WO2022204904A1 - Use of human amniotic epithelial cell in preparation of drug for treating and/or alleviating intrauterine adhesion diseases - Google Patents
Use of human amniotic epithelial cell in preparation of drug for treating and/or alleviating intrauterine adhesion diseases Download PDFInfo
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- WO2022204904A1 WO2022204904A1 PCT/CN2021/083698 CN2021083698W WO2022204904A1 WO 2022204904 A1 WO2022204904 A1 WO 2022204904A1 CN 2021083698 W CN2021083698 W CN 2021083698W WO 2022204904 A1 WO2022204904 A1 WO 2022204904A1
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- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/48—Reproductive organs
- A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
Definitions
- the invention belongs to the field of biotechnology, in particular to the use of human amniotic epithelial cells in the treatment of intrauterine adhesion diseases.
- the endometrium can undergo more than 400 times of hyperplasia, differentiation and shedding, showing an active and powerful regeneration ability. However, it is easily affected by many factors such as injury, inflammation, endocrine, etc., which leads to endometrial repair obstacles, resulting in endometrial basal layer damage, mutual adhesion between muscle walls, and damage to the normal anatomical shape of the uterine cavity.
- the occurrence of intrauterine adhesions, IUA The incidence of IUA is as high as 25%-30%, and it is one of the intractable diseases in the field of gynecology. Bleeding and other clinical symptoms.
- IUA The pathological features of IUA are thinning and atrophy of the normal intima, replaced by fibrotic stroma and inactive cuboidal columnar epithelium, indistinguishable functional and basal layers, and a small or complete absence of normal active endometrial glands. Body, etc., leading to intimal scar repair, adhesions, glandular hypofunction, poor response to hormones, and lack of interstitial blood vessels.
- endometrial glandular epithelial cells still face the pressure of exfoliation-regeneration, resulting in further repair obstacles.
- the withdrawal of progesterone causes the atrophy of the small spiral blood vessels of the endometrial glandular epithelium, and the increase in the level of oxidative stress in the endometrial glandular epithelial cells leads to apoptosis and shedding, which is part of the physiological process.
- endometrial gland epithelial cells due to the reduction of estrogen and progesterone receptors, the growth of small spiral blood vessels is limited, the new blood vessels can only maintain a small part of the thickening of the endometrium, and apoptosis causes a larger area endometrial loss.
- Endometrial stromal cells cause more mobilization of myofibroblasts on the basis of the original mechanical damage and necrosis, and the massive secretion of extracellular matrix squeezes the functional cell niche, which cannot provide good support for the recovery and regeneration of endometrial gland epithelial cells. .
- the technical problem to be solved by the present invention is to provide a new therapeutic drug or method for the treatment of the existing intrauterine adhesion disease.
- the present invention provides human amniotic epithelial cells (human amniotic epithelial cells, hAECs) or cell preparations thereof for treating intrauterine adhesion diseases, and provides a method for isolating human amniotic epithelial cells from amniotic tissue.
- the present invention provides a method for human amniotic epithelial cells (hAECs) for the treatment of intrauterine adhesion disease.
- the method described in the present invention not only increases the cell therapy effect of human amniotic epithelial cells, but also alleviates intrauterine damage caused by endometrial damage.
- Membrane thinness and fibrosis in damaged parts also significantly improved the physiological response function of estrogen and progesterone in neo-endometrial tissue. It was also found that hAECs treated with bioactive substances could promote the recovery of damaged endometrial tissue. Can be used to treat intrauterine adhesions.
- the present invention provides a method for treating intrauterine adhesion diseases with human amniotic epithelial cells (hAECs), wherein the human amniotic epithelial cells (hAECs) are subjected to hypoxic culture treatment during the culture stage.
- the fibrosis-related indexes (TGF- ⁇ , Collagen I, ⁇ -SMA) in the endometrium were significantly decreased, and the functional indexes of the endometrium were significantly affected by hormones.
- the degree of response of the treatment has increased significantly, showing a prominent therapeutic effect, so it can be used to repair the thin endometrium and fibrosis caused by the injury of patients with intrauterine adhesions, which can help patients increase the possibility of pregnancy.
- the present invention provides a method for treating intrauterine adhesion disease using an effective dose of human amniotic epithelial cells or cell preparations thereof, wherein the cell preparations include human amniotic epithelial cells and pharmaceutically acceptable accepted vector.
- the suitable amount of amniotic epithelial cells will vary depending on the age, sex, weight, health, and other factors of the patient. Typically, doses per administration range from about 103-109 cells , typically about 106-107 cells.
- the amniotic epithelial cells may be administered to a patient using any suitable method, such as intrauterine injection, vaginal administration to females, and the like.
- Another method is to seed the cells in a bioabsorbable material such as gelatin sponge, which is surgically implanted into the desired part of the uterus. The above two methods can be combined to achieve better curative effect.
- the present invention provides a method for isolating human amniotic epithelial cells from amniotic tissue, the method comprising the steps of:
- the washed amniotic membrane is digested with digestive enzymes, and the digested liquid is centrifuged to obtain human amniotic membrane epithelial cells.
- the present invention provides a method for culturing human amniotic epithelial cells under hypoxia, the method comprising the steps of:
- hypoxia culture can promote stem cell proliferation without changing the stemness of cells, the proliferation ability of stem cells is a great guarantee for the success of treatment.
- Fig. 1 The effect of hypoxic environment on the proliferation ability (Fig. 1.1) and stemness (Fig. 1.2) of human amniotic epithelial cells.
- Figure 1.1 shows that compared with normoxia culture, the proliferation rate of human amniotic epithelial stem cells increased after hypoxic culture, and the final number of cells was more than that of normoxia culture group.
- Figure 1.2 shows that the expression of stem cell markers SOX2, OCT4, and nanog in the hypoxia culture group increased compared with the normoxia culture group, showing better stemness potential.
- Figure 2 Model establishment and hAEC transplantation.
- Figure 3 Morphological structure of endometrium in different groups.
- Figure 3 Blank control group (A), sham operation group (B), IUA model group (C), human amniotic epithelial cell treatment group (D) cultured under normoxia, and human amniotic epithelial cell treatment group after hypoxia culture (E), HE staining results of rat uterus in each group.
- the results of HE staining in each group showed that human amniotic epithelial cells cultured in hypoxia had more advantages in promoting the thickening of the neonatal endometrium and the increase in the number of glands than those cultured in normoxia, showing a better treatment outcome.
- Figure 4 Effects of human blood amniotic epithelial cell treatment on regulation of fibrosis.
- Figure 4 Blank control group (A), sham operation group (B), IUA model group (C), human amniotic epithelial cell treatment group (D) cultured under normoxia, and human amniotic epithelial cell treatment group after hypoxia culture (E), after the treatment of human amniotic epithelial cells cultured in hypoxia, the degree of fibrosis was significantly decreased compared with other groups.
- Figure 5 Effects of human blood amniotic epithelial cell treatment on endometrial angiogenesis and proliferation (Figure 5-1, 5-2).
- Angiogenesis (VEGF) and hormone response (p-ER) showed higher expression, showing better therapeutic potential.
- FIG. 6 Implantation of GFP-labeled human amniotic epithelial cells in rat uterus.
- Figure 6 shows that human amniotic epithelial cells are chimeric into the damaged endometrial layer during the treatment process, showing the therapeutic properties of stem cells.
- FIG. 7 Residence time of human amniotic epithelial cells in rat uterus.
- Figure 7 PCR results showing the residence time of human amniotic epithelial stem cells in the uterus, showing a long-term residence and exerting a relevant therapeutic effect.
- amniotic membrane 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 encased in a sac called the amniotic membrane, which also covers the umbilical cord.
- the amniotic membrane contains amniotic fluid that maintains a homeostatic environment and protects the embryonic environment from the external environment. This barrier also protects the embryo from organisms, such as bacteria or viruses, that may travel up the vagina and potentially cause infection.
- placenta refers to both preterm placenta and term placenta.
- hAECs human amniotic epithelial cells
- IUA intrauterine adhesions
- Asheman syndrome is caused by trauma to the pregnant or non-pregnant uterus, resulting in damage to the basal lining of the endometrium, resulting in partial or complete occlusion of the uterine cavity. Abnormal menstruation, infertility or repeated miscarriage. Its essence is intimal fibrosis.
- isolated refers to the removal of material from its original environment, and thus has been altered “by hand” from its natural state.
- patient refers to an animal, usually a mammal, preferably a human, being treated with the cells or compositions provided herein.
- patient refers to that particular animal.
- treatment refers to any action that provides benefit to a patient at risk for a disease or in a disease state that can be ameliorated by the cellular compositions or cell preparations described herein. Treatment as used herein includes prophylactic and therapeutic management.
- formulation refers to a composition in a form that allows the biological activity of the active ingredient contained therein to be effective, and is free of other ingredients that would have unacceptable toxicity to the subject.
- pharmaceutically acceptable carrier refers to a component of a pharmaceutical formulation that is distinct from the active ingredient and that is not toxic to the subject.
- Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives, and the like.
- an effective amount refers to an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical disease.
- An effective amount also means an amount sufficient to enable or facilitate diagnosis.
- the effective amount for a particular subject may vary depending on factors such as the disease being treated, the general health of the patient, the method, route and dosage of administration, and the severity of side effects.
- An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
- % represents the cell density.
- % represents the cell density.
- the degree of confluence is expressed as %, which represents the cell density. For example, when the cells are evenly spread in a culture dish, they are in a single and independent state. As the cells grow, they will spread around to form clones one by one. Finally, the percentage of the area formed by cell clones to the total culture area is the cell confluence. %.
- amniotic epithelial cells have the functions of inhibiting inflammatory responses, reducing fibrosis and restoring tissue regeneration, the inventors of the present application hope to use the above characteristics of amniotic epithelial cells to explore whether they have therapeutic effects on intrauterine adhesions.
- the present invention provides the use of human amniotic epithelial cells (hAECs) or cell preparations thereof for treating and/or improving intrauterine adhesion disease.
- hAECs human amniotic epithelial cells
- the preferred and effective treatment for intrauterine adhesions is surgical treatment-hysteroscopic transcervical resection of adhesion (TCRA). Re-formation of fresh wounds during separation of adhesions, reduction of normal endometrium, destruction of endometrial basal layer, damage or loss of stem cells, and almost no longer have regenerative functions, so the recurrence rate after treatment can be as high as 62.5%, and pregnancy is successful The rate is only 22.5%-33.3%.
- TCRA transcervical resection of adhesion
- the method of the present invention not only increases the cell therapy effect of human amniotic epithelial cells, reduces the thin endometrial and fibrosis of damaged parts caused by endometrial damage, but also significantly The physiological response function of estrogen and progesterone of neonatal endometrial tissue is improved, and it is also found that hAECs treated with bioactive substances can promote the recovery of damaged endometrial tissue, and can be used for the treatment of intrauterine adhesions. Therefore, the inventors of the present application believe that hAECs are an effective method for the treatment of intrauterine adhesions, and can be used as a candidate source for cell therapy for this type of disease, thus completing the present invention.
- hAECs Human amniotic epithelial cells
- the placenta can be divided into three layers from the inside out: the amniotic epithelium, the chorion, and the decidua, and the origin of each layer is completely different.
- the decidua of the uterus is derived from the mother
- the chorion is derived from the trophoblast
- the amniotic epithelium is derived from the epiblast at eight days after fertilization, that is, like embryonic stem cells (ESCs) are derived from embryonic cells.
- Inner cell mass, hAECs can maintain pluripotency for a long time after differentiation into amniotic epithelium.
- hAECs can express the main surface maker (such as Oct4, Sox2, Nanog, SSEA-3, SSEA-4, etc.) of pluripotent stem cells (such as embryonic stem cells), indicating that it has the unique differentiation of embryonic stem cells into the three germ layers. potential.
- pluripotent stem cells such as embryonic stem cells
- hAECs had the potential to differentiate into three germ layers in vitro, both at the transcriptional and expression levels.
- hAECs were negative in the in vivo teratoma test, mainly because they had no telomerase activity and thus could not differentiate into tissue structures with three germ layers. Because of this, hAECs are used as cell therapy and are not tumorigenic (including benign tumors, sarcomas and carcinomas).
- Human amniotic epithelial cells have stem cell paracrine function and can secrete various cell growth factors such as FGF, VEGF, EGF, HGH; NGF, BDNF, NT-3 and other neurotrophic factors; dopamine, catecholamine, acetylcholine, norepinephrine , histamine, serotonin, urotensin, neurotensin and other neurotransmitters and conditioners.
- FGF FGF
- VEGF vascular endothelial growth factor
- EGF epithelial growth factor
- HGH vascular endoblast growth factor
- NGF BDNF
- NT-3 neurotrophic factor-3
- dopamine catecholamine
- acetylcholine norepinephrine
- histamine serotonin
- urotensin neurotensin and other neurotransmitters and conditioners.
- hAECs hardly express MHC II molecules on the surface of cells and do not cause inflammation, allergic and immune responses, so they have low immunogenicity and are suitable for transplantation cell therapy.
- the separation process of hAECs is relatively simple. Except for a small amount of blood cell clumps, there is almost no other type of cell contamination on the amniotic membrane after scraping.
- the blood cells are suspension cells in the unstimulated condition, so they can be cultured and replaced by medium. remove. According to the experimental statistics of the inventor and foreign scientists, there are about 80 million to 300 million hAECs cells in each human amniotic membrane.
- hAECs In the presence of EGF, hAECs have strong proliferation ability, can proliferate for one passage in about 36 hours, and can maintain strong proliferation ability in the previous passage (within about 10 passages). These advantages ensure that the application can obtain a sufficient number of single cell types to meet the requirements of clinical treatment.
- the invention discloses the use of human amniotic epithelial cells or cell preparations thereof in preparing medicines for treating and/or improving intrauterine adhesion diseases.
- an effective dose of human amniotic epithelial cells or cell preparations thereof can be used to treat and/or ameliorate intrauterine adhesion disease.
- An effective dose refers to an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical disease.
- the effective amount for a particular subject may vary depending on factors such as the disease being treated, the general health of the patient, the method, route and dosage of administration, and the severity of side effects.
- An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
- the inventors have also surprisingly found that human amniotic epithelial cells treated with short-term hypoxia culture have significant differences in increasing the number of neonatal endometrial glands and neonatal endometrial thickness (p ⁇ 0.05), after the treatment of human amniotic epithelial cells cultured with hypoxia, the fibrosis-related indexes (TGF- ⁇ , Collagen I, ⁇ -SMA) in the endometrium were significantly decreased, and the endometrial functional The response of the indicators to hormones increased significantly, showing a prominent therapeutic effect.
- TGF- ⁇ fibrosis-related indexes
- hypoxia culture can promote stem cell proliferation without changing the stemness of cells, the proliferation ability of stem cells is a great guarantee for the success of treatment.
- Hypoxic culture-treated human amniotic epithelial cells have dryness and low immunogenicity, so they can be used to repair endometrial thinness and fibrosis caused by injury in patients with intrauterine adhesions, and improve neonatal endometrial tissue. Sensitivity to progesterone, which can help patients increase their chances of becoming pregnant and have a greater chance of having children.
- hAECs cultured in hypoxia can better meet the needs of clinical treatment, and human amniotic epithelial cells or cell preparations cultured in hypoxia can be used to treat and/or improve intrauterine adhesion diseases. .
- the animal with intrauterine adhesion disease refers to a mammal.
- the animal is a cow, horse, sheep, monkey, dog, rat, mouse, rabbit or human.
- the animal with intrauterine adhesion disease refers to a human.
- the cell preparation includes human amniotic epithelial cells and a pharmaceutically acceptable carrier.
- the pharmaceutically acceptable carrier of the present invention refers to a substance with suitable benefit/risk rate that is suitable for use in humans and/or animals without excessive adverse side effects (such as toxicity, irritation and allergic reaction), for example, a pharmaceutically acceptable carrier. Acceptable solvents, suspending agents, or excipients, which facilitate cell survival, are capable of delivering formulated cells to humans or animals.
- the carrier is selected for the appropriately planned 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, and the like.
- Various artificial scaffolds may also be included, including but not limited to gelatin sponge, polyglycolic acid (PGA), polylactic acid (PLA) and their copolymers, and the like.
- amniotic epithelial cells in consideration of the type of disease to be treated: collected cells without any treatment (crude fraction); partially purified cells; purified cells and then expanded by culture increase.
- the amniotic epithelial cells can be administered to patients by in situ administration in utero, and the dose range for each administration is about 10 3 -10 9 cells, such as intrauterine injection, vaginal administration of female animals, and the like. Typically these cells are contained in a pharmaceutically acceptable liquid medium. Cell administration can be repeated or continuous. In general, multiple administrations are usually administered at least 7-10 days apart. Another method is to seed the cells in a bioabsorbable material such as gelatin sponge, which is surgically implanted into the desired part of the uterus. The above two methods can be combined to achieve better curative effect.
- a bioabsorbable material such as gelatin sponge
- a suitable amount of amniotic epithelial cells will vary depending on the age, sex, weight, health, and other factors of the patient. Typically, doses per administration range from about 103-109 cells , typically about 106-107 cells.
- the present invention applies human amniotic epithelial cell hAECs to the treatment of intrauterine adhesion diseases for the first time, and has good effects, providing a new solution for the current treatment of such diseases.
- a method for isolating amniotic epithelial cells from amniotic tissue comprising the steps of:
- the washed amniotic membrane is digested with digestive enzymes, and the digested liquid is centrifuged to obtain human amniotic membrane epithelial cells.
- the amniotic epithelial cells of the present invention are derived from human.
- the amniotic membrane can be isolated from an isolated human placenta, rinsed with physiological buffer to remove blood cells, and mechanically removed from residual chorion and blood vessels. Isolation refers to the removal of cells from a tissue sample and separation from other tissue. Single cells are isolated from intact human amniotic epithelial layer tissue using any conventional technique or method including mechanical force (mincing or shearing), treatment with one or a combination of proteases such as collagenase, trypsin, lipoprotein Enzymes, liberase and pepsin for enzymatic digestion or a combination of mechanical and enzymatic methods.
- proteases such as collagenase, trypsin, lipoprotein Enzymes, liberase and pepsin for enzymatic digestion or a combination of mechanical and enzymatic methods.
- the human amniotic membrane should be obtained after the authorization of the puerpera.
- the placental tissue of a healthy puerpera after cesarean section is taken, the placenta is cut with a cross knife, and the whole amniotic membrane is obtained by mechanical separation.
- the human amniotic epithelial cells obtained in step (2) can be continuously cultured, and the preferred culture conditions are: cells are cultured at a density of 1 ⁇ 10 6 -1 ⁇ 10 8 cells/plate. It was inoculated into the culture medium and placed in a carbon dioxide incubator for culture. After the human amniotic epithelial cells adhered to the wall, the culture medium was changed. After the cells were overgrown, the cells were digested for cryopreservation.
- the present invention provides a method for hypoxic culture of human amniotic epithelial cells, the method comprising the steps of:
- the cells were digested for cryopreservation for future use.
- the present invention provides a method for culturing human amniotic epithelial cells under hypoxia, the method comprising the steps of:
- the cells were digested for cryopreservation for future use.
- the oxygen content in the air is about 21%.
- the oxygen concentration is between 19.5% and 23.5%, it belongs to the normal oxygen environment.
- the cells are evenly spread in the culture container, they are in a single and independent state. As the cells grow, they will spread to the surroundings to form clones one by one. Finally, the percentage of the area formed by cell clones to the total area of the culture container is the confluence % , representing the cell density.
- a method for culturing human amniotic epithelial cells under hypoxia is provided. , placed in a carbon dioxide incubator and cultured in a carbon dioxide incubator. After the human amniotic epithelial cells adhered to the wall, the culture medium was changed. After the cells reached a confluence of 70%-80%, they were placed in an environment with an oxygen concentration of 3%-5% for 24-36 hours. Digest for cryopreservation.
- the active cell population can be concentrated by other methods known to those skilled in the art. These post-processing wash/concentration steps can be performed separately or simultaneously.
- the viable cell population can be further purified or enriched to reduce foreign and dead cells after cell washing or culturing. Isolation of cells in suspension can be accomplished by 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 performing them can be found in the prior art and commercial products.
- basal medium used in the present invention, as long as it can be used for cell culture.
- Preferred media include DMEM media and NPBM media.
- preferred components include F-12, FCS, and neural survival factors.
- amniotic epithelial cells can be derived from amniotic epithelial cells isolated from amniotic tissue, and also include the aforementioned human amniotic epithelial cells cultured under hypoxia.
- amniotic epithelial cells prepared by the present invention can be formulated into amniotic epithelial cell preparations by adding other pharmaceutically acceptable carriers such as pharmaceutically acceptable solvents, suspending agents or excipients, which are beneficial to cell survival and can deliver the formulated cells to people or animals.
- pharmaceutically acceptable carriers such as pharmaceutically acceptable solvents, suspending agents or excipients, which are beneficial to cell survival and can deliver the formulated cells to people or animals.
- the present invention uses human amniotic epithelial cells for the treatment of intrauterine adhesion diseases, and fully utilizes the advantages of human amniotic epithelial cells, wherein human amniotic epithelial cells mainly have the following advantages:
- MHC type II molecules are hardly expressed on the cell surface, so it will not cause inflammation, allergic and immune reactions, and the requirements for transplantation matching are also reduced accordingly;
- telomerase reverse transcriptase No expression of telomerase reverse transcriptase, no tumorigenicity (including benign tumors, sarcomas and carcinomas);
- the fetal placenta by caesarean section was selected. Due to the stimulation of labor signals after term, the amniotic membrane will undergo apoptosis, so preterm fetal placenta (before 38 weeks) should be used. With the authorization and consent of the puerperae, the placenta tissue from healthy puerperae (serological reactions such as HIV, syphilis, hepatitis A, hepatitis B, hepatitis C, etc. were all negative) after caesarean section were taken, the placenta was cut with a cross knife, and the whole amniotic membrane was obtained by mechanical separation.
- CMF-HBSS containing 1X penicillin-streptomycin and amphotericin
- amniotic membrane was transferred to a new container, and 25ml of trypsin/EDTA was incubated in a 37°C water bath for 40min, and the digestion solution was stored.
- the amniotic membrane was transferred to a new container, and 25ml of pancreatin/EDTA was incubated in a 37°C water bath for 40min, and the digestion solution was stored.
- F12/DMEM containing 5% FBS, 1 ⁇ L-glutamic acid, 1 ⁇ pyruvate An equal volume of digestion stop solution (F12/DMEM containing 5% FBS, 1 ⁇ L-glutamic acid, 1 ⁇ pyruvate) was added, and centrifuged at 400 g for 10 min. Discard the liquid and use complete amniotic membrane medium: F12/DMEM containing 5% KSR (KnockOut Serum Replacement), 1xL-glutamine, 1xpyruvate, 1Xps (Penicillin-Streptomycin), and resuspend the pellet.
- KSR KnockOut Serum Replacement
- 1xL-glutamine 1xpyruvate
- 1Xps Penicillin-Streptomycin
- the cells on the culture dish were blown down in the same direction with a micropipette, transferred to a 15ml centrifuge tube, centrifuged at 300g for 3min, and then the cells were collected and counted.
- the human amniotic epithelial cells were recovered to a 10cm culture dish and cultured in a complete medium with 20% oxygen. When the confluence of the cells reached 70%-80%, the oxygen concentration was reduced to 3%-5% until the amnion cells proliferated and became full (generally). The required time is often 24-36h).
- the vaginal smear method is used to determine the estrous cycle.
- the proestrus phase there are round nucleated epithelial cells or a small amount of keratinocytes
- the estrus phase there are needle-like non-nucleated keratinocytes or round cells with serrated edges and a small amount of epithelial cells.
- Rats during estrus were modeled ( Figure 2). After the animals were anesthetized, the abdomen was opened, a transverse incision of about 2 mm was made on the uterus, and a curette was inserted to make a model by scratching.
- the abdominal cavity was flushed twice with warm saline, and the incision was sutured after confirming that there was no bleeding.
- the muscle layer and abdominal wall were sutured with 6-0 absorbable suture, the needle distance was 3mm, and the skin was sutured with 6-0 nylon suture. An appropriate amount of penicillin was injected postoperatively.
- amniotic epithelial cells Preparation of amniotic epithelial cells.
- Select healthy and uncontaminated amniotic epithelial cells (P1 or P2 generation, Figure 2), digest when the cell confluence is about 70%, gently wash with PBS twice to remove the cell culture medium, and resuspend 1 ⁇ 10 6 cells
- a cell suspension was prepared in 50 ⁇ L of sterile normal saline. Temporarily stored at 4°C and injected into animals within 1 h.
- the width of the uterine cavity was different, the color of the tissue was slightly darker, the appearance structure was completed, and the length was slightly shorter than that of the blank control group and the sham operation group. It was shown that there were multiple adhesions of varying degrees in the cavity.
- the uterine tissue was complete, the appearance was smooth and shiny, the width of the uterine horns was uniform, and the bilateral development was intact and healthy.
- the tissue was complete, the appearance was smooth and shiny, the width of the uterine horns was uniform up and down, the bilateral development was intact and the tissue was healthy, and there was no inflammatory infiltration during laparotomy. .
- HE staining After vertical embedding of the uterus, paraffin was made into 6 ⁇ m sections, dewaxed to water, and HE staining was used to make slices. The films were read under an optical microscope, observed with a low magnification microscope, photographed and the number of glands in each group was counted ( Figure 3).
- the results of HE staining showed that the uterine cavity of the IUA model group was severely adhered, the uterine cavity almost disappeared, the boundary between the myometrium and the endometrial layer was not clear, and the glands and other uterine cavity structures were severely damaged.
- the structure of the uterine cavity was complete, the area of the uterine cavity was significantly preserved, the boundary between the base layer and the endometrial layer was clear, but the thickness of the endometrial layer was slightly uneven; a certain number of glands were discernible and the distribution was uneven.
- the sham operation group had little difference in the area of the uterine cavity, with abundant glands, thick endometrial layer, clear boundaries between the muscle layer and the endometrial layer, and a complete structure.
- Masson staining The paraffin sections were dewaxed to water and then prepared by Masson staining. Read the film under an optical microscope, observe with a low-height microscope, and photograph.
- the experimental results showed that surgical modeling resulted in a large area of fibrotic phenotype in the uterus. After the treatment of amniotic epithelial cells, the fibrosis was relieved, especially the endometrial near the end of the uterine cavity, the degree of fibrosis was greatly reduced.
- tissue After the tissue was taken out from the body, it was quickly placed in PBS to wash away the blood stains, and the tissue was carefully cut into appropriate sizes (be careful not to forcefully pick the tissue). Tissues were placed in cassettes containing frozen section embedding medium (OCT). Place the cassettes in dry ice-cold isopentane until the OCT and tissue are completely frozen.
- OCT frozen section embedding medium
- the embedded tissue was fixed in a cryostat, cut into 0.5um sections, and the sections were attached to adhesive glass slides.
- the sections Before staining, the sections should be placed at room temperature for 30 minutes to make the sections fully adhere to the glass slides.
- the samples were placed in acetone at -20°C, and fixed for 10 min (if the tissue was fixed before embedding, proceed directly to subsequent operations).
- the slices were taken out and washed three times with PBS for 5 min each time.
- the tissue was first circled with an oil pen, and then the primary antibody (primary antibody was diluted with 5% HBS + 1% BSA in PBS) was dropped on the tissue, and placed in a wet box at 4°C overnight;
- the slices were taken out and washed three times with PBS for 5 min each time.
- DAPI was diluted with PBS, dropped onto the slices, incubated at room temperature for 3 min, and washed twice with PBS for 5 min each time;
- Example 6 Implantation of GFP-labeled human amniotic epithelial cells in rat uterus.
- the rats were treated with intrauterine injection of GFP-labeled amniotic epithelial cell suspension, euthanized after 7 days of treatment, and the uterus was dissected for GFP labeling detection.
- Rats were treated with intrauterine injection of GFP-labeled amniotic epithelial cell suspension 7 days after modeling, euthanized after 2h and 24h of treatment, and the uterus was dissected for GFP protein detection by ordinary fluorescent PCR (Fig. 7). ).
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Abstract
Provided is the use of a human amniotic epithelial cell (hAECS) in preparation of a drug for treating and/or alleviating intrauterine adhesion diseases.
Description
本发明属于生物技术领域，具体涉及人羊膜上皮细胞在治疗宫腔粘连疾病中的用途。The invention belongs to the field of biotechnology, in particular to the use of human amniotic epithelial cells in the treatment of intrauterine adhesion diseases.
在女性生育期子宫内膜可发生超过400次的增生、分化及脱落，显现出活跃而强大的再生能力。然而它又极易受损伤、炎症、内分泌等诸多因素的影响，导致子宫内膜修复障碍，造成子宫内膜基底层损伤、肌壁间相互粘连、宫腔正常解剖形态破坏即发生宫腔粘连(intrauterine adhesions，IUA)的发生。IUA发生率高达25％-30％，是妇科领域难治性疾病之一，引起包括月经减少甚至闭经、不孕、反复性流产、周期性腹痛以及妊娠后胎盘植入、胎儿生长受限、产后出血等一系列临床症状。近年来剖宫产后瘢痕子宫和重复人工流产所致的子宫严重机械损伤使IUA发生率逐年升高，已成为女性月经量减少、继发不孕的主要原因。在不孕患者中约有20％-30％的人存在IUA，而患有IUA的患者约有30％-60％合并不孕，严重影响了女性的正常生理和生育能力。然而，目前治疗和预防IUA的手段还很局限，需要从新的角度探索。During the female reproductive period, the endometrium can undergo more than 400 times of hyperplasia, differentiation and shedding, showing an active and powerful regeneration ability. However, it is easily affected by many factors such as injury, inflammation, endocrine, etc., which leads to endometrial repair obstacles, resulting in endometrial basal layer damage, mutual adhesion between muscle walls, and damage to the normal anatomical shape of the uterine cavity. The occurrence of intrauterine adhesions, IUA). The incidence of IUA is as high as 25%-30%, and it is one of the intractable diseases in the field of gynecology. Bleeding and other clinical symptoms. In recent years, the scarred uterus after cesarean section and the severe mechanical injury of the uterus caused by repeated artificial abortion have increased the incidence of IUA year by year, and it has become the main reason for female menstrual reduction and secondary infertility. About 20%-30% of infertile patients have IUA, and about 30%-60% of patients with IUA are infertile, which seriously affects the normal physiology and fertility of women. However, the current means of treating and preventing IUA are still limited and need to be explored from new perspectives.
既往研究发现，子宫内膜脱落后的再生从未被退化组织覆盖的裸露基底层腺体区域开始，新的腺上皮组织逐渐长出，并延伸整合入周围表面完整的区域。受损腺上皮创面愈合是一个复杂的生物学过程,包括局部低度炎症、细胞增殖分化和组织新生，由免疫细胞、修复细胞、细胞外基质以及细胞因子等多因素共同参与并高度协调、相互调控。正常情况下，子宫内膜损伤后，腺上皮细胞迅速再生使其达到完全的无瘢痕修复。而创伤、感染、低雌激素状态会破坏这种有序的过程,引起子宫内膜慢性难愈性创面形成并纤维化的发生，导致子宫内膜修复障碍，最终造成IUA。IUA的病理学特征表现为正常内膜变薄、萎缩，被纤维化的间质和无活性的立方柱状上皮替代，功能层和基底层不能辨别，残留少量甚至完全缺 乏正常有活性的内膜腺体等，导致内膜瘢痕性修复、粘连发生，腺体功能减退，对激素反应不佳，以及间质血管缺乏等。Previous studies have found that regeneration after endometrial shedding begins in areas of bare basal glands that are not covered by degenerated tissue, and new glandular epithelial tissue gradually grows and extends and integrates into areas with intact surrounding surfaces. The wound healing of damaged glandular epithelium is a complex biological process, including local low-grade inflammation, cell proliferation and differentiation, and tissue regeneration. regulation. Under normal circumstances, after endometrial injury, glandular epithelial cells regenerate rapidly to achieve complete scar-free repair. Trauma, infection, and low estrogen status will disrupt this orderly process, causing the formation of chronic and refractory endometrial wounds and the occurrence of fibrosis, resulting in endometrial repair obstacles and ultimately causing IUA. The pathological features of IUA are thinning and atrophy of the normal intima, replaced by fibrotic stroma and inactive cuboidal columnar epithelium, indistinguishable functional and basal layers, and a small or complete absence of normal active endometrial glands. Body, etc., leading to intimal scar repair, adhesions, glandular hypofunction, poor response to hormones, and lack of interstitial blood vessels.
IUA发生的机制尚不完全明确，但已有研究提示其宫腔感染和宫腔操作引起的损伤炎症是主要的引发因素，造成内膜屏障的破坏，内膜螺旋小动脉新生受阻，从而导致内膜再生的抑制。同时上皮坏死水肿及炎性细胞大量侵润，进而导致生长因子与雌孕激素受体产生减少,角质形成细胞和成纤维细胞迁移至损伤部位大量增殖，细胞外基质沉积形成纤维组织。这种以结构修复为主的病理过程反而侵占正常生理功能细胞的生态位，子宫内膜肌层不能为新生内膜提供足够营养支持，最终造成子宫内膜的纤维化和粘连。The mechanism of IUA is not completely clear, but studies have shown that intrauterine infection and inflammation caused by intrauterine operation are the main triggering factors, resulting in the destruction of the intimal barrier and the obstruction of the new intimal spiral arterioles, which leads to the Inhibition of membrane regeneration. At the same time, epithelial necrosis and edema and a large number of inflammatory cells infiltrated, which led to the reduction of the production of growth factors and estrogen and progesterone receptors, keratinocytes and fibroblasts migrated to the injured site and proliferated, and the extracellular matrix was deposited to form fibrous tissue. This pathological process, which is mainly based on structural repair, invades the ecological niche of normal physiological function cells, and the endometrial myometrium cannot provide sufficient nutritional support for the neo-endometrium, which eventually causes endometrial fibrosis and adhesion.
在损伤后的恢复期，子宫内膜腺上皮细胞仍旧面临脱落-再生的压力，造成进一步的修复障碍。孕激素撤退引起子宫内膜腺上皮螺旋小血管萎缩，子宫内膜腺上皮细胞氧化应激水平增大引发凋亡脱落本是生理过程的一部分。但在已经受损状态下子宫内膜腺上皮细胞因雌、孕激素受体减少，微小螺旋血管生长局限，新生的血管只能维持一小部分内膜的增厚，细胞凋亡引起更大范围的内膜损失。子宫内膜基质细胞在原本机械损伤坏死的基础上引起更多的肌成纤维细胞动员，细胞外基质大量分泌挤占功能细胞生态位，为子宫内膜腺上皮细胞的恢复再生不能提供很好的支持。During the recovery period after injury, endometrial glandular epithelial cells still face the pressure of exfoliation-regeneration, resulting in further repair obstacles. The withdrawal of progesterone causes the atrophy of the small spiral blood vessels of the endometrial glandular epithelium, and the increase in the level of oxidative stress in the endometrial glandular epithelial cells leads to apoptosis and shedding, which is part of the physiological process. However, in the damaged state of endometrial gland epithelial cells due to the reduction of estrogen and progesterone receptors, the growth of small spiral blood vessels is limited, the new blood vessels can only maintain a small part of the thickening of the endometrium, and apoptosis causes a larger area endometrial loss. Endometrial stromal cells cause more mobilization of myofibroblasts on the basis of the original mechanical damage and necrosis, and the massive secretion of extracellular matrix squeezes the functional cell niche, which cannot provide good support for the recovery and regeneration of endometrial gland epithelial cells. .
发明概述SUMMARY OF THE INVENTION
本发明所要解决的技术问题是针对现有宫腔粘连疾病治疗难题，提供新的治疗药物或方法。本发明提供了人羊膜上皮细胞(human amniotic epithelial cells，hAECs)或其细胞制剂用于治疗宫腔粘连疾病，并提供了从羊膜组织中分离人羊膜上皮细胞的方法。The technical problem to be solved by the present invention is to provide a new therapeutic drug or method for the treatment of the existing intrauterine adhesion disease. The present invention provides human amniotic epithelial cells (human amniotic epithelial cells, hAECs) or cell preparations thereof for treating intrauterine adhesion diseases, and provides a method for isolating human amniotic epithelial cells from amniotic tissue.
一方面，本发明提供了人羊膜上皮细胞(hAECs用于治疗宫腔粘连疾病的方法。本发明所述的方法不仅增加人羊膜上皮细胞的细胞治疗效果、减轻子宫内膜受损导致的子宫内膜菲薄与受损部位纤维化情况，同时也明显提高了新生内膜组织的雌孕激素生理响应功能，同时还发现生物活性物质处理过的hAECs对受损子宫内膜组织具有促进恢复的作用，能够用于治疗宫腔粘连病。In one aspect, the present invention provides a method for human amniotic epithelial cells (hAECs) for the treatment of intrauterine adhesion disease. The method described in the present invention not only increases the cell therapy effect of human amniotic epithelial cells, but also alleviates intrauterine damage caused by endometrial damage. Membrane thinness and fibrosis in damaged parts also significantly improved the physiological response function of estrogen and progesterone in neo-endometrial tissue. It was also found that hAECs treated with bioactive substances could promote the recovery of damaged endometrial tissue. Can be used to treat intrauterine adhesions.
另一方面，本发明提供了人羊膜上皮细胞(hAECs)用于治疗宫腔粘连疾病 的方法，其中人羊膜上皮细胞(hAECs)在培养阶段经低氧培养处理。经低氧培养的人羊膜上皮细胞(hAECs)治疗后，子宫内膜内的纤维化相关指标(TGF-β、Collagen Ⅰ、α-SMA)等有明显下降，同时子宫内膜功能性指标对于激素的响应程度明显上升，显示出突出的治疗效果，因此可以作为修复宫腔粘连病人因损伤导致的子宫内膜菲薄与纤维化，可以帮助患者增加怀孕可能。In another aspect, the present invention provides a method for treating intrauterine adhesion diseases with human amniotic epithelial cells (hAECs), wherein the human amniotic epithelial cells (hAECs) are subjected to hypoxic culture treatment during the culture stage. After the treatment of human amniotic epithelial cells (hAECs) cultured with hypoxia, the fibrosis-related indexes (TGF-β, Collagen I, α-SMA) in the endometrium were significantly decreased, and the functional indexes of the endometrium were significantly affected by hormones. The degree of response of the treatment has increased significantly, showing a prominent therapeutic effect, so it can be used to repair the thin endometrium and fibrosis caused by the injury of patients with intrauterine adhesions, which can help patients increase the possibility of pregnancy.
在本发明另一实施方案中，本发明提供了使用有效剂量的人羊膜上皮细胞或其细胞制剂用于治疗宫腔粘连疾病的方法，其中，所述的细胞制剂包括人羊膜上皮细胞和药学可接受的载体。In another embodiment of the present invention, the present invention provides a method for treating intrauterine adhesion disease using an effective dose of human amniotic epithelial cells or cell preparations thereof, wherein the cell preparations include human amniotic epithelial cells and pharmaceutically acceptable accepted vector.
在本发明另一实施方案中，羊膜上皮细胞的适合用量将根据患者的年龄、性别、体重、健康状况以及其它因素而改变。通常，每次给予的剂量范围为大约10
3-10
9个细胞，典型的是大约10
6-10
7个细胞。
In another embodiment of the present invention, the suitable amount of amniotic epithelial cells will vary depending on the age, sex, weight, health, and other factors of the patient. Typically, doses per administration range from about 103-109 cells , typically about 106-107 cells.
在本发明另一实施方案中，可以采用任意适合的方法将羊膜上皮细胞给予患者，例如宫腔注射、雌性动物阴道给药等。另外一种方法为将细胞种植在生物可吸收材料如明胶海绵里，采用手术将种有细胞的生物可吸收材料植入子宫所需的部位。上述两种方法可结合应用，可取得更好的疗效。In another embodiment of the present invention, the amniotic epithelial cells may be administered to a patient using any suitable method, such as intrauterine injection, vaginal administration to females, and the like. Another method is to seed the cells in a bioabsorbable material such as gelatin sponge, which is surgically implanted into the desired part of the uterus. The above two methods can be combined to achieve better curative effect.
另一方面，本发明提供了一种从羊膜组织中分离人羊膜上皮细胞的方法，所述方法包括以下步骤：In another aspect, the present invention provides a method for isolating human amniotic epithelial cells from amniotic tissue, the method comprising the steps of:
(1)从胎盘组织通过机械分离得到羊膜；(1) Obtaining the amniotic membrane from placental tissue by mechanical separation;
(2)清洗后的羊膜用消化酶进行消化，将消化后的液体进行离心，即可获得人羊膜上皮细胞。(2) The washed amniotic membrane is digested with digestive enzymes, and the digested liquid is centrifuged to obtain human amniotic membrane epithelial cells.
另一方面，本发明提供了低氧培养人羊膜上皮细胞的方法，所述方法包括以下步骤：In another aspect, the present invention provides a method for culturing human amniotic epithelial cells under hypoxia, the method comprising the steps of:
(1)将人羊膜上皮细胞复苏后接种于培养液中，正常氧气环境下培养；(1) Inoculate the human amniotic epithelial cells in a culture medium after resuscitation, and cultivate in a normal oxygen environment;
(2)待细胞汇合度达60％-90％后降低氧气浓度至1％-10％培养12-48小时。(2) After the cell confluence reaches 60%-90%, the oxygen concentration is reduced to 1%-10% and cultured for 12-48 hours.
经短时间低氧培养处理后的人羊膜上皮细胞在提高新生子宫内膜腺体数目、新生子宫内膜厚度方面作用具有显著性差异,同时子宫内膜功能性指标对于激素的响应程度明显上升，显示突出的治疗效果。由于低氧培养可以在不改变细胞干性前提下促进干细胞增殖，干细胞的增殖能力对于治疗的成功是极大的保障。Human amniotic epithelial cells treated with short-term hypoxic culture had significant differences in increasing the number of neonatal endometrial glands and neonatal endometrial thickness, and the response of endometrial functional indicators to hormones was significantly increased. Shows outstanding therapeutic effect. Since hypoxia culture can promote stem cell proliferation without changing the stemness of cells, the proliferation ability of stem cells is a great guarantee for the success of treatment.
附图简要说明Brief Description of Drawings
图1低氧环境对人羊膜上皮细胞的增殖能力(图1.1)、干性(图1.2)的影响。图1.1显示相比较于常氧培养，低氧培养后人羊膜上皮干细胞的增值速度有增加，最终的细胞数量要多于常氧培养组。图1.2显示相比于常氧培养组，低氧培养组的干细胞标志物SOX2、OCT4、nanog表达量有所上升，表现出更好的干性潜力。Fig. 1 The effect of hypoxic environment on the proliferation ability (Fig. 1.1) and stemness (Fig. 1.2) of human amniotic epithelial cells. Figure 1.1 shows that compared with normoxia culture, the proliferation rate of human amniotic epithelial stem cells increased after hypoxic culture, and the final number of cells was more than that of normoxia culture group. Figure 1.2 shows that the expression of stem cell markers SOX2, OCT4, and nanog in the hypoxia culture group increased compared with the normoxia culture group, showing better stemness potential.
图2模型建立和hAEC移植。图2空白对照组(A)、假手术组(B)、IUA模型组(C)、常氧环境下培养的人羊膜上皮细胞治疗组(D)、低氧培养后的人羊膜上皮细胞治疗组(E)，可以看出经治疗后子宫长度有所恢复，外观恢复光滑平整。Figure 2 Model establishment and hAEC transplantation. Figure 2 Blank control group (A), sham operation group (B), IUA model group (C), human amniotic epithelial cell treatment group (D) cultured under normoxia, and human amniotic epithelial cell treatment group after hypoxia culture (E), it can be seen that the length of the uterus has recovered after treatment, and the appearance is restored to smoothness.
图3不同组别子宫内膜的形态结构。图3空白对照组(A)、假手术组(B)、IUA模型组(C)、常氧环境下培养的人羊膜上皮细胞治疗组(D)、低氧培养后的人羊膜上皮细胞治疗组(E)，各组大鼠子宫的HE染色结果。各组的HE染色结果显示，低氧培养比常氧培养的人羊膜上皮细胞在促进新生子宫内膜增厚和腺体数量的增加方面更有优势，显示出更好的治疗结果。Figure 3. Morphological structure of endometrium in different groups. Figure 3 Blank control group (A), sham operation group (B), IUA model group (C), human amniotic epithelial cell treatment group (D) cultured under normoxia, and human amniotic epithelial cell treatment group after hypoxia culture (E), HE staining results of rat uterus in each group. The results of HE staining in each group showed that human amniotic epithelial cells cultured in hypoxia had more advantages in promoting the thickening of the neonatal endometrium and the increase in the number of glands than those cultured in normoxia, showing a better treatment outcome.
图4人血羊膜上皮细胞治疗对纤维化调节的影响。图4空白对照组(A)、假手术组(B)、IUA模型组(C)、常氧环境下培养的人羊膜上皮细胞治疗组(D)、低氧培养后的人羊膜上皮细胞治疗组(E)，经低氧培养的人羊膜上皮细胞治疗后，纤维化程度相比较其他各组都有明显的下降。Figure 4 Effects of human blood amniotic epithelial cell treatment on regulation of fibrosis. Figure 4 Blank control group (A), sham operation group (B), IUA model group (C), human amniotic epithelial cell treatment group (D) cultured under normoxia, and human amniotic epithelial cell treatment group after hypoxia culture (E), after the treatment of human amniotic epithelial cells cultured in hypoxia, the degree of fibrosis was significantly decreased compared with other groups.
图5人血羊膜上皮细胞治疗对子宫内膜血管生成和增殖影响(图5-1，5-2)。图5空白对照组(A)、假手术组(B)、IUA模型组(C)、常氧环境下培养的人羊膜上皮细胞治疗组(D)、低氧培养后的人羊膜上皮细胞治疗组(E)，经低氧培养的人羊膜上皮细胞治疗后，纤维化相关蛋白(TGF-β、α-SMA、Collagen Ⅰ)相比较其他各组都有明显的下降。血管新生(VEGF)与激素响应程度(p-ER)则表现出更高的表达，表现出更好的治疗潜力。Figure 5 Effects of human blood amniotic epithelial cell treatment on endometrial angiogenesis and proliferation (Figure 5-1, 5-2). Figure 5 Blank control group (A), sham operation group (B), IUA model group (C), human amniotic epithelial cell treatment group (D) cultured under normoxia, and human amniotic epithelial cell treatment group after hypoxia culture (E), compared with other groups, fibrosis-related proteins (TGF-β, α-SMA, Collagen I) were significantly decreased after the treatment of hypoxic cultured human amniotic epithelial cells. Angiogenesis (VEGF) and hormone response (p-ER) showed higher expression, showing better therapeutic potential.
图6绿色荧光蛋白标记人羊膜上皮细胞在大鼠子宫内的植入。图6免疫荧光结果显示，人羊膜上皮细胞在治疗过程中嵌合至受损子宫内膜层之中，表现了干细胞的治疗特性。Fig. 6 Implantation of GFP-labeled human amniotic epithelial cells in rat uterus. Figure 6 shows that human amniotic epithelial cells are chimeric into the damaged endometrial layer during the treatment process, showing the therapeutic properties of stem cells.
图7人羊膜上皮细胞在大鼠子宫内的停驻时间。图7PCR结果显示人羊膜上皮干细胞在子宫内的停留时间，显示出长时间的驻留并发挥相关治疗作用。Fig. 7 Residence time of human amniotic epithelial cells in rat uterus. Figure 7 PCR results showing the residence time of human amniotic epithelial stem cells in the uterus, showing a long-term residence and exerting a relevant therapeutic effect.
发明详述Detailed description of the invention
定义definition
除非另有说明，本发明的实施将采用分子生物学、微生物学、细胞生物学、生物化学和免疫学的常规技术，这些都在本领域的技术范围内，这些技术在本领域的技术文献和通用教科书中有充分解释。Unless otherwise stated, the practice of the present invention will employ conventional techniques of molecular biology, microbiology, cell biology, biochemistry and immunology, which are within the skill of the art, and which are found in the technical literature and Fully explained in general textbooks.
本文中所用的术语“羊膜”指的是包裹发育中的哺乳动物胚胎的最内层膜囊。在妊娠期间，胎儿由称作羊水的液体包围并缓冲。这种液体连同胚胎和胎盘被包裹在称作羊膜的囊中，该囊还覆盖脐带。羊膜包含维持稳态环境的羊水，保护胚胎环境不受外部环境影响。这种屏障还保护胚胎不受可能沿阴道上行并可能引起感染的生物(如细菌或病毒)的影响。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 encased in a sac called the amniotic membrane, which also covers the umbilical cord. The amniotic membrane contains amniotic fluid that maintains a homeostatic environment and protects the embryonic environment from the external environment. This barrier also protects the embryo from organisms, such as bacteria or viruses, that may travel up the vagina and potentially cause infection.
本文中所用的术语“胎盘”是指早产胎盘和足月胎盘二者。The term "placenta" as used herein refers to both preterm placenta and term placenta.
本文中所用的术语“人羊膜上皮细胞(human amniotic epithelial cells，hAECs)”是从胎盘上最靠近胎儿一侧的羊膜上分离而来的细胞。The term "human amniotic epithelial cells (hAECs)" as used herein are cells isolated from the amniotic membrane on the side of the placenta closest to the fetus.
本文中所用的术语“宫腔粘连(intrauterine adhesions，IUA)”又称Asheman综合征，是由于妊娠或非妊娠子宫的创伤，导致子宫内膜基底层受损，使宫腔部分或全部闭塞从而导致月经异常、不孕或反复流产等。其本质是内膜纤维化。As used herein, the term "intrauterine adhesions (IUA)", also known as Asheman syndrome, is caused by trauma to the pregnant or non-pregnant uterus, resulting in damage to the basal lining of the endometrium, resulting in partial or complete occlusion of the uterine cavity. Abnormal menstruation, infertility or repeated miscarriage. Its essence is intimal fibrosis.
本文中所用的术语“分离的”是指将材料从其原始环境取出，因此是“通过手工”从其天然状态改变而来的。The term "isolated" as used herein refers to the removal of material from its original environment, and thus has been altered "by hand" from its natural state.
本文中所用的术语“患者”和“受试者”表示用本发明提供的细胞或其组合物进行治疗的动物，通常是指哺乳动物，优选是人。对于特定动物患者例如人类患者所特有的感染，病状和疾病状态的治疗，术语患者指的是该特定的动物。The terms "patient" and "subject" as used herein refer to an animal, usually a mammal, preferably a human, being treated with the cells or compositions provided herein. For the treatment of infections, conditions and disease states specific to a particular animal patient, eg, a human patient, the term patient refers to that particular animal.
本文中所用的术语“治疗”是指向具有患病风险或处于疾病状态的患者提供益处的任何行为，所述疾病状态可通过本发明所述细胞组合物或细胞制剂改善。文中使用的治疗包括预防和治疗性处理。As used herein, the term "treatment" refers to any action that provides benefit to a patient at risk for a disease or in a disease state that can be ameliorated by the cellular compositions or cell preparations described herein. Treatment as used herein includes prophylactic and therapeutic management.
本文中所用的术语“制剂”指所处形式使得允许其中含有的活性组分的生物学活性是有效的，且不含对受试者具有不可接受的毒性的其他成分的组合物。As used herein, the term "formulation" refers to a composition in a form that allows the biological activity of the active ingredient contained therein to be effective, and is free of other ingredients that would have unacceptable toxicity to the subject.
本文中所用的术语“药学可接受载体”指药物制剂中与活性成分不同，且对受试者无毒的组分。药学可接受载体包括但不限于缓冲剂、赋形剂、稳定剂、或防腐剂等。The term "pharmaceutically acceptable carrier" as used herein refers to a component of a pharmaceutical formulation that is distinct from the active ingredient and that is not toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives, and the like.
本文中所用的术语“有效量”是指足以改善或防止医学疾病的症状或病症的量。有效量还意指足以使得可以诊断或促进诊断的量。对具体受治疗者的有效量可视多种因素而变化，例如待治疗的疾病、患者的整体健康状况、给药的方法途径和剂量及副作用的严重性。有效量可为避免显著副作用或毒性作用的最大剂量或给药方案。The term "effective amount" as used herein refers to an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical disease. An effective amount also means an amount sufficient to enable or facilitate diagnosis. The effective amount for a particular subject may vary depending on factors such as the disease being treated, the general health of the patient, the method, route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
本文中所用的术语“汇合度”代表细胞密度。细胞在培养容器中中增殖时，出现在细胞间的粘连和排列状态，达到某种状态用汇合度％表示，代表细胞密度。例如当细胞平均铺在培养皿中，呈单个独立的状态，随着细胞生长，就会向周围平铺，形成一个一个克隆，最后细胞克隆形成的面积占培养总面积的百分比即为细胞汇合度％。The term "confluency" as used herein refers to cell density. When cells proliferate in a culture vessel, they appear in a state of adhesion and arrangement between cells, and when a certain state is reached, the degree of confluence is expressed as %, which represents the cell density. For example, when the cells are evenly spread in a culture dish, they are in a single and independent state. As the cells grow, they will spread around to form clones one by one. Finally, the percentage of the area formed by cell clones to the total culture area is the cell confluence. %.
本发明的各个方面将在下述内容中进一步详细描述。Various aspects of the invention are described in further detail below.
治疗宫腔粘连疾病Treatment of intrauterine adhesions
恢复子宫内膜细胞丰度与功能的干细胞移植是现今治愈难治性宫腔粘连的唯一手段。由于羊膜上皮细胞具有抑制炎症反应降低纤维化程度，恢复组织新生的功能，因此本申请发明人希望利用羊膜上皮细胞上述特点，探寻其是否对宫腔粘连病具有治疗效应。Stem cell transplantation to restore endometrial cell abundance and function is currently the only cure for refractory intrauterine adhesions. Since amniotic epithelial cells have the functions of inhibiting inflammatory responses, reducing fibrosis and restoring tissue regeneration, the inventors of the present application hope to use the above characteristics of amniotic epithelial cells to explore whether they have therapeutic effects on intrauterine adhesions.
一方面，本发明提供了人羊膜上皮细胞(hAECs)或其细胞制剂用于治疗和/或改善宫腔粘连疾病的用途。In one aspect, the present invention provides the use of human amniotic epithelial cells (hAECs) or cell preparations thereof for treating and/or improving intrauterine adhesion disease.
目前，宫腔粘连(intrauterine adhesions，IUA)首选有效的治疗方法为手术治疗-宫腔镜宫腔粘连分离术(transcervical resection of adhesion，TCRA)，但术后仅能尽量恢复宫腔形态，并在分离粘连过程中新鲜创面的再次形成，正常子宫内膜的减少，子宫内膜基底层受到破坏，干细胞受损或缺失，几乎不再具有再生功能，因此治疗后复发率可高达62.5％，妊娠成功率仅为22.5％-33.3％。同时，临床上也尝试了多种其他方案控制和预防粘连，包括：宫内节育器、支撑球囊、生物胶类材料等的宫腔内放置。虽然这些手段和措施不断的改进和革新，但是并未能明显改善IUA的发生和手术治疗后的复发率，对患者妊娠 结局及月经状况也未能明显改善。At present, the preferred and effective treatment for intrauterine adhesions (IUA) is surgical treatment-hysteroscopic transcervical resection of adhesion (TCRA). Re-formation of fresh wounds during separation of adhesions, reduction of normal endometrium, destruction of endometrial basal layer, damage or loss of stem cells, and almost no longer have regenerative functions, so the recurrence rate after treatment can be as high as 62.5%, and pregnancy is successful The rate is only 22.5%-33.3%. At the same time, a variety of other solutions have been tried clinically to control and prevent adhesions, including intrauterine placement of intrauterine devices, support balloons, and bio-glue materials. Although these means and measures have been continuously improved and innovated, they have not significantly improved the occurrence of IUA and the recurrence rate after surgical treatment, and the pregnancy outcome and menstrual status of patients have not been significantly improved.
通过系列研究，发明人惊喜地发现，本发明所述的方法不仅增加人羊膜上皮细胞的细胞治疗效果、减轻子宫内膜受损导致的子宫内膜菲薄与受损部位纤维化情况，同时也明显提高了新生内膜组织的雌孕激素生理响应功能，同时还发现生物活性物质处理过的hAECs对受损子宫内膜组织具有促进恢复的作用，能够用于治疗宫腔粘连病。因此本申请发明人认为hAECs是治疗宫腔粘连病的有效方法，可以作为该类疾病细胞治疗的候选来源，因此完成了本发明。Through a series of studies, the inventors were pleasantly surprised to find that the method of the present invention not only increases the cell therapy effect of human amniotic epithelial cells, reduces the thin endometrial and fibrosis of damaged parts caused by endometrial damage, but also significantly The physiological response function of estrogen and progesterone of neonatal endometrial tissue is improved, and it is also found that hAECs treated with bioactive substances can promote the recovery of damaged endometrial tissue, and can be used for the treatment of intrauterine adhesions. Therefore, the inventors of the present application believe that hAECs are an effective method for the treatment of intrauterine adhesions, and can be used as a candidate source for cell therapy for this type of disease, thus completing the present invention.
人羊膜上皮细胞(human amniotic epithelial cells，hAECs)是从胎盘上最靠近胎儿一侧的羊膜上分离而来的细胞。胎盘属于孕妇生产后的废弃物，故此来源的细胞不存在伦理问题。Human amniotic epithelial cells (hAECs) are cells isolated from the amniotic membrane on the side of the placenta closest to the fetus. The placenta belongs to the waste of pregnant women after giving birth, so there are no ethical issues with the cells from this source.
胎盘在解剖学上，从内向外主要可分为三层组织结构:羊膜上皮层,绒毛膜(chorion),和子宫蜕膜(decidua)而每一层组织的来源却截然不同。子宫蜕膜来自母体，绒毛膜源自滋养层(trophoblast)，而羊膜上皮层来自受精后八天的上胚层(epiblast)，即与胚胎干细胞一样(embryonic stem cells，ESCs)都来源于胚胎内细胞团(inner cell mass)，而hAECs在分化为羊膜上皮后，能长期保持多潜能性。Miki等证实hAECs可以表达多能干细胞(如胚胎干细胞)主要的表面maker(如Oct4，Sox2，Nanog,SSEA-3,SSEA-4等),表明它具有胚胎干细胞所特有的向三个胚层组织分化的潜能。体外分化实验显示，无论从转录水平还是表达水平均显示hAECs具有体外三胚层分化潜能。然而与ESCs不同的是，hAECs体内成畸胎瘤实验却成阴性，主要原因是因为其无端粒酶活性，因此无法分化成具有三胚层分化的组织结构。也正因如此hAECs用作细胞治疗，不具有致瘤性(包括良性瘤、肉瘤和癌)。Anatomically, the placenta can be divided into three layers from the inside out: the amniotic epithelium, the chorion, and the decidua, and the origin of each layer is completely different. The decidua of the uterus is derived from the mother, the chorion is derived from the trophoblast, and the amniotic epithelium is derived from the epiblast at eight days after fertilization, that is, like embryonic stem cells (ESCs) are derived from embryonic cells. Inner cell mass, hAECs can maintain pluripotency for a long time after differentiation into amniotic epithelium. Miki et al. confirmed that hAECs can express the main surface maker (such as Oct4, Sox2, Nanog, SSEA-3, SSEA-4, etc.) of pluripotent stem cells (such as embryonic stem cells), indicating that it has the unique differentiation of embryonic stem cells into the three germ layers. potential. In vitro differentiation experiments showed that hAECs had the potential to differentiate into three germ layers in vitro, both at the transcriptional and expression levels. However, unlike ESCs, hAECs were negative in the in vivo teratoma test, mainly because they had no telomerase activity and thus could not differentiate into tissue structures with three germ layers. Because of this, hAECs are used as cell therapy and are not tumorigenic (including benign tumors, sarcomas and carcinomas).
人羊膜上皮细胞具有干细胞旁分泌功能，能够分泌FGF、VEGF、EGF、HGH等多种细胞生长因子；NGF、BDNF、NT-3等多种神经营养因子；多巴胺、儿茶酚胺、乙酰胆碱、去甲肾上腺素、组胺、五羟色胺、尿紧张素、神经紧张素等多种神经递质与调质。既往已有研究证明，人羊膜上皮细胞作为滋养层有良好的应用效果，如临床已证实人羊膜上皮细胞作为滋养层可促进角膜缘上皮细胞的增殖和干性维持。Human amniotic epithelial cells have stem cell paracrine function and can secrete various cell growth factors such as FGF, VEGF, EGF, HGH; NGF, BDNF, NT-3 and other neurotrophic factors; dopamine, catecholamine, acetylcholine, norepinephrine , histamine, serotonin, urotensin, neurotensin and other neurotransmitters and conditioners. Previous studies have shown that human amniotic epithelial cells have good application effects as trophoblasts. For example, it has been clinically confirmed that human amniotic epithelial cells as trophoblasts can promote the proliferation and stemness maintenance of corneal limbal epithelial cells.
同时，hAECs细胞表面几乎不表达MHCII型分子，不会引起炎症、过敏和免 疫反应，因此免疫原性很低，适合移植细胞治疗。hAECs分离过程较为简单，在刮洗以后的羊膜上除了少量血液细胞团块几乎不存在其他类型细胞污染，而血液细胞在未受刺激的情况下属于悬浮细胞，因此可以通过细胞培养后，换液去除。根据发明人和国外科学家实验统计，每张人的羊膜约有8000万到3亿个hAECs细胞。在EGF存在的条件下，hAECs具有较强的增殖能力，约36小时可以增殖一个代次，并能在前代次(约10代内)保持旺盛的增殖能力。这些优势保证了本申请能够获取足够数量单一细胞类型,以满足临床治疗的要求。At the same time, hAECs hardly express MHC II molecules on the surface of cells and do not cause inflammation, allergic and immune responses, so they have low immunogenicity and are suitable for transplantation cell therapy. The separation process of hAECs is relatively simple. Except for a small amount of blood cell clumps, there is almost no other type of cell contamination on the amniotic membrane after scraping. The blood cells are suspension cells in the unstimulated condition, so they can be cultured and replaced by medium. remove. According to the experimental statistics of the inventor and foreign scientists, there are about 80 million to 300 million hAECs cells in each human amniotic membrane. In the presence of EGF, hAECs have strong proliferation ability, can proliferate for one passage in about 36 hours, and can maintain strong proliferation ability in the previous passage (within about 10 passages). These advantages ensure that the application can obtain a sufficient number of single cell types to meet the requirements of clinical treatment.
本发明公开了人羊膜上皮细胞或其细胞制剂在制备治疗和/或改善宫腔粘连疾病的药物中的用途。在本发明优选的实施方案中，可使用有效剂量的人羊膜上皮细胞或其细胞制剂进行治疗和/或改善宫腔粘连疾病。有效剂量是指足以改善或防止医学疾病的症状或病症的量。对具体受治疗者的有效量可视多种因素而变化，例如待治疗的疾病、患者的整体健康状况、给药的方法途径和剂量及副作用的严重性。有效量可为避免显著副作用或毒性作用的最大剂量或给药方案。The invention discloses the use of human amniotic epithelial cells or cell preparations thereof in preparing medicines for treating and/or improving intrauterine adhesion diseases. In a preferred embodiment of the present invention, an effective dose of human amniotic epithelial cells or cell preparations thereof can be used to treat and/or ameliorate intrauterine adhesion disease. An effective dose refers to an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical disease. The effective amount for a particular subject may vary depending on factors such as the disease being treated, the general health of the patient, the method, route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
在发明的另一实施方案中，发明人还惊讶的发现，经短时间低氧培养处理后的人羊膜上皮细胞在提高新生子宫内膜腺体数目、新生子宫内膜厚度方面作用具有显著性差异(p<0.05),经低氧培养的人羊膜上皮细胞治疗后，子宫内膜内的纤维化相关指标(TGF-β、Collagen Ⅰ、α-SMA)等有明显下降，同时子宫内膜功能性指标对于激素的响应程度明显上升，显出突出的治疗效果。In another embodiment of the invention, the inventors have also surprisingly found that human amniotic epithelial cells treated with short-term hypoxia culture have significant differences in increasing the number of neonatal endometrial glands and neonatal endometrial thickness (p<0.05), after the treatment of human amniotic epithelial cells cultured with hypoxia, the fibrosis-related indexes (TGF-β, Collagen Ⅰ, α-SMA) in the endometrium were significantly decreased, and the endometrial functional The response of the indicators to hormones increased significantly, showing a prominent therapeutic effect.
由于低氧培养可以在不改变细胞干性前提下促进干细胞增殖，干细胞的增殖能力对于治疗的成功是极大的保障。而低氧培养处理的人羊膜上皮细胞(hAECs)具有干性和低免疫原性，因此可以作为修复宫腔粘连病人因损伤导致的子宫内膜菲薄与纤维化，改善新生子宫内膜组织对于雌孕激素的敏感度，这可以帮助患者增加怀孕可能，获得更大的生育机会。故而，低氧培养的hAECs具有的生物学功效与安全性更能够满足临床治疗的需要，低氧培养的人羊膜上皮细胞或其细胞制剂可用于治疗和/或改善宫腔粘连疾病。。Since hypoxia culture can promote stem cell proliferation without changing the stemness of cells, the proliferation ability of stem cells is a great guarantee for the success of treatment. Hypoxic culture-treated human amniotic epithelial cells (hAECs) have dryness and low immunogenicity, so they can be used to repair endometrial thinness and fibrosis caused by injury in patients with intrauterine adhesions, and improve neonatal endometrial tissue. Sensitivity to progesterone, which can help patients increase their chances of becoming pregnant and have a greater chance of having children. Therefore, the biological efficacy and safety of hAECs cultured in hypoxia can better meet the needs of clinical treatment, and human amniotic epithelial cells or cell preparations cultured in hypoxia can be used to treat and/or improve intrauterine adhesion diseases. .
在本发明另一实施方案中，所述的具有宫腔粘连疾病的动物是指哺乳动物。在更为优选的实施方案中，所述的动物为牛、马、羊、猴子、狗、大鼠、小鼠、兔子或人类。在最优选的实施方案中，所述的具有宫腔粘连疾病的动物是指人类。In another embodiment of the present invention, the animal with intrauterine adhesion disease refers to a mammal. In a more preferred embodiment, the animal is a cow, horse, sheep, monkey, dog, rat, mouse, rabbit or human. In the most preferred embodiment, the animal with intrauterine adhesion disease refers to a human.
在发明的另一实施方案中，所述的细胞制剂包括人羊膜上皮细胞和药学可接受的载体。本发明所述的药学可接受的载体是指适合用于人和/或动物，无过度的不良副作用(如毒性、刺激性和过敏反应)的具有合适的有益/风险率的物质，例如药学可接受的溶剂、悬浮剂或赋形剂，有利于细胞存活，能递送所配制的细胞到人或动物。载体依合适地计划的给药方式而选择。本发明的载体包括但不限于各种生理缓冲液，如生理盐水、磷酸缓冲液、人工脑脊液或是全血清、脐带血清等。还可包括各种人工支架，包括但不限于明胶海绵、聚乙醇酸(PGA)、聚乳酸(PLA)及它们的共聚物等。In another embodiment of the invention, the cell preparation includes human amniotic epithelial cells and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier of the present invention refers to a substance with suitable benefit/risk rate that is suitable for use in humans and/or animals without excessive adverse side effects (such as toxicity, irritation and allergic reaction), for example, a pharmaceutically acceptable carrier. Acceptable solvents, suspending agents, or excipients, which facilitate cell survival, are capable of delivering formulated cells to humans or animals. The carrier is selected for the appropriately planned 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, and the like. Various artificial scaffolds may also be included, including but not limited to gelatin sponge, polyglycolic acid (PGA), polylactic acid (PLA) and their copolymers, and the like.
考虑到要治疗的疾病的类型，本领域的技术人员可以适当选择羊膜上皮细胞的合适状态：未经任何处理的收集的细胞(粗提部分)；部分纯化的细胞；纯化的细胞然后经培养扩增。Those skilled in the art can appropriately select the appropriate state of the amniotic epithelial cells in consideration of the type of disease to be treated: collected cells without any treatment (crude fraction); partially purified cells; purified cells and then expanded by culture increase.
可以采用子宫内原位给药的方法将羊膜上皮细胞给予患者，每次给予的剂量范围为大约10
3-10
9数量级细胞，例如宫腔注射、雌性动物阴道给药等。通常这些细胞是包含在药学上可接受的液体培养基中。细胞给予可以重复或连续进行。一般而言，多重给药方式通常要间隔至少7-10天分别使用。另外一种方法为将细胞种植在生物可吸收材料如明胶海绵里，采用手术将种有细胞的生物可吸收材料植入子宫所需的部位。上述两种方法可结合应用，可取得更好的疗效。
The amniotic epithelial cells can be administered to patients by in situ administration in utero, and the dose range for each administration is about 10 3 -10 9 cells, such as intrauterine injection, vaginal administration of female animals, and the like. Typically these cells are contained in a pharmaceutically acceptable liquid medium. Cell administration can be repeated or continuous. In general, multiple administrations are usually administered at least 7-10 days apart. Another method is to seed the cells in a bioabsorbable material such as gelatin sponge, which is surgically implanted into the desired part of the uterus. The above two methods can be combined to achieve better curative effect.
羊膜上皮细胞的适合用量将根据患者的年龄、性别、体重、健康状况以及其它因素而改变。通常，每次给予的剂量范围为大约10
3-10
9数量级细胞，典型的是大约10
6-10
7数量级细胞。
A suitable amount of amniotic epithelial cells will vary depending on the age, sex, weight, health, and other factors of the patient. Typically, doses per administration range from about 103-109 cells , typically about 106-107 cells.
综上，本发明首次将人羊膜上皮细胞hAECs应用于宫腔粘连疾病的治疗中，并且具有良好的效果，为当前该类疾病的治疗提供了一种新的方案。In conclusion, the present invention applies human amniotic epithelial cell hAECs to the treatment of intrauterine adhesion diseases for the first time, and has good effects, providing a new solution for the current treatment of such diseases.
羊膜上皮细胞的制备Preparation of Amniotic Epithelial Cells
在发明的一个优选实施方案中，提供了一种从羊膜组织中分离羊膜上皮细胞的方法，所述方法包括以下步骤：In a preferred embodiment of the invention, a method for isolating amniotic epithelial cells from amniotic tissue is provided, the method comprising the steps of:
(1)从胎盘组织通过机械分离得到羊膜；(1) Obtaining the amniotic membrane from placental tissue by mechanical separation;
(2)清洗后的羊膜用消化酶进行消化，将消化后的液体进行离心，即可获得人羊膜上皮细胞。(2) The washed amniotic membrane is digested with digestive enzymes, and the digested liquid is centrifuged to obtain human amniotic membrane epithelial cells.
本发明所述的羊膜上皮细胞来源于人类。可从离体的人胎盘分离羊膜，采用 生理缓冲液冲洗除去血细胞，机械剔除残留绒毛膜和血管。分离指的是从组织样品中移出细胞且与另外的组织分开。使用任何常规技术或方法从完整人羊膜上皮层组织分离出单细胞，这些技术或方法包括机械力(切碎力或剪切力)，用一种或组合的蛋白酶例如胶原酶、胰蛋白酶、脂肪酶、释放酶(liberase)和胃蛋白酶进行酶消化或机械和酶方法的组合。The amniotic epithelial cells of the present invention are derived from human. The amniotic membrane can be isolated from an isolated human placenta, rinsed with physiological buffer to remove blood cells, and mechanically removed from residual chorion and blood vessels. Isolation refers to the removal of cells from a tissue sample and separation from other tissue. Single cells are isolated from intact human amniotic epithelial layer tissue using any conventional technique or method including mechanical force (mincing or shearing), treatment with one or a combination of proteases such as collagenase, trypsin, lipoprotein Enzymes, liberase and pepsin for enzymatic digestion or a combination of mechanical and enzymatic methods.
在本发明一个优选的实施方案中，人羊膜的获取应经产妇授权同意后，取健康产妇剖腹产后的胎盘组织，十字刀切割胎盘，通过机械分离得到整张羊膜。In a preferred embodiment of the present invention, the human amniotic membrane should be obtained after the authorization of the puerpera. The placental tissue of a healthy puerpera after cesarean section is taken, the placenta is cut with a cross knife, and the whole amniotic membrane is obtained by mechanical separation.
在本发明另一个优选的实施方案中，可对步骤(2)中获得人羊膜上皮细胞继续培养，优选的培养条件为：以1×10
6-1×10
8个细胞/平板的密度将细胞接种于培养液中，放置于二氧化碳培养箱中培养，待人羊膜上皮细胞贴壁后换培养液，待细胞长满后将细胞消化下来进行冻存。
In another preferred embodiment of the present invention, the human amniotic epithelial cells obtained in step (2) can be continuously cultured, and the preferred culture conditions are: cells are cultured at a density of 1×10 6 -1×10 8 cells/plate. It was inoculated into the culture medium and placed in a carbon dioxide incubator for culture. After the human amniotic epithelial cells adhered to the wall, the culture medium was changed. After the cells were overgrown, the cells were digested for cryopreservation.
在发明的另一实施方案中，本发明提供了低氧培养人羊膜上皮细胞的方法，所述方法包括以下步骤：In another embodiment of the invention, the present invention provides a method for hypoxic culture of human amniotic epithelial cells, the method comprising the steps of:
(1)将人羊膜上皮细胞复苏后接种于培养液中，正常氧气环境下培养；(1) Inoculate the human amniotic epithelial cells in a culture medium after resuscitation, and cultivate in a normal oxygen environment;
(2)待细胞汇合度达60％-90％后降低氧气浓度至1％-10％培养12-48小时。(2) After the cell confluence reaches 60%-90%, the oxygen concentration is reduced to 1%-10% and cultured for 12-48 hours.
培养结束后，将细胞消化下来进行冻存，备用。After the culture, the cells were digested for cryopreservation for future use.
在发明的另一优选实施方案中，本发明提供了低氧培养人羊膜上皮细胞的方法，所述方法包括以下步骤：In another preferred embodiment of the invention, the present invention provides a method for culturing human amniotic epithelial cells under hypoxia, the method comprising the steps of:
(1)将人羊膜上皮细胞复苏后接种于培养液中，正常氧气环境下培养；(1) Inoculate the human amniotic epithelial cells in a culture medium after resuscitation, and cultivate in a normal oxygen environment;
(2)待细胞汇合度达70％-80％后降低氧气浓度至3％-5％培养24-36小时。(2) After the cell confluence reaches 70%-80%, the oxygen concentration is reduced to 3%-5% and cultured for 24-36 hours.
培养结束后，将细胞消化下来进行冻存，备用。After the culture, the cells were digested for cryopreservation for future use.
正常情况下，空气中含氧量为21％左右，当氧气浓度在19.5％至23.5％之间属于正常氧气环境。当细胞平均铺在培养容器中，呈单个独立的状态，随着细胞生长，就会向周围平铺，形成一个一个克隆，最后细胞克隆形成的面积占培养容器总面积的百分比即为汇合度％，代表细胞密度。Under normal circumstances, the oxygen content in the air is about 21%. When the oxygen concentration is between 19.5% and 23.5%, it belongs to the normal oxygen environment. When the cells are evenly spread in the culture container, they are in a single and independent state. As the cells grow, they will spread to the surroundings to form clones one by one. Finally, the percentage of the area formed by cell clones to the total area of the culture container is the confluence % , representing the cell density.
在发明的特别优选的实施方案中，提供了低氧培养人羊膜上皮细胞的方法，优选的培养条件为：以1×10
6-1×10
8个细胞/平板的密度将细胞接种培养液中，放置于二氧化碳培养箱中培养，待人羊膜上皮细胞贴壁后换培养液，细胞汇合度达70％-80％后放入氧气浓度3％-5％的环境中培养24-36h，之后将细胞消化下来进 行冻存。
In a particularly preferred embodiment of the invention, a method for culturing human amniotic epithelial cells under hypoxia is provided. , placed in a carbon dioxide incubator and cultured in a carbon dioxide incubator. After the human amniotic epithelial cells adhered to the wall, the culture medium was changed. After the cells reached a confluence of 70%-80%, they were placed in an environment with an oxygen concentration of 3%-5% for 24-36 hours. Digest for cryopreservation.
本领域技术人员可用已知的其它方法将活性细胞群体浓缩。这些加工后洗涤/浓缩步骤可单独或同时施行。除了上述方法以外，还可在细胞洗涤后或培养后，进一步纯化或富集活性细胞群体，减少杂细胞和死细胞。分离悬浮液中的细胞可通过以下技术来实现：浮力密度沉降离心、有差别的与固相的粘着和从固相上洗脱、免疫磁性珠、荧光激光细胞分选(FACS)或其它技术。这些不同技术以及进行这些技术的装置的实例可参见现有技术和上市商品。The active cell population can be concentrated by other methods known to those skilled in the art. These post-processing wash/concentration steps can be performed separately or simultaneously. In addition to the methods described above, the viable cell population can be further purified or enriched to reduce foreign and dead cells after cell washing or culturing. Isolation of cells in suspension can be accomplished by 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 performing them can be found in the prior art and commercial products.
对本发明使用的基础培养基的类型没有限制，只要可用于细胞培养的培养基即可。优选的培养基包括DMEM培养基和NPBM培养基。对上面提到的基础培养基中可能含有的其他组份类型没有限制，优选的成分包括F-12、FCS和神经存活因子等。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 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, and neural survival factors.
考虑到要治疗的疾病的类型，本领域的技术人员可以适当选择羊膜上皮细胞的合适状态：未经任何处理的收集的细胞(粗提部分)；部分纯化的细胞；纯化的细胞然后经培养扩增。所述的羊膜上皮细胞可来源于从羊膜组织中分离出的羊膜上皮细胞，也包括前述经低氧培养的人羊膜上皮细胞。Those skilled in the art can appropriately select the appropriate state of the amniotic epithelial cells in consideration of the type of disease to be treated: collected cells without any treatment (crude fraction); partially purified cells; purified cells and then expanded by culture increase. Said amniotic epithelial cells can be derived from amniotic epithelial cells isolated from amniotic tissue, and also include the aforementioned human amniotic epithelial cells cultured under hypoxia.
对于本发明制备的羊膜上皮细胞可通过添加其他药学上可接受的载体例如药学可接受的溶剂、悬浮剂或赋形剂等配制成羊膜上皮细胞制剂，有利于细胞存活，能递送所配制的细胞到人或动物。The amniotic epithelial cells prepared by the present invention can be formulated into amniotic epithelial cell preparations by adding other pharmaceutically acceptable carriers such as pharmaceutically acceptable solvents, suspending agents or excipients, which are beneficial to cell survival and can deliver the formulated cells to people or animals.
本发明将人羊膜上皮细胞用于宫腔粘连疾病的治疗，充分发挥了人羊膜上皮细胞的优点，其中人羊膜上皮细胞主要具有以下几个方面的优势：The present invention uses human amniotic epithelial cells for the treatment of intrauterine adhesion diseases, and fully utilizes the advantages of human amniotic epithelial cells, wherein human amniotic epithelial cells mainly have the following advantages:
(1)能长期保持多潜能性并具有胚胎干细胞所特有的向三个胚层组织分化的潜能；(1) It can maintain pluripotency for a long time and has the unique potential of embryonic stem cells to differentiate into three germ layers;
(2)细胞表面几乎不表达MHCII型分子，因此不会引起炎症、过敏和免疫反应，移植配型要求也相应降低；(2) MHC type II molecules are hardly expressed on the cell surface, so it will not cause inflammation, allergic and immune reactions, and the requirements for transplantation matching are also reduced accordingly;
(3)具有调节体内和体外免疫反应的能力，在体外培养时能分泌多种免疫调节因子、抗血管生成蛋白或抗炎因子相关蛋白；(3) It has the ability to regulate immune responses in vivo and in vitro, and can secrete a variety of immune regulatory factors, anti-angiogenic proteins or anti-inflammatory factor-related proteins when cultured in vitro;
(4)具有低免疫原性，可以看作是免疫赦免细胞，无抗原呈递的功能，移植后可减少免疫细胞来源，避免免疫排斥反应的发生；(4) It has low immunogenicity and can be regarded as immune amnesty cells without antigen-presenting function. After transplantation, it can reduce the source of immune cells and avoid the occurrence of immune rejection;
(5)不表达端粒酶逆转录酶，没有致瘤性(包括良性瘤、肉瘤和癌)；(5) No expression of telomerase reverse transcriptase, no tumorigenicity (including benign tumors, sarcomas and carcinomas);
(6)具有较强的增殖能力，并能在前代次(约10代内)保持旺盛的增殖能力；(6) It has strong proliferation ability, and can maintain strong proliferation ability in the previous generation (about 10 generations);
(7)来源广泛，取材容易，没有应用限制，不存在伦理问题。(7) Wide range of sources, easy to obtain materials, no application restrictions, and no ethical issues.
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照本领域常规方法和条件，或按照商品说明书选择。The present invention is further described below by way of examples, but the present invention is not limited to the scope of the described examples. In the following examples, the experimental methods without specific conditions are selected according to the conventional methods and conditions in the art, or according to the product description.
实施例1 原代羊膜上皮细胞分离及培养Example 1 Isolation and culture of primary amniotic epithelial cells
1、人羊膜的来源1. The source of human amniotic membrane
为了避免产道微生物污染，选用了剖腹产胎儿胎盘。由于足月后分娩信号刺激，羊膜会发生凋亡，因此宜用早产胎儿胎盘(38周以前)。经产妇授权同意后，取健康产妇(HIV、梅毒、甲肝、乙肝、丙肝等血清学反应均显示为阴性)剖腹产后的胎盘组织，十字刀切割胎盘，通过机械分离得到整张羊膜。In order to avoid microbial contamination of the birth canal, the fetal placenta by caesarean section was selected. Due to the stimulation of labor signals after term, the amniotic membrane will undergo apoptosis, so preterm fetal placenta (before 38 weeks) should be used. With the authorization and consent of the puerperae, the placenta tissue from healthy puerperae (serological reactions such as HIV, syphilis, hepatitis A, hepatitis B, hepatitis C, etc. were all negative) after caesarean section were taken, the placenta was cut with a cross knife, and the whole amniotic membrane was obtained by mechanical separation.
2、hAECs的分离(全程要求无菌操作)2. Separation of hAECs (the whole process requires aseptic operation)
获取39周以前剖腹产婴儿胎盘，从胎盘内面剥离羊膜，将之浸入含有F12/DMEM(含1X青霉素-链霉素和两性霉素)基础培养基的离心管中。4℃冷链运送至实验室细胞间。The placenta of infants delivered by caesarean section before 39 weeks was obtained, and the amniotic membrane was stripped from the inner surface of the placenta and immersed in a centrifuge tube containing F12/DMEM (containing 1X penicillin-streptomycin and amphotericin) basal medium. 4°C cold chain transport to laboratory cells.
将羊膜取出，并将每张羊膜置于40ml CMF-HBSS(含1X青霉素-链霉素和两性霉素)中清洗去粘液，并用镊子刮去贴近绒毛膜层的间充质层及粘液，重复3次，每次洗涤均换新容器和新HBSS液。Remove the amniotic membranes, and place each amniotic membrane in 40ml of CMF-HBSS (containing 1X penicillin-streptomycin and amphotericin) to wash and remove the mucus, and scrape off the mesenchymal layer and mucus close to the chorionic layer with tweezers, repeat 3 times, with a new container and new HBSS solution for each wash.
将洗净的羊膜转入新容器，加10ml 0.05％胰酶/EDTA，颠倒30s，弃液。Transfer the washed amniotic membrane to a new container, add 10ml of 0.05% trypsin/EDTA, invert for 30s, and discard the solution.
将羊膜转入新容器，加20ml 0.05％胰酶/EDTA，37℃水浴孵育10min弃液。Transfer the amniotic membrane to a new container, add 20 ml of 0.05% trypsin/EDTA, and incubate in a 37°C water bath for 10 min to discard the solution.
羊膜转入新容器，25ml胰酶/EDTA 37℃水浴孵育40min，保存消化液。The amniotic membrane was transferred to a new container, and 25ml of trypsin/EDTA was incubated in a 37°C water bath for 40min, and the digestion solution was stored.
初次消化后羊膜转入新容器，25ml胰酶/EDTA 37℃水浴孵育40min，保存消化液。After the initial digestion, the amniotic membrane was transferred to a new container, and 25ml of pancreatin/EDTA was incubated in a 37°C water bath for 40min, and the digestion solution was stored.
加入等体积消化终止液(F12/DMEM含5％FBS，1xL-谷氨酸，1x丙酮酸)，400g离心10min。弃液，用羊膜完全培养基：F12/DMEM含5％KSR(KnockOut Serum Replacement)，1xL-谷氨酰胺，1x丙酮酸,1Xps(Penicillin-Streptomycin), 重悬沉淀。An equal volume of digestion stop solution (F12/DMEM containing 5% FBS, 1×L-glutamic acid, 1×pyruvate) was added, and centrifuged at 400 g for 10 min. Discard the liquid and use complete amniotic membrane medium: F12/DMEM containing 5% KSR (KnockOut Serum Replacement), 1xL-glutamine, 1xpyruvate, 1Xps (Penicillin-Streptomycin), and resuspend the pellet.
加入等体积消化终止液，400g离心10min。弃液，完全培养基重悬沉淀。An equal volume of digestion stop solution was added and centrifuged at 400 g for 10 min. Discard the liquid and resuspend the pellet in complete medium.
过100um筛，计数，以10
5cells/cm2接种至培养皿或置于冻存液(90％FBS,10％DMSO)中液氮冻存备用。
Passed through a 100um sieve, counted, and inoculated to a petri dish at 10 5 cells/cm2 or placed in a freezing medium (90% FBS, 10% DMSO) for cryopreservation in liquid nitrogen.
3、hAECs的接种培养及冻存3. Inoculation, culture and cryopreservation of hAECs
细胞培养：接种1×10
7个细胞后，待hAECs贴壁后换培养液，之后三天换一次培养液。
Cell culture: After inoculating 1×10 7 cells, the culture medium was changed after the hAECs adhered, and the culture medium was changed every three days after that.
待细胞长满平板后将细胞消化下来进行冻存：15cm dish加5ml胰酶，10min后镜下观察，细胞变圆且平面晃动平皿时细胞全变为悬浮状态时加等量消化终止液终止消化。用微量移液器按同一方向将培养皿上的细胞吹下，移入15ml离心管，300g离心3min后收集细胞然后进行细胞计数。在冻存管内加入冻存液，标明冻存日期、批次及细胞数量之后将细胞放入冻存管，然后立刻将冻存管放入冻存盒中并将冻存盒放进-80℃冰箱，12h后取出冻存盒，将细胞移入液氮罐中保存。Digest the cells for cryopreservation after the cells are overgrown with the plate: add 5ml of trypsin to 15cm dish, observe under a microscope after 10min, add the same amount of digestion stop solution to stop the digestion when the cells become round and the plate is shaken and the cells all become suspended. . The cells on the culture dish were blown down in the same direction with a micropipette, transferred to a 15ml centrifuge tube, centrifuged at 300g for 3min, and then the cells were collected and counted. Add the freezing solution to the cryovial, indicate the date of cryopreservation, the batch and the number of cells, then put the cells into the cryovial, then immediately put the cryovial into the freezing box and put the freezing box into -80℃ Refrigerator, take out the freezing box after 12 hours, and transfer the cells to liquid nitrogen tank for preservation.
实施例2 人羊膜上皮细胞的预处理Example 2 Pretreatment of Human Amniotic Epithelial Cells
将人羊膜上皮细胞复苏至10cm培养皿，完全培养基20％氧气环境下培养，待细胞汇合度达70％-80％后降低氧气浓度至3％-5％，直至羊膜细胞增殖长满(一般所需时常为24-36h)。The human amniotic epithelial cells were recovered to a 10cm culture dish and cultured in a complete medium with 20% oxygen. When the confluence of the cells reached 70%-80%, the oxygen concentration was reduced to 3%-5% until the amnion cells proliferated and became full (generally). The required time is often 24-36h).
结果显示：经低氧处理过的人羊膜上皮细胞的增殖能力有较大上升，干性未发生改变(图1)。同时，低氧处理的人羊膜上皮干细胞在提高新生子宫内膜腺体数目、新生子宫内膜厚度方面作用具有显著性差异(p<0.05),经低氧培养的人羊膜上皮细胞治疗后，子宫内膜内的纤维化相关指标(TGF-β、Collagen Ⅰ、α-SMA)等有明显下降，同时子宫内膜功能性指标对于激素的响应程度明显上升。显出突出的治疗效果。The results showed that the proliferation ability of human amniotic epithelial cells treated with hypoxia was greatly increased, and the dryness did not change (Figure 1). At the same time, the effects of hypoxia-treated human amniotic epithelial stem cells in increasing the number of neonatal endometrial glands and neonatal endometrial thickness were significantly different (p<0.05). Endometrial fibrosis-related indicators (TGF-β, Collagen I, α-SMA) were significantly decreased, while the response of endometrial functional indicators to hormones was significantly increased. Shows outstanding therapeutic effect.
实施例3 宫腔粘连模型的建立与羊膜上皮细胞注射Example 3 Establishment of intrauterine adhesion model and injection of amniotic epithelial cells
1.大鼠宫腔粘连IUA造模1. IUA modeling of intrauterine adhesions in rats
阴道涂片法确定动情周期，动情前期均为圆形有核上皮细胞间或少量角化 细胞，动情期均为针样的无核角化细胞或锯齿样边缘的圆形细胞兼有少量上皮细胞。取动情期间的大鼠建模(图2)。麻醉动物后后开腹，在子宫上开一个约2mm的横切口，伸入刮匙进行搔刮造模。确保所有的器官都回到它们的解剖位置后用温热的生理盐水冲洗腹腔两遍，在确认无出血点之后缝合切口。6-0可吸收线缝合肌层和腹壁，针距3mm,6-0尼龙缝线缝合皮肤。术后注射适当量青霉素。The vaginal smear method is used to determine the estrous cycle. In the proestrus phase, there are round nucleated epithelial cells or a small amount of keratinocytes, and in the estrus phase, there are needle-like non-nucleated keratinocytes or round cells with serrated edges and a small amount of epithelial cells. Rats during estrus were modeled (Figure 2). After the animals were anesthetized, the abdomen was opened, a transverse incision of about 2 mm was made on the uterus, and a curette was inserted to make a model by scratching. After ensuring that all organs were returned to their anatomical positions, the abdominal cavity was flushed twice with warm saline, and the incision was sutured after confirming that there was no bleeding. The muscle layer and abdominal wall were sutured with 6-0 absorbable suture, the needle distance was 3mm, and the skin was sutured with 6-0 nylon suture. An appropriate amount of penicillin was injected postoperatively.
2.羊膜上皮细胞宫腔内注射2. Intrauterine Injection of Amniotic Epithelial Cells
羊膜上皮细胞的准备。选取健康状态良好无污染的羊膜上皮细胞(P1或P2代，图2)，于细胞汇合度约70％时消化，PBS轻柔清洗两次以去除细胞培养液，取1×10
6个细胞重悬于50μL无菌生理盐水制成细胞悬液。4℃暂时保存，于1h内注射至动物体内。
Preparation of amniotic epithelial cells. Select healthy and uncontaminated amniotic epithelial cells (P1 or P2 generation, Figure 2), digest when the cell confluence is about 70%, gently wash with PBS twice to remove the cell culture medium, and resuspend 1×10 6 cells A cell suspension was prepared in 50 μL of sterile normal saline. Temporarily stored at 4°C and injected into animals within 1 h.
羊膜上皮细胞的注射。造模后一周麻醉动物，消毒备皮，延原先缝合处剪开组织开腹，尽量减小动物损伤。开一小口暴露子宫。将细胞悬液小心吸入1mL无菌医用注射针筒(针头换成最细的头皮针头)，轻微弹去气泡，每侧宫角给予1×10
6个羊膜上皮细胞。于子宫角约中间靠近卵巢一侧进针，确保针头达到子宫腔内后轻轻推入细胞悬液，在针头退出组织时用镊子夹住进针口帮助组织收缩，确保没有多余的液体渗出。将子宫放回腹腔，缝合。将未醒动物头低脚高放回笼内，并观察术后动物状态。(图2)
Injection of amniotic epithelial cells. One week after the modeling, the animals were anesthetized, the skin was sterilized, and the tissue was cut along the original sutures to open the abdomen to minimize animal damage. A small opening is made to expose the uterus. The cell suspension was carefully inhaled into a 1 mL sterile medical syringe (the needle was replaced with the thinnest scalp needle), the air bubbles were slightly deflected, and 1×10 6 amniotic epithelial cells were administered to each uterine horn. Insert the needle at about the middle of the uterine horn near the ovary. Make sure the needle reaches the uterine cavity and gently push the cell suspension. When the needle exits the tissue, use tweezers to hold the needle inlet to help the tissue shrink and ensure that no excess fluid leaks out. . The uterus is placed back into the abdominal cavity and sutured. The awake animals were put back into the cage with their heads lowered, and the state of the animals after the operation was observed. (figure 2)
结果显示：视野下可见羊膜上皮细胞治疗组，组织结构完整，外表光滑有光泽，子宫角上下宽度均一，近宫口处稍有水肿，其余与空白对照与假手术组无异。而宫腔粘连(IUA)组，组织略水肿相较于对照组，宫腔宽窄不一，组织外表颜色稍深，外观结构完成整，长度较空白对照组与假手术组稍短，后续切片结果显示，腔内有程度不一的多处粘连。空白对照子宫组织完整，外表光滑有光泽，子宫角上下宽度均一，双侧发育均完好组织健康。假手术组组织完整，外表光滑有光泽，子宫角上下宽度均一，双侧发育均完好组织健康，开腹时未见炎症浸润，后续研究结果发现假手术并未对实验鼠子宫结构和功能造成影响。The results showed that the amniotic epithelial cells in the treatment group were visible under the visual field. The tissue structure was complete, the appearance was smooth and shiny, the width of the upper and lower uterine horns was uniform, and there was slight edema near the cervix. The rest were no different from the blank control and the sham operation group. In the intrauterine adhesion (IUA) group, the tissue was slightly edematous. Compared with the control group, the width of the uterine cavity was different, the color of the tissue was slightly darker, the appearance structure was completed, and the length was slightly shorter than that of the blank control group and the sham operation group. It was shown that there were multiple adhesions of varying degrees in the cavity. In the blank control, the uterine tissue was complete, the appearance was smooth and shiny, the width of the uterine horns was uniform, and the bilateral development was intact and healthy. In the sham operation group, the tissue was complete, the appearance was smooth and shiny, the width of the uterine horns was uniform up and down, the bilateral development was intact and the tissue was healthy, and there was no inflammatory infiltration during laparotomy. .
经后续石蜡切片检测发现IUA模型组子宫腔多处粘连，造模成功。而空白对照组，假手术组，IUA羊膜上皮治疗组子宫内未发现粘连。各组子宫外观大体一致，外表光滑完整无破损溃烂等病理变化。外观不能成为评价内部粘连与否和严重程度的评价标准，本实验各组均未对子宫除内腔之外的部位产生损伤。Subsequent detection of paraffin sections revealed multiple adhesions in the uterine cavity of the IUA model group, and the modeling was successful. In the blank control group, the sham operation group, and the IUA amniotic epithelium treatment group, no intrauterine adhesions were found. The appearance of the uterus in each group was generally the same, and the appearance was smooth and complete without pathological changes such as breakage and ulceration. Appearance cannot be used as an evaluation standard for evaluating the internal adhesion and its severity. In this experiment, no damage was caused to the parts of the uterus other than the inner cavity of the uterus in each group.
实施例4 组织学染色Example 4 Histological staining
1.HE染色：垂直包埋子宫后将石蜡制成6μm切片，脱腊至水，采用HE染色制片。于光学显微镜下读片，以低倍镜观察，摄片并统计各组腺体数目(图3)。1. HE staining: After vertical embedding of the uterus, paraffin was made into 6 μm sections, dewaxed to water, and HE staining was used to make slices. The films were read under an optical microscope, observed with a low magnification microscope, photographed and the number of glands in each group was counted (Figure 3).
HE染色结果可见：IUA造模组宫腔严重粘连，子宫内腔几乎消失，肌层与内膜层界限不清晰，腺体和其他宫腔组织结构破坏严重。羊膜上皮细胞治疗组宫腔结构完整，子宫内腔面积显著保留，基层与内膜层界限清晰，但内膜层厚度略有不均一；有一定数量腺体可辨，分布不均匀。正常组宫腔结构完整，子宫内膜丰厚，内膜结构有序，腺体结构清晰分布均匀，肌层与内膜层分界清晰，子宫内腔较大。而假手术组与空白对照组相比子宫内腔面积差异不大，腺体丰富，内膜层较厚，肌层与内膜层界限清晰，结构完整。The results of HE staining showed that the uterine cavity of the IUA model group was severely adhered, the uterine cavity almost disappeared, the boundary between the myometrium and the endometrial layer was not clear, and the glands and other uterine cavity structures were severely damaged. In the amniotic epithelial cell treatment group, the structure of the uterine cavity was complete, the area of the uterine cavity was significantly preserved, the boundary between the base layer and the endometrial layer was clear, but the thickness of the endometrial layer was slightly uneven; a certain number of glands were discernible and the distribution was uneven. In the normal group, the structure of the uterine cavity was complete, the endometrium was rich, the structure of the endometrium was orderly, the structure of the glands was clear and evenly distributed, the boundary between the myometrium and the endometrium was clear, and the endometrium was larger. Compared with the blank control group, the sham operation group had little difference in the area of the uterine cavity, with abundant glands, thick endometrial layer, clear boundaries between the muscle layer and the endometrial layer, and a complete structure.
2.子宫内膜腺体数量与内膜厚度定量：采用ImageJ图像处理软件测量子宫内膜内腺体数量子宫内膜厚度(即子宫内膜基层交界处至宫腔的垂直距离，每张切片随机选取5个部位，取其平均值作为子宫内膜厚度)。2. Quantification of the number of endometrial glands and endometrial thickness: ImageJ image processing software was used to measure the number of endometrial glands and endometrial thickness (ie the vertical distance from the junction of the endometrial base to the uterine cavity, each slice was randomly selected) 5 sites were selected and the average value was taken as the endometrial thickness).
实验结果显示，IUA造模后，大鼠子宫内膜厚度明显降低，腺体显著减少；经羊膜上皮细胞治疗后，子宫内膜厚度显著增加，但各部位增幅程度不一，腺体数目也有所恢复，但与健康子宫有一定差距(图3)。假手术组和空白对照组相比没有显著性差异，假手术并没有对大鼠子宫结构功能产生影响。The experimental results showed that after IUA modeling, the endometrial thickness and glands of the rats were significantly reduced; after treatment with amniotic epithelial cells, the endometrial thickness was significantly increased, but the degree of increase was different in each part, and the number of glands was also different. Recovery, but there is a certain gap with the healthy uterus (Figure 3). There was no significant difference between the sham operation group and the blank control group, and the sham operation did not affect the structure and function of the rat uterus.
3.Masson染色：石蜡切片脱腊至水后采用Masson染色制片。于光学显微镜下读片，以低高镜观察，摄片。3. Masson staining: The paraffin sections were dewaxed to water and then prepared by Masson staining. Read the film under an optical microscope, observe with a low-height microscope, and photograph.
测量子宫内膜纤维化程度：经Masson染色后每张切片取四个高倍镜视野观察子宫内膜纤维化程度，采用ImageJ图像处理软件计算每个高倍镜视野下纤维化面积占总内膜面积的百分比，计算平均值(图4)。To measure the degree of endometrial fibrosis: After Masson staining, four high-power fields of view were taken from each section to observe the degree of endometrial fibrosis, and ImageJ image processing software was used to calculate the proportion of the fibrosis area in each high-power field of view to the total endometrial area Percentages were calculated as mean values (Figure 4).
实验结果显示，手术造模造成子宫内大面积的纤维化表型。在羊膜上皮细胞治疗后，纤维化有所缓解，特别是子宫内膜靠近宫腔一端，纤维化程度大大降低。The experimental results showed that surgical modeling resulted in a large area of fibrotic phenotype in the uterus. After the treatment of amniotic epithelial cells, the fibrosis was relieved, especially the endometrial near the end of the uterine cavity, the degree of fibrosis was greatly reduced.
实施例5 组织免疫荧光Example 5 Tissue Immunofluorescence
组织自体内取出后，迅速置于用PBS中洗去血污，小心将组织切分为合适大小(注意不要用力捏取组织)。将组织置于装有冰冻切片包埋剂(OCT)的包埋盒 中。将包埋盒置于干冰预冷的异戊烷中，直至OCT和组织完全冻结。After the tissue was taken out from the body, it was quickly placed in PBS to wash away the blood stains, and the tissue was carefully cut into appropriate sizes (be careful not to forcefully pick the tissue). Tissues were placed in cassettes containing frozen section embedding medium (OCT). Place the cassettes in dry ice-cold isopentane until the OCT and tissue are completely frozen.
将包埋好的组织固定于冰冻切片机，切为0.5um的切片，并将切片贴于粘附性载玻片上。The embedded tissue was fixed in a cryostat, cut into 0.5um sections, and the sections were attached to adhesive glass slides.
切片染色前需室温放置30min，以使切片与玻片充分粘附。Before staining, the sections should be placed at room temperature for 30 minutes to make the sections fully adhere to the glass slides.
将样品放于丙酮中，-20℃，固定10min(若组织在包埋前已固定，则直接进行后续操作)。The samples were placed in acetone at -20°C, and fixed for 10 min (if the tissue was fixed before embedding, proceed directly to subsequent operations).
切片取出，用PBS洗3次，每次5min。The slices were taken out and washed three times with PBS for 5 min each time.
组织先用油性笔圈住，后在组织上滴加一抗(一抗用5％HBS+1％BSA的PBS稀释)，置于湿盒4℃过夜；The tissue was first circled with an oil pen, and then the primary antibody (primary antibody was diluted with 5% HBS + 1% BSA in PBS) was dropped on the tissue, and placed in a wet box at 4°C overnight;
切片取出，用PBS洗3次，每次5min；The slices were taken out and washed 3 times with PBS, 5 min each time;
除去切片表明水分，滴加荧光偶联二抗(用5％HBS+1％BSA的PBS稀释)，置于湿盒室温避光孵育1h。(此步开始的后续操作均避光操作)Remove the moisture indicated by the section, add fluorescently conjugated secondary antibody (diluted with 5% HBS + 1% BSA in PBS) dropwise, and incubate in a humid box at room temperature for 1 h in the dark. (Subsequent operations from this step are all protected from light)
切片取出，用PBS洗3次，每次5min。The slices were taken out and washed three times with PBS for 5 min each time.
DAPI用PBS稀释后，滴加至切片上，室温孵育3min，后用PBS清洗2次，每次5min；DAPI was diluted with PBS, dropped onto the slices, incubated at room temperature for 3 min, and washed twice with PBS for 5 min each time;
封片液封片，荧光显微镜镜检(图5)。The slides were sealed with mounting fluid and examined by a fluorescence microscope (Figure 5).
实施例6 绿色荧光蛋白标记人羊膜上皮细胞在大鼠子宫内的植入。Example 6 Implantation of GFP-labeled human amniotic epithelial cells in rat uterus.
在大鼠造模术后7天后予以宫腔内注射具有GFP标记的羊膜上皮细胞悬液治疗，在治疗7天后予以安乐死处理，剖取子宫后进行GFP标记检测。After 7 days of modeling, the rats were treated with intrauterine injection of GFP-labeled amniotic epithelial cell suspension, euthanized after 7 days of treatment, and the uterus was dissected for GFP labeling detection.
结果显示在子宫恢复期间人羊膜上皮细胞能够存留并发挥其生物学作用(图6)。The results showed that human amniotic epithelial cells were able to persist and exert their biological roles during uterine recovery (Figure 6).
实施例7 人羊膜上皮细胞在子宫内的停驻Example 7 Intrauterine resident of human amniotic epithelial cells
在大鼠造模术后7天后予以宫腔内注射具有GFP标记的羊膜上皮细胞悬液治疗，在治疗2h与24h后予以安乐死处理，剖取子宫后进行GFP蛋白的普通荧光PCR检测(图7)。Rats were treated with intrauterine injection of GFP-labeled amniotic epithelial cell suspension 7 days after modeling, euthanized after 2h and 24h of treatment, and the uterus was dissected for GFP protein detection by ordinary fluorescent PCR (Fig. 7). ).
结果显示在子宫恢复期间人羊膜上皮细胞能够存留并发挥其生物学作用。The results show that human amniotic epithelial cells are able to persist and exert their biological roles during uterine recovery.
Claims (26)
- 人羊膜上皮细胞(hAECs)或其细胞制剂用于治疗和/或改善宫腔粘连疾病的用途。Use of human amniotic epithelial cells (hAECs) or cell preparations thereof for treating and/or improving intrauterine adhesion diseases.
- 根据权利要求1所述的用途，其特征在于：所述的人羊膜上皮细胞(hAECs)经过低氧培养处理。The use according to claim 1, wherein the human amniotic epithelial cells (hAECs) are subjected to hypoxia culture treatment.
- 根据权利要求1或2所述的用途，其特征在于：所述的具有宫腔粘连疾病的动物是指哺乳动物。The use according to claim 1 or 2, wherein the animal with intrauterine adhesion disease refers to a mammal.
- 根据权利要求3所述的用途，其特征在于：所述的动物为牛、马、羊、猴子、狗、大鼠、小鼠、兔子或人类。The use according to claim 3, wherein the animal is a cow, a horse, a sheep, a monkey, a dog, a rat, a mouse, a rabbit or a human.
- 根据权利要求4所述的用途，其特征在于：所述的动物是指人类。The use according to claim 4, wherein the animal is a human.
- 根据权利要求1所述的用途，其特征在于：所述的细胞制剂包括人羊膜上皮细胞和药学可接受的载体。The use according to claim 1, wherein the cell preparation comprises human amniotic epithelial cells and a pharmaceutically acceptable carrier.
- 根据权利要求1或2所述的用途，其特征在于：所述的羊膜上皮细胞的合适状态为未经任何处理的收集的细胞、部分纯化的细胞或纯化的细胞然后经培养扩增。The use according to claim 1 or 2, wherein the suitable state of the amniotic epithelial cells is the collected cells without any treatment, partially purified cells or purified cells and then cultured and expanded.
- 根据权利要求1或2所述的用途，其特征在于：可以采用子宫内原位给药的方法将羊膜上皮细胞给予患者，每次给予的剂量范围为大约10 3-10 9数量级细胞。 The use according to claim 1 or 2, characterized in that the amniotic epithelial cells can be administered to the patient by in situ administration in utero, and the dose range for each administration is about 10 3 -10 9 cells.
- 根据权利要求8所述的用途，其特征在于：可以采用子宫内原位给药的方法将羊膜上皮细胞给予患者，每次给予的剂量范围为大约大约10 6-10 7数量级细胞。 8. The use of claim 8 , wherein the amniotic epithelial cells can be administered to the patient by in situ administration in utero, in a dose range of about 106-107 cells per administration.
- 根据权利要求8所述的用途，其特征在于：所述的子宫内原位给药方法为宫腔注射、雌性动物阴道给药等。The use according to claim 8, wherein the intrauterine in situ administration method is intrauterine injection, female animal vaginal administration and the like.
- 根据权利要求8所述的用途，其特征在于：所述的子宫内原位给药方法为将细胞种植在生物可吸收材料里，采用手术将种有细胞的生物可吸收材料植入子宫所需的部位。The use according to claim 8, characterized in that: the method of in situ administration in uterus is to plant cells in bioabsorbable materials, and implant the bioabsorbable materials with cells into the uterus by surgery. part.
- 根据权利要求1所述的用途，其特征在于：所述的羊膜上皮细胞由包括以下步骤的方法制备得到：The use according to claim 1, wherein the amniotic epithelial cells are prepared by a method comprising the following steps:(1)从胎盘组织通过机械分离得到羊膜；(1) Obtaining the amniotic membrane from placental tissue by mechanical separation;(2)清洗后的羊膜用消化酶进行消化，将消化后的液体进行离心，即可获得人羊膜上皮细胞。(2) The washed amniotic membrane is digested with digestive enzymes, and the digested liquid is centrifuged to obtain human amniotic membrane epithelial cells.
- 根据权利要求12所述的用途，其特征在于：所述方法步骤(1)可从离体的人胎盘分离羊膜，采用生理缓冲液冲洗除去血细胞，机械剔除残留绒毛膜和血管。The use according to claim 12, wherein the method step (1) can separate the amniotic membrane from the isolated human placenta, rinse with a physiological buffer to remove blood cells, and mechanically remove residual chorion and blood vessels.
- 根据权利要求13所述的用途，其特征在于：人羊膜的获取应经产妇授权同意后，取健康产妇剖腹产后的胎盘组织，通过机械分离得到整张羊膜。The use according to claim 13, wherein the human amniotic membrane should be obtained with the authorization and consent of the puerpera, and the placental tissue of the healthy puerperae after cesarean section is taken, and the whole amniotic membrane is obtained by mechanical separation.
- 根据权利要求12所述的用途，其特征在于：所述方法步骤(2)使用任何常规技术或方法从完整人羊膜上皮层组织分离出单细胞，这些技术或方法包括机械力，用一种或组合的选自胶原酶、胰蛋白酶、脂肪酶、释放酶和胃蛋白酶的蛋白酶进行酶消化或机械和酶方法的组合。The use according to claim 12, wherein the method step (2) uses any conventional technique or method to isolate single cells from intact human amniotic epithelium tissue, these techniques or methods include mechanical force, use one or A combined protease selected from collagenase, trypsin, lipase, libidase and pepsin performs enzymatic digestion or a combination of mechanical and enzymatic methods.
- 根据权利要求15所述的用途，其特征在于：对步骤2中获得人羊膜上皮细胞继续培养，优选的培养条件为：以1×10 6-1×10 8个细胞/平板的密度将细胞接种于培养皿中，放置于二氧化碳培养箱中培养，待人羊膜上皮细胞贴壁后换培养液，待细胞长满平板后将细胞消化下来进行冻存。 The use according to claim 15, characterized in that the human amniotic epithelial cells obtained in step 2 are continued to be cultured, and the preferred culture conditions are: inoculate the cells at a density of 1×10 6 -1×10 8 cells/plate In a petri dish, placed in a carbon dioxide incubator for culture, after the human amniotic epithelial cells adhered to the wall, the culture medium was changed, and the cells were digested and cryopreserved after the cells were covered with the plate.
- 根据权利要求16所述的用途，其特征在于：培养人羊膜上皮细胞的培养基为DMEM培养基或NPBM培养基。The use according to claim 16, wherein the medium for culturing human amniotic epithelial cells is DMEM medium or NPBM medium.
- 根据权利要求14所述的用途，其特征在于：可在基础培养基中添加F-12、FCS或神经存活因子等。The use according to claim 14, characterized in that: F-12, FCS or nerve survival factor and the like can be added to the basal medium.
- 根据权利要求1-18任一项所述的用途，其特征在于，所述的羊膜上皮细胞由包括以下步骤的方法经低氧培养制备得到：The use according to any one of claims 1-18, wherein the amniotic epithelial cells are prepared by hypoxic culture by a method comprising the following steps:(1)将人羊膜上皮细胞复苏后接种于培养液中，正常氧气环境下培养；(1) Inoculate the human amniotic epithelial cells in a culture medium after resuscitation, and cultivate in a normal oxygen environment;(2)待细胞汇合度达60％-90％后降低氧气浓度至1％-10％培养12-48小时。(2) After the cell confluence reaches 60%-90%, the oxygen concentration is reduced to 1%-10% and cultured for 12-48 hours.
- 根据权利要求19所述的用途，其特征在于：所述的羊膜上皮细胞由包括以下步骤的方法经低氧培养制备得到：The use according to claim 19, wherein the amniotic epithelial cells are prepared by hypoxic culture by a method comprising the following steps:(1)将人羊膜上皮细胞复苏后接种于培养液中，正常氧气环境下培养；(1) Inoculate the human amniotic epithelial cells in a culture medium after resuscitation, and cultivate in a normal oxygen environment;(2)待细胞汇合度达70％-80％后降低氧气浓度至3％-5％培养24-36小时。(2) After the cell confluence reaches 70%-80%, the oxygen concentration is reduced to 3%-5% and cultured for 24-36 hours.
- 根据权利要求19或20所述的用途，其特征在于：培养结束后，将细胞消化下来进行冻存，备用。The use according to claim 19 or 20, characterized in that: after culturing, the cells are digested for cryopreservation and used for later use.
- 根据权利要求19或20所述的用途，其特征在于：所述的正常氧气环境为氧 气浓度在19.5％至23.5％之间。The use according to claim 19 or 20, wherein the normal oxygen environment is an oxygen concentration between 19.5% and 23.5%.
- 根据权利要求19或20所述的用途，其特征在于：所述方法的优选培养条件为：以1×10 6-1×10 8个细胞/平板的密度将细胞接种培养液中，放置于二氧化碳培养箱中培养，待人羊膜上皮细胞贴壁后换培养液，细胞汇合度达70％-80％后放入氧气浓度3％-5％的环境中培养24-36h，之后将细胞消化下来进行冻存。 The use according to claim 19 or 20, wherein the preferred culture conditions of the method are as follows: cells are inoculated into the culture medium at a density of 1×10 6 -1×10 8 cells/plate, placed in carbon dioxide Culture in an incubator, change the culture medium after the human amniotic epithelial cells adhere to the wall, and place the cells in an environment with an oxygen concentration of 3%-5% for 24-36 hours after the confluence of the cells reaches 70%-80%, and then digest the cells for freezing. live.
- 人羊膜上皮细胞或其细胞制剂在制备治疗和/或改善宫腔粘连疾病的药物中的用途。Use of human amniotic epithelial cells or cell preparations thereof in the preparation of medicaments for treating and/or improving intrauterine adhesion diseases.
- 根据权利要求24所述的用途，其特征在于：所述的羊膜上皮细胞由包括以下步骤的方法经低氧培养制备得到：The use according to claim 24, characterized in that: the amniotic epithelial cells are prepared by hypoxic culture by a method comprising the following steps:(1)将人羊膜上皮细胞复苏后接种于培养液中，正常氧气环境下培养；(1) Inoculate the human amniotic epithelial cells in a culture medium after resuscitation, and cultivate in a normal oxygen environment;(2)待细胞汇合度达60％-90％后降低氧气浓度至1％-10％培养12-48小时。(2) After the cell confluence reaches 60%-90%, the oxygen concentration is reduced to 1%-10% and cultured for 12-48 hours.
- 根据权利要求25所述的用途，其特征在于：所述的羊膜上皮细胞由包括以下步骤的方法经低氧培养制备得到：The use according to claim 25, wherein the amniotic epithelial cells are prepared by hypoxic culture by a method comprising the following steps:(1)将人羊膜上皮细胞复苏后接种于培养液中，正常氧气环境下培养；(1) Inoculate the human amniotic epithelial cells in a culture medium after resuscitation, and cultivate in a normal oxygen environment;(2)待细胞汇合度达70％-80％后降低氧气浓度至3％-5％培养24-36小时。(2) After the cell confluence reaches 70%-80%, the oxygen concentration is reduced to 3%-5% and cultured for 24-36 hours.
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