CN114470001A

CN114470001A - Therapeutic use of human amniotic epithelial cells in autoimmune diseases

Info

Publication number: CN114470001A
Application number: CN202111282950.4A
Authority: CN
Inventors: 余路阳; 张传宇; 谭冰; 李金英; 袁惟芯; 郭礼和; 邵小燕; 刘佳
Original assignee: Shanghai Icell Biotechnology Co ltd; Zhejiang University ZJU
Current assignee: Shanghai Icell Biotechnology Co ltd; Zhejiang University ZJU
Priority date: 2018-04-18
Filing date: 2019-04-17
Publication date: 2022-05-13
Also published as: CN110090228A

Abstract

The present invention relates to the use of human amniotic epithelial cells (hAECs) for the treatment of autoimmune diseases. The invention discloses a method for treating and/or improving autoimmune diseases, including hashimoto's thyroiditis, uveitis and lupus erythematosus, by using an effective dose of amniotic epithelial cells or a cell preparation containing the amniotic epithelial cells alone or in combination with other drugs. The amniotic membrane can be administered by local injection or intravenous injectionEpithelial cells are administered to the patient in a dose range of about 10 per administration³‑10⁹The cell makes up the defects of the existing method to a certain extent, produces good treatment effect and provides a new clinical treatment scheme for the current autoimmune diseases.

Description

Therapeutic use of human amniotic epithelial cells in autoimmune diseases

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a therapeutic application of human amniotic epithelial cells in autoimmune diseases.

Background

Immune diseases refer to diseases caused by the influence of the imbalance of immune regulation on the immune response of the body. Immune diseases in the broader sense also include structural or functional abnormalities of the immune system caused by congenital or acquired causes. Autoimmune diseases (autoimmune diseases) refer to diseases caused by the damage of tissues caused by the immune response of human body to self-antigen. The main characteristics are that the etiology is unknown, the etiology is related to heredity and various environmental factors, the course of disease is long, attacks frequently occur, and great pain is caused to the life of a patient. Common autoimmune diseases include hashimoto's thyroiditis, uveitis, lupus erythematosus, and the like.

Among them, hashimoto's thyroiditis is the most common endocrine dyscrasia disease and autoimmune disease in humans. The disease is caused by the adverse effects of related sex steroids and X chromosome on thyroid gland and immune system, female incidence is higher than male incidence and is 5-10 times higher than male patients, incidence increases with age, and peak is between 45-65 years old.

The current cause of hashimoto thyroiditis is unclear and is generally thought to be caused by the breakdown of self-tolerance of genetically susceptible individuals caused by environmental factors. Hashimoto thyroiditis is mainly characterized in that a large number of lymphocytes are infiltrated in thyroid gland, thyroid gland specific self antibodies are generated, and thyroid gland apoptosis is performed. HT is clinically manifested in various forms, has occult diseases and slow progression, and many patients with HT have no obvious symptom and are usually found 1 to 2 years or even longer after the patients suffer from the HT. The patient may be accompanied by four conditions: 1. hypothyroidism; 2. the thyroid gland functions normally; 3. hyperthyroidism; 4. hypothyroidism or hyperthyroidism occur alternately, and patients have difficulty in controlling hormone levels, accompanied by diffuse goiter or thyroid nodules, etc., due to thyroid fibrosis. Other clinical features are manifested in various aspects of the human body, including abnormalities of the digestive system, lesions of the skin and the accessory structures, lesions of the cardiovascular system, and related symptoms affecting the respiratory system, the hematopoietic system, the reproductive system, the nervous system and the mental system, which have great influence on the quality of life and the happiness index of patients. Current approaches to hashimoto's thyroiditis-related treatment are mainly divided into: thyroid hormone replacement therapy, iodine-limiting or selenium-supplementing therapy, laser therapy and surgical resection treatment, but the above methods all have the problems of complications of a large sample and poor treatment effect.

In addition, autoimmune diseases of the eye can occur in various tissues of the eye, including conjunctiva, cornea, sclera, uvea, retina, etc., which severely affect vision. Among them, uveitis, also known as uveitis, includes inflammation of iris, ciliary body, choroid, retina, retinal blood vessels and vitreous body, and is a common ophthalmological blinding disease with recurrent attacks. The grape membrane is rich in melanin-related antigens, and the choroidal blood flow is rich and slow, so that the grape membrane is easily influenced by factors such as autoimmunity and the like, and becomes one of the most common autoimmune diseases in clinical ophthalmology. Statistics show that about 4% -10% of blindness is due to uveitis. Uveitis occurs mostly in young adults 20-50 years of age, with more men than women.

Uveitis results from a T cell-mediated autoimmune response to specific immune antigens of the eye, but its specific pathogenesis is not completely understood. In order to better study uveitis, Wacker et al successfully induced an Experimental Autoimmune Uvea (EAU) animal model, thereby building a good platform for better study of uveitis. EAU has similar clinical manifestations and disease course with human uveitis, and pathological changes have great similarity with human. EAU is a helper T cell (Th) mediated autoimmune response, and four major cell populations, mainly Th1, Th17, Th2 and regulatory T cells (treg), play important roles in EAU development, wherein Th1 and Th17 are major effector cell populations, and interact under different conditions to exert pathogenic effects separately or simultaneously. Th2 cells are mainly protective, suppressing immune response, and protecting tissues from inflammatory factor damage. Treg cells are the main immunosuppressive cell population and play an important role in the recovery process of EAU. Complex and subtle regulatory relationships exist among Th1, Th17, Th2 and Treg cells, which participate in the EAU process and play different roles in various stages of the disease. Moreover, the disease has the characteristic of repeated attack, and causes great pain and burden to patients. At present, hormone and immunosuppressant are mainly used for treating uveitis, but the hormone and immunosuppressant have obvious side effects and bring serious complications to patients while treating diseases. How to treat uveitis more effectively and safely is one of the hot spots in research.

Another common autoimmune disease is Systemic Lupus Erythematosus (SLE). SLE is a multiple organ, multiple system, severely compromised autoimmune disease that seriously compromises human health, primarily affecting women of childbearing age with an incidence rate of 9:1 in women versus men. Patients develop self-antibodies that bind to a variety of nuclear antigens including double-stranded DNA, Ribonucleoproteins (RNPs), sm (small nuclear ribonucleoprotein), and the like. These autoantibodies are deposited in various organs including the kidney, skin, joints, and the like, resulting in the development of inflammation. Systemic lupus erythematosus is clinically manifested in a variety of forms, including erythematous skin rash, canker sores, arthritis, serositis, vasculitis, nephritis, neurological, pulmonary and cardiac abnormalities, and the like. Disease activity, infection, lupus nephritis, neurological disorders are often associated with high morbidity and mortality in SLE.

The exact etiology and pathogenesis of systemic lupus erythematosus are not clear, the systemic lupus erythematosus is a multigenic complex disease, and 30 genetic loci are known to be related to the pathogenesis of SLE. The etiology is complex and includes environmental factors, sex hormones, genetic factors, random events, and the like. So far, no specific effective treatment means exists at home and abroad, and the disease can not be cured. The consequences of the disease to the patient, family and society, namely economic loss (villar lost), drug intoxication (drug toxicity), disability (disability), distress (disconfort) and death (death) can be described by the 5D disease description.

In conclusion, in view of the complex and various pathogenesis of autoimmune diseases such as thyroiditis, uveitis and lupus erythematosus, the specific pathogenesis is still not completely understood, and the existing treatment methods such as hormones and immunosuppressants have obvious side effects or poor treatment effects, so a safer, effective and economic treatment method is found, and the method has important significance for improving the life quality of patients and reducing the mortality or disability rate.

Disclosure of Invention

In order to solve the technical problem that the existing treatment method for the autoimmune disease has obvious side effect or poor treatment effect, the invention provides a treatment medicament or a method for treating the autoimmune disease by using amniotic epithelial cells.

In one aspect, the invention provides a use of human amnionic epithelial cells (hAECs) for treating autoimmune diseases.

In another embodiment of the invention, the autoimmune diseases include hashimoto's thyroiditis, uveitis, lupus erythematosus and the like.

The human amniotic epithelial cells are derived from the amniotic membrane of the postpartum waste placenta of the newborn. The placenta is oval or round in shape, has different diameters (15-20 cm) and thicknesses (2-3 cm), 500-600g, and is composed of amnion, chorion (fetal part) and decidua (maternal part). Wherein the decidua comes from the mother's endometrium, the amnion and chorion come from the fetus, wherein the side close to the mother's decidua is chorion, and the amnion is located on the surface of fetal chorion, is connected with umbilical cord and infantile skin, and wraps amniotic fluid and fetus, so is also called fetal membrane, is the early product of embryo development closely related with the developing fetus, and is the important tissue for material communication between the mother and fetus.

Genetically, amniotic epithelial cells are generated from the inner cell mass formed when the fertilized egg begins to develop. Morulae formed early in zygote development, not before implantation into the uterus (3-4 days after fertilization), and consisted of approximately 100 more cells. Tens of cells in the outer layer become trophoblasts and eventually form chorions, and tens of cells in the inner layer are the inner cell population and develop into embryos and amnions in the future. Approximately 8 days after fertilization, the human blastocyst is partially implanted into the endometrial stroma. The blastocyst outer layer cells (trophoblasts) differentiate into two layers embedded within the stroma, and the inner cell mass also differentiates into two layers: the epiblast and the hypoblast. The upper embryonic layer is the source of all three germ layers, ultimately forming the developing embryo. At the same time, the amniotic cavity appears in the epiblast, the epiblast of the adjacent cell trophoblastThe layer cells are called amniotic cells. The amniotic cavity expands with time, and forms a layer with a thickness of about 0.02-0.05mm and an area of about 700-²The amnion has no blood vessel, nerve, muscle and lymphatic vessel, has certain toughness and elasticity, and is divided into five layers from inside to outside. The amnion consists of five layers, namely an amniotic epithelial layer (epithelium), a basement membrane (basement membrane), a compact layer (compact layer), a fibroblast layer (fibroblast layer) and a sponge layer (sponge layer), wherein the innermost layer of the amnion is amniotic epithelial cells which face the amniotic cavity and wrap amniotic fluid.

The amniotic epithelial cells and the embryonic stem cells have the same development tissue source and are differentiated from the inner cell mass of the sac embryo from the development of the fertilized eggs to the 8 th day, so that the characteristics of the embryonic stem cells are retained, and the amniotic epithelial cells and the embryonic stem cells have multipotential dryness and stronger differentiation capacity and plasticity. hAECs typically express a variety of embryonic stem cell-associated markers, including stage specific embryonic antigen-3 (SSEA-3), stage specific embryonic antigen-4 (SSEA-4), tumor rejection antigen-60 (TRA 1-60), and tumor rejection antigen-81 (TRA 1-81). Meanwhile, specific transcription factors OCT-4, SOX-2, Nanog, FGF4 and REX-1 of the pluripotent stem cells are also expressed.

In practical application, the human amniotic epithelial cells are derived from the amniotic membrane on the postpartum waste placenta of the newborn, so that the source is wide, the materials are easily obtained, the price is low, the application limitation is avoided, no harm is caused to the baby or the mother, and obviously, the ethical problem caused by the application of the embryonic stem cells is avoided. Meanwhile, the human amniotic epithelial cells have the capacity of regulating in-vivo and in-vitro immune reactions, and researches show that the amniotic epithelial cells do not express or express the HLA-A, B, C gene in a low way; the expression of HLA-Ib (HLA-E, HLA-G); MHC II gene: HLA-DP, DQ, DR are low or not expressed; does not express beta₂Microglobulin; co-stimulatory factors CD80, CD86 were not expressed. Since hAECs secrete a variety of immunomodulatory factors, anti-angiogenic proteins, or anti-inflammatory factor-related proteins when cultured in vitro, human amniotic epithelial cells can be considered as immune privileged cells without the function of antigen presentation and can reduce immune cells after transplantationThe cell source avoids the occurrence of immunological rejection. Based on these considerations, human amniotic epithelial cells are the most suitable source and type of seed cells for clinical cell therapy and regenerative medicine among various sources and kinds of pluripotent cells.

In one aspect, the invention discloses the use of human amniotic epithelial cells or cell preparations thereof in the preparation of a medicament for treating and/or ameliorating an autoimmune disease. The human amniotic epithelial cells or cell preparations thereof can be used alone or in combination with other drugs to treat and/or ameliorate autoimmune diseases. An effective dose refers to an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount for a particular subject may vary depending on a number of factors, such as the disease to be treated, the overall health of the patient, the method of administration and the dosage and severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

In another embodiment of the present invention, the animal with autoimmune disease is a mammal. In a more preferred embodiment, the animal is a cow, horse, sheep, monkey, dog, rat, mouse, rabbit or human. In a most preferred embodiment, the animal having an autoimmune disease is a human.

In another embodiment of the invention, the cell preparation comprises human amniotic epithelial cells and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier of the present invention refers to a substance having a suitable benefit/risk ratio, e.g. a pharmaceutically acceptable solution, suspension or excipient, suitable for use in humans and/or animals, without undue adverse side effects (such as toxicity, irritation and allergic response), which facilitates cell survival and enables delivery of the formulated cells to humans or animals. The carrier is selected according to the appropriately intended mode of administration. The carrier of the present invention includes, but is not limited to, various physiological buffers such as physiological saline, phosphate buffer, artificial cerebrospinal fluid or whole serum, umbilical cord serum, etc. Various artificial scaffolds may also be included, including but not limited to gelatin sponge, demineralized bone, polyglycolic acid (PGA), polylactic acid (PLA), and copolymers thereof.

The appropriate state of the amniotic epithelial cells can be appropriately selected by those skilled in the art, taking into account the type of disease to be treated: collected cells without any treatment (crude fraction); partially purified cells; the purified cells are then expanded by culture.

In a preferred embodiment of the present invention, the autoimmune diseases include hashimoto's thyroiditis, uveitis, lupus erythematosus and the like.

In a preferred embodiment of the invention, the invention provides a method of treating Hashimoto's thyroiditis with amniotic epithelial cells. EAT is a classical model of Tg-induced research in human hashimoto's thyroiditis and has many of the same characteristics as hashimoto's thyroiditis. The method provided by the invention can improve the thyroid function of EAT mice, reduce the concentration of serum autoantibodies, reduce lymphocyte infiltration in thyroid, and regulate immune system, and is an effective method for treating pTg (porcine thyroglobulin) -induced autoimmune thyroiditis. By using the model, the method for treating the Hashimoto thyroiditis by using the amniotic epithelial cells can be popularized to human patients.

In another preferred embodiment of the invention, the invention provides a method of treating autoimmune uveitis using amniotic epithelial cells. Experimental Autoimmune Uveitis (EAU) has similar clinical manifestations and disease course as human uveitis, and pathological changes are also very similar to those of human. The method can obviously inhibit the occurrence and development of the diseases of the EAU mouse. Using this model, methods of amniotic epithelial cell therapy for uveitis can be generalized to human patients.

In another preferred embodiment of the invention, the invention provides a method of treating red lupus erythematosus with amniotic epithelial cells. The invention researches the potential capability of the human amniotic epithelial cells in the treatment of SLE and excavates the treatment mechanism of the SLE. When hAECs are injected after mouse SLE occurs, diseases can be obviously improved and even cured, serum ANA and anti-dsDNA antibodies are converted from positive to negative, and the antibody levels of IgG1, IgG2a and IgG3 are obviously reduced. And hAECs are found to restore the immune balance of SLE mice by regulating the proportion of T cell subsets and the level of cytokines. By using the model, the method for treating lupus erythematosus by using the amniotic epithelial cells can be popularized to human patients.

In a preferred embodiment of the invention, there is provided a method of isolating amniotic epithelial cells from amniotic tissue, the method comprising the steps of:

(1) mechanically separating the placenta tissue to obtain an amniotic membrane;

(2) and digesting the washed amniotic membrane by using digestive enzyme, and centrifuging the digested liquid to obtain the human amniotic epithelial cells.

In another embodiment of the invention, the amniotic epithelial cells of the invention are of human origin. The amniotic membrane may be isolated from isolated human placenta, washed with physiological buffer to remove blood cells, and mechanically removed of residual chorion and blood vessels. Isolation refers to removing cells from a tissue sample and separating them from additional non-tissue stem cells. Intact tissue is isolated as single cells using any conventional technique or method, including mechanical forces (shredding or shearing forces), enzymatic digestion with one or a combination of proteases, such as collagenase, trypsin, lipase, liberase and pepsin, or a combination of mechanical and enzymatic methods.

In a preferred embodiment of the invention, the human amniotic membrane is obtained by mechanically separating placenta tissue from a healthy parturient after cesarean section, with the consent of the parturient.

In another preferred embodiment of the present invention, the human amniotic epithelial cells obtained in step 2 may be cultured continuously, preferably under the following conditions: at 1 × 10⁶-1×10⁸Inoculating cells into a culture dish according to the density of each cell/plate, placing the culture dish in a carbon dioxide incubator for culture, replacing culture solution after human amniotic epithelial cells are attached to the wall, and digesting the cells after the plates are full of the cells for cryopreservation.

Other methods known to those skilled in the art can be used to concentrate the active cell population. These post-processing washing/concentration steps may be performed separately or simultaneously. In addition to the above methods, the viable cell population can be further purified or enriched after cell washing or after culture to reduce both contaminating and dead cells. Separation of cells in suspension can be achieved by the following techniques: buoyant density sedimentation centrifugation, differential adhesion to and elution from solid phases, immunomagnetic beads, fluorescent laser cell sorting (FACS), or other techniques. Examples of these different techniques and apparatus for performing these techniques can be found in the prior art and in commercial products.

The type of the basic medium used in the present invention is not limited as long as the medium can be used for cell culture. Preferred media include DMEM media and NPBM media. There is no limitation on the types of other components that may be contained in the above-mentioned basal medium, and preferred components include F-12, FCS, nerve survival factor, and the like.

In another preferred embodiment of the present invention, bFGF (basic fibroblast growth factor) or EGF (epidermal growth factor) is added to the above-mentioned basal medium. In this case, either one or both of them may be added. Exemplary concentrations of bFGF or EGF mentioned above are 1ng/ml to 100ng/ml, with a preferred concentration of 10 ng/ml. There is no limitation on the time and method of addition. Preferably, the agent is added daily when the above-mentioned amniotic epithelial cells are cultured in a basal medium.

The amniotic epithelial cells can be administered to the patient by any suitable method, such as local injection at the site of disease, sub-retinal injection, intravenous injection, or intraspinal injection, and the like. Typically, these cells are contained in a pharmaceutically acceptable liquid medium. Cell administration may be repeated or continuous (e.g., by continuous infusion into the cerebrospinal fluid). In general, multiple administrations are usually given at intervals of at least 7-10 days. Another method is to implant cells in a bioabsorbable material such as gelatin sponge and surgically implant the bioabsorbable material with the cells in the desired location. The two methods can be combined for application, and can obtain better curative effect.

Epithelial thin of amnionThe appropriate amount of cells will vary depending on the age, sex, weight, health of the patient and other factors. Typically, the dosage range for each administration is about 10³-10⁹Cells, typically about 10⁶-10⁷A cell.

In some embodiments of the invention, the amniotic epithelial cells are administered to the patient with one or more drugs.

The invention firstly uses the human amniotic epithelial cells for the treatment of autoimmune diseases such as hashimoto thyroiditis, uveitis, lupus erythematosus and the like, makes up the defects of the existing method to a certain extent, produces good treatment effect and provides a new clinical treatment scheme for the current autoimmune diseases. The technical scheme of the invention fully exerts the advantages of the human amniotic epithelial cells, wherein the human amniotic epithelial cells mainly have the following advantages in several aspects:

(1) the product has the advantages of being dry, strong in differentiation capacity and plasticity, strong in differentiation capacity in vitro, and capable of differentiating into three germ layers;

(2) has the capability of regulating in vivo and in vitro immune response, and can secrete a plurality of immune regulatory factors, anti-angiogenesis proteins or anti-inflammatory factor related proteins during in vitro culture;

(3) the immune cells have low immunogenicity, can be regarded as immune privileged cells, have no function of antigen presentation, can reduce the sources of the immune cells after transplantation, and avoid the occurrence of immune rejection;

(4) does not express telomerase reverse transcriptase and has no tumorigenicity;

(5) wide source, easily obtained materials, no application limitation and no ethical problem.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical means of the present invention more clearly understood and to be implemented according to the content of the description, the following detailed description is made with reference to the accompanying drawings by using preferred embodiments of the present invention.

Drawings

FIG. 1a mesenchymal Stem cell marker expression from hAECs

FIG. 1b expression of MHC markers for hAECs

FIG. 1c expression of hematopoietic Stem cells and endothelial cell markers by hAECs

FIG. 2a light mirror structural HE staining of thyroid follicles in control CBA/J mice with thyroiditis score 0+ (. times.40) (Uniform thyroid follicles indicated by arrows)

FIG. 2b light-microscopic HE staining of thyroid follicles in CBA/J mice in EAT group, thyroiditis score 1+ (. times.40) (diffuse lymphocyte infiltration as shown by arrows)

FIG. 2c light-microscopic HE staining of thyroid follicles in CBA/J mice in EAT group, thyroiditis score 2+ (. times.40) (arrows indicate aggregated lymphocytes, lesions up to one size of thyroid follicle)

FIG. 2d light-mirror structural HE staining of thyroid follicles in CBA/J mice in EAT group, thyroiditis score 3+ (. times.40) (diffuse and aggregated lymphocytes shown by arrows)

FIG. 2e light-microscopic HE staining of thyroid follicles in CBA/J mice in EAT group, thyroiditis score 4+ (. times.40) (perivascular aggregated lymphocytes are shown by arrows)

FIG. 3 pathological scoring of mice at various times after modeling

FIG. 4 TGAb and TMAb concentrations in serum of WT and EAT mice

FIG. 5 the serum concentrations of TT3, TT4, TSH in the WT and EAT groups of mice

FIG. 6 TGAb concentration in EAT and EAT + hAECs group mice serum

FIG. 7 serum TMAB concentrations in EAT and EAT + hAECs group mice

FIG. 8 concentration of serum TT3 in EAT and EAT + hAECs group mice

FIG. 9 concentration of serum TT4 in EAT and EAT + hAECs group mice

FIG. 10 serum TSH concentrations in EAT and EAT + hAECs group mice

FIG. 11 Effect of different treatment times on EAT mouse disease score

FIG. 12 intrathyroid infiltrating NK and B cells (infiltrating lymphocytes shown by arrow B, C, infiltrating NK cells shown by F, G, infiltrating B cells shown by J, K)

FIG. 13 variation of the proportion of Treg, Th17 and Breg cells in different groups

FIG. 14 is an overall flow chart of the experiment. The prevention group on day D0 was treated with cells at the same time as the model, the treatment group on day D6 was injected with hAECs 6 days after immunization, and the control group was injected with BSS at the corresponding time. Samples of aqueous humor, eyeball, spleen lymph node and the like are collected at 12 th and 18 th days after immunization for relevant detection in each group.

Figure 15 slit lamp observations and inflammation scores for each group. (A) Slit

lamp fundus images

12 and 18 days after immunization of the D0 day prevention group and the D6 day treatment group and the corresponding control group; (B) statistics of slit

lamp fundus images

12 and 18 days after immunization in the D0 day prevention group and the D6 day treatment group and inflammation scores of corresponding control groups; (C) dynamic change chart of preventing group slit lamp inflammation score in D0 day; (D) dynamic change chart of slit lamp inflammation score of treatment group in D6 days; (E) fundus map of normal Lewis rat. (xp <0.0001, x P <0.001, x P <0.05) n ═ 6, scale ═ 1mm.

Fig. 16 histopathological observations and scores. (A) Representative pictures of HE staining of retinal structures and inflammatory cell infiltration of D0-hAECs in the prevention group and D6-hAECs in the control group; (B) each group of histology score statistical plots; (C) statistical plots of retinal and ectonuclear thickness in each group 18 days post-immunization; (D) normal retinal architecture. (xp <0.0001, x P <0.001, x P <0.05) n ═ 6, scale ═ 100 μm.

FIG. 17 infiltration of macrophages and T cells in each group. (A-C) infiltration and quantitative statistics of D0-hAECs prevention group and D6-hAECs treatment group on day 12 with corresponding control group CD68+ (macrophage) CD3+ (T cell) cells; (D-F) infiltration of cells from D0-hAECs prevention group and D6-hAECs treatment group on day 18 and CD68+ (macrophages) CD3+ (T cells) control group and quantitative statistics. (xp <0.0001, x P <0.001, x P <0.05) n ═ 6, scale ═ 100 μm.

Figure 18 effect of hAECs on Th17 cell population and Treg cell population. (A) A dynamic change chart of the proportion of IL-17+/CD4+ T cells of a D0-hAECs prevention group and a D6-hAECs treatment group to a control group is detected in a flow mode; (B) a dynamic change chart of the ratio of FoxP3+/CD4+ CD25+ T cells of a D0-hAECs prevention group and a D6-hAECs treatment group to a control group is detected in a flow mode; (C) statistical plots of the variation of Th17 cell populations on

days

12 and 18 in each group; (D) statistical plots of the change in the Tregs cell population on

days

12 and 18 in each group; (E) statistical plots of the variation of the Tregs/Th17 ratio at

days

12 and 18. (. P <0.0001,. P <0.001,. P <0.05) n ═ 6

FIG. 19 Effect of hAECs on aqueous humor and splenic lymph node monocyte-associated immune factors. (A-D) dynamic changes of the expression levels of MCP-1, IFN-gamma, IL-17 and IL-10 in the aqueous humor of rats in each group; (E-F) dynamic changes in the expression levels of IL-17 and IL-10 in the culture supernatants of splenic lymph node mononuclear cells from rats in each group. (. P <0.0001,. P <0.001,. P <0.05) n ═ 6

FIG. 20 hAECs convert SLE mouse serum ANA and anti-dsDNA antibodies from positive to negative. Two weeks after injection, serum was isolated from Control, SLE and SLE + hAECs and immunofluorescence was used to detect levels of ANAs and anti-dsDNA antibodies in mice. The graphs show the detection of ANAs, control mice (A), SLE mice (B), SLE + hAECs (C) on the basis of Hep-2 cells and the mean MFI statistics of each mouse (D) and the detection of anti-dsDNA antibodies, control mice (E), SLE mice (F), SLE + hAECs (G) on the basis of Crithia luciliae kinetoplast and the mean MFI statistics of each mouse (H). Arrow, nucleus (B), kinetoplast (F). Data are expressed as mean ± SD, derived from three independent experiments of 5 independent samples. P < 0.05; p < 0.01; p <0.001, data analysis using one-way analysis of variance and Tukey multiple comparisons.

FIG. 21 hAECs reduce serum IgG Isotypes concentrations in SLE mice. MRL-Faslpr (SLE) mice develop an immune response to self-cellular nuclear antigens (dsDNA, etc.) and continue to produce auto-IgG antibodies to these nuclear antigens. Autoantibodies form a large number of immune complexes with antigens that can deposit on joints, vessel walls and glomeruli. To further verify the therapeutic effect of hAECs on SLE, serum IgG1, IgG2a, IgG3 concentrations were measured. As shown, the serum IgG1(p ═ 0.0065), IgG2a (p ═ 0.0010), IgG3(p ═ 0.0017) were significantly higher in the SLE group than in the Control group, and the serum IgG1(p ═ 0.0375), IgG2a (p ═ 0.0001 ×), and IgG3(p ═ 0.0001 ×) were significantly lower in the SLE group than in the SLE group after the injection of hAECs. The results show that the injection of hAECs can obviously reduce the activation of B lymphocytes and reduce the circulating IgG isotype level in SLE mice.

FIG. 22 shows that the level of proinflammatory factors in the treated group is reduced after hAECs are administered. Figure 22-1 is a significant decrease in serum IL-17A concentration (p 0.0067) two weeks after injection of hAECs, consistent with changes in the proportion of Th17 cells in the mouse spleen. FIG. 22-2 shows that hAECs modulate the mouse serum IFN-. gamma./IL-4 balance in SLE primarily by reducing IFN-. gamma.concentration. Fig. 22-3 shows that the serum IL-10 concentration of SLE group mice was significantly higher than that of Control group (p ═ 0.0307), the serum TGF- β concentration of SLE group mice was significantly lower than that of Control group (p ═ 0.0231), and the serum TGF- β concentration was significantly higher after injection of hAECs (p ═ 0.0101). hAECs act immunoregulatory in SLE primarily by upregulating TGF- β concentrations, with less effect on IL-10 concentrations.

FIG. 23 hAECs improved the splenic Th17/Treg cell balance in SLE mice. Figure 23-1 shows that Th17 and Tregs balance affect the immune system, and it is known that Th17/Treg cell balance is disrupted in various autoimmune diseases. As shown in the figure, the results of the detection of Th17 cells significantly increased the proportion of splenic Th17 cells in SLE mice after onset (p ═ 0.0084), and significantly decreased Th17 cells two weeks after hAECs treatment (p ═ 0.0023). FIG. 23-2 shows a synergistic reduction in the Th17 cell fraction and an increase in the Tregs cell fraction following treatment, hAECs can improve the immune imbalance in SLE mice by modulating the Th17/Treg cell fraction.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

The first part of human amniotic epithelial cells are used for treating hashimoto thyroiditis

Example 1-1 construction of an Experimental autoimmune thyroiditis model

1. Purchasing and raising of laboratory animals

CBA/J mice, SPF grade, 30-40g, female, animals were provided by Shanghai south-Square model animal center. The mice of the same group are placed in the same cage, 6 mice in each cage are only raised in the experimental animal center of Zhejiang university, the room temperature is controlled to be 23-26 ℃, the relative humidity is controlled to be within 55 +/-10%, the illumination is carried out in a day-night period of 12 hours, and the mice are fed with food and drinking water.

2. Experimental medicine

(1) Complete Freund's Adjuvant (CFA), Sigma in USA

(2) Incomplete Freund's Adjuvant (IFA) Sigma, USA

(3) Porcine thyroglobulin (pTg) Sigma in the United states

3. Grouping of laboratory animals

The treatment was divided into groups of at least three replicates per group at different treatment times (Table 1), with 9 replicates per group tested at one time, 3 model-treated groups (EAT + hAECs group), 3 model-only groups (EAT group), and 3 normal controls (WT group), respectively.

TABLE 1

4. Primary immunization

Female CBA/J mice of 6-8 weeks were selected, pTg was dissolved in sterile PBS to a final concentration of 2mg/ml, CFA was mixed with pTg solution in equal volumes and emulsified thoroughly. CBA/J mice were fixed and injected subcutaneously in multiple points (neck, upper back, etc.) with 100. mu.l of emulsifier, pTg 100. mu.g, per mouse. Normal control mice were injected subcutaneously with equal doses of sterile PBS solution.

5. Boosting immunity

The 14 th day after the primary immunization was boosted, IFA and pTg solution were mixed in equal volume, emulsified thoroughly, and injected into the mice of the simple model group and the model treatment group at 100. mu.l each, i.e., pTg of 100. mu.g, at the subcutaneous multiple injection points of the lower back. Normal control mice were injected subcutaneously with equal doses of sterile PBS solution.

EXAMPLE 1-2 preparation of human amniotic cell test solution

1. Preparing an amniotic epithelial cell culture solution: 500ml of DMEM/F12 to which 56ml of KSR was added; 6ml L-Glutamine; 6ml of Sodium Pyruvate; 6ml of MEMNEAA; 600. mu.l of 2-ME; 2000 × EGF and 100 × P/S were added before use;

2. 2000 × EGF preparation: adding 1ml of sterile ddH2O into an EGF packaging tube, standing for 5-10min for dissolving, adding 4ml of diluent (5% Trehalose PBS), mixing, and subpackaging into 1.5ml of EP tubes with 100 mu l per tube;

3. preparing a digestion stop solution: DMEM/F12+ 10% FBS;

4. preparing a frozen stock solution: 40% FBS + 50% culture medium + 10% DMSO.

Examples 1-3 isolation of human amniotic epithelial cells

1. Source of human amniotic membrane

After the authorization of the lying-in woman, placenta tissues after the cesarean section of healthy lying-in women (serological reactions such as HIV, syphilis, hepatitis A, hepatitis B, hepatitis C and the like are all shown to be negative) are taken, the placenta is cut by a cross knife, and the whole amnion is obtained by mechanical separation.

2. Isolation of hAECs

The amniotic membrane was washed three times with sterile PBS solution with double antibody (P/S), blood and other impurities were washed off, and the amniotic membrane was transferred to a 50ml centrifuge tube.

10ml of 0.25% pancreatin (bathed at 37 ℃ C. earlier) was added for digestion for 30s, inverted 20 times and the amniotic membrane was transferred to another 50ml centrifuge tube.

15ml of 0.25% pancreatin (bathed at 37 ℃ in advance) was added to the centrifuge tube, and after digestion in a water bath at 37 ℃ for 10min, the amniotic membrane was transferred to another 50ml centrifuge tube.

Adding 25ml of 0.25% pancreatin into a centrifuge tube, digesting in water bath at 37 ℃ for 40min, shaking 10 times every 10 minutes, forcibly reversing 10 times after finishing, adding an equal volume of digestion stop solution to stop digestion, rotating at the speed of 500g, centrifuging at room temperature for 10min, collecting cells, and re-suspending with 1ml of culture solution.

Transferring amnion into another 50ml centrifuge tube, adding 25ml of 0.25% pancreatin, digesting at 37 ℃ for 40min, mixing 10 times every 10min, turning over 10 times, adding equal volume of digestion stop solution to stop digestion, rotating at a speed of 500g, centrifuging at room temperature for 10min, collecting cells, and resuspending with 1ml culture solution.

Mixing the two re-suspended cells, adding 18ml culture solution (adding the double antibody and EGF in the culture solution in advance), uniformly mixing, sieving with a 200-mesh sieve, and sieving with a 400-mesh sieve.

Examples 1-4 inoculation culture and cryopreservation of human amniotic epithelial cells

Cell counting culture:

one plate inoculation 1X 10⁷And (4) cells. And changing the culture solution after the hAECs are attached to the wall, and changing the culture solution once three days later.

After the cells grew over the plate, the cells were digested and cryopreserved: 5ml of pancreatin is added into 15cm dish, observation is carried out under a mirror after 10min, when the cells become round and the cells are in a suspension state when the plate is shaken in a plane, the digestion is stopped by adding the same amount of digestion stop solution. The cells on the culture dish were blown down by a micropipette in the same direction, transferred into a 15ml centrifuge tube, centrifuged at 300g for 3min, collected and then counted. And adding the freezing solution into the freezing tube, marking the freezing date, the freezing batch and the number of the cells, putting the cells into the freezing tube, immediately putting the freezing tube into a freezing box, putting the freezing box into a refrigerator at minus 80 ℃, taking out the freezing box after 12 hours, and transferring the cells into a liquid nitrogen tank for storage.

Examples 1-5 identification of hAECs cell surface markers by flow cytometry

The 1 st generation hAECs were digested, centrifuged at 1000rpm for 5min at 4 ℃, the supernatant was discarded, and 1ml PBS was added to resuspend and dispense into 1.5ml centrifuge tubes. After centrifugation at 1000rpm for 5min at 4 ℃ the supernatant was discarded and 100. mu.l of flow Buffer (PBS + 2% FBS) was added.

1, HLA-DQ-FITC (MHC class II), CD34-PE (endothelial cell marker), CD90-APC (mesenchymal stem cell marker) is added into each tube of cells; HLA-ABC-FITC (MHC class I), CD31-APC (endothelial cell marker); 3. CD73-FITC (mesenchymal stem cell marker), HLA-DR-PE (MHC class II); 5. mu.l each of CD45-FITC (hematopoietic stem cell marker) and CD166-PE (mesenchymal stem cell marker) was mixed; 5. blank control; 6. homotypic control; 7. and (5) single labeling.

Incubating at 4 ℃ in the dark for 30min, centrifuging at 1000rpm for 5min, discarding the supernatant, washing with 1ml PBS once, adding 500 μ l PBS containing 1% paraformaldehyde into each tube, mixing, standing at 4 ℃ in the dark, and detecting and analyzing by a flow cytometer within 24 hours.

The number of cells collected in each sample is more than or equal to 10⁴Phenotypic analysis was performed using Flow JO software. Isotype control antibodies were corresponding fluorescein-labeled mouse IgG.

Examples 1-6 mice tail vein injection of hAECs

Mice of the model treatment group (EAT + hAECs) were treated by tail vein injection of hAECs, and the number of cells per injection was 1.5X 10⁶Per (concentration is 1.5X 10)⁷Cell suspension per ml, 100. mu.l each injection).

In operation, a place with sufficient light is selected, the mouse is fixed by a mouse fixer, a l ml insulin syringe is prepared, 100 mul cell suspension is sucked each time, and the cells are prevented from depositing in the syringe.

Clamping the root of the rat tail to expand the blood vessel, selecting an obvious side vein, wiping the obvious side vein with 75% alcohol until the blood vessel is obviously expanded by naked eyes, inserting a needle at the far end of the rat tail, and withdrawing blood to show that the vein of the mouse is successfully punctured, and injecting the cell suspension into the tail vein of the mouse.

Mice in the normal control group and the EAT model group were injected with the same amount of sterile PBS in tail vein at the same time.

Examples 1-7 thyroid function assay in mice

1. Mouse serum isolation

Collecting blood from orbital venous plexus of mouse, standing the blood at room temperature for 1 hr, standing at 4 deg.C for 30min, centrifuging at 4 deg.C at 3600rpm for 10min, separating serum, packaging, and freezing at-80 deg.C. After collecting the whole group of sera, the concentrations of TSH, TT3 and TT4 in the mouse sera were measured by ELISA method.

2. ELISA for detecting the concentrations of TSH, TT3 and TT4 in mouse serum

Dilution of standards

The highest concentration of the standard substance is as follows: TSH (80pg/ml), TT3(32pmol/L), TT4 (40. mu.g/L);

2.1 sample adding: each group is provided with a blank hole (without adding a sample and an enzyme-labeled reagent), a standard sample hole and a sample hole to be detected. And adding 50 mul of standard sample and sample to be detected respectively (the sample is diluted by 5 times by using a sample diluent) on the enzyme-labeled coated plate. Adding a sample to the bottom of an enzyme-labeled plate hole, keeping the sample from touching the hole wall as much as possible, and slightly shaking and uniformly mixing the sample and the hole wall;

2.2 incubation: sealing with viscous sealing membrane (not used for cross use), and oven drying at 37 deg.C for 30 min;

2.3 liquid preparation: diluting 30 times of the concentrated washing solution by using distilled water for later use;

2.4 washing: pouring liquid, spin-drying with force, beating on dust-free paper, filling each hole with washing liquid, waiting for 30 seconds, discarding for 5 times, and beating to dry;

2.5 adding enzyme: 50 mul of enzyme labeling reagent is added in each hole except for blank holes;

2.6 incubation: the operation is the same as 2;

2.7 washing: the operation is the same as 4;

2.8 color development: 50. mu.l of color-developing agent A and 50. mu.l of color-developing agent B were added to each well. Shaking gently to avoid liquid splash to cause pollution, and developing in oven at 37 deg.C in dark for 10 min;

2.9 termination: the reaction was stopped by adding 50. mu.l of stop solution to each well (at this time, the blue color immediately became yellow), and the absorbance (OD value) of each well was measured at a wavelength of 450nm within 15 min;

2.10 calculation: drawing a standard curve (the concentration of the standard substance is an abscissa, and the OD value is an ordinate), calculating a linear regression equation of the standard curve by using the OD value and the concentration of the standard substance, calculating the concentration of the sample during detection according to the OD value of the sample, and multiplying the concentration by the dilution factor to obtain the actual concentration of the sample.

Examples 1-8 detection of mouse thyroid autoantibody levels

1. Mouse serum isolation

The procedure is as in examples 1-7, and the levels of the autoantibodies TGAb and TMAb in the mouse serum are determined after the serum of the whole group is collected.

2. ELISA detection of thyroid autoantibody levels in mouse serum

The highest concentration of the standard substance is as follows: TGAb (120IU/ml), TMAb (48pg/ml), detection method same as example 1-7.

Examples 1-9 pathological grading of mouse thyroid

1. Isolation of thyroid tissue

Blood-treated mice were sterilized with alcohol, perfused with PBS and mechanically isolated for bilateral thyroid glands. Cleaning the nail gland tissue with PBS, fixing in 4% PFA (volume ratio of 1:20), fixing at room temperature for 8h, removing PFA, cleaning with PBS, adding 70% alcohol, and storing for a long time.

2. Solution preparation:

4% PFA: 180ml of ddH2O were heated to 65 ℃ and 20. mu.l of 5M NaOH were added and 8g of PFA (paraformaldehyde) were added and the mixture was heated continuously until dissolved (stirring with a rotor), cooled to room temperature and 10ml of 20 XPBS were added to bring the volume to 200 ml.

Hydrochloric acid ethanol solution: 7ml of HCl (37%) +252ml of 70% ethanol.

Diluted ammonia water: 200ml of H2O was added with 8 drops of ammonia water to obtain 0.05% dilute ammonia water.

3. Histopathological examination of thyroid gland

3.1 dehydration and wax penetration:

thyroid tissue was placed in an embedding cassette sequentially through a dehydration jar containing the following solutions:

and (3) dehydrating: sequentially passing the embedding box filled with thyroid tissue through 70% ethanol, 80% ethanol and 90% ethanol for 15min respectively, placing in two 95% ethanol dehydration cylinders for 30min, and placing in two 100% ethanol dehydration cylinders for 30 min;

and (3) transparency: sequentially placing in dehydration jar containing 50% xylene and 50% alcohol for 60min, and placing in two xylene dehydration jars for 60 min;

wax penetration: passing through two dehydration cylinders containing pure wax for 60 min.

3.2 embedding:

taking out the embedding box by using tweezers, putting the embedding box and a paraffin mold into a wax cylinder, clamping the paraffin mold by using the tweezers, putting the paraffin mold at a wax dripping position, putting the paraffin mold at a 4 ℃ position after dripping a drop of wax, putting the tissue on the wax (the section faces downwards), putting a mould at the wax dripping position, covering the embedding box, adding the paraffin, putting the embedding box at the 4 ℃ position for solidification, and then supplementing the paraffin.

3.3 slicing:

the wax block is trimmed according to the tissue part, and redundant wax blocks are cut off. The slicer is adjusted to make the blade and the wax block have proper distance and angle. The thickness of the slice was adjusted to 50 μm, the thickness of the slice was adjusted to 4 μm after the sample was cut, whether the desired tissue was cut or not was examined under the microscope, and then the slice was cut at a thickness of 4 μm. Carefully cut the wax tape to a certain length, and then cut the wax tape by a blade according to the distance of 5 samples.

3.4 spreading, pasting and baking:

the water temperature was maintained at 42 ℃ by opening the slide-out apparatus, the wax tape was held with a pair of tweezers and placed on the water surface, and the slide was spread in the water. And taking a clean glass slide, carefully fishing out the unfolded slice, and marking the date and the sample number on the ground surface at the other end. And adjusting the temperature of the sheet baking machine to 37 ℃, and putting the slices on the sheet baking machine for baking so that the slices are attached to the glass sheet. Baking for 4h, and collecting the slices, wherein the slices can be stored for a long time.

3.5 thyroid section HE staining:

the cut thyroid pathological section sequentially passes through the following containers:

3.5.1 dewaxing: xylene 10min × 3;

3.5.2 hydration: 100% ethanol 5min × 2-95% ethanol 2 min; washing with slow tap water flow for 5 min;

3.5.3 staining of the nuclei: hematoxylin (45S to minutes, depending on the staining, the nucleus stains blue); washing with slow tap water flow for 5 min;

3.5.4 differentiation: hydrochloric acid ethanol solution for 2 s; washing with slow tap water flow for 5 min;

3.5.5 Rebluing: putting into dilute ammonia water for 8 times; washing with slow tap water flow for 6 min; 95% ethanol for 4 min;

3.5.6 staining cytoplasm: eosin (45S, cytoplasmic staining red);

3.5.7 dehydration: 95% ethanol 2min x 2-100% ethanol 4min x 2;

3.5.8 transparency: xylene 5min × 3, after staining was completed the sections were placed in a fume hood with air-dried residual xylene and blocked with neutral resin.

3.6 thyroid section score:

the basis for the histopathological score of the thyroid is the percentage of thyroid follicles that are infiltrated by lymphocytes. The infiltration degree of inflammatory cells (including lymphocytes, plasma cells and neutrophils) of the thyroid tissue is observed under a light microscope, and the ratio of the inflammatory cells to the thyroid tissue is calculated by using Image-Pro Plus Image analysis software after photographing.

Reference to the staging criteria for inflammation by Tang H [105], grade 1: inflammatory cells accumulate between two or more thyroid follicles; and 2, stage: the focus of the inflammatory cells can reach the size of thyroid follicle; and 3, level: 10% -40% of the thyroid tissue is replaced by inflammatory cells; 4, level: more than 40% of the thyroid tissue is replaced by inflammatory cells.

Examples 1-10 phenotypic identification of infiltrating intrathyroid lymphocytes

1. Solution preparation (all solutions are ready for use)

1.1 antigen retrieval solution: adding solution A, 0.1M citric acid solution and 21.01g of citric acid into 1L of distilled water; solution B, 0.1M trisodium citrate solution, 29.41g trisodium citrate is added into 1L of distilled water; the citric acid repair solution, 9ml of solution A and 41ml of solution B were added together to 450ml of distilled water.

1.2 blocking serum: HBS 5% (horse serum 20-fold dilution).

1.33% H2O2 methanol solution: commercial H2O2 was 30% concentrated and diluted 10-fold with methanol.

1.4 ABC solution preparation: 5ml PBS +2 drop A +2 drop B (prepared 30min before).

1.5 AEC solution preparation (care is taken to avoid light during use): 5ml of distilled water +2 drop buffer storage solution +3 drop AEC storage solution +2 drop hydroperoxide solution.

2. Immunohistochemistry for Paraffin section (avoid drying section in whole process)

2.1 dewaxing: xylene 10min × 3;

2.2 hydration: 100% ethanol 5min × 2, 95% ethanol 2min, and tap water washing for 5 min;

2.3 antigen retrieval: placing the slices in an antigen repairing solution at 95-97 ℃ for 10min, and immersing the slices in PBS for 5min after the whole body is restored to the room temperature; the oily pen circles the tissue (to avoid marginal effects, the circle may be appropriately larger);

2.4 removal of endogenous peroxidase Activity: 3% H2O2 methanol solution for 10 min; soaking and washing with PBS for 5 min;

2.5 incubation Primary antibody: after 30min, PBS is used for immersion washing for 5min multiplied by 3;

2.6 incubation secondary antibody: after 30min, PBS is used for immersion washing for 5min and multiplied by 3;

2.7 ABC solution incubation for 30 min; soaking and washing with PBS for 5 min;

2.8 incubation and color development of AEC reagent for 10-30min, and microscopic examination after ten minutes to determine the color development time; washing with slow water flow for 5 min;

2.9 Restaining cell nucleus with hematoxylin for 45s-3 min; washing with slow tap water flow for 5 min;

2.10 aqueous mounting tape.

Examples 1-11 flow cytometry for detecting the ratio of Th17, Tregs and Bregs in mouse spleen cells

1. Solution preparation

1.1 preparation of PMA solution

Storage solution: the concentration is 0.1mg/ml, and the mixture is stored in the dark at the temperature of minus 80 ℃;

working fluid: 1:10 diluted in RPMI1640 with 10% FBS, at a concentration of 10. mu.g/ml;

final working concentration: 25ng/ml, namely adding 1.25 mul of PMA into every 500 mul of culture solution;

1.2 preparation of Ionomycin solution

Storage solution: the concentration is 1mg/ml, and the product is preserved at minus 80 ℃ in the dark;

working fluid: 1:2 diluted in RPMI1640 containing 10% FBS at a concentration of 500. mu.g/ml;

final working concentration: 1 mu g/ml, namely adding 1 mu l of Ionomycin into every 500 mu l of culture solution;

1.3 preparation of Monensin solution

Storage solution: the concentration is 50mg/ml, and the product is stored at minus 80 ℃ in a dark place;

working fluid: 1:50 diluted in 10% FBS-containing RPMI1640 at a concentration of 1000. mu.g/ml;

final working concentration: 1.7 mu g/ml, namely adding 0.85 mu l of Monensin into every 500 mu l of culture solution;

1.4 preparation of lymphocyte culture solution:

RPMI1640(430ml) + 10% FBS (50ml) + (1000X) 50. mu.M. beta. mercaptoethanol (500. mu.l) + (100X) 100mM Sodium Sodium Pyruvate Pyruvate (5ml) + (50X) 1M HEPES (10ml) + (100X) double antibody 5 ml.

1.5 erythrocyte lysate:

1.8675g of NH4Cl and 0.65g of Tris were added to 250ml of ddH2O, after which the pH was adjusted to 7.2 and filter sterilized at 0.22 mm.

1.6 flow-related buffer preparation

1.6.1 machine and Wash Buffer: 5ml FBS +245ml PBS (2% FBS).

1.6.2 fixed Buffer: 30ml Buffer +10ml (4% PFA) (1% PFA).

1.6.3 transmembrane 1 × Permeabilization Buffer: 5ml (5 × Permeabilization Buffer) +45ml dH 2O.

1.6.4 Foxp3 Fixation/Permeabilization working solution：Concentrate(1part) +Diluent(3part)。

2. Preparation method of mouse spleen cell suspension

Spleens were removed aseptically, placed in centrifuge tubes containing a double-antibody PBS solution, and placed on ice. After all the spleens of the mice are taken out, the spleens are placed in a 6-well plate, 4ml and 2ml of PBS solution containing the double antibody are added into the 6-well plate in advance, the mixture is washed by 4ml of PBS solution, and then 2ml of PBS solution per well is ground by a glass sheet to obtain the lymph suspension. The suspension was transferred to a 15ml centrifuge tube (tissue discarded), centrifuged at 1400rpm for 5min at 4 ℃ and the supernatant carefully aspirated by a vacuum pump. Adding 2ml erythrocyte lysate, mixing, standing at room temperature for 3min, immediately adding 3 times volume of PBS solution, mixing, centrifuging at 1400rpm for 5min at 4 deg.C, and carefully sucking off the supernatant with vacuum pump. The remaining cell pellet was blown down well by adding 2ml of PBS solution, and after filtration, 1. mu.l of cell suspension was diluted 100-fold and counted by trypan blue cells. After centrifugation, the cells were resuspended in lymphocyte culture medium to a concentration of 1X 107 cells/ml.

3. Flow cytometric staining

3.1Th17 cells (intracellular cytokine: IL-17)

25ng/ml PMA and 1. mu.g/ml ionomycin were added 5 hours before the experiment and 1.7. mu.g/ml monensin was added 2 hours later. After 3 hours, the cells were collected in a 1.5ml centrifuge tube, centrifuged at 1000rpm at 4 ℃ for 5min and the supernatant discarded. Washed once with 1ml Buffer, centrifuged at 1000rpm at 4 ℃ for 5min and the supernatant discarded. Mu.l of Buffer, 0.5. mu.l of Anti-mouse CD4-FITC antibody was added and incubated at 4 ℃ for 30min in the absence of light. Centrifuging at 1000rpm and 4 deg.C for 5min, adding 1ml Buffer, resuspending, centrifuging at 1000rpm and room temperature for 5min, removing supernatant, adding 200 μ L IC hybridization Buffer, mixing, and keeping away from light at room temperature for 20 min. The cells were washed twice with 1mL, 1 × Permeabilization Buffer, centrifuged at 1000rpm for 5min at room temperature, and the supernatant was discarded. Add 100. mu.L, 1 × Permeabilization Buffer for resuspension, add 0.625. mu.l Anti-mouse/Rat IL-17A-PE antibody, and protect from light at room temperature for 20 min. The cells were washed by adding 1mL of 1 × Permeabilization Buffer, centrifuged at 1000rpm for 5min at room temperature, and the supernatant was discarded. Add 1mL, Buffer washing cells, room temperature, 1000rpm centrifugation for 5min, discard the supernatant, 500 u L Buffer heavy suspension cells on the machine.

3.2 Tregs cell (nuclear transcription factor: FOXP3)

The cells were collected in a 1.5ml centrifuge tube, centrifuged at 1000rpm for 5min at 4 ℃ and the supernatant discarded, 100. mu.l of Buffer and 0.5. mu.l of Anti-mouse CD4-FITC antibody were added, and incubated for 30min at 4 ℃ in the absence of light. Centrifuging at 1000rpm and 4 deg.C for 5min, discarding supernatant, adding 1ml Buffer for resuspension, centrifuging at 1000rpm and 4 deg.C for 5min, and discarding supernatant. Add 200. mu.l Foxp3 Fixation/Permeabilization purification solution and mix well, protect from light for 30min at room temperature. The cells were washed twice with 1mL Buffer, centrifuged at 1000rpm for 5min at room temperature, and the supernatant was discarded. Add 100u l Buffer heavy suspension, adding Anti-mouse/Rat Foxp3-PE antibody 2.5 u l, room temperature light protection for 30 min. Adding 1mLBuffer to wash the cells twice, centrifuging at 1000rpm for 5min at room temperature, discarding the supernatant, and resuspending the cells in 500. mu.L Buffer

3.3Bregs cells

The cells were collected in a 1.5ml centrifuge tube, centrifuged at 1000rpm at 4 ℃ for 5min and the supernatant discarded. Mu.l of Buffer, 0.625. mu.l of Anti-mouse CD1d-PE antibody, 0.625. mu.l of Anti-mouse CD19-APC antibody, 1. mu.l of Anti-mouse CD5-FITC antibody were added and incubated at 4 ℃ for 30min in the absence of light. Centrifuging at 1000rpm and 4 ℃ for 5min, discarding the supernatant, adding 1ml Buffer to resuspend and wash the residual antibody, centrifuging at 1000rpm and 4 ℃ for 5min, discarding the supernatant, and resuspending the cells with 500. mu.L Buffer on a machine.

Examples 1-12 ELISA detection of cytokines in mouse serum

1. Mouse serum isolation

The method was the same as in example 7, and the level of cytokines in the mouse serum was measured.

2. ELISA for detecting cytokine level in mouse serum

Dilution of standards

The highest concentration of the standard substance is as follows: IFN-gamma (800ng/L), IL-4(240pg/ml), IL-10(1000pg/ml), IL-17(120pg/ml), TGF-beta (240 ng/L);

2.1 sample adding: each group is provided with a blank hole (without adding a sample and an enzyme-labeled reagent), a standard sample hole and a sample hole to be detected. And adding 50 mul of standard sample and sample to be detected respectively (the sample is diluted by 5 times by using a sample diluent) on the enzyme-labeled coated plate. Adding the sample to the bottom of the hole of the enzyme label plate, keeping the sample from touching the hole wall as much as possible, and slightly shaking and uniformly mixing the sample and the hole wall;

2.4 washing: pouring liquid, spin-drying with force, beating on dust-free paper, filling each hole with cleaning solution, waiting for 30 seconds, discarding for 5 times, and beating to dry;

2.6 incubation: the operation is the same as 2;

2.7 washing: the operation is the same as 4;

2.8 color development: 50. mu.l of color-developing agent A and 50. mu.l of color-developing agent B were added to each well. Shaking gently to avoid liquid splash to cause pollution, and developing in oven at 37 deg.C in dark for 10min (TGF-beta should develop for 15 min);

This experiment investigated the potential of human amniotic epithelial cells (hAECs) in Experimental Autoimmune Thyroiditis (EAT) treatment and explored its therapeutic mechanism. When the disease index begins to rise (21d) and the disease reaches the severity (35d), hAECs are injected, the number of lymphocytes infiltrated in the thyroid is reduced remarkably, and the disease score cannot be reduced remarkably by injecting hAECs at other times, which shows that hAECs have remarkable treatment effect on hashimoto thyroiditis, but the effect of the hAECs injected at different disease stages on EAT treatment is different. The experiment applies the human amniotic epithelial cells to the treatment of experimental autoimmune thyroiditis for the first time, has good effect and provides a new treatment scheme for the current autoimmune diseases.

Second part of human amniotic epithelial cells for treating uveitis

Example 2-1 construction of an Experimental autoimmune uveitis model

1. Purchasing and raising of laboratory animals

The experimental animal is selected from male Lewis rats with the age of 6-8 weeks, the weight of 160-. Feeding in experimental animal center of Zhejiang university, controlling room temperature at 23-26 deg.C with air conditioner, controlling relative humidity within 55 + -10%, lighting for 12 hr, and taking food and drinking water freely.

2. Main experimental medicine

(1) Complete Freund's Adjuvant (CFA), Sigma in USA

(2) Interphotoreceptor vitamin binding protein (IRBP) Shanghai bioengineering Co

(3) Chloral hydrate Sigma USA

3. Preparation of the principal agent

(1) Preparation of 8% chloral hydrate solution

Weighing 4g of chloral hydrate powder, adding 10ml of tri-distilled water, shaking until the chloral hydrate powder is completely dissolved, adding the tri-distilled water, and carrying out volume fixing to 50ml to obtain an 8% chloral hydrate solution, and carrying out filter sterilization by using a filter membrane for later use.

(2) Preparation of 4% paraformaldehyde fixing liquid

Weighing 4g of paraformaldehyde, adding 80ml of 0.1mol/L phosphate buffer solution, heating to about 60 ℃, continuously stirring, adding a little 1mol/L NaOH for clarification after the powder is completely dissolved, cooling, adding 5ml of glacial acetic acid, 10ml of acetone and 0.1mol/L phosphate buffer solution for constant volume to 100 ml.

4. Grouping of laboratory animals

Animals are numbered in sequence, according to the principle of layering and randomization, the animals are firstly divided into an EAU group (70 animals) and a normal group (5 animals) by adopting a stochastic digital table method, then the EAU group animals are randomly divided into a 0-day experimental group (50 animals) and a 6-day experimental group (20 animals), the 0-day experimental group is divided into a control group (25 animals) and a 0-day hAECs treatment group (25 animals), and the 6-day experimental group is divided into a control group (10 animals) and a 6-day hAECs treatment group (10 animals).

5. Construction of experimental animal model

Taking 1 count of IRBP powder (1 mg/count), adding 1ml PBS, mixing uniformly, fully dissolving to prepare PBS solution containing IRBP with concentration of 1000 mug/ml, and standing for later use. The 2 screw injectors are connected with a three-way valve, 0.6ml of PBS solution containing IRBP, 1.4ml of PBS and 2ml of Complete Freund's Adjuvant (CFA) are sequentially added, and the mixture is repeatedly injected and fully emulsified to obtain 4ml of mixed emulsion. The needle is inserted into the middle part of the foot pad of the rat subcutaneously and is submerged upwards to the subcutaneous part of the upper end of the tibia, 0.2ml of mixed emulsion (the final immune amount of each rat is 30 mug of IRBP polypeptide) is injected slowly, and the needle is taken out and pressed into the needle to prevent the emulsion from overflowing. The above subcutaneous injection procedure was continued and 20 rats were modeled with EAU. The above operations were repeated 4 times, and 70 rats in total were subjected to EAU animal model creation.

Example 2-2 isolation and culture of Primary amniotic epithelial cells

1. Source of human amniotic membrane

In order to avoid the microbial contamination of the birth canal, we chose to deliver foetal placenta by caesarean section. Since apoptosis of amnion occurs due to stimulation of delivery signals after term, the placenta of a premature fetus (before 38 weeks) is preferably used. After the puerpera gives consent, placenta tissue of healthy puerpera (serological reactions such as HIV, syphilis, hepatitis A, hepatitis B, hepatitis C and the like are all shown to be negative) after cesarean is taken, the placenta is cut by a cross knife, and the whole amnion is obtained by mechanical separation.

2. Isolation of hAECs (sterile procedure is required throughout)

Placenta of an infant delivered by caesarean section for 38 weeks was harvested, the amniotic membrane was dissected from the inner surface of the placenta and immersed in a centrifuge tube containing DMEM/F12 (containing 1% penicillin-streptomycin) basal medium. Cold chain transport to the laboratory cells at 4 ℃.

The amniotic membranes were removed and each membrane was washed in 40ml CMF-HBSS (containing 1% penicillin-streptomycin) to remove mucus, and the interstitial matrix layer adjacent to the chorionic villus layer and mucus were scraped off with forceps and repeated 3 times with each wash being replaced with a new container and new HBSS solution.

Transferring the cleaned amnion into a new container, adding 10ml of 0.25% pancreatin/EDTA, reversing for 30s, and discarding the solution.

Transferring amnion into new container, adding 20ml 0.25% pancreatin/EDTA, incubating in water bath at 37 deg.C for 10min, and discarding solution.

Transferring amnion into new container, incubating in 25ml 0.25% pancreatin/EDTA water bath at 37 deg.C for 40min, and preserving digestive juice.

Transferring the amnion after primary digestion into a new container, incubating in 25ml pancreatin/EDTA water bath at 37 ℃ for 40min, and preserving digestive juice.

Equal volume of digestion stop solution (F12/DMEM with 10% FBS, 2mM L-glutamic acid, 1mM sodium pyruvate, 1% non-essential amino acids) was added and centrifuged at 400g for 10 min. Discarding liquid, using amnion complete culture medium: F12/DMEM contained 10% KSR (KnockOut Serum replacement), 2mM L-glutamine, 1% non-essential amino acids, 1mM sodium pyruvate, 100U/mL Penicillin-Streptomycin (Penicillin-Streptomycin),10ng/mL hEGF in a resuspension.

Sieving with 100um sieve, and counting

The culture dish is inoculated with 10^5cells/cm2 or placed in frozen stock solution (90% FBS, 10% DMSO) and frozen in liquid nitrogen for standby.

3. Inoculation culture and cryopreservation of hAECs

Cell counting culture: 1X 10⁷Individual cells were plated onto 15cm dishes. Changing the culture solution after the hAECs are attached to the wall, and changing the culture solution once three days later.

After the cells grew over the plate, the cells were digested and cryopreserved: 5ml pancreatin is added into a 15cm culture dish, observation is carried out under a mirror after 10min, when cells become round and the whole cells become a suspension state when the dish is shaken in a plane, the digestion is stopped by adding the same amount of digestion stop solution. The cells on the culture dish were blown down by a micropipette in the same direction, transferred into a 15ml centrifuge tube, centrifuged at 300g for 3min, collected and then counted. And adding the freezing solution into the freezing tube, marking the freezing date, the freezing batch and the number of the cells, putting the cells into the freezing tube, immediately putting the freezing tube into a freezing box, putting the freezing box into a refrigerator at minus 80 ℃, taking out the freezing box after 12 hours, and transferring the cells into a liquid nitrogen tank for storage.

Example 2-3 rat Ocular slit Lamp Observation and inflammation Scoring

The groups of rats were observed daily under slit lamp for ocular EAU inflammatory manifestations starting on day 4 post-immunization and by reference to the Caspi clinical scale for inflammatory scoring, to day 18 post-immunization. The specific scoring criteria were as follows, score 0: no inflammation reaction exists, and the fundus red light is reflected normally; 0.5 min: iris blood vessels were slightly dilated and hyperemic; 1 minute: moderate congestion of iris vessels, miosis; and 2, dividing: the aqueous humor is slightly turbid, and the red light reflection of the fundus is weakened; and 3, dividing: moderate turbidity of aqueous humor, reduced red light reflection of fundus oculi; and 4, dividing: pus is accumulated in the anterior chamber, the pupillary membrane is closed, and the red light reflection of the fundus disappears.

The results show (FIG. 15), that D0-hAECs in the rats with eye inflammation significantly reduced at the same time point, the onset of EAU was delayed, the aqueous humor in the anterior chamber was moderately turbid on the 12 th day after immunization, the iris congestion was increased, and the fundus red light was reduced, compared with the D0-BSS control group; on day 18 after immunization, inflammation declined, aqueous humor cleared, and fundus red light reflex was essentially normal. Meanwhile, compared with the D6-BSS control group, the eye inflammation score of the rats in the D6-hAECs treatment group is also reduced, but the difference of the D0-hAECs prevention group is not obvious. Demonstrating that hAECs treatment reduced the inflammation score observed with ocular slit lamps.

Example 2-4 rat fundus hAECs injection

Taking hAECs with good growth state and 90% fusion, pouring out the culture solution in an ultra-clean bench, rinsing with PBS for 1 time, adding about 5ml of 0.25% trypsin digestion solution, observing under a microscope, stopping digestion with 0.5ml fetal calf serum after cell retraction and rounding, gently blowing, transferring the cell suspension to a 15ml centrifuge tube, centrifuging for 5 minutes at 1500 rpm, removing supernatant, resuspending the cells with PBS, and counting the cells. The rats in the 0-day hAECs treatment group were injected with a solution containing about 1X 10 molecules from the subretinal space at the same time as immunization ⁵2. mu.l of a cell suspension of one hAECs. The control group was injected with the same volume of BSS. Rats treated with hAECs on day 6 after immunization were treated with infusion of hAECs into the fundus oculi at day 6, and injected with a solution containing about 1X 10 of hAECs into the subretinal space ⁵2. mu.l of a cell suspension of one hAECs. The control group was injected with the same volume of BSS.

Examples 2-5 histopathological observations and histological scoring

(1) Fixing: on

days

6, 9, 12, 15 and 20 after immunization, 8% chloral hydrate (70 mul/10 g of body weight) is adopted to carry out intraperitoneal injection on each group of rats, after complete anesthesia, 0.3% oxybivacaine eye drops are dripped on rat eyes to carry out sufficient surface anesthesia, eyelids are slightly separated, and eyeballs are removed. The left and right eyes were soaked in 4% paraformaldehyde neutral buffer solution for fixation for 24 hours.

(2) Dehydrating and wax dipping: taking out the eyeball from the fixative, cutting two sides of the eyeball with sharp blade along visual axis direction, cutting off two side parts of eyeball wall, carefully peeling off the crystalline lens, dehydrating at room temperature, and soaking the whole eyeball in ethanol with volume fraction of 55%, 65%, 75%, 85%, 95% and 100% for 1h respectively. Mixed paraffin (stearic acid and soft wax volume ratio is 3: 7) for 2h, mixed paraffin (stearic acid and soft wax volume ratio is 2: 8) for 1h, soft wax for 1.5h, and hard wax for 1.5 h.

(3) Embedding the section: the eyeball after being soaked in wax faces to the bottom of the embedding box by a horizontal section and is embedded by hard wax. The embedded eyeball specimen is serially sliced with the retina parallel to the sagittal axis of the optic nerve and in the plane of the sagittal axis to form a section with a thickness of about 4 μm

(4) And (3) staining the section: and (4) performing conventional HE dyeing and mounting. The photographs were observed under a microscope. Rat retinal tissue structures were observed under an optical microscope and scored with reference to the Caspi histopathological grading. 0 minute: no inflammation, normal retinal structure; 0.5 min: inflammatory cells lightly infiltrate the retina with or without photoreceptor damage; 1 minute: inflammatory cells lightly infiltrate damaged retina and/or photoreceptor outer segments; and 2, dividing: infiltration of mild to moderate inflammatory cells and/or extension of damaged sites into the outer nuclear layer; and 3, dividing: moderate to marked inflammatory cell infiltration and/or involvement of damaged sites into the inner limiting membrane: and 4, dividing: severe inflammatory cell infiltration and/or destruction and damage of the entire retinal layer.

The results shown in the figure (fig. 16) show that the rats in the control group have obvious inflammatory cell infiltration and retinal structure damage, while the rats in the treatment group injected with hAECs at the 6 th day of the disease have greatly reduced inflammatory cell invasion and retinal damage, which indicates that hAECs can inhibit inflammation and reduce retinal structure disorder.

Examples 2-6 Collection of aqueous humor of rat eye

Injecting 8% chloral hydrate (70 mu l/10G of body weight) into abdominal cavity respectively at 12 th and 18 th days after immunization, after satisfactory anesthesia is achieved, dropping mouse eye with 0.3% oxybivacaine eye drops to fully perform surface anesthesia, slightly separating eyelid, performing anterior chamber puncture of mouse eye with 30G needle under microscope, keeping the needle in the anterior chamber for a while, withdrawing the needle, placing 1ml empty needle, collecting the obtained aqueous humor in a sterile EP tube, and freezing and storing at-80 ℃ ultra-low temperature refrigerator.

Example 2-7 flow cytometry determination of the proportion of Th17, Treg cell populations in splenic lymph node mononuclear cells

1. Th17 cell flow assay procedure

1.1 pipette 100. mu.l of spleen lymph node mononuclear cell suspension into flow tubes at different time points for each group of rats.

1.2 mu.l of FITC-labeled anti-rat CD4 fluorescent antibody was added, and 2. mu.l of FITC-labeled isotype control antibody was added to the negative control tube. Add 2. mu.l of CD4 fluorescent antibody or isotype control antibody into single positive tube, shake and mix well.

1.3 incubate for 30 minutes at room temperature in the dark.

1.4 adding 1ml of membrane rupture fixed permeabilization solution, and carrying out membrane rupture in a dark place for about 40 minutes at room temperature.

1.51800 spin for 5 minutes and discard the supernatant.

1.6 adding PE marked anti-rat IL-17 fluorescent antibody 2 mul, adding PE marked isotype control antibody 2 mul in negative control tube, shaking and mixing evenly. Add IL-17 fluorescent antibody or isotype control antibody 2. mu.l into single positive tube, and mix them by shaking.

1.7 incubate for 30 minutes in the dark.

1.8 adding 1ml of the permeabilization solution, shaking, resuspending, centrifuging for 5 minutes at 1800 rpm, and repeating the step 2 times.

1.9 Add 2% paraformaldehyde 0.5ml per tube for fixation, and store at 4 deg.C in dark.

1.10 on a flow cytometer.

2. Treg cell flow assay procedure

2.1 pipette 100. mu.l of spleen lymph node mononuclear cell suspension into flow tubes at different time points for each group of rats.

2.2 mu.l FITC-labeled anti-rat CD4 fluorescent antibody and 0.6. mu.l PE-labeled CD25 antibody were added, and 2. mu.l FITC-labeled isotype control antibody and 0.6. mu.l PE-labeled isotype control antibody were added to the negative control tube. A single positive tube was added with either CD4 fluorescent antibody or CD25 antibody or the corresponding isotype control antibody. Shaking and mixing evenly.

2.3 incubate for 30 minutes at room temperature in the dark.

2.4 adding 1ml of membrane rupture fixed permeabilization solution, and carrying out membrane rupture in a dark place for about 40 minutes at room temperature.

2.51800 spin for 5 minutes and discard the supernatant.

2.6 adding 2 mul of APC-labeled anti-rat Foxp3 fluorescent antibody, adding 2 mul of APC-labeled isotype control antibody into a negative control tube, and shaking and mixing uniformly. 2. mu.l of Foxp3 fluorescent anti-or isotype control antibody was added to the single positive tube and mixed well with shaking.

2.7 incubate for 30 minutes in the dark.

2.8 Add 1ml of permeabilization solution, resuspend the cells after shaking, centrifuge for 5min at 1800 rpm, repeat step 2 times.

2.9 Each tube was fixed by adding 0.5ml of 2% paraformaldehyde, and stored at 4 ℃ in the dark.

2.10 on a flow cytometer.

The experimental results show (fig. 18) that hAECs are injected at the same time of immunization or during the onset period of EAU, the proportion of Th17 cell population in the course of EAU is inhibited, the proportion of Treg cell population is increased, and the effect of relieving the condition of EAU is achieved, but the effect of hAECs cell treatment is more obvious when immunization is carried out at the same time.

Examples 2-8 ELISA detection of immunocytokines

Spleen lymph node mononuclear cells separated and counted from each group at different time points at 2x10^⁶And (3) paving the cells/well into a 12-well plate, culturing the cells in a culture medium containing IRBP30ug/ml for 72 hours, collecting supernatant, sequentially numbering according to classification, and performing ELISA detection on each factor according to the specific requirements of an ELISA kit.

1. Preparation before ELISA experiments

1.1 preparing a standard solution: before use, distilled water is added and mixed to prepare a solution with a certain proportion, standard 8 tubes are arranged, a certain amount of specimen diluent is added into each tube according to the requirements of specific specifications for double dilution, and the last tube is a blank control.

1.210 × specimen dilution: distilled water is used as a solvent for 1:10 times dilution.

1.3 washing solution: using double distilled water as a solvent 1: and (4) diluting by 20 times.

2. Specific experimental procedures

2.1 sample adding: add 100 mul of standard or sample to be tested into each well, mix the reaction plate well and put it at 37 deg.C for 120 minutes.

2.2 washing the plate: the reaction plate was washed thoroughly 4-6 times with washing solution and printed dry on filter paper.

2.3 Add 100. mu.l of the first antibody solution to each well, mix the reaction plate well and then place at 37 ℃ for 60 minutes.

2.4 washing the plate: the reaction plate was washed thoroughly 4-6 times with washing solution and printed dry on filter paper.

2.5 Add 100. mu.l of the enzyme-labeled antibody solution to each well, and place the plate at 37 ℃ for 30 minutes.

2.6 plate washing: the reaction plate was washed thoroughly 4-6 times with washing solution and printed dry on filter paper.

2.7 substrate working solution (100. mu.l) was added to each well, and the mixture was left to react at 37 ℃ in the dark for 15 minutes.

2.8 Add stop solution 100. mu.l per well and mix well.

The absorbance was measured at 450nm with a microplate reader for 2.930 minutes.

The results show (FIG. 19) that the level of anti-inflammatory factor (IL-10) in the culture supernatant of splenic lymph node mononuclear cells of the treated rats is obviously increased, and the level of proinflammatory cytokine (IL-17) is reduced, which indicates that hAECs can reduce proinflammatory factors secreted by Th1/Th17 cells and increase anti-inflammatory factors secreted by Treg cells.

EXAMPLE 2-9 immunofluorescence of eye sections

9.1, fixing: freezing section of eyeball, immersing in acetone, and fixing at-20 deg.C for 10 min.

9.2 the sections were washed with 1xPBS solution and the residual acetone was washed.

9.3, sealing:

preparing a sealing liquid: 5ml PBS +0.05BSA powder + 250. mu.l HBS.

And (3) sealing: the blocking solution was dropped onto the tissue and blocked for 1 h.

9.4 Primary antibody incubation:

the blocking solution was aspirated and washed with 1xPBS solution, primary antibody was dispensed with the blocking solution at a ratio of 1:200, and primary antibody was dropped onto the tissue and incubated overnight at 4 ℃. The following procedures were carried out in the dark as much as possible.

9.5 second antibody incubation:

the primary antibody was aspirated and washed with 1xPBS solution, the secondary antibody was mixed with 1xPBS solution at a ratio of 1:500, and the secondary antibody was dropped on the tissue and incubated at room temperature for 1 hour.

9.6DAPI staining of nuclei:

the secondary antibody was aspirated and washed with 1xPBS solution, the DAPI and 1xPBS solution were prepared at a ratio of 1:1000, DAPI was dropped on the tissue for 1min and then aspirated, and then washed with 1xPBS solution.

9.7, sealing sheet:

and sealing by using sealing liquid.

9.8 Observation under fluorescent microscope

The results are shown in the figure (FIG. 17), and a large amount of macrophage and T cell invasion and retinal tissue structure (nuclear layers) disorder were observed at day 12 after immunization of the control groups (D0-BSS and D6-BSS), and by day 18, the number of infiltrated cells was reduced and the retinal structure was slightly disturbed. Compared with a control group, the D0-hAECs prevention group can obviously reduce the infiltration of macrophages and T cells, a small amount of infiltration of macrophages and T cells can be seen on the 12 th day after immunization, the retinal structure is slightly disordered, and the retinal structure is basically normal and has almost no inflammatory cell infiltration on the 18 th day after immunization. The D6-hAECs treatment group can obviously reduce the infiltration of macrophages and T cells compared with the control group at the same time point, but the treatment effect is not obvious compared with that of the D0-hAECs prevention group. Suggesting that hAECs treatment reduced macrophage and T cell infiltration.

This experiment investigated the potential of human amniotic epithelial cells (hAECs) in the treatment of Experimental Autoimmune Uveitis (EAU) and explored its therapeutic mechanism. According to the experiment, the inventor thinks that the hAECs can obviously inhibit the expression of proinflammatory cytokines and promote the secretion of anti-inflammatory factors, and simultaneously balance the micro-physiological environment of in vivo immune regulation, thereby playing a role in inhibiting the occurrence and development of experimental autoimmune uveitis, and being a new way for treating diseases. The experiment applies the human amniotic epithelial cells to the treatment of experimental autoimmune uveitis for the first time, has good effect, and provides a new treatment scheme for the current diseases.

The third part of human amniotic epithelial cells for treating lupus erythematosus

Example 3-1 construction of an Experimental model for autoimmune lupus erythematosus

MRL-Faslpr mice, SPF grade, 30-40g, animals were provided by Nanjing university-Nanjing Biopharmaceutical research institute. The mice are raised in cages, 6 mice in each cage are raised in the experimental animal center of Zhejiang university, the room temperature is controlled to be 23-26 ℃, the relative humidity is controlled to be within 55 +/-10%, the illumination is carried out in a day-night period of 12 hours, and the mice can be freely taken by food and water. Mice were established to develop disease (SLE mice) when autoantibodies were detected starting 12 weeks after MRL-Faslpr mice and serum ANA and anti-dsDNA antibodies were positive, at which time hAECs were injected (preparation of hAECs as disclosed in the first example). Mice with negative anti-dsDNA antibody in the serum of MRL-Faslpr mice were randomly selected as the Control group.

Example 3-2 mice tail vein injection of hAECs

Mice of the model treatment group (SLE + hAECs) were treated by tail vein injection of hAECs, with a cell count of 1.5X 10 per injection per mouse⁶Per (concentration is 1.5X 10)⁷Cell suspension per ml, 100. mu.l each injection).

When the operation is carried out, a place with sufficient light is selected, the mouse is fixed by the mouse fixer, a l ml insulin syringe is prepared, and 100 mu l of cell suspension is sucked each time to prevent the cells from depositing in the syringe.

Example 3-3 immunofluorescence detection of MRL-Fas^lprMouse serum ANA and anti-dsDNA antibodies

1. Mouse serum isolation

Collecting blood from orbital venous plexus of mouse, standing the blood at room temperature for 1 hr, standing at 4 deg.C for 30min, centrifuging at 4 deg.C at 3600rpm for 10min, separating serum, packaging, and freezing at-80 deg.C.

1. Primary reagent

Antinuclear antibody (ANA) mosaic indirect immunofluorescence assay kit, FA 1510-1, Euromeng,

kit for detecting the short membrane worm (nDNA) of green fly by indirect immunofluorescence method, FA 1572, Euromeng,

goat Anti-mouse IgG H & L (FITC), ab6785, Abcam.

2. Experimental methods

(1) Sample, reagent preparation

1) Blood was collected into 200. mu.l tube by cutting one millimeter of the rat tail. After standing at room temperature for 1h, the mixture was left at 4 ℃ for 30 min. Centrifuging at 4 deg.C and 3600rpm for 10min to separate serum, and testing the serum at 4 deg.C for 14 days, but testing the diluted serum on the same day.

2) The sample application plate was washed and examined to see if the reaction area was hydrophilic and the periphery hydrophobic.

3) Diluting the sample 10 times before detecting anti-dsDNA antibody; samples were diluted 40-fold before detection of ANA.

4) The slide glass with the biological film is used after being placed for 30min at room temperature.

5) For initial use, the secondary antibody, negative and positive control sera were mixed well with a sample applicator.

6) Adding 1L of distilled water into a packet of PBS salt, adding 2ml of Tween 20, mixing well, and storing at 4 ℃.

(2) And sample adding: the sample adding plate is placed on a foam plate, and 25 mul of diluted serum is respectively dripped to each reaction area of the sample adding plate according to the detection sequence, so that bubbles are avoided.

(3) And incubation: the side of the slide coated with the biological sheet faces downwards, the slide is covered in the groove of the sample adding plate, the reaction is immediately started, and the slide is incubated at room temperature for 30 min.

(4) And washing: the glass slide was washed with running water containing PBS Tween in a beaker, and immediately immersed for 5min in a wash cup containing PBS Tween (16 sheets at most).

(5) And sample adding: mu.l of FITC-labeled anti-mouse globulin (fluorescent secondary antibody) was added dropwise to the reaction area of the clean sample addition plate using a line gun, and the next incubation was performed after all the fluorescent secondary antibody was added completely.

(6) And incubation: the slide was removed from the wash cup and immediately covered in the wells of the template after wiping off the water on the back and edge with absorbent paper (without wiping the reaction zone gap). Ensure good contact of the biological sheet with the drop, then continue the next sheet, incubate for 30min at room temperature in the dark.

(7) And washing: the glass slide is washed by running water containing PBS Tween buffer solution in a beaker, and then immediately immersed in the washing cup containing the PBS Tween buffer solution for 5 min.

(8) And sealing sheet: place the coverslip directly into the recess of the foam plate, drop the blocking tablet (glycerol/PBS) to the coverslip: each reaction zone contained 10. mu.l. The slide is taken out, after water on the back and the edge is wiped off by absorbent paper (the gap of the reaction area is not wiped), the side, coated with the biological slices, of the slide is placed on the prepared cover glass downwards, and the cover glass is immediately checked and slightly adjusted to be embedded into the groove of the slide.

(9) And observing under a microscope: exciting a filter disc: 488nm, spectral filter: 510nm, blocking filter: at 520nm, the ANA and anti-dsDNA antibodies were observed to be positive under fluorescence. When the serum ANA of the mouse is positive, the anti-dsDNA antibody of the mouse is detected, and when both antibodies are positive, the MRL-Fas is determined^lprSLE has been characterized, judged to have developed, can be treated by intravenous injection of hAECs into the tail vein of mice. After two weeks of treatment, the mice serum ANA and anti-dsDNA antibodies were tested to determine the effect of treatment.

(10) Mean Fluorescence Intensity (MFI) measurement using Image Pro Plus 6.0 software, 10cells/sample or more.

Example 3-4 ELISA detection of IgG Isotypes and cytokine concentrations in serum of MRL-Faslpr mice

1. Primary reagent

Mouse IgG1 ELISA kit (HY2407),

mouse IgG2a ELISA kit (HY2410),

mouse IgG3 ELISA kit was constant (HY 2414).

2. Experimental methods

The whole blood serum separation method is the same as the above, and the ELISA method is adopted to detect the concentrations of IgG1, IgG2a, IgG3, IL-17 alpha, IFN-gamma, IL-4, IL-10 and TGF-beta in the mouse serum after the serum of all mice is collected. The standard solution was prepared as follows.

Dilution of standards

The highest concentration of the standard substance is as follows: IgG1(800ng/L), IgG2a (120ng/L), IgG3(240ng/L), IFN-gamma (800ng/L), IL-4(240pg/ml), IL-10(1000pg/ml), IL-17 alpha (120pg/ml), TGF-beta (240 ng/L);

2.6 incubation: the operation is the same as 2.2;

2.7 washing: the operation is the same as 4;

2.9 termination: the reaction was stopped by adding 50. mu.l of stop solution to each well (at which time the blue color immediately became yellow), and the absorbance (OD value) of each well was measured at a wavelength of 450nm within 15 min;

Example 3-5 flow cytometry detection of immune cell balance in spleen of SLE mice

1. Experimental materials and main reagents

Experimental Material

Based on the results of the previous experiments, spleens of mice in the Control, SLE, SLE + hAECs group from the disease peak (35day) treatment, 49day sampling group were selected for analysis.

SLE mice serum ANA, anti-dsDNA antibody positive, injection hAECs for treatment. Two weeks later, the mice were sacrificed and splenocytes were removed for flow analysis.

Primary reagent

Anti-mouse CD4 FITC(GK1.5，eBioseienee，USA)，

Anti-mouse/Rat Foxp3 PE(FJK-16s，eBiosciene，USA)，

Anti-mouse/Rat IL-17A PE(17B7，eBiosciene，USA)，

Anti-mouse CD1d PE(1B1，eBiosciene，USA)，

Anti-mouse CD5 FITC(53-7.3，eBiosciene，USA)，

Anti-mouse CD19 APC(1D3，eBiosciene，USA)，

Rat IgG2b kappa Isotype Control PE(eBiosciene，USA)，

Rat IgG2a kappa Isotype Control PE(eBiosciene，USA)，

Immobilization/permeation solution (eBioscience, USA), permeation buffer (eBioscience, USA), phorbol acetate (phorbol myrtle acetate, PMA, synbiotics), ionomycin (ionomycin, synbiotics), monensin (monensin, synbiotics), RPMI1640 (GIBCO), fbs (GIBCO), Sodium pyrolute (GIBCO), 1M HEPES (GIBCO), NH₄Cl (Producer organism), Tris (Producer organism).

2. Experimental methods

Solution preparation

(1) Preparation of PMA solution

Storage solution: the concentration is 0.1mg/ml, and the product is stored at minus 80 ℃ in a dark place;

final working concentration: 25ng/ml, namely adding 1.25 mu l of PMA per 500 mu l of culture solution;

(2) preparation of Ionomycin solution

(3) preparation of Monensin solution

working fluid: 1:50 dilution in RPMI1640 with 10% FBS, at a concentration of 1000. mu.g/ml;

final working concentration: 1.7 mu g/ml, namely adding 0.85 mu l of Monensin to every 500 mu l of culture solution;

(4) preparing a lymphocyte culture solution: RPMI1640(430ml) + 10% FBS (50ml) + (1000X) 50. mu.M.beta.mercaptoethanol (500. mu.l) + (100X) 100mM Sodium Pyruvate (5ml) + (50X) 1M HEPES (10ml) + (100X) dual antibody (5 ml).

(5) Preparing a red blood cell lysate: 1.8675g of NH4Cl and 0.65g of Tris were added to 250ml of ddH2O, after which the pH was adjusted to 7.2 and filter sterilized at 0.22 mm.

(6) Flow dependent buffer allocation

1) Loading and washing Buffer: 5ml FBS +245ml PBS (2% FBS).

2) Transmembrane 1 × Permeabilization Buffer: 5ml of 5 × Permeabilization Buffer +45ml dH₂O。

3).Foxp3 Fixation/Permeabilization working solution：Concentrate(1part) +Diluent(3part)。

3. Preparation method of mouse spleen cell suspension

The spleens were aseptically removed, placed in 6-well plates, washed with a P/S-containing PBS solution, and then ground with a glass slide to obtain a lymph suspension. The suspension was transferred into a 15ml centrifuge tube (tissue discarded), centrifuged at 1400rpm for 5min at 4 ℃ and the supernatant was aspirated off by a vacuum pump. Adding 2ml erythrocyte lysate, mixing, standing at room temperature for 3min, immediately adding 3 times volume of PBS solution, mixing, centrifuging at 1400rpm at 4 deg.C for 5min, and sucking off supernatant with vacuum pump. The remaining cell pellet was blown down well by adding 2ml of PBS solution, and 1. mu.l of cell suspension trypan blue was detectedAfter filtration, 1. mu.l of the cell suspension was diluted 100-fold and the cells were counted. After centrifugation, the cells were resuspended in lymphocyte culture medium to a concentration of 1X 10⁷ Cell size 1X 10 cells/ml, required for flow cytostaining^5-6And (4) respectively.

4. Flow cytostaining procedure

(1) Th17 cell (intracellular cytokine: IL-17A)

Before the experiment, 25ng/ml phorbol ester and 1 mug/ml ionomycin are added, and after 2 hours, 1.7 mug/ml monensin is added. After further culturing for 3 hours, the cells were collected in a 1.5ml centrifuge tube, centrifuged at 1000rpm at 4 ℃ for 5min and the supernatant discarded. Washed once with 1ml Buffer, centrifuged at 1000rpm for 5min at 4 ℃ and the supernatant discarded. Mu.l of Buffer, 0.5. mu.l of Anti-mouse CD4 FITC antibody was added and incubated at 4 ℃ for 30min in the absence of light. Centrifuging at 1000rpm and 4 deg.C for 5min, discarding the supernatant, adding 1ml Buffer for resuspension, centrifuging at 1000rpm and 4 deg.C for 5min, discarding the supernatant, adding 200 μ L IC Fixation Buffer fixed cells, mixing, and keeping in the dark at room temperature for 20 min. The cells were washed twice with 1mL, 1 × Permeabilization Buffer, centrifuged at 1000rpm for 5min at room temperature, and the supernatant was discarded. Add 100u L, 1x Permeabilization Buffer heavy suspension, adding Anti-mouse/Rat IL-17A PE antibody 0.625 u L or Isotype, room temperature light protection for 20 min. The cells were washed by adding 1mL of 1 × Permeabilization Buffer, centrifuged at 1000rpm for 5min at room temperature, and the supernatant was discarded. Add 1mL, Buffer washing cells, room temperature, 1000rpm centrifugation for 5min, discard the supernatant, 500 u L Buffer heavy suspension cells on the machine.

(2) Treg cells (nuclear transcription factor: FOXP3)

The cells were collected in a 1.5ml centrifuge tube, centrifuged at 1000rpm for 5min at 4 ℃ and the supernatant discarded, 100. mu.l of Buffer and 0.5. mu.l of Anti-mouse CD4 FITC antibody were added, and incubated at 4 ℃ for 30min in the absence of light. Centrifuging at 1000rpm and 4 deg.C for 5min, discarding supernatant, adding 1ml Buffer for resuspension, centrifuging at 1000rpm and 4 deg.C for 5min, and discarding supernatant. Add 200. mu.l Foxp3 Fixation/Permeabilization purification solution and mix well, protect from light for 30min at room temperature. The cells were washed twice with 1mL Buffer, centrifuged at 1000rpm for 5min at room temperature, and the supernatant was discarded. Add 100u l Buffer to re-suspend, add Anti-mouse/Rat Foxp3 PE antibody 2.5 u l or Isotype, room temperature and light protection for 30 min. The cells were washed twice with 1mL Buffer, centrifuged at 1000rpm for 5min at room temperature, the supernatant was discarded, and 500. mu.L of Buffer was used to resuspend the cells on the cell plate. Flow cytometric results were analyzed using Kaluza Analysis 1.5a software.

This experiment investigated the potential of human amniotic epithelial cells (hAECs) in the treatment of SLE and explored their therapeutic mechanisms. hACs is injected after SLE occurs in mice, diseases can be obviously improved and even cured, serum ANA and anti-dsDNA antibodies are changed from positive to negative, and the antibody levels of IgG1, IgG2a and IgG3 are obviously reduced. And hAECs are found to restore the immune balance of SLE mice by regulating the proportion of T cell subsets and the level of cytokines. The experiment applies the human amniotic epithelial cells to the treatment of the systemic lupus erythematosus for the first time, has good effect, and provides a new treatment scheme for the current autoimmune diseases.

In this specification the invention has been described with reference to specific embodiments, which are intended only to assist in understanding the method of the invention and its core ideas. The present invention has been described in an illustrative rather than a restrictive sense, and it will be readily apparent to those skilled in the art that modifications and variations may be made without departing from the spirit of the invention and those modifications and variations are intended to be included within the scope of the appended claims.

Claims

1. The application of the human amniotic epithelial cells or cell preparations thereof in preparing medicaments for treating and/or improving autoimmune diseases, wherein the autoimmune diseases comprise uveitis and lupus erythematosus.

2. Use according to claim 1, characterized in that: an effective amount of human amniotic epithelial cells or cell preparations thereof are used alone or in combination with other drugs to treat and/or ameliorate autoimmune diseases.

3. Use according to claim 1 or 2, characterized in that: in the treatment of autoimmune diseases, the cellular administration may be repeated or continuous, and multiple administrations are usually given at intervals of at least 7-10 days.

4. Use according to claim 1, characterized in that: the animal with autoimmune disease refers to mammals.

5. Use according to claim 1, characterized in that: the amniotic epithelial cells are suitably harvested cells without any treatment, partially purified cells or purified cells which are then expanded in culture.

6. Use according to claim 1, characterized in that: the amniotic epithelial cells can be administered to the patient by any suitable method, including local injection at the site of disease, sub-retinal injection, intravenous injection, or intraspinal injection.

7. Use according to claim 1, characterized in that: the dosage range of each administration of the amniotic epithelial cells is 10³-10⁹A cell.

8. Use according to any one of claims 1 to 7, characterized in that: the amniotic epithelial cells are prepared by the method comprising the following steps:

(1) mechanically separating the placenta tissue to obtain an amniotic membrane;

9. Use according to claim 8, characterized in that: continuously culturing the human amniotic epithelial cells obtained in the step 2, wherein the preferable culture conditions are as follows: at 1 × 10⁶-1×10⁸Inoculating cells into a culture dish according to the density of each cell/flat plate, placing the culture dish in a carbon dioxide incubator for culture, replacing culture solution after the human amniotic epithelial cells are attached to the wall, digesting the cells after the flat plate is full of the cells, and freezing and storing the cells.

10. Use according to claim 9, characterized in that: bFGF (basic fibroblast growth factor) or EGF (epidermal growth factor) may be added to the basal medium.