Application of human amniotic epithelial stem cells in preparation of medicine for treating Parkinson's disease

Abstract

The invention discloses an application of human amniotic epithelial stem cells in preparation of a medicine for treating Parkinson's disease, and the effect of the human amniotic epithelial stem cells in treatment of Parkinson's disease is clear by using a 6-OHDA induced Parkinson's rat model. It is found that human amniotic epithelial stem cell transplantation can significantly improve dyskinesia of a Parkinson model rat and promote remodeling of dopaminergic neurons in a rat nigra-striatum pathway. The specific action mechanism of the human amniotic epithelial stem cell is further explored, and the human amniotic epithelial stem cell is found to be capable of improving the immune microenvironment in the brain by regulating and controlling neuroinflammation related to microglia-astrocyte axes, so that on one hand, the apoptosis of neurons is reduced, on the other hand, the neogenesis of the neurons is increased, and the neuroprotection effect is achieved. The application of the human amniotic epithelial stem cells in preparing the medicine for treating the Parkinson's disease can be used for treating the Parkinson's disease and has a good clinical transformation prospect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to the application of human amniotic epithelial stem cells in treating Parkinson's disease and research on its mechanism. Technical Background

[0002] Parkinson's disease (PD) is a serious chronic neurodegenerative disease that occurs in the central nervous system. The prevalence of Parkinson's disease in people over 60 years old worldwide exceeds 1%. The pathogenesis of Parkinson's disease is the loss of dopaminergic neurons caused by factors such as neuroinflammation. As the disease progresses, multiple dopamine pathways in the brain are successively affected, causing patients to experience a series of motor and non-motor symptoms, which not only seriously affects the quality of life of patients and their families, but also brings serious medical and economic burdens to society. At present, the clinical treatment of Parkinson's disease is still mainly based on dopaminergic drugs. Although increasing the content of dopamine in the brain by administering drugs such as levodopa can improve some Parkinson's symptoms, the progressive course of the disease cannot be delayed, and long-term use of drugs can cause a series of side effects including levodopa-induced movement disorders, gastrointestinal dysfunction and mental disorders. Therefore, new treatment methods for Parkinson's disease that are safe, effective and have few side effects are urgently needed.

[0003] Cell therapy is considered a new approach to treat Parkinson's disease. Traditional cell replacement therapy based on human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) has achieved certain results, but issues such as tumorigenicity, transplant rejection and ethics limit its further clinical transformation. In addition, due to the harsh brain immune microenvironment of Parkinson's disease, the survival rate of transplanted cells is often poor.

[0004] Recent studies have revealed the important role of neuroinflammation regulated by the microglia-astrocyte axis in the development of Parkinson's disease. When the central nervous system is damaged or diseased, microglia are activated. Activated neuroinflammatory microglia further induce astrocyte activation into reactive astrocytes by secreting cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), and mainly transform into the A1 phenotype. A1 astrocytes are neurotoxic. They not only lose their original functions of promoting neuronal survival, growth and synapse formation, but also can induce the death of neurons and other neural cells. In addition, immunohistochemical staining analysis of brain tissues of Parkinson's patients found that compared with healthy people, the number of complement component 3 (C3)-positive A1 astrocytes in the substantia nigra of Parkinson's patients increased significantly, further proving the important role of neuroinflammation regulated by the microglia-astrocyte axis in Parkinson's disease. Therefore, regulating the immune microenvironment in the brain may be a new idea for treating Parkinson's disease. Developing treatments that can regulate the microglia-astrocyte axis in the brain and reduce neurotoxic A1 astrocytes is expected to solve the challenges currently faced in the treatment of Parkinson's disease.

[0005] Human amniotic epithelial stem cells (hAESCs) can be isolated from placental tissue abandoned by full-term mothers after cesarean section. Therefore, compared with hESCs and hiPSCs, it has the advantages of easy access, economy and no ethical issues. In addition, the placenta is a neonatal accessory tissue with less DNA damage, which is an ideal choice for regenerative medicine. In addition to the above properties, hAESCs also have the advantages of non-tumorigenicity and low immunogenicity. In addition, the immunomodulatory and anti-inflammatory functions of hAESCs have been confirmed in multiple in vitro and in vivo experiments. Studies have shown that hAESCs can regulate multiple physiological processes of immune cells such as T cells, B cells, natural killer cells and macrophages by secreting a variety of immunomodulatory factors. In addition, hAESCs also have neural-like properties. Experiments have shown that it expresses specific markers of neurons and glial cells, and can synthesize and secrete a variety of neurotransmitters and neurotrophic factors. Therefore, hAESCs are ideal candidate cells for cell therapy of Parkinson's disease.

[0006] Based on the above advantages of hAESCs, the present invention has demonstrated the significant therapeutic effect of hAESCs derived from discarded placentas on Parkinson's disease in Parkinson's model animals, and has conducted in-depth research on the specific treatment mechanism by taking neuroinflammation regulated by the microglia-astrocyte axis as the starting point. The present invention realizes the reuse of biological waste, and hAESCs have the advantages of no ethical issues and high biosafety. This method enriches the preclinical research related to the treatment of Parkinson's disease, and has a good prospect for clinical transformation, providing new ideas for the future clinical treatment of Parkinson's disease. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a new therapeutic drug or method for the difficult problem of Parkinson's disease treatment.

[0008] In order to achieve the above-mentioned object, the present invention provides an application of human amniotic epithelial stem cells (hAESCs) in the preparation of a drug for treating Parkinson's disease, and explores its specific therapeutic mechanism.

[0009] The above-mentioned human amniotic epithelial stem cells expressed epithelial cadherin (E-cadherin), tight junction protein-1 (Zonula occludens-1, ZO-1) and keratin (Pan-cytokeratin, Pan-CK) positively, while CD34, CD45, CD31 and CD144 were negatively expressed.

[0010] After the human amniotic epithelial stem cells were cultured in soft agar for 21 days, no clones were formed.

[0011] The above human amniotic epithelial stem cells do not express telomerase.

[0012] The human amniotic epithelial stem cells do not express HLA class II antigens HLA-DQ and HLA-DR, but express the non-classical HLA class I antigen HLA-G.

[0013] The above human amniotic epithelial stem cells are able to synthesize and secrete a variety of anti-inflammatory and neurotrophic factors.

[0014] The transplantation of human amniotic epithelial stem cells can significantly improve the motor dysfunction of 6-OHDA-induced Parkinson's model rats and promote the remodeling of dopaminergic neurons in the substantia nigra-striatum pathway of rats.

[0015] The human amniotic epithelial stem cells can improve the brain immune microenvironment of Parkinson's rats by reducing the activation level of microglia, significantly reducing the activation of astrocytes and neurotoxic A1 astrocytes in the substantia nigra, thereby reducing neuronal apoptosis on the one hand and increasing neuronal regeneration on the other hand, thereby playing a neuroprotective role.

[0016] The human amniotic epithelial stem cells are human amniotic epithelial stem cells of passage P1.

[0017] The human amniotic epithelial stem cells are prepared by a method comprising the following steps:

[0018] (1) mechanically separating the amniotic membrane from the inner surface of discarded placental tissue;

[0019] (2) After the obtained amniotic membrane is carefully cleaned, it is digested with trypsin, centrifuged, and resuspended to harvest human amniotic epithelial stem cells.

[0020] In the method, the amniotic epithelial stem cells are derived from humans, and refer to human amniotic epithelial stem cells.

[0021] In the method, fresh amniotic membrane is collected from placental tissue abandoned by healthy pregnant women (negative for syphilis treponema, HIV, hepatitis A, hepatitis B, etc.) after cesarean section, and all collections are obtained with written informed consent from the donors.

[0022] The human amniotic epithelial stem cells are resuspended in a phenol red-free DMEM / F12 culture medium to obtain a cell injection solution.

[0023] The human amniotic epithelial stem cells or their cell preparations are used in the preparation of treatment and / or improvement of Parkinson's disease. The cell preparations include human amniotic epithelial stem cells and various pharmaceutically acceptable carriers.

[0024] Based on the above results, the present invention further discloses the therapeutic effect of transplanting human amniotic epithelial stem cells on Parkinson's disease, and the method comprises the following steps:

[0025] (1) Injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle (MFB) of the right brain of rats induced dopaminergic neuron damage in the substantia nigra-striatum pathway of the right brain of rats;

[0026] (2) Select rats with successful modeling and perform two human amniotic epithelial stem cell transplantations into their right striatum;

[0027] (3) Behavioral assessment and histological staining were performed to evaluate the therapeutic effect.

[0028] After clarifying the significant therapeutic effect of human amniotic epithelial stem cell transplantation on Parkinson's disease, the present invention further discloses its specific therapeutic mechanism. The method comprises the following steps:

[0029] (1) Histological staining was performed to investigate the activation of microglia in the substantia nigra of rats before and after transplantation.

[0030] (2) Histological staining was performed to investigate the activation of astrocytes and the number of neurotoxic A1 astrocytes in the substantia nigra of rats before and after transplantation.

[0031] (3) TUNEL and Fluoro-Jade C staining were performed to investigate the apoptosis / degeneration of dopaminergic neurons in the substantia nigra-striatum pathway of rats before and after transplantation.

[0032] (4) Histological staining was performed to investigate the new generation of dopaminergic neurons in the substantia nigra-striatum pathway of rats before and after transplantation.

[0033] One of the technical solutions adopted by the present invention is: a method for isolating human amniotic epithelial stem cells from placental tissue abandoned after cesarean section, the method comprising the following steps:

[0034] Step 1: Obtain the placenta in the obstetric operating room, mechanically peel off the amniotic membrane from the inner surface of the discarded placental tissue, and then quickly transfer it to the laboratory biosafety cabinet through cold chain transportation. The above process obtained written informed consent from all pregnant women and passed ethical review;

[0035] Step 2: In a biosafety cabinet, transfer the amniotic membrane to a sterile container and wash the amniotic membrane several times with phosphate-balanced saline (PBS) supplemented with antibiotics to remove blood clots and mucus attached to the surface of the amniotic membrane. Then use surgical scissors to cut the amniotic membrane into small pieces, place them in 0.25% trypsin digestion solution, and digest them in a 37°C water bath. After terminating the digestion, centrifuge to obtain the human amniotic epithelial stem cell precipitate;

[0036] Step 3: Resuspend human amniotic epithelial stem cells in culture medium and count them at 1.5×10 7 Inoculate each cell in a 15 cm culture dish and culture in an incubator for 48-72 hours. After the cells adhere to the wall, replace with new culture medium and continue culturing.

[0037] Step 4: When the human amniotic epithelial stem cells grow to basically cover the entire 15 cm culture dish, use 0.25% trypsin digestion solution to digest them. After terminating the digestion, centrifuge to obtain the human amniotic epithelial stem cell pellet, resuspend the cells in freezing solution, and press 5×10 6 The samples were packaged into individual / cryotubes, placed in a cryobox and slowly frozen in a -80°C refrigerator overnight, and then transferred to a liquid nitrogen tank for storage.

[0038] In step 1 of the method, the amniotic membrane is usually placed in a sterile conical flask containing 150 ml of pre-cooled DMEM / F12 culture medium and quickly transported to a biosafety cabinet through a cold chain.

[0039] The cleaning solution in step 2 of the method refers to a PBS solution added with 1% penicillin-streptomycin mixture, and the amniotic membrane is generally cleaned more than 3 times to substantially remove the blood clots and mucus attached to the surface of the amniotic membrane.

[0040] The digestion process in step 2 of the method is specifically described as follows: use surgical scissors to cut the amniotic membrane into small pieces, transfer to a new sterile 50 ml centrifuge tube, add three times the volume of 0.25% trypsin digestion solution of the amniotic membrane tissue, and digest in a 37°C water bath for 20-30 minutes, during which the centrifuge tube is turned upside down 4-5 times, and the digestion is judged to be complete according to the turbidity of the digestion solution. After the digestion is completed, the amniotic membrane tissue is discarded, and 10% fetal bovine serum (Fetal bovine serum, FBS) is added to the digestion solution to terminate the digestion.

[0041] The specific description of the process of obtaining human amniotic epithelial stem cell precipitation by centrifugation in step 2 of the method is as follows: centrifuge at 500×g for 6 minutes at room temperature, discard the supernatant, and harvest the cell precipitation.

[0042] The culture medium in step 3 of the method refers to human amniotic epithelial stem cell culture medium, and the specific formula is DMEM / F12 culture medium supplemented with 15% Knockout TM Serum Replacement (KSR), 2mM L-glutamine, 1% non-essential amino acids, 1% sodium pyruvate, 1% penicillin-streptomycin mixture, 10ng / ml human epidermal growth factor.

[0043] The freezing solution in step 4 of the method refers to a mixture obtained by mixing FBS and dimethyl sulfoxide (DMSO) in a volume ratio of 9:1.

[0044] The second technical solution adopted by the present invention is: detecting the separation purity, tumorigenicity, immunogenicity, and neurotrophic and immunoregulatory functions of the isolated human amniotic epithelial stem cells, and the method comprises the following steps:

[0045] Step 1: Using immunofluorescence and flow cytometry to detect the purity of the isolated human amniotic epithelial stem cells;

[0046] Step 2: Perform soft agar colony formation assay and enzyme-linked immunosorbent assay (ELISA) to detect the tumorigenicity of the isolated human amniotic epithelial stem cells;

[0047] Step 3: Using flow cytometry to detect the immunogenicity of the isolated human amniotic epithelial stem cells;

[0048] Step 4: Use RNA-Seq and proteomic analysis to investigate the neurotrophic and immunoregulatory functions of isolated human amniotic epithelial stem cells.

[0049] The indicators detected by immunofluorescence method in step 1 of the method are E-cadherin, ZO-1 and Pan-CK.

[0050] The indicators detected by flow cytometry analysis in step 1 of the method are CD34, CD45, CD31 and CD144.

[0051] The indicator detected by the ELISA experiment in step 2 of the method is the expression of human telomerase.

[0052] The indicators detected by flow cytometry in step 3 of the method are human leukocyte antigen-DQ (HLA-DQ), human leukocyte antigen-DR (HLA-DR) and human leukocyte antigen-G (HLA-G).

[0053] The third technical solution adopted by the present invention is: obtaining a Parkinson's model rat by injecting 6-OHDA, and using the model rat to explore the therapeutic effect of human amniotic epithelial stem cell transplantation on Parkinson's disease, the method comprises the following steps:

[0054] Step 1: Purchase adult female Sprague Dawley rats weighing 220 to 240 g from Shanghai Slake Company. The rats were kept in a ventilated environment and given adequate food and water;

[0055] Step 2: All animals were randomly divided into 2 groups: a control group and a human amniotic epithelial stem cell transplantation group.

[0056] Step 3: Preparation of 6-OHDA solution: Protect from light, accurately weigh 0.012 g of 6-OHDA powder using an analytical balance, dissolve in 2 ml of physiological saline containing 0.2% ascorbic acid to obtain a 6-OHDA solution with a concentration of 6 mg / ml, dispense into 200 μl EP tubes at 50 μl / tube, and freeze in a -80°C refrigerator.

[0057] Step 4: At week -4, a digital stereotaxic instrument was used with a microinjection needle to inject 6-OHDA solution into the right brain MFB of the rats. At week 0, the rats injected with 6-OHDA were subjected to apomorphine-induced rotation test, and the rats with successful modeling were selected for subsequent experiments;

[0058] Step 5: Cell therapy group: At week 0, human amniotic epithelial stem cells (resuspended in resuspension culture medium) were injected into two coordinate areas of the right striatum of Parkinson's model rats using a digital stereotaxic instrument with a microinjection needle; control group: the same volume of resuspension culture medium was injected into two coordinate areas of the right striatum of Parkinson's model rats using a digital stereotaxic instrument with a microinjection needle;

[0059] Step 6: After surgery, each rat was injected intramuscularly with 1.2×10 5 unit of penicillin to prevent postoperative infection, and the rats were placed on a warming pad until awake;

[0060] Step 7: Repeat step 5 in the second week;

[0061] Step 8: Conduct three behavioral tests, namely, apomorphine-induced rotation test, cylinder test, and gait test, at 0, 2, and 6 weeks, and perform statistics;

[0062] Step 9: After the behavioral assessment at week 6, the animals were euthanized and samples were collected for subsequent experiments.

[0063] In steps 4 and 5 of the method, propofol is injected intraperitoneally to anesthetize the rats, and the injection dose is 100 mg / kg.

[0064] In steps 4 and 5 of the method, the injection speed of the 6-OHDA solution, human amniotic epithelial stem cells and resuspension culture medium is 0.4 μl / min. After the injection is completed, the needle is stopped at the original position for 10 minutes and then slowly withdrawn.

[0065] In step 5 of the method, the resuspension medium is a DMEM / F12 medium without phenol red.

[0066] In step 5 of the method, the coordinates of the two injection points in the right striatum of the rat are: 1.2 mm in front of the anterior fontanelle, 2.6 mm lateral to the midline, 4.4 mm below the dura mater; and 0.5 mm in front of the anterior fontanelle, 3.0 mm lateral to the midline, 4.0 mm below the dura mater. 3 μl and 2 μl of cell suspension / resuspended culture medium were injected at these two locations, respectively, wherein the density of the cell suspension was 1.5×10 5 Pieces / μl.

[0067] Subsequent experiments in step 9 of the method include immunohistochemical staining and immunofluorescence staining of brain slices.

[0068] The fourth technical solution adopted by the present invention is to explore the specific mechanism of action of human amniotic epithelial stem cell transplantation in treating Parkinson's disease, and the method comprises the following steps:

[0069] Step 1: Immunohistochemical staining and immunofluorescence were used to investigate the activation of microglia in the substantia nigra of rats before and after transplantation;

[0070] Step 2: Immunohistochemical staining and immunofluorescence were used to investigate the activation of astrocytes and the number of neurotoxic A1 astrocytes in the substantia nigra of rats before and after transplantation.

[0071] Step 3: TUNEL and Fluoro-Jade C staining were performed to investigate the apoptosis / degeneration of dopaminergic neurons in the substantia nigra-striatum pathway of rats before and after transplantation.

[0072] Step 4: Use immunofluorescence to investigate the new generation of dopaminergic neurons in the substantia nigra-striatum pathway of rats before and after transplantation.

[0073] In step 1 of the method, the index detected by immunohistochemical staining is the microglia-specific marker ionized calcium binding adaptor molecule-1 (IBA-1), and the index detected by immunofluorescence is the neuroinflammation marker translocator protein (TSPO);

[0074] In step 2 of the method, the index detected by immunohistochemical staining is glial fibrillary acidic protein (GFAP), a specific marker of astrocytes, and the index detected by immunofluorescence is C3d, a representative marker of type A1 astrocytes;

[0075] In step 3 of the method, a TUNEL staining kit and a Fluoro-Jade C staining kit were used to detect the apoptosis / degeneration of dopaminergic neurons, and the experimental steps followed the instructions of the kits;

[0076] The indicators of immunofluorescence detection in step 4 of the method are respectively the neural progenitor cell-specific markers nestin and sex-determining region Y box transcription factor 2 (SOX-2), and the migrating neural progenitor cell-specific marker polysialic acid-neural cell adhesion molecule (PSA-NCAM).

[0077] Compared with the prior art, the present invention has the following advantages:

[0078] The present invention develops an application of human amniotic epithelial stem cells in the preparation of drugs for the treatment of Parkinson's disease and conducts in-depth research on its specific therapeutic mechanism. For the first time, based on the immunomodulatory and neurotrophic properties of human amniotic epithelial stem cells, the present invention clarifies the significant therapeutic effect of human amniotic epithelial stem cells on Parkinson's disease, targeting the neuroinflammation regulated by the microglia-astrocyte axis, which plays an important role in the occurrence and development of Parkinson's disease. Further exploring the therapeutic mechanism, the present invention first discovered that human amniotic epithelial stem cells can improve the brain immune microenvironment of Parkinson's rats by reducing the activation level of microglia, significantly reducing the activation of astrocytes and neurotoxic A1 astrocytes in the substantia nigra, thereby reducing neuronal apoptosis on the one hand and increasing neuronal regeneration on the other hand, playing a neuroprotective role. Compared with the prior art, due to the advantages of human amniotic epithelial stem cells such as easy access, no ethical issues, no tumorigenicity and low immunogenicity, the present invention has a broader prospect for clinical application in the treatment of Parkinson's disease, and provides a new idea for the clinical treatment of Parkinson's disease in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] In order to make the purpose, technical solution and test results of the present invention clearer, the present invention provides the following drawings for illustration:

[0080] Figure 1:Morphological and phenotypic characteristics of human amniotic epithelial stem cells, scale bar, 100um, *P<0.05; **P<0.01; ***P<0.001; ns, no statistical difference, F: Compared with hAESCs, one-way ANOVA followed by Tukey's multiple comparison test, G: Compared with HepG2, Student's t test. (A) Morphology of human amniotic epithelial stem cells in serum-free culture system. (B) Expression of epithelial cell-specific markers Epithelial cadherin, Pan-cytokeratin and Zonula occludens-1 by human amniotic epithelial stem cells. (C) FCM analysis of hematopoietic stem cell markers CD34 and CD45 expressed by human amniotic epithelial stem cells in hAESCs. (D) Expression of endothelial markers CD31 and CD144 by human amniotic epithelial stem cells. (E) Clonal formation of human AES, human cervical cancer cells HeLa and human liver cancer cells HepG2 after 21 days of culture in soft agar. (F) Quantitative analysis of clone formation in E (n=3). (G) Quantitative analysis of telomerase expression in human AES and human liver cancer cells HepG2 (n=3). (H) Expression of HLA class II antigens HLA-DQ and HLA-DR, and non-classical HLA class I antigen HLA-G in human AES after 72 hours of treatment with 10 ng / ml IFN-γ. (I) Quantitative statistics of HLA-DQ-positive, HLA-DR-positive and HLA-G-positive hAESCs under normal / inflammatory conditions (n=3). (J) RNAomic analysis of human AES (n=3). (K) Proteomic analysis of conditioned medium of human AES (n=3).

[0081] Figure 2:Therapeutic effect of human amniotic epithelial stem cell transplantation on 6-OHDA-induced parkinsonian rats, scale bar, 500 μm, *P<0.05; **P<0.01; ***P<0.001, compared with the control group, Student's t test. (A) Schematic diagram of the in vivo human amniotic epithelial stem cell transplantation experiment. The schematic diagram was drawn using BioRender.com. (BD) Behavioral experiments were performed in the control group (n=6) and the human amniotic epithelial stem cell transplantation group (n=8), including apomorphine-induced rotation test (B), cylinder test (C), and gait test (D). (E) Coronal brain slice images containing tyrosine hydroxylase (TH)-positive dopaminergic neurons in the striatum (Caudate putamen, CPu) and substantia nigra pars compacta (SNpc). (F) Enlarged images of i, ii, and iii in (E), showing TH in SNpc of the uninjured side, the injured but untransplanted side, and the transplanted side, respectively. + (G) TH in SNpc of rats in the control group (n=6) and human amniotic epithelial stem cell transplantation group (n=6) + Semi-quantitative analysis of neurons. (H) Enlarged images of iv, v, and vi in ​​(E), showing TH in CPu of the uninjured side, the injured but untransplanted side, and the transplanted side, respectively. + Nerve fibers. (I) TH in CPu of rats in the control group (n=6) and human amniotic epithelial stem cell transplantation group (n=6) + Semiquantitative analysis of nerve fiber density.

[0082] Figure 3 :Human amniotic epithelial stem cells regulate the microglia-astrocyte axis in the brain of Parkinson's rats, scale bar, 100 μm, *P<0.05; **P<0.01; ***P<0.001, compared with the control group, Student's t test. (A) Representative immunohistochemical images showing IBA-1 in SNpc of the uninjured side, the injured but untransplanted side, and the transplanted side + Microglia. (B) Semi-quantitative analysis of microglia density in SNpc of rats in control group (n=6) and human amniotic epithelial stem cell transplantation group (n=8). (C) Semi-quantitative analysis of IBA-1 level in SNpc of rats in control group (n=6) and human amniotic epithelial stem cell transplantation group (n=8). (D) Immunofluorescence staining to detect the co-localization of TSPO and IBA-1 in SNpc of uninjured side, injured non-transplanted side and transplanted side. (E) Semi-quantitative analysis of TSPO in SNpc of rats in control group (n=6) and human amniotic epithelial stem cell transplantation group (n=8). +Semi-quantitative analysis of microglia. (F) Representative immunohistochemical images showing GFAP in SNpc of the uninjured side, the injured but untransplanted side, and the transplanted side + Astrocytes. (G) Semi-quantitative analysis of astrocyte density in the SNpc of rats in the control group (n=6) and human amniotic epithelial stem cell transplantation group (n=8). (H) Semi-quantitative analysis of GFAP levels in the SNpc of rats in the control group (n=6) and human amniotic epithelial stem cell transplantation group (n=8). (I) Immunofluorescence staining to detect the co-localization of C3d and GFAP in the SNpc of the uninjured side, the injured non-transplanted side, and the transplanted side. (J) Semi-quantitative analysis of C3d in the SNpc of rats in the control group (n=6) and human amniotic epithelial stem cell transplantation group (n=8). + Semiquantitative analysis of astrocytes.

[0083] Figure 4 : Human amniotic epithelial stem cells play an anti-apoptotic and pro-regenerative role by regulating the microglia-astrocyte axis. Scale bar, 100 μm. *P<0.05; **P<0.01; ***P<0.001, compared with the control group, Student's t test. (A) Immunofluorescence staining to detect the co-localization of TH and TUNEL in the SNpc of the uninjured side, the injured but not transplanted side, and the transplanted side. (B) TUNEL in the SNpc of rats in the control group (n=6) and the human amniotic epithelial stem cell transplantation group (n=8) + Semi-quantitative analysis of dopaminergic neurons. (C) Immunofluorescence staining to detect the co-localization of TH and Fluoro-Jade C in the SNpc of the uninjured side, the injured but not transplanted side, and the transplanted side. (D) Fluoro-Jade C in the SNpc of rats in the control group (n=6) and the human amniotic epithelial stem cell transplantation group (n=8) + Semi-quantitative analysis of dopaminergic neurons. (E) Immunofluorescence staining to detect the co-localization of nestin, Sox-2, and Ki67 in the SNpc of the uninjured side, the injured but not transplanted side, and the transplanted side. (F) Immunofluorescence staining to detect the co-localization of PSA-NCAM and TH in the SNpc of the uninjured side, the injured but not transplanted side, and the transplanted side. + Dopaminergic neurons are indicated by white arrows. (G) PSA-NCAM in SNpc of rats in the control group (n=6) and human amniotic epithelial stem cell transplantation group (n=8) + Quantitative analysis of dopaminergic neurons. DETAILED DESCRIPTION

[0084] The preferred embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings. The experimental conditions and methods not specified in the embodiments are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0085] The purpose of the present invention is to address the difficulty in treating Parkinson's disease, develop an application of human amniotic epithelial stem cells in the preparation of a drug for treating Parkinson's disease, and explore its specific treatment mechanism.

[0086] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0087] 1. Isolate primary human amniotic epithelial stem cells from discarded placental tissue after cesarean section and culture them in vitro;

[0088] 2. To determine the therapeutic effect of human amniotic epithelial stem cell transplantation on 6-OHDA-induced Parkinson's disease in rats;

[0089] 3. To explore the specific mechanism of human amniotic epithelial stem cells in treating Parkinson’s disease.

[0090] Example 1 Isolation and culture of human amniotic epithelial stem cells

[0091] As mentioned above, human amniotic epithelial stem cells are derived from placental tissue discarded by healthy full-term pregnant women (negative for syphilis, HIV, hepatitis A, and hepatitis B, etc.) after cesarean section, and all collections were obtained with written informed consent from the donors.

[0092] 1. Obtain the placenta in the obstetric operating room, mechanically peel off the amniotic membrane from the inner surface of the discarded placental tissue, place it in pre-cooled DMEM / F12 culture medium containing 1% penicillin-streptomycin mixture, and quickly transfer it to the laboratory biosafety cabinet through cold chain transportation;

[0093] 2. In a biosafety cabinet, transfer the amniotic membrane to a sterile container and wash the amniotic membrane several times with PBS solution containing antibiotics to remove blood clots and mucus attached to the surface of the amniotic membrane;

[0094] 3. Cut the amniotic membrane into smaller pieces, place them in a 50ml centrifuge tube, add three times the volume of the tissue 0.25% pancreatic enzyme digestion solution, and pre-digest in a 37°C constant temperature water bath for 10 minutes, gently shaking the centrifuge tube several times during the pre-digestion.

[0095] 4. After the pre-digestion, discard the pre-digestion solution, transfer the amniotic membrane tissue to a new sterile 50ml centrifuge tube, add three times the volume of the tissue 0.25% trypsin digestion solution again, and perform formal digestion in a 37℃ constant temperature water bath for 20-30 minutes. During this period, turn the centrifuge tube upside down 4-5 times to determine whether the digestion is complete based on the turbidity of the digestion solution;

[0096] 5. After the formal digestion is completed, the amniotic tissue is discarded, FBS is added to the digestion solution to terminate the digestion, and then centrifuged at 500×g for 6 minutes to harvest the human amniotic epithelial stem cell pellet. After resuspending the cells in human amniotic epithelial stem cell culture medium, count them and calculate the number of cells to 1.5×10 7 Inoculate cells / 15 cm culture dish, culture in the incubator for 48-72 hours. After the cells adhere to the wall, replace with new culture medium and continue culturing.

[0097] Human amniotic epithelial stem cell culture medium: DMEM / F12 medium supplemented with 15% KnockoutTM Serum Replacement (KSR), 2mM L-glutamine, 1% non-essential amino acids, 1% sodium pyruvate, 1% penicillin-streptomycin mixture, and 10ng / ml human epidermal growth factor;

[0098] 6. When the human amniotic epithelial stem cells grow to basically cover the entire 15cm culture dish, freeze the cells: discard the culture medium, wash the adherent cells 2-3 times with sterile PBS solution, add 5ml of 0.25% trypsin digestion solution, place in a 37℃ incubator for digestion for 8-10min, and add FBS to terminate digestion when the cell morphology becomes round and some cells begin to fall off the surface of the culture dish under the microscope. Collect the cell suspension, centrifuge at 500×g for 6min, harvest the human amniotic epithelial stem cell pellet, resuspend the cells in cell freezing solution (FBS:DMSO=9:1), and add 5×10 6 Pack into individual pieces / cryotubes, place in a cryo box and slowly freeze in a -80℃ refrigerator overnight, then transfer to a liquid nitrogen tank for storage;

[0099] 7. When using human amniotic epithelial stem cells later, take out the frozen cells from the liquid nitrogen tank for resuscitation.

[0100] Figure 1 The results showed that in the serum-free culture system, the adherent human amniotic epithelial stem cells took on an oval "paving stone" shape, which is a typical epithelial cell morphology.

[0101] Example 2 Cell Immunofluorescence Staining (IF)

[0102] 1. Take out the cell culture dish from the incubator, discard the culture medium, wash the cells 2-3 times with PBS solution, and then add 4% paraformaldehyde solution to fix for 15 minutes;

[0103] 2. Discard the fixative solution, wash with PBS solution 3 times, and add 0.3% Triton-X-100 solution for permeabilization for 5 minutes;

[0104] 3. Discard the permeabilization solution, wash with PBS solution for 3 times, add blocking solution (PBS solution containing 5% goat serum and 1% bovine serum albumin) and block at room temperature for 2 hours;

[0105] 4. Use the above blocking solution to dilute the primary antibody according to the instructions, invert the cell slide in the primary antibody dilution solution, and incubate at 4°C overnight;

[0106] 5. Discard the primary antibody diluent, wash the cell slides 3 times with PBS solution, add fluorescent conjugated secondary antibody diluted in PBS solution, and incubate at room temperature in the dark for 1 hour;

[0107] 6. Discard the secondary antibody diluent, wash the cell slides 3 times with PBS solution, and then seal the slides with anti-fluorescence quenching sealing medium containing DAPI;

[0108] 7. Use an upright fluorescence microscope or an inverted confocal fluorescence microscope FV3000 to collect images and use Image J software to analyze the images.

[0109] Figure 1 The results showed that human amniotic epithelial stem cells growing adherently expressed epithelial cell-specific markers E-cadherin, tight junction protein-1 and keratin, further demonstrating that the isolated human amniotic epithelial stem cells exhibited epithelial cell characteristics, and the positive rate was nearly 100%, demonstrating a relatively ideal separation purity.

[0110] Example 3 Flow cytometry analysis (FCM)

[0111] 1. Prepare washing buffer: sterile PBS solution containing 2% FBS;

[0112] 2. Add 0.25% trypsin digestion solution to the cell culture dish and place it in a 37°C incubator for digestion. After the digestion is stopped, collect the cells in a centrifuge tube and centrifuge at 4°C, 500×g for 5 minutes;

[0113] 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;

[0114] 4. Discard the supernatant, add 1 ml of washing buffer to resuspend the cells, and centrifuge at 4°C, 500×g for 3 min;

[0115] 5. Discard the supernatant, add 50 μl of washing buffer to each tube to resuspend the cells, then add an appropriate amount of fluorescent-labeled antibodies, resuspend again, and incubate at 4°C in the dark for 30 minutes;

[0116] 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;

[0117] 7. Repeat step 6 twice;

[0118] 8. Add 500 μl of washing buffer to each tube to resuspend the cells, then transfer the cell suspension to the flow cytometry tube for on-line testing.

[0119] 9. Use FlowJo analysis software to process the data.

[0120] Figure 1 The results showed that the isolated and cultured human amniotic epithelial stem cells did not express blood cell-specific markers CD34 and CD45 and endothelial cell-specific markers CD31 and CD144, indicating that the isolated and cultured cells were pure and uniform epithelial cells without contamination of blood cells and endothelial cells.

[0121] Figure 1 The results showed that under IFN-γ stimulation, human amniotic epithelial stem cells did not express HLA class II antigens HLA-DQ and HLA-DR, but expressed the non-classical HLA class I antigen HLA-G, indicating that human amniotic epithelial stem cells have very low immunogenicity and are not likely to cause strong host rejection reactions in allogeneic transplantation.

[0122] Example 4 Soft agar colony formation experiment

[0123] 1. Prepare 1.2% and 0.7% agar solutions respectively, sterilize them in a high temperature and high pressure sterilizer, and place them in a 42℃ water bath to prevent solidification;

[0124] 2. Take the freshly prepared cell culture medium into an ultrafiltration concentration tube, centrifuge at 4°C, 1000×g for 10 min to obtain 2× concentrated cell culture medium;

[0125] 3. Mix 1.2% agar solution and 2× concentrated cell culture medium in a volume ratio of 1:1, add 1.5 ml of 1.2% agar-culture medium mixture to each well of a six-well cell culture dish, shake the dish quickly after adding so that the mixture spreads on the entire bottom of the dish, and incubate in a 37°C incubator for 30 minutes until the mixture is completely solidified;

[0126] 4. Take out the culture dish of adherent cells from the incubator, discard the culture medium, wash the adherent cells 2-3 times with sterile PBS solution, add appropriate amount of 0.25% trypsin digestion solution, digest at 37℃ for appropriate time, collect the cell suspension in a 15ml centrifuge tube, centrifuge at 500×g for 5min. Discard the supernatant, resuspend with 2× concentrated cell culture medium, count, and adjust the cell suspension density to 6.7×10 3 Pieces / ml;

[0127] 5. Mix 0.7% agar solution and single cell suspension in a volume ratio of 1:1, add 1.5ml of 0.7% agar-single cell suspension mixture to each well of the six-well cell culture dish as the upper soft agar, incubate in a 37℃ incubator for 1.5h until the mixture is completely solidified, then add 2ml of cell culture medium to each well. Incubate in a 37℃ incubator, and replace the culture medium every 3 days;

[0128] 6. On the 21st day of culture, discard the culture medium, wash twice with sterile PBS solution, and record the colony formation under a camera and optical microscope respectively.

[0129] Figure 1 The results showed that after 21 days of culture in soft agar, human amniotic epithelial stem cells did not form any clones compared to human liver cancer cells HepG2 and human cervical cancer cells HeLa, which formed many clones, indicating that human amniotic epithelial stem cells are not tumorigenic.

[0130] Example 5 Enzyme-linked immunosorbent assay (ELISA)

[0131] The expression of telomerase in human amniotic epithelial stem cells was detected using an ELISA kit. All operations followed the instructions of the kit, as follows:

[0132] 1. Collect the conditioned medium of human amniotic epithelial stem cells and human liver cancer cells HepG2 for 48 hours, centrifuge at 700×g, 4℃ for 10 minutes to remove cell debris, collect the supernatant, take part of it for ELISA detection, and freeze the remaining supernatant at -80℃;

[0133] 2. Place all reagents in the kit at room temperature for about 30 minutes;

[0134] 3. Preparation of standard curve samples: Before opening the cover, immediately separate the tube containing the standard powder, add the sample diluent as indicated in the instructions, and gently pipette to fully dissolve it to obtain a standard solution with a concentration of 20ng / ml. Take the 20ng / ml standard solution and use the sample diluent to dilute it in a gradient of 1:1 to obtain six concentrations of standard solutions: 10ng / ml, 5ng / ml, 2.5ng / ml, 1.25ng / ml, 0.625ng / ml, and 0.312ng / ml. Use the sample diluent as the zero concentration of the standard curve;

[0135] 4. Dilution of the sample to be tested: Use sample diluent to dilute the conditioned medium of human liver cancer cell HepG2 by 6 times, and do not dilute the conditioned medium of human amniotic epithelial stem cells;

[0136] 5. Add 100 μl of standard or sample to each well of the ELISA plate, gently shake to mix, apply sealing film, and incubate at 37°C for 2 hours;

[0137] 6. Discard the liquid in the wells and pat the ELISA plate dry on absorbent paper without washing;

[0138] 7. Add 100 μl of biotin-labeled antibody working solution to each well, cover with sealing film, and incubate at 37°C for 1 hour;

[0139] 8. Discard the liquid in the wells, pat the ELISA plate dry on absorbent paper, add 200 μl of washing solution to each well, soak for 2 minutes, repeat the washing 3 times, discard the washing solution, and pat the ELISA plate dry on absorbent paper;

[0140] 9. Add 100 μl of horseradish peroxidase-labeled avidin working solution to each well, cover with sealing film, and incubate at 37°C for 1 hour;

[0141] 10. Discard the liquid in the wells, pat the ELISA plate dry on absorbent paper, add 200 μl of washing solution to each well, soak for 2 minutes, repeat the washing 5 times, discard the washing solution, and pat the ELISA plate dry on absorbent paper;

[0142] 11. Add 90 μl of chromogenic substrate to each well and incubate at 37°C in the dark for about 30 minutes;

[0143] 12. When the sample wells of the standard curve show a clear gradient blue, add 50 μl of stop solution to each well, and then use an ELISA reader to detect the OD value of each well at a wavelength of 450 nm;

[0144] 13. Collect data, draw standard curve, process data using Excel software, and draw graphs using Graphpad software.

[0145] Figure 1 The results showed that compared with human liver cancer cells HepG2 that highly expressed telomerase, human amniotic epithelial stem cells did not express telomerase, indicating that the proliferation capacity of human amniotic epithelial stem cells is limited and they will not form a three-germ layer structure after transplantation into the body, thus ensuring their biological safety.

[0146] Example 6 RNA-omics detection of human amniotic epithelial stem cells

[0147] 1. Take three different batches of frozen human amniotic epithelial stem cells from the liquid nitrogen tank, revive and plate them in six-well cell culture dishes at a density of 1×10 6 Each well was cultured in an incubator for 24 hours until the cells adhered to the wall and spread out in a "paving stone" shape, and new culture medium was replaced to continue culturing for 48 hours until the human amniotic epithelial stem cells grew to basically cover the entire well;

[0148] 2. Discard the culture medium, wash the cells 2-3 times with sterile PBS solution, add 1 ml Trizol to each well, let stand for 1 min, use a micropipette to repeatedly blow until the cells are completely lysed, collect the cell lysate in a 1.5 ml EP tube, and freeze it in a -80°C refrigerator;

[0149] 3. The samples were subsequently handed over to Kidio Biotechnology Co., Ltd. for RNA-Seq testing.

[0150] Example 7 Proteomic Detection of Conditioned Medium of Human Amniotic Epithelial Stem Cells

[0151] 1. Take three different batches of frozen human amniotic epithelial stem cells from the liquid nitrogen tank, resuscitate and plate them in a 10 cm cell culture dish at a density of 5×10 6 10 cm culture dish, culture in an incubator for 24 hours until the cells adhere to the wall and spread out in a "paving stone" shape, replace with new culture medium and continue to culture until the human amniotic epithelial stem cells grow to cover 80% of the bottom of the culture dish;

[0152] 2. Discard the old culture medium and add fresh human amniotic epithelial stem cell culture medium without human epidermal growth factor. Continue culturing for 48 hours, then collect the cell-conditioned medium, centrifuge at 700×g, 4°C for 10 minutes to remove cell debris, collect the supernatant, and store in a -80°C refrigerator;

[0153] 3. The samples will then be handed over to Lianchuan Biotechnology Co., Ltd. for proteomics testing.

[0154] Figure 1 The results showed that human amniotic epithelial stem cells can synthesize and secrete a variety of neurotrophic factors, indicating that human amniotic epithelial stem cells have the potential for neuroprotection. At the same time, human amniotic epithelial stem cells can synthesize and secrete a variety of anti-inflammatory factors, indicating that human amniotic epithelial stem cells can inhibit the activation, recruitment and migration of immune cells such as macrophages, T cells and B cells, and have strong immunoregulatory potential.

[0155] Example 8 Obtaining a unilaterally damaged Parkinson's disease model rat

[0156] 1. Preparation of 6-OHDA solution: Protect from light, accurately weigh 0.012 g 6-OHDA powder using an analytical balance, dissolve in 2 ml of physiological saline containing 0.2% ascorbic acid to obtain a 6 mg / ml 6-OHDA solution, dispense into 200 μl EP tubes at 50 μl / tube, and freeze at -80°C;

[0157] 2. Rats were anesthetized by intraperitoneal injection of propofol at a dose of 100 mg / kg;

[0158] 3. Remove the hair from the rat's head and fix it on a digital stereotaxic apparatus;

[0159] 4. Use iodine to disinfect the rat's head, then use a scalpel to cut the scalp along the middle of the head to expose the skull, find the anterior fontanelle, and mark it with a marker as the origin;

[0160] 5. According to the coordinates: 4.4mm behind the anterior fontanelle, 1.2mm lateral to the midline, and 7.8mm below the dura mater, find the position of the medial forebrain bundle (MFB) in the right brain of the rat and make a mark there with a marker;

[0161] 6. Use a dental drill to carefully drill the skull at the MFB location. After the microinjection needle draws up the 6-OHDA solution, slowly insert the needle to the corresponding position and inject 3.66μl of 6-OHDA solution at a speed of 0.4μl / min. After the injection is completed, stop the needle at the original location for 10 minutes and then slowly withdraw the needle.

[0162] 7. The rat scalp was sutured and the wound and surrounding area were disinfected with iodine. In addition, each rat was injected intramuscularly with 1.2×10 5 Unit penicillin to prevent postoperative infection. Place the rat on a warming mat until it wakes up. Monitor the weight of the rat after surgery and pay close attention to its status, such as eating habits;

[0163] 8. Four weeks after surgery, rats injected with 6-OHDA were subjected to apomorphine-induced rotation test, and rats that rotated more than 300 times per hour were selected for subsequent cell transplantation experiments.

[0164] Example 9 Human amniotic epithelial stem cell transplantation in Parkinson's rats

[0165] 1. Take out the frozen human amniotic epithelial stem cells from the liquid nitrogen tank, revive and plate them in a six-well cell culture dish at a density of 1×10 6 Each well was cultured in an incubator for 24 hours until the cells adhered to the wall and spread out in a "paving stone" shape, and new culture medium was replaced to continue culturing for 48 hours until the human amniotic epithelial stem cells grew to basically cover the entire well;

[0166] 2. Abandon the culture medium, wash the adherent cells 2-3 times with sterile Dulbecco's phosphate-buffered saline (DPBS), add 1 ml of Accutase cell digestion solution to each well, digest in a 37°C incubator for 10-15 min, collect the human amniotic epithelial stem cell suspension in a 15 ml centrifuge tube, and centrifuge at 500×g for 5 min;

[0167] 3. Discard the supernatant and resuspend the human amniotic epithelial stem cells in DMEM / F12 medium without phenol red. Pass the cell suspension through a 70 μm pore size cell filter to remove large cell clumps.

[0168] 4. Count and adjust the cell suspension density to 1.5×10 5 pcs / μl, used for cell transplantation;

[0169] 5. Intraperitoneal injection of propofol was performed to anesthetize the rats at a dose of 100 mg / kg;

[0170] 6. Remove the hair from the rat's head and fix it on a digital stereotaxic apparatus;

[0171] 7. Use iodine to disinfect the rat's head, then use a scalpel to cut the scalp along the middle of the head to expose the skull, find the anterior fontanelle, and mark it with a marker as the origin;

[0172] 8. According to the coordinates: 1.2mm in front of the anterior fontanelle, 2.6mm lateral to the midline, 4.4mm below the dura mater; 0.5mm in front of the anterior fontanelle, 3.0mm lateral to the midline, 4.0mm below the dura mater, find the locations of the two injection points in the right striatum of the rat, and mark them with a marker pen;

[0173] 9. Use a dental drill to carefully drill open the skull at these two locations. After the microinjection needle absorbs the cell suspension / resuspended culture medium, slowly insert the needle to the corresponding position and inject 3μl and 2μl of cell suspension / resuspended culture medium at these two locations respectively. The injection speed is 0.4μl / min. After the injection is completed, stop the needle at the original location for 10 minutes and then slowly withdraw the needle.

[0174] 10. The rat scalp was sutured and the wound and surrounding area were disinfected with iodine. In addition, 1.2×10 5 Unit penicillin was used to prevent postoperative infection. The rats were placed on a warming pad until they woke up. The weight of the rats was monitored after surgery, and their status, such as eating, was closely monitored.

[0175] Example 10 Apomorphine-induced rotation test

[0176] 1. Preparation of apomorphine solution: Protect from light, use an analytical balance to accurately weigh an appropriate amount of apomorphine hydrochloride powder, dissolve it in physiological saline to obtain a 1 mg / ml apomorphine solution, store it on ice, and prepare it fresh each time;

[0177] 2. Place the rat in a quiet testing area and allow it to adapt to the surrounding environment for 30 minutes;

[0178] 3. Weigh the rats, calculate the injection volume for each rat based on a dose of 1 mg / kg, and inject apomorphine solution subcutaneously through the back of the neck to induce rotational behavior in the rats;

[0179] 4. Rotation to the undamaged side (i.e., the left side) was assigned a positive value, and only each completed whole-body rotation was recorded. The number of rotations of each rat within 60 minutes was recorded, and finally expressed as the net number of rotations per hour. Rats that rotated more than 300 times per hour were considered to have met the Parkinson's model rats that met the modeling success criteria and were used for subsequent experiments.

[0180] Example 11 Cylinder Test

[0181] 1. Place the rat in a quiet testing area and allow it to adapt to the surrounding environment for 30 minutes;

[0182] 2. Place the rat in a glass cylinder and record the number of times its front paws on the undamaged and damaged sides touch the cylinder wall for 10 minutes;

[0183] 3. After the test of one mouse is finished, clean the cylinder with water first, then spray 75% ethanol to remove the odor traces, and wait for the cylinder to dry before testing the next mouse;

[0184] 4. Calculate the percentage of the number of times the front paw on the undamaged side touches the cylinder wall / the number of times the front paw on the damaged side touches the cylinder wall.

[0185] Example 12 Gait Test

[0186] 1. Place the rat in a quiet testing area and allow it to adapt to the surrounding environment for 30 minutes;

[0187] 2. Pick up a rat, place it on a flat table, gently lift its tail, lift its hind legs off the table, and leave only its front paws touching the table;

[0188] 3. Pull the rat back along the tabletop for 1 m at a speed of 0.25 m / s, and record the number of adjustment steps of the front paws on the undamaged and damaged sides. Repeat the above operation 3 times for each rat.

[0189] 4. After the test of one mouse is finished, wipe the table with water and 75% ethanol to remove the odor traces. After the table is dry, conduct the test of the next mouse;

[0190] 5. Calculate the percentage of the undamaged side adjustment steps / damaged side adjustment steps.

[0191] Figure 2 The results showed that compared with the control group, the number of rotations per hour induced by apomorphine in rats transplanted with human amniotic epithelial stem cells was significantly reduced. In addition, the results of the cylinder test and gait test showed that the transplantation of human amniotic epithelial stem cells could significantly improve the impairment of forelimb function of rats caused by 6-OHDA damage. In summary, the results of the three behavioral tests all showed that the transplantation of human amniotic epithelial stem cells could significantly improve the motor dysfunction of Parkinson's rats.

[0192] Example 13: Collection and Slicing of Rat Brain Slices

[0193] 1. Preparation of antifreeze buffer for brain slices: Use an electronic balance to weigh 2.5 g PVP-40, 0.322 g Na 2 HPO 4 -12H 2 O, 1.302 g NaH 2 PO 4 -2H 2 O and 75g sucrose in a beaker, add 75ml ethylene glycol, add appropriate amount of ddHO 2 O, stir and dissolve on a magnetic stirrer, and then adjust the volume to 250 ml. The prepared antifreeze buffer for brain slices is stored at 4°C;

[0194] 2. Anesthetize the rats by intraperitoneal injection of propofol;

[0195] 3. Remove the hair from the rat's chest, cut the chest skin and ribs, expose the heart, and fix the brain tissue by aortic perfusion through the left ventricle: first use 100 ml of pre-cooled PBS solution to perfuse until the liquid flowing out of the right atrial appendage is clear, and then use 200 ml of 4% formaldehyde solution for perfusion;

[0196] 4. Remove the brain, place it in 4% formaldehyde solution, and continue to fix it at 4°C for 3 days;

[0197] 5. After 3 days, transfer the brain tissue to 20% and then 30% sucrose solution for gradient precipitation;

[0198] 6. Use a surgical blade to remove half of the cerebellum tissue, place the remaining brain tissue in an embedding container at a suitable angle, embed it with OCT embedding medium, and then carefully transfer it to a -80℃ freezer;

[0199] 7. Take out the embedded brain tissue from the -80℃ freezer, slice it using a freezing microtome, and continuously collect coronal brain slices covering the entire striatum and substantia nigra, with a slice thickness of 30μm / sheet;

[0200] 8. The collected brain slices were placed in antifreeze buffer and stored in a -20°C refrigerator.

[0201] Example 14 Immunohistochemistry (IHC) staining of brain slices

[0202] 1. Take out the brain slices from the antifreeze buffer and place them in a twelve-well plate, 3 brain slices per well;

[0203] 2. Wash the brain slices 3 times with PBS solution, 3 minutes each time;

[0204] 3. Add 4 ml of 3% HO to each well. 2 O2 PBS solution, incubate in dark for 15 min;

[0205] 4. Wash the brain slices 3 times with PBS solution, 3 minutes each time;

[0206] 5. Add 4 ml of blocking solution (PBS solution containing 3% bovine serum albumin, 1% Triton-X-100 and 5% goat serum) to each well and block for 2 hours at room temperature;

[0207] 6. Use antibody diluent (PBS solution containing 1% bovine serum albumin and 0.3% Triton-X-100) to dilute the primary antibody. The dilution ratio refers to the antibody instructions. Add an appropriate amount of diluted primary antibody to each well and incubate at 4°C overnight.

[0208] 7. Discard the primary antibody and wash the brain slices with PBS solution 3 times, 3 minutes each time;

[0209] 8. Use antibody diluent to dilute the peroxidase-conjugated secondary antibody. The dilution ratio refers to the antibody instructions. Add an appropriate amount of diluted secondary antibody to each well and incubate at room temperature for 2 hours.

[0210] 9. Discard the secondary antibody and wash the brain slices with PBS solution 3 times, 3 minutes each time;

[0211] 10. Use the DAB peroxidase substrate kit for color development. The specific steps are as follows: protect from light, mix the DAB concentrate and diluent according to the ratio specified in the instructions to obtain the DAB working solution, add 500 μl of DAB working solution to one well of a 24-well plate, put in the brain slice, and gently shake the plate. After the color development is completed, take out the brain slice and wash it with PBS solution 3 times, 5 minutes each time;

[0212] 11. Place the patch on an adhesive slide and place in a cool, ventilated place to dry overnight;

[0213] 12. Dehydrate with 75% ethanol for 2 minutes, 95% ethanol for 2 minutes, anhydrous ethanol for 2 minutes, and xylene for 2 minutes to make it transparent. Take out the slides, dry them in a fume hood, and then seal them with neutral gum.

[0214] 13. Use the digital scanning system VS200 to collect images and use Image J software to analyze the images.

[0215] Figure 2 The results showed that compared with the untransplanted side, the expression of TH in the substantia nigra and striatum of the transplanted side increased significantly, indicating that the number of dopaminergic neurons and nerve fiber density in the substantia nigra-striatum pathway of the transplanted side increased significantly. This result echoes the results of behavioral experiments, indicating that the transplantation of human amniotic epithelial stem cells promotes the remodeling of dopaminergic neurons in the substantia nigra-striatum pathway of Parkinson's rats.

[0216] Figure 3 The results showed that the density of microglia in the substantia nigra on the injured and untransplanted side was significantly increased compared with the uninjured side, and the expression level of IBA-1 was also significantly increased. In addition, we observed the typical microglial activation morphology of "enlarged cell bodies and shortened processes" on the injured and untransplanted side, and the transplantation of human amniotic epithelial stem cells significantly reduced the aggregation and activation of these microglia.

[0217] Figure 3 The results showed that compared with the uninjured side, the expression level of GFAP on the injured non-transplanted side was significantly increased, and the cell bodies of astrocytes were significantly enlarged, showing a typical activation morphology; while the expression level of GFAP on the transplanted side was significantly lower than that on the untransplanted side, indicating that the transplantation of human amniotic epithelial stem cells significantly reduced the degree of activation of astrocytes in the substantia nigra region of Parkinson's rats.

[0218] Example 15 Immunofluorescence staining of brain slices

[0219] 1. Take out the brain slices from the antifreeze buffer and place them in a twelve-well plate, 3 brain slices per well;

[0220] 2. Wash the brain slices 3 times with PBS solution, 3 minutes each time;

[0221] 3. Add 4 ml of blocking solution (PBS solution containing 3% bovine serum albumin, 1% Triton-X-100 and 5% goat serum) to each well and block for 2 hours at room temperature;

[0222] 4. Use antibody diluent (PBS solution containing 1% bovine serum albumin and 0.3% Triton-X-100) to dilute the primary antibody. The dilution ratio refers to the antibody instructions. Add an appropriate amount of diluted primary antibody to each well and incubate at 4°C overnight.

[0223] 5. Discard the primary antibody and wash the brain slices with PBS solution 3 times, 5 minutes each time;

[0224] 6. Use antibody diluent to dilute the secondary antibody with fluorescent label. The dilution ratio refers to the antibody instructions. Add an appropriate amount of diluted secondary antibody to each well and incubate at room temperature in the dark for 2 hours.

[0225] 7. Discard the secondary antibody and wash the brain slices with PBS solution 3 times, 5 minutes each time;

[0226] 8. Place the patch on an adhesive slide and place it in a dark and ventilated place to dry;

[0227] 9. Drop about 60 μl of anti-fluorescence quenching mounting medium containing DAPI on each slide and cover it with a coverslip;

[0228] 10. Use an upright fluorescence microscope or an inverted confocal fluorescence microscope FV3000 to collect images and use Image J software to analyze the images.

[0229] Figure 3 The results showed that compared with the untransplanted side, the TSPO expression level in the substantia nigra region of the transplanted side was significantly reduced, indicating that the transplantation of human amniotic epithelial stem cells reduced the activation of microglia and the level of neuroinflammation, which was consistent with the results of immunohistochemical staining.

[0230] Figure 3 The results showed that the number of C3d-positive astrocytes on the injured non-transplanted side was increased compared with the uninjured side, which was consistent with the clinical situation of Parkinson's patients described in the background section, while the transplantation of human amniotic epithelial stem cells reduced C3d-positive neurotoxic A1 type astrocytes in the substantia nigra.

[0231] Figure 4 The results showed that compared with the untransplanted side, the number of Nestin-Sox-2 double-positive neural progenitor cells in the substantia nigra on the transplanted side increased significantly, indicating that the transplantation of human amniotic epithelial stem cells promoted the migration of neural progenitor cells from the subventricular and hippocampal dentate gyrus to the substantia nigra. In addition, the increase in the Ki67 positive rate of neural progenitor cells on the transplanted side indicated that human amniotic epithelial stem cell transplantation promoted their proliferation.

[0232] Figure 4 The results showed that compared with the non-transplanted side, the PSA-NCAM + The increase in dopaminergic neurons (as indicated by white arrows), that is, the increase in newly generated dopaminergic neurons derived from neural progenitor cells, indicates that human amniotic epithelial stem cell transplantation promotes the regeneration of dopaminergic neurons in the substantia nigra-striatum pathway of Parkinson's rats by promoting the migration and proliferation of neural progenitor cells to the substantia nigra.

[0233] Example 16 TUNEL neuronal apoptosis detection

[0234] The TUNEL apoptosis detection kit was used to detect the apoptosis of dopaminergic neurons in the substantia nigra-striatum pathway of Parkinson's rats. All operations followed the instructions of the kit, as follows:

[0235] 1. Take out the brain slices from the antifreeze buffer and place them in a twelve-well plate, 3 brain slices per well;

[0236] 2. Wash the brain slices 3 times with PBS solution, 3 minutes each time;

[0237] 3. Add 4 ml of blocking solution (PBS solution containing 3% bovine serum albumin, 1% Triton-X-100 and 5% goat serum) to each well and block for 2 hours at room temperature;

[0238] 4. Use antibody diluent (PBS solution containing 1% bovine serum albumin and 0.3% Triton-X-100) to dilute Anti-TH antibody. The dilution ratio refers to the antibody instructions. Add an appropriate amount of diluted primary antibody to each well and incubate at 4°C overnight.

[0239] 5. Discard the primary antibody and wash the brain slices with PBS solution 3 times, 5 minutes each time;

[0240] 6. Use antibody diluent to dilute the secondary antibody with fluorescent label. The dilution ratio refers to the antibody instructions. Add an appropriate amount of diluted secondary antibody to each well and incubate at room temperature in the dark for 2 hours.

[0241] 7. Discard the secondary antibody and wash the brain slices with PBS solution 3 times, 5 minutes each time;

[0242] 8. According to the instructions of the kit, prepare TUNEL detection solution at a ratio of TdT enzyme: fluorescent labeling solution = 1:9. Add appropriate amount of TUNEL detection solution to each well and incubate at 37℃ in the dark for 60 minutes;

[0243] 9. Discard the TUNEL detection solution and wash the brain slices with PBS solution 3 times, 5 minutes each time;

[0244] 10. Place the patch on an adhesive slide and place it in a dark and ventilated place to dry;

[0245] 11. Drop about 60 μl of anti-fluorescence quenching mounting medium containing DAPI on each slide and cover it with a coverslip;

[0246] 12. Use an upright fluorescence microscope or an inverted confocal fluorescence microscope FV3000 to collect images and use Image J software to analyze the images.

[0247] Figure 4 The results showed that compared with the uninjured side, the apoptosis level of dopaminergic neurons in the substantia nigra on the injured and non-transplanted side was increased, while the transplantation of human amniotic epithelial stem cells reduced the apoptosis of neurons on the injured side.

[0248] Example 17 Fluoro-Jade C fluorescence staining detection of degenerated neurons

[0249] Fluoro-Jade C (FJC) fluorescent staining kit was used to detect degenerated dopaminergic neurons in the substantia nigra-striatum pathway of Parkinson's rats. All operations followed the instructions of the kit, as follows:

[0250] 1. Take out the brain slices from the antifreeze buffer and place them in a twelve-well plate, 3 brain slices per well;

[0251] 2. Wash the brain slices 3 times with PBS solution, 3 minutes each time;

[0252] 3. Add 4 ml of blocking solution (PBS solution containing 3% bovine serum albumin, 1% Triton-X-100 and 5% goat serum) to each well and block for 2 hours at room temperature;

[0253] 4. Use antibody diluent (PBS solution containing 1% bovine serum albumin and 0.3% Triton-X-100) to dilute Anti-TH antibody. The dilution ratio refers to the antibody instructions. Add an appropriate amount of diluted primary antibody to each well and incubate at 4°C overnight.

[0254] 5. Discard the primary antibody and wash the brain slices with PBS solution 3 times, 5 minutes each time;

[0255] 6. Use antibody diluent to dilute the secondary antibody with fluorescent label. The dilution ratio refers to the antibody instructions. Add an appropriate amount of diluted secondary antibody to each well and incubate at room temperature in the dark for 2 hours.

[0256] 7. Discard the secondary antibody and wash the brain slices with PBS solution 3 times, 5 minutes each time;

[0257] 8. Place the patch on the adhesive slide and place it in a dark and ventilated place to dry, then place it in a 50℃ oven for 30 minutes until it is completely dry;

[0258] 9. Protect from light and add 1 ml of ddH 2 O, incubate for 2 min;

[0259] 10. Use ddH 2 Dilute Solution B (potassium permanganate) 10 times. Protect from light and discard ddHO. 2 O, add 1 ml of Solution B diluent to each slide and incubate for 10 min;

[0260] 11. Protect from light, discard Solution B diluent, and add 1 ml of ddH 2 O, incubate for 2 min;

[0261] 12. Protect from light and use ddH 2 Dilute Solution C (Fluoro-Jade C staining solution) 10 times and discard ddHO. 2 O, add 1 ml of Solution C diluent to each slide and incubate for 10 min;

[0262] 13. Protect from light, discard Solution C diluent, and add 1 ml of ddHO to each slide. 2 O, incubate for 1 min, repeated 3 times;

[0263] 14. Protect from light and use ddH 2Dilute Solution D (DAPI) 10 times and discard ddHO. 2 O, add 1 ml of Solution D diluent to each slide and incubate for 10 min;

[0264] 15. Protect from light, discard Solution D diluent, and add 1 ml of ddHO to each slide. 2 O, incubate for 1 min, repeated 3 times;

[0265] 16. Protect from light, place the slide in a 50°C oven to dry for 5 minutes, then transparentize in xylene for 2 minutes;

[0266] 17. Take out the slide, dry it in a fume hood, and then seal it with DPX sealing medium;

[0267] 18. Use an upright fluorescence microscope or an inverted confocal fluorescence microscope FV3000 to collect images and use Image J software to analyze the images.

[0268] Figure 4 The results showed that the results of FJC staining were consistent with those of TUNEL staining.

[0269] The human amniotic epithelial stem cells isolated and cultured by the present invention exhibit an oval "paving stone" shape in a serum-free culture system, which is a typical epithelial cell morphology. In addition, they express epithelial cell-specific markers at a high positive rate of nearly 100%, but do not express blood cell and endothelial cell-specific markers, indicating that the isolated and cultured cells are simple and uniform epithelial cells. As a cell isolated from a discarded placenta, human amniotic epithelial stem cells have the advantages of being easy to obtain and free of ethical issues compared to human embryonic stem cells and human induced pluripotent stem cells. Further exploration of its basic properties revealed that human amniotic epithelial stem cells also have the advantages of being non-tumorigenic and low immunogenic, and can synthesize and secrete a variety of anti-inflammatory and neurotrophic factors, making them ideal candidate cells for cell therapy of Parkinson's disease. Based on the above-mentioned advantageous properties, the present invention first clarified the therapeutic effect of human amniotic epithelial stem cells on Parkinson's disease, and found that human amniotic epithelial stem cell transplantation can significantly improve the motor dysfunction of 6-OHDA-induced Parkinson's model rats, and promote the remodeling of dopaminergic neurons in the substantia nigra-striatum pathway of rats. Further research on the mechanism found that human amniotic epithelial stem cells can significantly reduce the activation of astrocytes and neurotoxic A1 astrocytes in the substantia nigra by reducing the activation level of microglia, thereby improving the brain immune microenvironment of Parkinson's rats, thereby reducing neuronal apoptosis on the one hand and increasing neuronal regeneration on the other hand, playing a neuroprotective role. In summary, the present invention aims at the difficult problem of Parkinson's disease treatment, develops a method of using human amniotic epithelial stem cells in the preparation of a drug for the treatment of Parkinson's disease, and explores its specific treatment mechanism, which has good prospects for clinical transformation.

Claims

1. Application of human amniotic epithelial stem cells in the preparation of drugs for the treatment of Parkinson's disease.

2. The use according to claim 1, characterized in that: The human amniotic epithelial stem cells are positive for E-cadherin, tight junction protein-1 and keratin, but negative for CD34, CD45, CD31 and CD144.

3. The use according to claim 1, characterized in that: The human amniotic epithelial stem cells do not express HLA class II antigens HLA-DQ and HLA-DR, but express non-classical HLA class I antigen HLA-G.

4. The use according to claim 1, characterized in that: The human amniotic epithelial stem cells are human amniotic epithelial stem cells of passage P1.

5. The use according to claim 1, characterized in that: The steps of isolating human amniotic epithelial stem cells are as follows: (1) mechanically separating the amniotic membrane from the inner surface of discarded placental tissue; (2) After the obtained amniotic membrane is carefully cleaned, it is digested with trypsin, centrifuged, and resuspended to harvest human amniotic epithelial stem cells.

6. The use according to claim 1, characterized in that: The human amniotic epithelial stem cells are resuspended in a phenol red-free DMEM / F12 culture medium to obtain a cell injection solution.

7. Use of human amniotic epithelial stem cell preparations in the preparation of drugs for treating and / or improving Parkinson's disease.

8. The use according to claim 7, characterized in that: The described materials include human amniotic epithelial stem cells and various pharmaceutically acceptable carriers.

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