using Nano Drop, and quality control was performed using an Agilent 2100 bioanalyzer. If minimal RNA degradation was observed, library construction could proceed. Total RNA was denatured at an appropriate temperature to open its secondary structure, and mRNA was enriched using oligo(dT) magnetic beads. A fragmentation reagent was added to the obtained mRNA, and the reaction was carried out at a suitable temperature for a specific time to fragment the mRNA. The pre-prepared first-strand synthesis reaction system was added to the fragmented mRNA, and first-strand cDNA was synthesized using a PCR instrument according to the corresponding program. The second-strand synthesis reaction system was prepared, and the reaction was carried out at a suitable temperature for a specific time to synthesize second-strand cDNA. The double-stranded cDNA ends were repaired by preparing a reaction system and reacting at a suitable temperature for a specific time, adding an A base to the 3' end. The adapter ligation reaction system was prepared, and the adapter was ligated to the cDNA by reacting at a suitable temperature for a specific time. The PCR reaction system was prepared, and the reaction program was set to amplify the cDNA ligation product. After denaturing the PCR product into single strands, a cyclization reaction system was prepared, thoroughly mixed, and reacted at a suitable temperature to obtain a single-stranded circular product. After digesting the uncirculated linear DNA molecules, the final library was obtained.

[0089] The concentration and quality of the obtained library were determined using an Agilent 2100 bioanalyzer.

[0090] Target gene detection: Single-stranded circular DNA molecules undergo rolling circle replication to form DNA nanospheres (DNBs) containing more than 200 copies. Using high-density DNA nanochip technology, the resulting DNBs are added into the mesh wells on the chip. Sequencing is performed using combined probe-anchored polymerization (cPAS) technology, yielding sequencing reads of 50bp / 100bp / 150bp.

[0091] Data analysis and statistics: The obtained RNA-seq data were analyzed on the BGI Dr.TOM website system.

[0092] Experimental results are as follows Figure 9 As shown, the results indicate that comparative analysis of transcriptome sequencing results between the depression group and the human amniotic epithelial stem cell treatment group revealed that human amniotic epithelial stem cells effectively reversed depression-related neuroinflammatory states and promoted the recovery of neural network function by regulating immune inflammation and related neural pathways in the brain, thereby rebuilding brain homeostasis.

[0093] Therefore, the hAESCs provided by this invention have the advantage of being readily available and have good clinical application prospects in the development of drugs for the treatment of depression.

[0094] The above specific embodiments are used to explain and illustrate the present invention, but not to limit the present invention. Any modifications and changes made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.

[0095] The above description is only a preferred embodiment of the present invention. Therefore, all equivalent changes or modifications made to the structure, features and principles described in the claims of this patent application are included in the scope of this patent application.

Claims

1. The application of human amniotic epithelial stem cells, characterized in that, Application in the preparation of drugs for treating depression.

2. The application as described in claim 1, characterized in that, The human amniotic epithelial stem cells were prepared in the following manner: (1) The amnion was separated from the inner surface of the discarded placental tissue by mechanical means; (2) After cleaning the obtained amnion, human amniotic epithelial stem cells were harvested by sequentially undergoing trypsin digestion, centrifugation, and resuspension.

3. The application as described in claim 1, characterized in that, The human amniotic epithelial stem cells mentioned are first- or second-generation cells.

4. A pharmaceutical composition for treating depression, characterized in that, It contains human amniotic epithelial stem cells and a pharmaceutically acceptable carrier.

5. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutically acceptable carrier is selected from at least one of microcapsules, microspheres, liposomes, micelles or sustained-release materials, as well as other pharmaceutically acceptable carriers.

6. The pharmaceutical composition according to claim 4 or 5, characterized in that, The human amniotic epithelial stem cells mentioned are first- or second-generation cells.

7. A cell preparation for treating depression, characterized in that, It contains an effective therapeutic dose of human amniotic epithelial stem cells and a pharmaceutically acceptable carrier.

8. The cell preparation according to claim 8, characterized in that, The cell preparation is in the form of an injection, and the route of administration includes intravenous injection, intrathecal injection, or nasal delivery.

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