Human NLRP7 mutation decidua-like organ and application thereof

Abstract

The invention discloses a human NLRP7 mutation decidua type organ and application thereof, and belongs to the technical field of construction of type organs. The human NLRP7 mutant decidua organ is preserved in the China Center for Type Culture Collection, and is named as the human NLRP7 mutant decidua organ N7LLY (Homo sapiens), the preservation number is CCTCC (China Center for Type Culture Collection) NO: C2024375, and the preservation date is November 06, 2024. The human NLRP7 mutant decidua-like organ is applied to construction of a model for simulating an in-vivo NLRP7 mutant embryo decidua-like organ, and is applied to screening of drugs with prevention or treatment effects on grape embryo or embryo implantation failure. The human NLRP7 mutant decidua-like organ provided by the invention has endometrial epithelial cells and interstitial cells at the same time, expresses receptivity-related markers, and can retain pathological mutation and disease phenotypes of patients in vitro.

Description

Technical Field

[0001] The present invention relates to a human NLRP7 mutant decidual organoid and application thereof, belonging to the technical field of organoid construction. Background Art

[0002] Hydatidiform mole (HM) is a typical abnormal pregnancy disease that can lead to repeated implantation failure, miscarriage, stillbirth or progression to invasive gestational trophoblastic tumors, seriously affecting female fertility and health. Maternal NLRP7 gene mutations are significantly associated with the incidence of recurrent hydatidiform mole and repeated implantation failure. The characteristics of the patient's abortion tissue are trophoblastic hyperplasia and differentiation, and embryonic development defects, but the pathogenic mechanism remains unclear. NLRP7 is mainly expressed in the endometrium of pregnant women and is less expressed in non-pregnant endometrial cells. Exploring the role of NLRP7 mutations in the occurrence and development of HM will help to accurately predict and intervene in it. As a typical disease that affects early embryonic development and causes implantation failure or early miscarriage, clarifying its pathogenic mechanism will help enhance our understanding of early embryo implantation failure and abnormal embryo development, and provide new ideas for saving and preventing early pregnancy loss.

[0003] The human endometrium is composed of various types of cells, which undergo shedding (menstrual phase), regeneration (proliferative phase), and differentiation (secretory phase) under the regulation of estrogen and progesterone during the menstrual cycle. Cyclic changes in the endometrium similar to those in humans are only found in apes, Old World monkeys, bats, and spiny mice, but not in mice, which makes typical mouse models insufficient to accurately simulate the human endometrium. Existing endometrial cell lines, such as Ishikawa cells and HEEC cells, are composed of only a single cell type and therefore cannot replicate the complex physiological structure and function of the endometrium. In addition, the study of NLRP7 has been limited by the research model due to the lack of a homologous gene corresponding to NLRP7 in mice and the pathological phenotype similar to hydatidiform mole. Therefore, there is currently no reliable model that can simulate the human endometrium with NLRP7 mutations.

[0004] Replicating and reconstructing human organs has become a necessity for exploring tissue physiology and function. Organoids are self-assembled 3D structures that closely resemble in vivo tissues or organs. They have high scalability, phenotypic and functional properties, and are powerful tools for studying tissue physiology and disease. In 2017, the first long-term culturable and hormone-responsive human endometrial organoids were established using adult stem cells from endometrial biopsies. Based on this, Margherita Y. Turco et al. developed a non-invasive method for constructing endometrial organoids using menstrual blood. Summary of the invention

[0005] In view of the above-mentioned prior art, the present invention provides a human NLRP7 mutant decidual organoid and application thereof, belonging to the technical field of organoid construction.

[0006] The present invention is achieved through the following technical solutions: A human NLRP7 mutant decidual organoid, which is deposited in the China Center for Type Culture Collection and named human NLRP7 mutant decidual organoid N7_LLY (Homo sapiens), with a deposit number of CCTCC NO: C2024375 and a deposit date of November 6, 2024.

[0007] The human NLRP7 mutant decidual organoids are used to construct a model that simulates the in vivo NLRP7 mutant embryonic decidual organoids.

[0008] The human NLRP7 mutant decidual organoids are used to screen for drugs that have preventive or therapeutic effects on hydatidiform mole or embryo implantation failure.

[0009] The human NLRP7 mutant decidual organoids of the present invention have both endometrial epithelial cells and stromal cells and express receptivity-related markers, and can retain the patient's pathological mutations and disease phenotypes in vitro. The present invention has the following advantages: (1) This invention is the first to construct an organoid of NLRP7 mutant embryonic decidua that can simulate the in vivo endometrium. Its structure is highly similar to the endometrium in vivo and its function can preserve the patient's pathological mutation and disease phenotype in vitro. This model is not only of great theoretical significance, but also promotes the development and innovation of related technologies.

[0010] (2) The present invention provides a reliable model for studying recurrent hydatidiform mole and implantation failure in patients with NLRP7 mutations: The model constructed by the present invention can study the specific mechanism of recurrent hydatidiform mole, recurrent implantation failure and miscarriage in patients with NLRP7 mutations without the need for animals and in an ethical manner, and explore and clarify the key targets in the occurrence and development of the disease; compared with traditional two-dimensional culture models, organoids have the advantages of being closer to the composition and behavior of physiological cells, a more stable genome, and being more suitable for biological transfection and high-throughput screening; compared with animal evaluation systems, organoids can reduce experimental complexity, are suitable for real-time imaging technology, and are low-cost; and they solve the problem of species differences and can be applied to the study of various aspects of human development and disease.

[0011] (3) This invention lays the foundation for finding treatment methods, in vitro intervention, and clinical transformation: organoids can be expanded in vitro for high-throughput screening of drugs, efficiently selecting drugs with preventive or rescue functions. The FDA has approved drug developers to use alternative methods, such as organoids and artificial intelligence, where feasible, to conduct preclinical testing of drugs, thereby personalizing the prevention and treatment strategies for patients. From the perspective of drug screening, organoids have advantages in terms of success rate, throughput, and clinical relevance.

[0012] Various terms and phrases used herein have the general meanings that are well known to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The human NLRP7 mutant decidual organoid of the present invention is deposited in the China Center for Type Culture Collection and named as human NLRP7 mutant decidual organoid N7_LLY (Homo sapiens), with a deposit number of CCTCC NO: C2024375 and a deposit date of November 6, 2024. The deposit address is: Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code: 430072.

[0014] Figure 1 : Pedigree of a family with heterozygous NLRP7 mutation.

[0015] Figure 2 : Genetic testing results of I1, I2, Ⅱ1 and Ⅱ3.

[0016] Figure 3 : Overall view of endometrial organoids at day 19 of culture.

[0017] Figure 4 : Photographs of endometrial organoids at 4 days in culture.

[0018] Figure 5 : Photographs of endometrial organoids at day 19 of culture.

[0019] Figure 6 : Immunofluorescence detection results of cell nuclei (marked by DAPI).

[0020] Figure 7 : Results of immunofluorescence detection of receptivity (marked by HAND2).

[0021] Figure 8 : Expression of NLRP7.

[0022] Fig. 9 : Figure 6 , Figure 7 and Figure 8 Overlay display.

[0023] Fig.10 : Schematic diagram of NLRP7 expression detection results.

[0024] Fig.11 : Schematic diagram of the expression level detection results of the receptivity marker HAND2.

[0025] Fig.12 : Immunofluorescence detection results of cell nuclei (marked by DAPI) corresponding to cell type staining.

[0026] Fig.13 :The expression of glandular epithelial cell marker E-cadherin.

[0027] Fig.14 :The expression of mesenchymal cell marker vimentin.

[0028] Fig.15 : Fig.12 , Fig.13 and Fig.14 Overlay display.

[0029] Fig.16 : Immunofluorescence detection results of cell nuclei corresponding to receptivity marker staining (marked by DAPI).

[0030] Fig.17 :Positive expression of receptivity marker FoxO1.

[0031] Fig.18 :Positive expression of receptivity marker IGFBP1.

[0032] Fig.19 : Fig.16 , Fig.17 and Fig.18 Overlay display. DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0034] The instruments, reagents, materials, etc. involved in the following embodiments, unless otherwise specified, are all conventional instruments, reagents, materials, etc. in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, are all conventional experimental methods, detection methods, etc. in the prior art.

[0035] Example 1 Construction of human NLRP7 mutant decidual organoids Recurrent hydatidiform mole and implantation failure caused by NLRP7 mutation are rare diseases. In clinical practice, the present invention discovered a very rare case, and the patient's decidual sample was extremely precious. On this basis, the present invention constructed human NLRP7 mutant decidual organoids, and the specific process is described below.

[0036] (1) Basic information of patients with NLRP7 mutation The patient was 40 years old and was admitted to the Reproductive Hospital Affiliated to Shandong University in July 2017 for "recurrent miscarriage". The patient had regular menstruation, no dysmenorrhea, and normal menstrual flow. The patient got married in November 2005 and had regular sexual life. She had two miscarriages in February 2006 and August 2010. The pathology was "complete hydatidiform mole, degenerative necrotic tissue and decidual tissue"; (uterine cavity) consistent with vesicular fetal mass, high edema of villi, mild to moderate hyperplasia of trophoblast cells, immunohistochemistry suggested: ki67(+) P57(-), consistent with the diagnosis of complete hydatidiform mole. The patient had three pregnancies in 2012, 2014 and 2017. All the cases underwent uterine curettage due to "embryo arrest" at more than 60 days of gestation. All the cases underwent embryonic tissue examination. Pathology showed "partial vesicular fetal mass (in the uterine cavity), active local trophoblastic proliferation, immunohistochemistry: P57+, Ki67+<10%". In March 2017, the STR results of chorionic villus tissue indicated that the chorionic villus tissue was of biparental origin. Histopathology suggested that (in the uterine cavity) was consistent with partial vesicular fetal mass, and trophoblastic cells showed moderate proliferation, immunohistochemistry: ki67+, P57+. Combined with the medical history, routine morphology, immunohistochemistry and STR results, the patient was diagnosed with partial hydatidiform mole. The results of the patient's gene mutation test showed: NLRP7, NM_001127255; compound heterozygous c.2471+1G>A, c.C2161T; splicing point, p.R721W; father: heterozygous c.2471+1G>A, mother: heterozygous c.C2161T. There is a sister who also has a history of recurrent hydatidiform mole, and the genetic test results are the same. Comprehensive medical history, routine morphology, P57 immunohistochemistry, STR genotyping and mutation test results, it was a complete hydatidiform mole in 2011 and a partial hydatidiform mole in 2017. These two types of hydatidiform mole can be seen in different pregnancies of the same patient. The pedigree of NLRP7 heterozygous mutation is shown in the figure below. Figure 1 As shown in Figure 2, the gene test results of I1, I2, Ⅱ1 and Ⅱ3 are as follows Figure 2 As shown, based on the results of genetic testing, the patient was highly suspected of having familial recurrent complete hydatidiform mole.

[0037] The patient underwent ICSI in July 2018, July 2019, and August 2020, respectively. The sperm quality was acceptable, and the embryos did not form cysts in vitro. The donated eggs were thawed on August 4, 2020, and ICSI was performed, and the embryos were frozen. One 5BC embryo was transferred on August 18, 2021, and the biochemical pregnancy was followed up on August 31, and the clinical pregnancy was followed up on September 22. There were no complications during pregnancy, and a full-term cesarean section was performed on June 21, 2022. The patient gave informed consent, and the placental decidua was collected during the cesarean section for scientific research.

[0038] (2) Construction of human NLRP7 mutant decidual organoids ① Obtaining endometrial tissue The decidua from the implantation and non-implantation sides of the embryos of NLRP7 mutation patients were collected aseptically and immediately placed in pre-cooled collection medium, which was DMEM / F12 medium supplemented with 10% FBS and 1% antibiotic-antimycotic drugs, temporarily stored at 4°C, and quickly transferred to a sterile laboratory.

[0039] ②Use digestive fluid to dissociate endometrial tissue into single cells After washing with pre-cooled DPBS and removing blood clots, the endometrial tissue was minced and transferred to a digestion solution containing collagenase V, dispase II, and DNase I. It was digested at 37°C for 30 min, and then an equal volume of neutralization medium was added to prevent further digestion.

[0040] The composition of the neutralization medium is the same as that of the collection medium in step ①.

[0041] The digestion solution is composed of the following components: water; collagenase V, 0.4 mg / mL; dispase II, 1.5 U / mL; deoxyribonuclease I (DNase I), 15 μg / mL.

[0042] During digestion, the incubation container was rotated at 15 rpm and shaken at 1 rpm, and the mixture was blown every 10 min to make it more evenly dispersed.

[0043] ③Inoculate single cells into matrix gel for three-dimensional culture to establish endometrial organoid model The mixed solution in step ② was vortexed thoroughly and allowed to stand for 1 min to precipitate undigested tissue fragments. The supernatant was passed through a cell sieve with a pore size of 40 μm to retain as many cells in the endometrial microenvironment as possible, such as stromal cells and immune cells. The cell sieve was inverted on a culture dish and backwashed with pre-cooled DPBS to collect cell clusters to obtain a cell suspension.

[0044] The cell suspension was centrifuged (400 g, 5 min), the cell pellet was resuspended in DMEM / F12 medium, centrifuged again (400 g, 5 min), the cell pellet was resuspended in DMEM / F12 medium, mixed with Matrigel (Corning 536231), and plated. The volume ratio of DMEM / F12 medium to Matrigel was 1:3. The cells were placed in an incubator at 37°C and 5% CO 2 Incubate for more than 30 min under the same conditions to allow the matrix gel to solidify.

[0045] After the matrix gel solidified, it was spread on the cell well plate, and the endometrial organoid culture medium was added and placed in an incubator at 37°C and 5% CO 2 The culture medium was changed every 2 days under the same conditions for 19 days. After the endometrial organoids were assembled, they were observed under a bright field microscope. The overall picture of the endometrial organoids at day 19 of culture is shown in the figure below. Figure 3 As shown, the photo of endometrial organoids at 4 days of culture is shown Figure 4 As shown, the photo of endometrial organoids at 19 days of culture is shown Figure 5 As shown by Figure 3 , Figure 4 , Figure 5 It can be seen that endometrial organoids are composed of vesicle-like glands, stromal cells and other surrounding cells, and gradually develop into three-dimensional structures under the support of matrix gel.

[0046] The endometrial organoid culture medium is a DMEM / F12 culture medium supplemented with antibiotic-antimycotic (100X), insulin-transferrin-selenium (ITS), L-glutamine supplement (GlutaMAX), nicotinamide (Nicotinamide), B27, N2, recombinant human Noggin, recombinant human EGF, recombinant human FGF2, recombinant human Wnt-3A, recombinant human R-sponding-1, A83-01, N-acetyl-L-cysteine ​​(N-acetyl-L-cysteine), and p38 inhibitor SB202190, wherein the concentration, purchase source, and product number of each component are shown in Table 1.

[0047] Table 1 Concentration, purchase source and product number of each component in the culture medium

[0048] (3) Immunofluorescence detection of NLRP7 mutant decidual organoids Here are the steps: ① Fix the organoids in 4% PFA (paraformaldehyde solution) at room temperature for 30 min, wash three times with PBS, dehydrate in 20% sucrose solution at 4°C overnight, embed the organoids with OCT, and cut into 10μm thick slices using a freezing microtome.

[0049] ② The frozen sections were returned to room temperature, fixed with 4% PFA for 5 min, and permeabilized with PBS containing 0.3% TritonX-100 for 20 min. Antigen retrieval was performed with sodium citrate at 95°C for 20 min. After returning to room temperature, the sections were blocked in immunostaining blocking buffer (Beyotime, P0260) for 20 min.

[0050] ③ The sections were incubated with primary antibodies at 4°C overnight, and then washed three times with PBS containing 0.1% TritonX-100. The secondary antibodies were incubated at room temperature for 2 hours and washed three times with PBS containing 0.1% TritonX-100. DAPI (Beyotime, C1002) was incubated for 15 min and the sections were sealed with anti-fluorescence quencher. Among them, the primary antibodies included E-Cadherin (4A2) Mouse mAb, Vimentin (D21H3) XP® Rabbit mAb, Forkhead box protein O1 (FoxO1 (D7C1H) Mouse mAb, Anti-insulin-like growth factor binding protein 1 (Anti-IGFBP1), NALP7 antibody (C-8) and recombinant Anti-HAND2 antibody (EPR19451). The suppliers, product numbers and dilution ratios of the primary antibodies are shown in Table 2. The secondary antibodies included donkey anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody [Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 555] and donkey anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody [Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 488]. The suppliers, product numbers and dilution ratios of the secondary antibodies are shown in Table 3.

[0051] Table 2 Suppliers, product numbers and dilution ratios of primary antibodies

[0052] Table 3 Suppliers, product numbers and dilution ratios of secondary antibodies

[0053] ④ Images were collected using a confocal laser scanning microscope (Andor Dragonfly 200) and processed using Imaris x649.0.1.

[0054] (4) Identification of NLRP7 expression in NLRP7 mutant decidual organoids The results of immunofluorescence detection of cell nuclei (marked by DAPI) are as follows Figure 6 As shown in Figure 2, the results of immunofluorescence detection of receptivity markers (with HAND2 as a marker) are as follows: Figure 7 As shown in Figure 8 As shown, Figure 6 , Figure 7 and Figure 8 The superposition display is as follows Fig. 9 The expression level of NLRP7 was detected as shown in Fig.10 As shown in Figure 2, the expression level of the receptivity marker HAND2 was detected. Fig.11 As shown. Figures 6 to 11 It can be seen that the decidual organoids constructed in the present invention express NLRP7 and the receptivity marker HAND2.

[0055] (5) Structural identification of NLRP7 mutant decidual organoids The immunofluorescence detection results of the cell nuclei (marked by DAPI) corresponding to the cell type staining are as follows Fig.12 The expression of glandular epithelial cell marker E-cadherin is shown in Fig.13 The expression of mesenchymal cell marker vimentin is shown in Fig.14 As shown, Fig.12 , Fig.13 and Fig.14 The superposition display is as follows Fig.15 As shown. Figure 12 to Figure 15 It can be seen that E-cadherin and vimentin are expressed in the endometrial organoids cultured in the present invention.

[0056] (6) Functional characterization of NLRP7 mutant decidual organoids The immunofluorescence detection results of the cell nuclei corresponding to the receptivity marker staining (marked by DAPI) are as follows Fig.16 As shown in Figure 2, the positive expression of the receptivity marker FoxO1 is as follows Fig.17 As shown in Figure 2, the positive expression of the receptivity marker IGFBP1 is as follows Fig.18 As shown, Fig.16 , Fig.17 and Fig.18 The superposition display is as follows Fig.19 As shown. Figure 16 to Figure 19 It can be seen that the decidual organoids constructed in the present invention express the receptivity-related proteins IGFBP1, FOXO1 and HAND2.

[0057] In summary, the human NLRP7 mutant decidual organoids constructed by the present invention have glandular epithelial and stromal cells (glandular epithelial marker E-cadherin, stromal cell marker vimentin), express NLRP7, and have tolerance (tolerance marker HAND2).

[0058] The present invention preserves the human NLRP7 mutant decidual organoid constructed above and deposits it in the China Center for Type Culture Collection, named human NLRP7 mutant decidual organoid N7_LLY (Homo sapiens), with a deposit number of CCTCC NO: C2024375, and a deposit date of November 6, 2024. The deposit address is: Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code: 430072.

[0059] The above examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. A human NLRP7 mutant decidual organoid, characterized in that: It was deposited in the China Center for Type Culture Collection and named human NLRP7 mutant decidual organoid N7_LLY (Homo sapiens), with the deposit number of CCTCC NO: C2024375 and the deposit date of November 6, 2024.

2. Use of the human NLRP7 mutant decidual organoids described in claim 1 in constructing a model simulating the in vivo NLRP7 mutant embryonic decidual organoids.

3. Use of the human NLRP7 mutant decidual organoid according to claim 1 in screening for drugs that have preventive or therapeutic effects on hydatidiform mole or embryo implantation failure.

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