CN116981467A - Application of human amniotic epithelial cells in preparation of medicines for treating and/or improving uterine cavity adhesion diseases - Google Patents
Application of human amniotic epithelial cells in preparation of medicines for treating and/or improving uterine cavity adhesion diseases Download PDFInfo
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- CN116981467A CN116981467A CN202180092158.4A CN202180092158A CN116981467A CN 116981467 A CN116981467 A CN 116981467A CN 202180092158 A CN202180092158 A CN 202180092158A CN 116981467 A CN116981467 A CN 116981467A
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Abstract
There is provided the use of human amniotic epithelial cells (hAECS) in the manufacture of a medicament for the treatment and/or amelioration of a uterine cavity adhesion disease.
Description
The invention belongs to the technical field of biology, and particularly relates to application of human amniotic epithelial cells in treating intrauterine adhesion diseases.
Endometrium can be proliferated, differentiated and shed for more than 400 times in female fertility period, and active and strong regeneration capacity is shown. However, it is also very susceptible to many factors such as injury, inflammation, endocrine, etc., resulting in endometrial repair disorders, resulting in endometrial basal layer injury, inter-muscular-wall adhesion, and the occurrence of intrauterine adhesion (intrauterine adhesions, IUA) as a result of the disruption of the normal anatomical morphology of the uterine cavity. The incidence rate of IUA is as high as 25% -30%, which is one of refractory diseases in the gynaecology field, and causes a series of clinical symptoms including menorrhagia, even amenorrhea, infertility, recurrent abortion, periodic abdominal pain, placenta implantation after pregnancy, fetal growth restriction, postpartum hemorrhage and the like. In recent years, the occurrence rate of IUA is increased year by year due to severe mechanical injury of uterus caused by scar uterus after cesarean section and repeated artificial abortion, and the IUA has become a main cause of female menstrual flow reduction and secondary infertility. IUA is present in about 20% -30% of infertility patients, while 30% -60% of patients with IUA are combined with infertility, severely affecting the normal physiology and fertility of females. However, current means of treatment and prevention of IUA are still limited and need to be explored from a new perspective.
Previous studies have found that regeneration after endometrial ablation begins with areas of the exposed basal lamina glands not covered by degenerated tissue, and new glandular epithelial tissue grows and extends into areas of intact surrounding surfaces. Damaged glandular epithelial wound healing is a complex biological process including local low-grade inflammation, cell proliferation differentiation and tissue regeneration, and is jointly involved in and highly coordinated and mutually regulated by immune cells, repair cells, extracellular matrix, cytokines and other factors. Normally, after endometrial injury, glandular epithelial cells are rapidly regenerated to achieve complete scar-free repair. The wound, infection and low estrogen state can destroy the ordered process, cause the chronic refractory wound surface of the endometrium to form and fibrosis to occur, lead to the repair disorder of the endometrium and finally lead to IUA. The pathological characteristics of IUA are represented by normal intimal thinning, atrophy, replacement by fibrotic interstitial and inactive cubic columnar epithelium, indistinguishable functional and basal layers, residual small amounts or even complete lack of normal active intimal glands, etc., leading to intimal scar repair, adhesions, reduced gland function, poor response to hormones, and interstitial vascular deficiencies, etc.
The mechanism by which IUA occurs is not completely understood, but studies have suggested that infection of the uterine cavity and inflammation of lesions caused by operation of the uterine cavity are major causative factors, causing disruption of the intimal barrier, and blockage of neogenesis of the intimal spiral arterioles, leading to inhibition of intimal regeneration. At the same time, the epithelial necrosis edema and inflammatory cells infiltrate in a large amount, which results in reduced production of growth factors and estrogen receptors, migration of keratinocytes and fibroblasts to the site of injury and proliferation of the large amount, and deposition of extracellular matrix to form fibrous tissue. The pathological process mainly based on structural repair instead invades the ecological niche of normal physiological function cells, and the endometrium muscle layer cannot provide enough nutrition support for the neointima, so that fibrosis and adhesion of the endometrium are finally caused.
In the convalescence after injury, endometrial gland epithelial cells still face shedding-regenerating pressure, causing further repair barriers. The increase of oxidative stress level of endometrial gland epithelial cells, which causes apoptosis and shedding, is part of physiological process, which is caused by the shrinkage of the spiral small blood vessel of the endometrial gland epithelium by the withdrawal of progestogen. However, in the already damaged state, the endometrial gland epithelial cells are reduced by the estrogen and progestogen receptors, the growth of tiny spiral blood vessels is limited, the new blood vessels can only maintain the thickening of a small part of the intima, and the apoptosis causes the greater range of intimal loss. The endometrial stromal cells cause more myofibroblast mobilization on the basis of the original mechanical injury necrosis, and the extracellular matrix is secreted in a large amount to occupy the functional cell niche, so that good support can not be provided for the recovery and regeneration of the endometrial gland epithelial cells.
Summary of The Invention
The invention aims to solve the technical problem of providing a novel therapeutic drug or method for solving the existing difficult problem of treatment of the intrauterine adhesion diseases. The present invention provides human amniotic epithelial cells (human amniotic epithelial cells, hAECs) or cell preparations thereof for use in the treatment of uterine cavity adhesion diseases and methods for isolating human amniotic epithelial cells from amniotic tissue.
In one aspect, the invention provides a method for treating intrauterine adhesion disease by using human amniotic epithelial cells (hAECs), which not only increases the cell treatment effect of the human amniotic epithelial cells and reduces the endometrial film and the fibrosis condition of the damaged part caused by the damage of the endometrium, but also obviously improves the physiological response function of the estrogens of the new-born endometrium tissue, and simultaneously discovers that the hAECs treated by the bioactive substances have the effect of promoting recovery of the damaged endometrium tissue and can be used for treating the intrauterine adhesion disease.
In another aspect, the invention provides methods for treating a uterine cavity adhesion disorder using human amniotic epithelial cells (hAECs), wherein the human amniotic epithelial cells (hAECs) are subjected to a hypoxic culture during the culture phase. After the human amniotic epithelial cells (hAECs) are treated by hypoxia culture, the related indexes (TGF-beta, collagenI, alpha-SMA) and the like of the endometrial fibrosis are obviously reduced, and the response degree of the functional indexes of the endometrial fibrosis to hormone is obviously increased, so that the human amniotic epithelial cells (hAECs) show outstanding treatment effects, and can be used for repairing the endometrial film and fibrosis of a patient suffering from uterine cavity adhesion caused by injury, and can help the patient to increase pregnancy possibility.
In another embodiment, the invention provides a method for treating a uterine cavity adhesion disease using an effective dose of human amniotic epithelial cells or a cell preparation thereof, wherein the cell preparation comprises human amniotic epithelial cells and a pharmaceutically acceptable carrier.
In another embodiment of the invention, the appropriate amount of amniotic epithelial cells will vary according to the age, sex, weight, health condition of the patient, and other factors. Typically, the dosage range for each administration is about 10 3 -10 9 Individual cells, typically about 10 6 -10 7 Individual cells.
In another embodiment of the invention, the amniotic epithelial cells may be administered to the patient by any suitable method, such as intrauterine injection, vaginal administration to a female, and the like. Another method is to implant cells in a bioabsorbable material such as gelatin sponge, and to surgically implant the cell-seeded bioabsorbable material into the desired location in the uterus. The two methods can be combined and applied to obtain better curative effect.
In another aspect, the invention provides a method of isolating human amniotic epithelial cells from amniotic tissue, the method comprising the steps of:
(1) Obtaining an amniotic membrane from placenta tissue by mechanical separation;
(2) The washed amniotic membrane is digested by digestive enzyme, and the digested liquid is centrifuged to obtain the human amniotic epithelial cells.
In another aspect, the invention provides a method of hypoxia culturing human amniotic epithelial cells, the method comprising the steps of:
(1) Resuscitating the human amniotic epithelial cells, inoculating the resuscitated human amniotic epithelial cells into a culture solution, and culturing under a normal oxygen environment;
(2) And (3) reducing the oxygen concentration to 1% -10% after the cell confluence reaches 60% -90%, and culturing for 12-48 hours.
The human amniotic epithelial cells treated by short-time hypoxia culture have remarkable differences in the functions of improving the number of new-born endometrial glands and the thickness of new-born endometrial membranes, and the response degree of the endometrial functional index to hormone is obviously increased, so that the remarkable treatment effect is shown. The proliferation capacity of the stem cells is greatly ensured for the success of treatment because the hypoxia culture can promote the proliferation of the stem cells on the premise of not changing the stem property of the cells.
Brief description of the drawings
FIG. 1 shows the effect of hypoxia on human amniotic epithelial cells on proliferation capacity (FIG. 1.1) and dryness (FIG. 1.2). FIG. 1.1 shows an increase in proliferation rate of human amniotic epithelial stem cells after hypoxia culture compared to Chang Yang culture, with a final cell number greater than that of normoxic culture. FIG. 1.2 shows that the expression levels of SOX2, OCT4 and nanog, which are stem cell markers, are increased in the hypoxia culture group, and the stem cell markers have better drying potential compared with the normoxic culture group.
FIG. 2 model creation and hAEC transplantation. Fig. 2 shows that the uterus length is recovered after treatment, and the appearance is smooth and flat, as shown in the blank control group (a), the sham operation group (B), the IUA model group (C), the human amniotic epithelial cell treatment group (D) cultured in the normoxic environment, and the human amniotic epithelial cell treatment group (E) cultured in the hypoxia environment.
Figure 3 shows the morphological structure of the endometrium of different groups. Fig. 3 results of HE staining of the uterus of rats in the blank control group (a), the sham operation group (B), the IUA model group (C), the human amniotic epithelial cells treated group (D) cultured under normoxic conditions, the human amniotic epithelial cells treated group (E) after hypoxia culture. HE staining results for each group showed that hypoxia culture was more advantageous than normoxic human amniotic epithelial cells in promoting neo-endometrial thickening and increase in gland numbers, showing better therapeutic results.
Figure 4 effect of human amniotic epithelial cell treatment on fibrotic regulation. FIG. 4 shows a marked decrease in fibrosis after hypoxia-cultured human amniotic epithelial cells treatment compared to the other groups, as compared to the control group (A), the sham group (B), the IUA model group (C), the human amniotic epithelial cells treated group (D) cultured in normoxic conditions, and the human amniotic epithelial cells treated group (E) cultured in hypoxia.
FIG. 5 effects of human amniotic epithelial cell treatment on endometrial angiogenesis and proliferation (FIGS. 5-1, 5-2). FIG. 5 shows a marked decrease in fibrosis-related proteins (TGF-. Beta.,. Alpha. -SMA, collagen I) in comparison to the other groups in the blank (A), sham (B), IUA model (C), human amniotic epithelial cell treated group cultured under normoxic conditions (D), human amniotic epithelial cell treated group cultured under hypoxic conditions (E) and human amniotic epithelial cell treated by hypoxia. Angiogenesis (VEGF) and the degree of hormone response (p-ER) exhibit higher expression and better therapeutic potential.
FIG. 6 implantation of green fluorescent protein-labeled human amniotic epithelial cells in the uterus of rats. The immunofluorescence results of fig. 6 show that human amniotic epithelial cells are chimeric into damaged endometrial layers during treatment, exhibiting therapeutic properties of stem cells.
Figure 7 residence time of human amniotic epithelial cells in rat uterus. FIG. 7 shows the PCR results showing the residence time of human amniotic epithelial stem cells in utero, showing prolonged residence and associated therapeutic effects.
Detailed Description
Definition of the definition
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry and immunology, which are within the skill of the art, which are fully explained in the technical literature and in general textbooks of the art.
The term "amniotic membrane" as used herein refers to the innermost membrane sac that encloses the developing mammalian embryo. During pregnancy, the fetus is surrounded and buffered by a fluid called amniotic fluid. This fluid, along with the embryo and placenta, is enclosed in a sac called the amniotic membrane, which also covers the umbilical cord. The amniotic membrane comprises amniotic fluid which maintains a steady state environment and protects the embryo environment from the external environment. This barrier also protects the embryo from organisms (e.g., bacteria or viruses) that may travel up the vagina and may cause infection.
The term "placenta" as used herein refers to both premature and term placenta.
As used herein, the term "human amniotic epithelial cells (human amniotic epithelial cells, hAECs)" is a cell isolated from the amniotic membrane on the side of the placenta closest to the fetus.
The term "intrauterine adhesion (intrauterine adhesions, IUA)" as used herein is also known as Asheman syndrome, and is due to trauma of pregnant or non-pregnant uterus, resulting in damage to the basal endometrium layer, partial or total occlusion of the uterine cavity, resulting in abnormal menstruation, infertility or recurrent abortion, etc. The essence is intimal fibrosis.
The term "isolated" as used herein refers to the removal of a material from its original environment, and thus is "altered by hand" from its natural state.
The terms "patient" and "subject" as used herein refer to an animal, typically a mammal, preferably a human, treated with the cells or compositions thereof provided herein. For the treatment of infections, conditions and disease states specific to a particular animal patient, e.g., a human patient, the term patient refers to that particular animal.
The term "treatment" as used herein refers to any action that provides benefit to a patient at risk of developing or in a disease state that may be ameliorated by the cell composition or cell preparation of the invention. Treatment as used herein includes prophylactic and therapeutic treatments.
The term "formulation" as used herein refers to a composition in a form that allows the biological activity of the active ingredient contained therein to be effective and free of other ingredients having unacceptable toxicity to the subject.
The term "pharmaceutically acceptable carrier" as used herein refers to a component of a pharmaceutical formulation that is different from the active ingredient and is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives, and the like.
The term "effective amount" as used herein refers to an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount also means an amount sufficient to allow diagnosis or facilitate diagnosis. The effective amount for a particular subject can vary depending upon a variety of factors, such as the disease to be treated, the overall health of the patient, the route of administration, and the dosage and severity of the side effects. An effective amount may be the maximum dose or regimen that avoids significant side effects or toxic effects.
The term "confluence" as used herein represents the cell density. When cells proliferate in a culture vessel, adhesion and alignment state between cells occur, and a state of reaching a certain state is expressed as% confluency, representing cell density. For example, when cells are spread on average in a culture dish and are in a single independent state, the cells are spread around along with growth to form a clone, and finally the percentage of the total area of the culture formed by the clone of the cells is the percentage of the confluency of the cells.
Various aspects of the application will be described in further detail below.
Treating intrauterine adhesion disease
Stem cell transplantation, which restores endometrial cell abundance and function, is the only means to cure refractory intrauterine adhesions today. Because the amniotic epithelial cells have the functions of inhibiting inflammatory reaction, reducing fibrosis degree and recovering tissue regeneration, the inventor hopes to search whether the amniotic epithelial cells have therapeutic effects on intrauterine adhesion diseases by utilizing the characteristics of the amniotic epithelial cells.
In one aspect, the application provides the use of human amniotic epithelial cells (hAECs) or cell preparations thereof for the treatment and/or amelioration of a uterine cavity adhesion disease.
At present, the first effective treatment method for the intrauterine adhesion (intrauterine adhesions, IUA) is surgical treatment-hysteroscopic intrauterine adhesion separation (transcervical resection of adhesion, TCRA), but the intrauterine morphology can only be recovered as much as possible after the operation, fresh wound surfaces are formed again in the separation adhesion process, the normal endometrium is reduced, the basal lamina of the endometrium is damaged, stem cells are damaged or lost, and the regeneration function is hardly realized, so the recurrence rate after the treatment can reach 62.5 percent, and the success rate of pregnancy is only 22.5-33.3 percent. At the same time, various other protocols have been tried clinically to control and prevent adhesions, including: intrauterine devices, support balloons, bio-gel materials, etc. Although these means and measures are continually improved and innovated, the occurrence of IUA and the recurrence rate after surgical treatment are not significantly improved nor are pregnancy outcomes and menstrual conditions in patients significantly improved.
Through series of researches, the inventor surprisingly discovers that the method not only increases the cell treatment effect of human amniotic epithelial cells, reduces the endometrial film and the fibrosis condition of the damaged part caused by the damage of the endometrium, but also obviously improves the physiological response function of the estrogens of the new-born endometrium tissue, and simultaneously discovers that hAECs treated by bioactive substances have the effect of promoting recovery of the damaged endometrium tissue and can be used for treating the intrauterine adhesion disease. The present inventors therefore considered that hAECs are effective methods for treating intrauterine adhesion disease and could be a candidate source for cell therapy for such diseases, and thus completed the present application.
Human amniotic epithelial cells (human amniotic epithelial cells, hAECs) are cells isolated from the amniotic membrane on the side of the placenta closest to the fetus. Placenta belongs to the waste after the pregnant woman is produced, so that the cells from the placenta have no ethical problem.
The placenta is anatomically divided into three layers, namely, amniotic epithelial layer, chorion (chorion), and decidua (decidua), from inside to outside, with each layer of tissue being of a distinct origin. The decidua of the uterus is from the mother, the chorion is from the trophoblast (trophoblast), and the amniotic epithelial layer is from the epiblast (epiblast) eight days after fertilization, i.e. as embryonic stem cells (embryonic stem cells, ESCs) are all derived from the intra-embryonic cell mass (inner mass), whereas hAECs retain multipotency for a long period after differentiation into amniotic epithelium. Miki et al demonstrated that hAECs expressed the major surface maker (e.g., oct4, sox2, nanog, SSEA-3, SSEA-4, etc.) of pluripotent stem cells (e.g., embryonic stem cells), indicating its potential to differentiate into three germ layer tissue that is characteristic of embryonic stem cells. In vitro differentiation experiments showed that hAECs have in vitro tricodermic differentiation potential, both from transcriptional and from expression levels. However, unlike ESCs, the in vivo teratoma formation assay of hAECs is negative, mainly because it has no telomerase activity and therefore cannot differentiate into tissue structures with tricodermic differentiation. As such hAECs are also used as cell therapies, and are not tumorigenic (including benign tumors, sarcomas, and carcinomas).
The human amniotic epithelial cells have a paracrine function of stem cells, and can secrete a plurality of cell growth factors such as FGF, VEGF, EGF, HGH; NGF, BDNF, NT-3 and other neurotrophic factors; various neurotransmitters and modulators such as dopamine, catecholamines, acetylcholine, norepinephrine, histamine, serotonin, angiotensin, and neurotensin. The prior researches prove that the human amniotic epithelial cells have good application effect as the trophoblasts, and for example, clinical experiments prove that the human amniotic epithelial cells can promote proliferation and stem maintenance of the limbal epithelial cells as the trophoblasts.
Meanwhile, the hAECs hardly express MHCII type molecules on the cell surface, and cannot cause inflammation, allergy and immune response, so that the hAECs have low immunogenicity and are suitable for treatment of transplanted cells. The hAECs separation process is simple, and after scraping, the amniotic membrane has little cell pollution of other types except a small amount of blood cell aggregates, and the blood cells belong to suspension cells under the condition of no stimulation, so that the blood cells can be removed by changing the liquid after cell culture. According to experimental statistics of the inventors and foreign scientists, about 8000 ten thousand to 3 hundred million hAECs cells are found per human amniotic membrane. In the presence of EGF, hAECs have strong proliferation capacity, can proliferate for one generation in about 36 hours, and can maintain vigorous proliferation capacity in the previous generation (within about 10 generations). These advantages ensure that the present application is able to obtain a sufficient number of single cell types to meet the requirements of clinical treatment.
The invention discloses application of human amniotic epithelial cells or cell preparations thereof in preparing medicines for treating and/or improving uterine cavity adhesion diseases. In a preferred embodiment of the invention, an effective dose of human amniotic epithelial cells or cell preparations thereof may be used to treat and/or ameliorate a uterine cavity adhesion disease. An effective dose refers to an amount sufficient to ameliorate or prevent a symptom or condition of a medical disease. The effective amount for a particular subject can vary depending upon a variety of factors, such as the disease to be treated, the overall health of the patient, the route of administration, and the dosage and severity of the side effects. An effective amount may be the maximum dose or regimen that avoids significant side effects or toxic effects.
In another embodiment of the invention, the inventors have surprisingly found that the human amniotic epithelial cells treated by the short-time hypoxia culture have significant differences (p < 0.05) in actions of increasing the number of neoendometrial glands and the thickness of neoendometrial membranes, and that after the treatment of the human amniotic epithelial cells treated by the hypoxia culture, fibrosis-related indexes (TGF-beta, collagen I, alpha-SMA) and the like in endometrium are obviously reduced, and the response degree of the endometrial function indexes to hormone is obviously increased, so that a remarkable treatment effect is achieved.
The proliferation capacity of the stem cells is greatly ensured for the success of treatment because the hypoxia culture can promote the proliferation of the stem cells on the premise of not changing the stem property of the cells. The human amniotic epithelial cells (hAECs) treated by hypoxia culture have dryness and low immunogenicity, so that the human amniotic epithelial cells (hAECs) can be used for repairing endometrium films and fibrosis of patients suffering from intrauterine adhesion caused by injury, and improving the sensitivity of new-born endometrium tissues to estrogens, thereby helping the patients to increase pregnancy possibility and obtaining a larger chance of fertility. Therefore, the biological efficacy and safety of the hypoxia-cultured hAECs can meet the clinical treatment requirement, and the hypoxia-cultured human amniotic epithelial cells or cell preparations thereof can be used for treating and/or improving the intrauterine adhesion diseases. .
In another embodiment of the present invention, the animal having a uterine cavity adhesion 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 a uterine cavity adhesion disorder is a human.
In another embodiment of the invention, the cell preparation comprises human amniotic epithelial cells and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carrier according to the present invention means a substance suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation and allergic response) commensurate with a suitable benefit/risk ratio, such as a pharmaceutically acceptable solvent, suspending agent or vehicle, for example, which facilitates cell survival, and which is capable of delivering the formulated cells to a human or animal. The carrier is selected according to the mode of administration which is appropriately planned. 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, polyglycolic acid (PGA), polylactic acid (PLA), copolymers thereof, and the like.
The appropriate state of the amniotic epithelial cells can be selected by those skilled in the art as appropriate, 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.
Amniotic epithelial cells may be administered to the patient in an intrauterine in situ manner, each administration being at a dose ranging from about 10 3 -10 9 Cells of the order of magnitude, such as intrauterine injection, vaginal administration to females, and the like. Typically, these cells are contained in a pharmaceutically acceptable liquid medium. Cell administration may be repeated or performed continuously. In general, multiple modes of administration are typically administered separately at least 7-10 days apart. Another method is to implant cells in a bioabsorbable material such as gelatin sponge, and to surgically implant the cell-seeded bioabsorbable material into the desired location in the uterus. The two methods can be combined and applied to obtain better curative effect.
The appropriate amount of amniotic epithelial cells will vary depending on the age, sex, weight, health condition of the patient, and other factors. Typically, the dosage range for each administration is about 10 3 -10 9 Cells of the order of magnitude, typically about 10 6 -10 7 Cells of the order of magnitude.
In summary, the invention applies the human amniotic epithelial cells hAECs to the treatment of intrauterine adhesion diseases for the first time, has good effect, and provides a new scheme for the current treatment of the diseases.
Preparation of amniotic epithelial cells
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) Obtaining an amniotic membrane from placenta tissue by mechanical separation;
(2) The washed amniotic membrane is digested by digestive enzyme, and the digested liquid is centrifuged to obtain the human amniotic epithelial cells.
The amniotic epithelial cells of the present invention are derived from humans. The amniotic membrane can be separated from the isolated human placenta, and the blood cells are removed by flushing with physiological buffer solution, so that the residual chorion and blood vessels are mechanically removed. Isolation refers to removing cells from a tissue sample and separating from additional tissue. Single cells are isolated from intact human amniotic epithelial tissue using any conventional technique or method, including mechanical (shredding or shearing) forces, enzymatic digestion with one or a combination of proteases such as collagenase, trypsin, lipase, release enzyme (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 taking placenta tissue of a healthy parturient after caesarean section after approval of the obstetrics and by cutting the placenta with a cross knife, and the whole amniotic membrane is obtained by mechanical separation.
In another preferred embodiment of the present invention, the human amniotic epithelial cells obtained in step (2) may be further cultured, preferably under the following culture conditions: at 1X 10 6 -1×10 8 The density of individual cells/plates is that the cells are inoculated in a culture solution, placed in a carbon dioxide incubator for culture, the culture solution is changed after the human amniotic epithelial cells are attached, and the cells are digested and frozen after the cells are grown up.
In another embodiment of the invention, the invention provides a method of hypoxia culturing human amniotic epithelial cells, the method comprising the steps of:
(1) Resuscitating the human amniotic epithelial cells, inoculating the resuscitated human amniotic epithelial cells into a culture solution, and culturing under a normal oxygen environment;
(2) And (3) reducing the oxygen concentration to 1% -10% after the cell confluence reaches 60% -90%, and culturing for 12-48 hours.
After the culture is finished, the cells are digested and frozen for later use.
In another preferred embodiment of the invention, the invention provides a method of hypoxia culturing human amniotic epithelial cells, the method comprising the steps of:
(1) Resuscitating the human amniotic epithelial cells, inoculating the resuscitated human amniotic epithelial cells into a culture solution, and culturing under a normal oxygen environment;
(2) And (3) reducing the oxygen concentration to 3% -5% after the cell confluence reaches 70% -80%, and culturing for 24-36 hours.
After the culture is finished, the cells are digested and frozen for later use.
Normally, the oxygen content in air is about 21%, and the oxygen concentration is between 19.5% and 23.5% in a normal oxygen environment. When the cells are evenly spread in the culture container and are in a single independent state, the cells are spread around along with growth to form one clone, and finally the percentage of the area formed by the cell clone to the total area of the culture container is confluence, which represents the cell density.
In a particularly preferred embodiment of the invention, there is provided a method of hypoxia culturing human amniotic epithelial cells, preferably under the following culture conditions: at 1X 10 6 -1×10 8 The density of individual cells/flat plates is that the cells are inoculated into a culture solution, placed in a carbon dioxide incubator for culture, the culture solution is changed after the human amniotic epithelial cells are adhered, the cell confluence is 70% -80%, then the cells are placed into an environment with the oxygen concentration of 3% -5% for culture for 24-36h, and then the cells are digested for freezing storage.
The active cell population can be concentrated by other methods known to those skilled in the art. These post-processing washing/concentrating steps may be performed separately or simultaneously. In addition to the above methods, the active cell population can be further purified or enriched after cell washing or after culturing to reduce the number of mixed cells and dead cells. The separation of cells in suspension can be achieved by the following technique: buoyant density sedimentation centrifugation, differential adhesion to and elution from a solid phase, immunomagnetic beads, fluorescence laser cell sorting (FACS), or other techniques. Examples of these different techniques and devices for carrying out these techniques can be found in the prior art and in commercial products.
There is no limitation on the type of basal medium used in the present invention, as long as it can be used for cell culture. Preferred media include DMEM medium and NPBM medium. There is no limitation on the types of other components that may be contained in the above-mentioned basal medium, and preferable components include F-12, FCS, nerve survival factor, and the like.
The appropriate state of the amniotic epithelial cells can be selected by those skilled in the art as appropriate, 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. The amniotic epithelial cells may be derived from amniotic epithelial cells isolated from amniotic tissue, and include the hypoxia-cultured human amniotic epithelial cells described above.
The amniotic epithelial cells prepared by the invention can be prepared into amniotic epithelial cell preparations by adding other pharmaceutically acceptable carriers such as pharmaceutically acceptable solvents, suspending agents or excipients, and the like, which are beneficial to cell survival and can deliver the prepared cells to human or animals.
The invention uses the human amniotic epithelial cells for treating the uterine cavity adhesion diseases, fully exerts the advantages of the human amniotic epithelial cells, wherein the human amniotic epithelial cells mainly have the following advantages:
(1) Can maintain multipotency for a long period of time and has the specific potential of differentiating into three germ layer tissues of embryonic stem cells;
(2) Almost no MHCII type molecules are expressed on the cell surface, so that inflammation, allergy and immune response are not caused, and the requirement for transplantation matching is correspondingly reduced;
(3) Has the ability to regulate immune responses in vivo and in vitro, and can secrete various immune regulation factors, anti-angiogenic proteins or anti-inflammatory factor related proteins during in vitro culture;
(4) The vaccine has low immunogenicity, can be regarded as immune privilege cells, has no antigen presentation function, can reduce the sources of immune cells after transplantation, and avoids the occurrence of immune rejection reaction;
(5) No telomerase reverse transcriptase is expressed, no tumorigenicity (including benign tumors, sarcomas, and carcinomas);
(6) Has stronger proliferation capability and can keep vigorous proliferation capability in the previous generation (about 10 generations);
(7) The source is wide, the materials are easy to obtain, the application limit is not existed, and the ethical problem is not existed.
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental procedures, without specific conditions noted in the examples below, were selected according to methods and conditions conventional in the art, or according to the commercial specifications.
EXAMPLE 1 Primary amniotic epithelial cells were isolated and cultured
1. Human amniotic membrane source
To avoid microbial contamination of the birth canal, the fetal placenta from caesarean section is selected. Premature fetal placenta (38 weeks ago) is preferred because of the apoptosis of amniotic membrane due to the stimulation of delivery signals after term. After the parturient is authorized to agree, placenta tissues after the abdomen of a healthy parturient (serological reactions such as HIV, syphilis, hepatitis A, hepatitis B, hepatitis C and the like are all negative) are taken, placenta is cut by a cross knife, and the whole amniotic membrane is obtained through mechanical separation.
2. Isolation of hAECs (Whole process requires sterile operation)
The placenta of the infant delivered by caesarean section 39 weeks ago was obtained, the amniotic membrane was peeled off from the inner surface of the placenta and immersed in a centrifuge tube containing F12/DMEM (containing 1 Xpenicillin-streptomycin and amphotericin) basal medium. Cold chain transport at 4 ℃ to laboratory intercellular spaces.
The amniotic membrane was removed and each amniotic membrane was placed in 40ml of CMF-HBSS (containing 1X penicillin-streptomycin and amphotericin) and washed to remove mucus, and the mesenchymal layers and mucus adjacent to the chorionic layer were scraped off with forceps, repeated 3 times, with each wash being replaced with a new container and new HBSS solution.
The washed amniotic membrane was transferred to a new container, 10ml of 0.05% pancreatin/EDTA was added, inverted for 30s, and discarded.
The amniotic membrane was transferred to a new vessel, 20ml of 0.05% pancreatin/EDTA was added, and incubated in a 37℃water bath for 10min to discard the solution.
The amniotic membrane was transferred to a new container, incubated in 25ml pancreatin/EDTA 37℃water bath for 40min, and the digests were preserved.
After primary digestion, the amniotic membrane is transferred to a new container, 25ml of pancreatin/EDTA is incubated in a 37 ℃ water bath for 40min, and the digestive juice is preserved.
An equal volume of digestion stop solution (F12/DMEM containing 5% FBS,1 xL-glutamic acid, 1 xpyruvic acid) was added and centrifuged at 400g for 10min. Discard, complete medium with amniotic membrane: F12/DMEM contains 5% KSR (KnockOut Serum Replacement), 1 xL-glutamine, 1 xpyruvic acid, 1Xps (Penicillin-Streptomycin), and the pellet is resuspended.
An equal volume of digestion stop solution was added and centrifuged at 400g for 10min. The solution was discarded and the pellet was resuspended in complete medium.
Sieving with 100um sieve, counting to obtain 10 5 cells/cm2 were inoculated to a petri dish or placed in a frozen stock solution (90% FBS,10% DMSO) for liquid nitrogen frozen stock.
3. Inoculation culture and cryopreservation of hAECs
Cell culture: inoculation of 1X 10 7 After the cells were grown, the culture medium was changed after hAECs had adhered to the wall, and then the culture medium was changed three days later.
After the cells grow up on the plate, the cells are digested and frozen: 15cm dish was added with 5ml pancreatin, observed under a rear mirror for 10min, and the digestion was stopped by adding an equivalent amount of digestion stop solution when the cells became round and the cells became suspended when the plate was shaken on a plane. Cells on the dish were blown down in the same direction with a micropipette, transferred into a 15ml centrifuge tube, centrifuged at 300g for 3min, and then collected and counted. Adding a freezing solution into a freezing tube, marking the freezing date, batch and cell number, then placing the cells into the freezing tube, then immediately placing the freezing tube into a freezing box, placing the freezing box into a refrigerator at-80 ℃, taking out the freezing box after 12 hours, and transferring the cells into a liquid nitrogen tank for preservation.
EXAMPLE 2 pretreatment of human amniotic epithelial cells
Resuscitates the human amniotic epithelial cells to a 10cm culture dish, cultures the human amniotic epithelial cells in a complete culture medium under an oxygen environment with 20 percent, and reduces the oxygen concentration to 3 percent to 5 percent after the cell confluency reaches 70 percent to 80 percent until the amniotic cells are fully proliferated (generally, the required time is 24 to 36 hours).
The results show that: the proliferation capacity of the human amniotic epithelial cells treated by hypoxia is greatly improved, and the dryness is not changed (figure 1). Meanwhile, the functions of the hypoxia-treated human amniotic epithelial stem cells in terms of improving the number of new-born endometrial glands and the new-born endometrial thickness are obviously different (p is less than 0.05), after the hypoxia-cultured human amniotic epithelial cells are treated, fibrosis related indexes (TGF-beta, collagen I, alpha-SMA) in the endometrium and the like are obviously reduced, and the response degree of the endometrial functional indexes to hormone is obviously increased. Showing outstanding therapeutic effects.
Example 3 construction of intrauterine adhesion model and amniotic epithelial cell injection
1. IUA model for rat intrauterine adhesion
The vaginal smear method determines estrus cycle, the estrus is round nucleated epithelial cells or few keratinocytes, and the estrus is needle-like non-nucleated keratinocytes or round cells with sawtooth-like edges and a small number of epithelial cells. Rat modeling during estrus was taken (fig. 2). After anesthetizing the animal, the abdomen is opened, a transverse incision of about 2mm is made on the uterus, and a curette is inserted to scratch and mould. Ensure that all organs return to their anatomical position and the abdominal cavity is flushed twice with warm saline, and the incision is sutured after confirming no bleeding points. 6-0 absorbable suture was used to suture the muscular layer and abdominal wall, 3mm gauge, 6-0 nylon suture was used to suture the skin. A suitable amount of penicillin is injected post-operatively.
2. Intrauterine injection of amniotic epithelial cells
Preparation of amniotic epithelial cells. Selecting pollution-free amniotic epithelial cells (P1 or P2 generation, figure 2) with good health state, when cell confluency is about 70%Digestion, PBS and gentle washing twice to remove cell culture fluid, 1X 10 6 The individual cells were resuspended in 50. Mu.L of sterile physiological saline to prepare a cell suspension. Temporarily stored at 4 ℃ and injected into animals within 1 hour.
Injection of amniotic epithelial cells. One week after molding, animals are anesthetized, skin is sterilized and prepared, and the original suture is cut off to open the abdomen, so that the damage of the animals is reduced as much as possible. A small opening is made to expose the uterus. The cell suspension was carefully aspirated into a 1mL sterile medical syringe (needle replaced with the finest scalp needle), gently flicked off of air bubbles, and given 1X 10 per uterine horn 6 And amniotic epithelial cells. The needle is placed near one side of the ovary in the middle of the uterine horn, so that the cell suspension is gently pushed into the uterine cavity after the needle reaches the uterine cavity, and the forceps are used for clamping the needle inlet to help the tissue to shrink when the needle exits the tissue, so that no redundant liquid is oozed. The uterus is placed back into the abdominal cavity and sutured. The lower feet of the non-awake animal are put back into the cage, and the state of the animal after operation is observed. (FIG. 2)
The results show that: the amniotic epithelial cell treatment group is visible under the visual field, the tissue structure is complete, the appearance is smooth and glossy, the upper and lower width of the uterine horn is uniform, the near uterine orifice is slightly edematous, and the rest is not different from the blank control group and the sham operation group. And the uterine cavity adhesion (IUA) group has slightly edema tissue compared with the control group, the uterine cavity is different in width, the tissue is slightly dark in appearance color, the appearance structure is complete, the length is slightly shorter than that of the blank control group and the sham operation group, and the subsequent slicing results show that a plurality of adhesion positions with different degrees exist in the uterine cavity. The blank control uterus has complete tissue, smooth and glossy appearance, uniform upper and lower width of uterine horns and healthy tissue with perfect bilateral development. The tissue of the false operation group is complete, the appearance is smooth and glossy, the upper and lower width of uterine horns is uniform, the tissue is healthy with perfect development of both sides, no inflammatory infiltration is seen during the abdomen opening, and the subsequent research results show that the false operation does not influence the uterine structure and function of the experimental mice.
Subsequent paraffin section detection shows that the IUA model group has multiple adhesion parts of uterine cavity and successful molding. Whereas in the blank control group, in the sham operation group, no adhesion was found in the uterus of the IUA amniotic epithelium treatment group. The appearance of each group of uterus is generally consistent, and the uterus has smooth and complete appearance and no pathological changes such as damage and ulcer. The appearance cannot be an evaluation standard for evaluating the internal adhesion or not and the severity, and each group in the experiment did not damage the uterus except the inner cavity.
EXAMPLE 4 histological staining
He staining: after the uterus is vertically embedded, paraffin is made into 6 mu m slices, the slices are dewaxed to water, and HE staining is adopted for tabletting. The plates were read under an optical microscope, observed with a low power microscope, photographed and counted for each group of glands (fig. 3).
HE staining results were seen: IUA building set has serious intrauterine adhesion, almost vanishing intrauterine cavity, indistinct boundary between myometrium and endometrium, and serious damage to gland and other intrauterine tissue structures. The amniotic epithelial cell treatment group has complete uterine cavity structure, the area of the uterine cavity is obviously reserved, the boundary between the basal layer and the intima layer is clear, but the thickness of the intima layer is slightly nonuniform; the glands are distinguishable and distributed unevenly. The normal uterine cavity has complete structure, plump endometrium, ordered endometrium structure, clear and even glandular structure distribution, clear boundary between myometrium and endometrium, and larger uterine cavity. Compared with the blank control group, the artificial operation group has the advantages of small difference of the areas of the uterine cavities, rich glands, thicker intima layer, clear limit between the myometrium and the intima layer and complete structure.
2. Endometrial gland number and intima thickness quantification: the number of endometrial glands is measured by ImageJ image processing software (i.e. the perpendicular distance from the endometrial base border to the uterine cavity, 5 sites per slice are randomly selected and the average value is taken as the endometrial thickness).
Experimental results show that after IUA modeling, the endometrium of the rat is obviously reduced, and the glands are obviously reduced; after treatment with amniotic epithelial cells, the endometrium was significantly increased, but the degree of amplification was different from site to site, and the number of glands was also restored, but at a distance from healthy uterus (fig. 3). There was no significant difference between sham and placebo, sham did not affect the uterine structural function of rats.
Masson staining: paraffin sections were dewaxed to water and then pelleted using Masson stain. The film was read under an optical microscope, observed with a low-high microscope, and photographed.
Measurement of endometrial fibrosis: four high power mirror fields were taken from each section after Masson staining to observe the degree of endometrial fibrosis, and the percentage of the fibrosis area in each high power mirror field to the total intima area was calculated using ImageJ image processing software to calculate the average (fig. 4).
Experimental results show that surgical modeling creates a large area fibrotic phenotype in the uterus. After amniotic epithelial cell treatment, fibrosis is relieved, and particularly, the fibrosis degree is greatly reduced when the endometrium is close to one end of the uterine cavity.
EXAMPLE 5 tissue immunofluorescence
After the tissue was removed from the body, the tissue was quickly placed in PBS to wash out blood stains, and the tissue was carefully dissected to the appropriate size (care was taken not to pinch the tissue with force). Tissues were placed in an embedding cassette containing frozen section embedding medium (OCT). The cassette was placed in dry ice precooled isopentane until OCT and tissue were completely frozen.
The embedded tissue was fixed in a frozen microtome, cut into 0.5um sections, and the sections were attached to an adhesive slide.
The sections were left at room temperature for 30min before staining to allow the sections to adhere well to the slides.
The samples were placed in acetone at-20℃and fixed for 10min (if the tissue was fixed before embedding, the subsequent work was directly performed).
The sections were removed and washed 3 times with 5min each with PBS.
Firstly, the tissue is surrounded by an oily pen, then, primary antibody (the primary antibody is diluted by PBS of 5% HBS+1% BSA) is dripped on the tissue, and the tissue is placed in a wet box at 4 ℃ overnight;
taking out the slices, washing with PBS for 3 times, each time for 5min;
The sections were removed to reveal moisture, fluorescent conjugated secondary antibodies (diluted with 5% HBS+1% BSA in PBS) were added dropwise and incubated in a wet box at room temperature for 1h in the absence of light. (the subsequent operations started in this step are all performed in the dark)
The sections were removed and washed 3 times with 5min each with PBS.
DAPI was diluted with PBS and added dropwise to the sections, incubated at room temperature for 3min, and then washed with PBS 2 times for 5min each;
sealing liquid sealing, fluorescence microscopy (fig. 5).
Example 6 implantation of green fluorescent protein-labeled human amniotic epithelial cells in the uterus of rats.
Intrauterine injection of amniotic epithelial cell suspension with GFP label was performed 7 days after rat molding, euthanasia was performed 7 days after treatment, and GFP-labeled detection was performed after dissecting uterus.
The results show that human amniotic epithelial cells are able to persist and exert their biological effects during uterine recovery (fig. 6).
EXAMPLE 7 parking of human amniotic epithelial cells in utero
The amniotic epithelial cell suspension with GFP label was injected into the uterine cavity 7 days after the rat molding operation, euthanized 2h and 24h after the treatment, and the normal fluorescence PCR detection of GFP protein was performed after dissecting the uterus (FIG. 7).
The results show that human amniotic epithelial cells are able to persist and exert their biological effects during uterine recovery.
Claims (26)
- Use of human amniotic epithelial cells (hAECs) or cell preparations thereof for the treatment and/or amelioration of a uterine cavity adhesion disease.
- Use according to claim 1, characterized in that: the human amniotic epithelial cells (hAECs) are subjected to hypoxia culture.
- Use according to claim 1 or 2, characterized in that: the animal with the uterine cavity adhesion disease refers to a mammal.
- Use according to claim 3, characterized in that: the animal is cow, horse, sheep, monkey, dog, rat, mouse, rabbit or human.
- Use according to claim 4, characterized in that: the animal is human.
- Use according to claim 1, characterized in that: the cell preparation comprises human amniotic epithelial cells and a pharmaceutically acceptable carrier.
- Use according to claim 1 or 2, characterized in that: the amniotic epithelial cells are suitably collected cells without any treatment, partially purified cells or purified cells which are then expanded by culture.
- Use according to claim 1 or 2, characterized in that: amniotic epithelial cells may be administered to the patient in an intrauterine in situ manner, each administration being at a dose ranging from about 10 3 -10 9 Cells of the order of magnitude.
- Use according to claim 8, characterized in that: amniotic epithelial cells may be administered to the patient in an intrauterine in situ manner, each administration being at a dosage ranging from about 10 6 -10 7 Cells of the order of magnitude.
- Use according to claim 8, characterized in that: the intrauterine in-situ administration method is uterine cavity injection, female animal vaginal administration and the like.
- Use according to claim 8, characterized in that: the intrauterine in-situ administration method is to implant cells in a bioabsorbable material, and implant the bioabsorbable material with the cells into the required part of the uterus by adopting an operation.
- Use according to claim 1, characterized in that: the amniotic epithelial cells are prepared by a method comprising the following steps:(1) Obtaining an amniotic membrane from placenta tissue by mechanical separation;(2) The washed amniotic membrane is digested by digestive enzyme, and the digested liquid is centrifuged to obtain the human amniotic epithelial cells.
- Use according to claim 12, characterized in that: the method step (1) can separate amniotic membrane from isolated human placenta, wash and remove blood cells by using physiological buffer solution, and mechanically remove residual chorion and blood vessel.
- Use according to claim 13, characterized in that: after the human amniotic membrane is obtained by approval and agreement of obstetrics and gynecology, placenta tissues after the Caesarean of a healthy puerpera are taken, and the whole amniotic membrane is obtained by mechanical separation.
- Use according to claim 12, characterized in that: the method step (2) separates single cells from the intact human amniotic epithelial layer tissue using any conventional technique or method including mechanical force, enzymatic digestion with one or a combination of proteases selected from collagenase, trypsin, lipase, releaser and pepsin, or a combination of mechanical and enzymatic methods.
- Use according to claim 15, characterized in that: continuing culturing the human amniotic epithelial cells obtained in the step 2, wherein the preferable culture conditions are as follows: at 1X 10 6 -1×10 8 The density of individual cells/plates is that the cells are inoculated in a culture dish, placed in a carbon dioxide incubator for culture, the culture solution is changed after the human amniotic epithelial cells are attached, and the cells are digested and frozen after the cells grow up the plates.
- Use according to claim 16, characterized in that: the culture medium for culturing the human amniotic epithelial cells is DMEM culture medium or NPBM culture medium.
- Use according to claim 14, characterized in that: f-12, FCS, nerve survival factor, etc. can be added to the basal medium.
- The use according to any one of claims 1 to 18, wherein the amniotic epithelial cells are prepared by hypoxia culture by a method comprising the steps of:(1) Resuscitating the human amniotic epithelial cells, inoculating the resuscitated human amniotic epithelial cells into a culture solution, and culturing under a normal oxygen environment;(2) And (3) reducing the oxygen concentration to 1% -10% after the cell confluence reaches 60% -90%, and culturing for 12-48 hours.
- Use according to claim 19, characterized in that: the amniotic epithelial cells are prepared by hypoxia culture according to the following steps:(1) Resuscitating the human amniotic epithelial cells, inoculating the resuscitated human amniotic epithelial cells into a culture solution, and culturing under a normal oxygen environment;(2) And (3) reducing the oxygen concentration to 3% -5% after the cell confluence reaches 70% -80%, and culturing for 24-36 hours.
- Use according to claim 19 or 20, characterized in that: after the culture is finished, the cells are digested and frozen for later use.
- Use according to claim 19 or 20, characterized in that: the normal oxygen environment is that the oxygen concentration is between 19.5% and 23.5%.
- Use according to claim 19 or 20, characterized in that: preferred culture conditions for the method are: at 1X 10 6 -1×10 8 The density of individual cells/flat plates is that the cells are inoculated into a culture solution, placed in a carbon dioxide incubator for culture, the culture solution is changed after the human amniotic epithelial cells are attached, the cell confluence is 70% -80%, then the cells are placed into an environment with the oxygen concentration of 3% -5% for culture for 24-36h, and then the cells are digested for freezing.
- Use of human amniotic epithelial cells or cell preparations thereof in the preparation of a medicament for treating and/or ameliorating a uterine cavity adhesion disorder.
- Use according to claim 24, characterized in that: the amniotic epithelial cells are prepared by hypoxia culture according to the following steps:(1) Resuscitating the human amniotic epithelial cells, inoculating the resuscitated human amniotic epithelial cells into a culture solution, and culturing under a normal oxygen environment;(2) And (3) reducing the oxygen concentration to 1% -10% after the cell confluence reaches 60% -90%, and culturing for 12-48 hours.
- Use according to claim 25, characterized in that: the amniotic epithelial cells are prepared by hypoxia culture according to the following steps:(1) Resuscitating the human amniotic epithelial cells, inoculating the resuscitated human amniotic epithelial cells into a culture solution, and culturing under a normal oxygen environment;(2) And (3) reducing the oxygen concentration to 3% -5% after the cell confluence reaches 70% -80%, and culturing for 24-36 hours.
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