L-NAME induced trophoblast organ injury model as well as construction method and application thereof

Abstract

The invention is suitable for the technical field of biology, and provides an L-NAME induced trophoblast organ injury model and a construction method and application thereof, and the construction method of the trophoblast organ injury model comprises the following steps: constructing a trophoblast organ by using human villus tissue; the method comprises the following steps: carrying out culture and passage treatment on a trophoblast organ, and then adding L-NAME with the final concentration of 15-60mM to carry out induced injury, so as to obtain the trophoblast organ injury model. The basic model used in the invention is a trophoblast-like organ which can be subcultured for a long time, so that the problems of difficult acquisition of placenta samples and ethics limitation in the clinical early stage are reduced. Meanwhile, L-NAME is used for performing drug-induced injury, a trophoblast organ injury model for simulating the placental pathological state of preeclampsia can be constructed, and compared with traditional hypoxia-induced injury, the method is more convenient, and the limitation of instruments and equipment is reduced.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to an L-NAME-induced trophoblast organoid injury model, its construction method, and its application. Background Technology

[0002] Preeclampsia (PE) is a common complication of pregnancy with a high global incidence. Its pathogenesis is unclear, and suitable treatments are lacking. Current research suggests that PE is related to placental developmental abnormalities, such as insufficient development of the uterine spiral arterioles (also known as shallow placental implantation). Trophoblast organoids are complex three-dimensional structures grown from trophoblast cells isolated from human placental villus samples. They can be used to simulate placental physiological functions in vitro and can be passaged multiple times and cultured long-term. However, existing trophoblast organoid models can only simulate normal pregnancy and cannot be used for disease research or drug screening.

[0003] Since the pathogenesis of preeclampsia is closely related to placental development, placental research models of preeclampsia are crucial for studying the pathogenesis and treatment of preeclampsia. Obtaining early human placental specimens is often limited by practical and ethical constraints, making the study of the human placenta very challenging. Most in vitro trophoblast models used in laboratories have the following limitations: (1) short lifespan, such as primary cells and cells with chromosomal abnormalities; (2) not fully possessing all the characteristics of trophoblast cells in early pregnancy in vivo. Although studies using animal models can provide a wealth of information, the results of animal experiments cannot be easily extrapolated to humans, because the characteristics of the human placenta are only fully reproduced in apes. Trophoblast organoids are complex three-dimensional structures grown from trophoblast cells isolated from human villus samples. They are anatomically and functionally very similar to placental villi in vivo and can be passaged multiple times and cultured for a long period of time. The emergence of trophoblast organoids provides a higher quality experimental model for studying human placental development and trophoblast biology.

[0004] Regarding the construction of trophoblast organoid models for preeclampsia, existing research employs two main approaches: First, a 2023 article published in *Advanced Science* by a team from the Third Affiliated Hospital of Guangzhou Medical University describes the use of placental samples from patients with early-onset preeclampsia for the construction of preeclampsia placental villiorganoids (PE-POVs). The study compared PE-POVs with POVs developed by the team for normal pregnancy, finding that PE-POVs possessed the morphological and molecular characteristics of villi, and exhibited consistent levels of gene expression, secreted proteins, and inflammatory factors associated with preeclampsia. Second, a 2025 article published in *Hypertension* by a team from East China Normal University describes how hypoxic treatment (5% CO2, 5% O2) of trophoblast organoids can produce a pathological state similar to preeclampsia placental tissue, and this model was applied to research on the mechanism of aspirin in preventing preeclampsia.

[0005] However, in the aforementioned prior art one: since preeclampsia needs to be diagnosed after 20 weeks of gestation, the placental samples used in this study were from the mid-to-late stages of pregnancy, resulting in poor villous organoid models with a lifespan of only <30 days; furthermore, the function and structure of placental villous organoids constructed by this method change after passage. In prior art two: this method does not study the effects of different hypoxia concentrations and durations on trophoblast organoids; while the selection of hypoxia concentration has some basis, it is somewhat coincidental; additionally, this method is not suitable for high-throughput drug screening, as the most definitive indicator after hypoxia, HIF-1α, was not detected after drug administration, possibly because preeclampsia treatment drugs do not significantly affect HIF-1α, but can significantly improve the expression of the proliferation-related protein KI67.

[0006] Therefore, given the current lack of clarity regarding the pathogenesis of preeclampsia and the limited availability of therapeutic drugs, there is an urgent need to develop a preeclampsia trophoblast organoid model that can be cultured long-term and used for high-throughput in vitro drug screening. Summary of the Invention

[0007] The purpose of this invention is to provide a method for constructing an L-NAME-induced trophoblast organoid injury model, aiming to solve the problems mentioned in the background art.

[0008] This invention is implemented by providing a method for constructing an L-NAME-induced trophoblast organoid injury model, comprising the following steps:

[0009] Using human villous tissue to construct trophoblast organoids;

[0010] After culturing and passaged trophoblast organoids, L-NAME at a final concentration of 15-60 mM was added to induce damage, thus obtaining a trophoblast organoid injury model.

[0011] Furthermore, the final concentration of the L-NAME is 30 mM.

[0012] Furthermore, the steps for constructing trophoblast organoids using human villous tissue specifically include:

[0013] Human chorionic villus tissue was sequentially digested with trypsin and collagenase V to obtain cells; the human chorionic villus tissue was human chorionic villus tissue from 6-9 weeks of gestation.

[0014] Cells were mixed with matrix gel and cultured using a special culture medium for trophoblast organoids to obtain trophoblast organoids.

[0015] Further, the human chorionic villus tissue is subjected to trypsin digestion and collagenase V digestion sequentially to obtain cells, specifically including:

[0016] Human chorionic villus tissue was transferred to a solution containing 0.25 wt% EDTA-trypsin for enzymatic hydrolysis for 3-5 minutes;

[0017] The enzymatically hydrolyzed solution and tissue were filtered through a 70-100μm cell sieve, and the filtrate was retained and labeled as filtrate #1.

[0018] The tissue on the cell sieve was digested a second time for 3-5 minutes using 0.8-1.2 mg / mL collagenase V, and then filtered through a 70-100 μm cell sieve. The filtrate was retained and labeled as filtrate #2.

[0019] Centrifuge filtrate #1 and filtrate #2 separately, discard the supernatant, combine the precipitates, resuspend and wash in DMEM / F12 medium, centrifuge again, discard the supernatant, and obtain the cells.

[0020] Further, the steps of mixing cells with matrix gel and culturing them using a trophoblast organoid-specific culture medium to obtain trophoblast organoids specifically include:

[0021] The cells were mixed with pre-thawed matrix gel, and the cell clumps were broken up to obtain a cell-matrix mixture;

[0022] Add the cell-Matrix gel mixture to a culture plate that has been preheated to 37°C;

[0023] The culture plate is placed in an incubator and incubated for 10-20 minutes to allow the matrix gel to solidify. Then, a special culture medium for trophoblast organoids is added for culturing to obtain trophoblast organoids. The special culture medium for trophoblast organoids includes WNT activator at a concentration of 1.5-5 μM, prostaglandin E2 at a concentration of 2.5-10 μM, and ROCK inhibitor at a concentration of 2-10 μM. It should be noted that, except for the above-mentioned components, the composition of the special culture medium for trophoblast organoids can be the same as that of existing trophoblast organoid culture media.

[0024] Furthermore, the method for culturing and passageing trophoblast organoids includes the following steps:

[0025] The trophoblast organoids were cultured in a special culture medium for trophoblast organoids. The medium was changed every 2-3 days, and the morphological changes of the trophoblast organoids were observed. When 50% of the trophoblast organoids in each well reached a diameter of 200-300 μm, they were passaged.

[0026] During the subculture process, the culture medium is not removed. Instead, a low-absorption pipette tip is used to scrape the growth surface of each well back and forth to separate the matrix gel into the culture medium, resulting in a matrix gel-culture medium suspension.

[0027] The matrix gel-medium suspension was centrifuged, the supernatant was discarded, and DMEM / F12 medium was added to the precipitate for agitation to separate the trophoblast organoids and matrix gel. Then, DMEM / F12 medium was added and centrifuged again, and the supernatant was discarded. Then, Accutase digestive enzyme preheated at 37°C was added for digestion. After digestion, DMEM / F12 medium was added again and centrifuged multiple times, the supernatant was discarded, and matrix gel was added at a 1:2 ratio. After mixing, the trophoblast organoid-medium gel suspension was obtained and inoculated into new culture plates for culture.

[0028] Furthermore, the method for constructing the L-NAME-induced trophoblast organoid injury model further includes the following steps:

[0029] The successful construction of the trophoblast organoid injury model was verified by detecting the expression level of the proliferation-related protein KI67 in the trophoblast organoids.

[0030] Another objective of this invention is to provide an L-NAME-induced trophoblast organoid injury model obtained using the above-described construction method.

[0031] Another object of the present invention is to provide an application of the above-mentioned L-NAME-induced trophoblast organoid injury model in screening drugs for the prevention and / or treatment of preeclampsia.

[0032] Furthermore, the drug includes isorhizine; the screening index for the drug is: fluorescence detection of proliferation-associated protein KI67; the screening method for the drug includes the following steps:

[0033] Different concentration gradients of the drugs to be screened were added to the trophoblast organoid injury model to obtain the drug-treated trophoblast organoid injury model.

[0034] Immunofluorescence staining was performed on the trophoblast organoid injury model after drug treatment to detect the expression level of KI67 protein. Fluorescence images and data were acquired using a high-content imaging system to obtain the detection data of KI67 protein expression level.

[0035] Based on the detection data of KI67 protein expression level, the therapeutic effect of the drugs to be screened on preeclampsia was evaluated by comparing them with an untreated trophoblast organoid injury model.

[0036] The basic model used in this invention is a trophoblast organoid that can be passaged and cultured for a long time, reducing the difficulties of obtaining placental samples in early clinical settings and addressing ethical restrictions. Simultaneously, this invention uses L-NAME for drug-induced injury, enabling the construction of a trophoblast organoid injury model to simulate the pathological state of the preeclamptic placenta. This is more convenient than traditional hypoxia-induced injury and reduces limitations on equipment. Furthermore, this invention compares the effects of L-NAME-induced trophoblast organoid injury on proliferation and differentiation-related indicators, clarifying that KI67 can be used as an indicator for subsequent drug screening, providing a foundation for applying the model to high-throughput drug screening. Attached Figure Description

[0037] Figure 1 To observe the diameter changes of trophoblast organoids (TO) cultured in bright field for 5 to 8 days (D5-D8), growing from approximately 50 μm to 200 μm; scale bar: 200 μm.

[0038] Figure 2 Results of expression of trophoblast cell-specific proteins KRT7, proliferation-related proteins KI67 and EPCAM, and cell cycle-related protein P63 in trophoblast organoids. (A) Immunohistochemical detection of KI67, KRT7, and P63 in trophoblast organoids and human villous tissue; scale bar: 100 μm. (B) Immunofluorescence detection of KRT7 and EPCAM in trophoblast organoids, and their three-dimensional structure displayed by confocal microscopy tomography. (C) Immunofluorescence detection of KI67 in trophoblast organoids, expression, and staining results at different magnifications displayed by confocal microscopy; scale bar: 25 μm.

[0039] Figure 3Figure 1 shows the experimental results of constructing a L-NAME-induced trophoblast organoid injury model. (A) Construction flowchart: L-NAME was added to the trophoblast organoids on day 2 after seeding, and immunofluorescence staining was performed on day 4 to detect relevant indicators; or the drug was administered on day 4, and immunofluorescence staining was performed on day 6. (B) IC50 detection of L-NAME on the trophoblast cell line (HTR-8 / SVneo) showed that L-NAME at a concentration of 15.27 mM caused half-maximal inhibition of HTR-8 / SVneo.

[0040] Figure 4 The results of KI67 and EPCAM staining for L-NAME-induced damage to trophoblast organoids. (A) KI67, EPCAM, and nuclear DAPI are red, green, and blue, respectively; under 60 mM L-NAME treatment, trophoblast organoids cannot maintain their morphology and structure, resulting in irreversible damage; KI67 expression gradually decreases with increasing L-NAME concentration; EPCAM expression shows no significant change after L-NAME treatment. (B) Average fluorescence intensity analysis results show that KI67 at 15 mM ( ** P<0.01) and 30mM L-NAME ( *** (P<0.001) The fluorescence intensity of EPCAM was significantly reduced at 15 mM (P<0.001). (C) The average fluorescence intensity analysis results showed that EPCAM at 15 mM (P<0.001) was significantly reduced. ns P>0.05) and 30mM L-NAME ( ns No significant changes were observed when P>0.05 was applied; scale bar: 200μm.

[0041] Figure 5 HLA-G and F-actin staining results for L-NAME-induced trophoblast organoid damage. (A) F-actin, HLA-G, and nuclear DAPI are red, green, and blue, respectively; under 60 mM L-NAME treatment, trophoblast organoids cannot maintain their morphology and structure, resulting in irreversible damage; HLA-G expression gradually decreases with increasing L-NAME concentration; F-actin expression shows no significant change after L-NAME treatment. (B) Average fluorescence intensity analysis results show that HLA-G expression increases with increasing L-NAME concentration at 30 mM L-NAME (… * The effect of P<0.05 was significantly reduced at 15 mM L-NAME ( ns (P>0.05) showed no significant change. (C) Average fluorescence intensity analysis results indicated that F-actin at 15 mM (P>0.05) showed no significant change. ns P>0.05) and 30mM L-NAME ( ns No significant changes were observed when P>0.05 was applied; scale bar: 200μm.

[0042] Figure 6The figure shows the safety and efficacy evaluation results of isorhizine in HTR-8 / SVneo cells. (A) Isorhizine ( *** (P<0.001) At concentrations above 100 μM, both significantly inhibited the proliferation of HTR-8 / SVneo cells. (B) Isorhizine at 50 (P<0.001) # P<0.05) and 100μM ( # When P < 0.05, the proliferation inhibition caused by L-NAME showed a significant reversal.

[0043] Figure 7 Figure 1 shows the safety and efficacy evaluation results of isorhamnetin in L-NAME-induced trophoblast organoid injury models. (A) High-content detection of KI67 expression in L-NAME-induced trophoblast organoid injury models; the diameter of trophoblast organoids was significantly reduced in the 30 and 60 μM isorhamnetin administration groups. (BD) Efficacy study results of trophoblast organoid injury models from three different human villous tissue sources; 30 mM L-NAME significantly inhibited KI67 expression in all three organoids. *** P<0.001); In the trophoblast organoid injury model #1, isorhamnetin at 15 μM ( # KI67 expression was significantly reduced at P<0.05; in the trophoblast organoid injury model #2, isorhamnetin at 15 μM ( # KI67 expression was significantly reduced at P<0.05; in the trophoblast organoid injury model #3, isorhamnetin at 15 μM ( ## P<0.01) and 30μM ( ## Significantly reduced KI67 expression when P < 0.01. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0045] This invention introduces a trophoblast organoid injury model by introducing the nitric oxide synthase inhibitor N'-nitro-L-arginine methyl ester hydrochloride (NG-Nitro-L-arginine Methyl Ester, L-NAME) to simulate the pathological state of the placenta in preeclampsia. High-content technology is then applied to drug screening based on the trophoblast organoid injury model to achieve high-throughput, multi-indicator drug screening for preeclampsia, providing more options for the clinical treatment of preeclampsia.

[0046] Specifically, in one embodiment of the present invention, a method for constructing an L-NAME-induced trophoblast organoid injury model is provided, comprising the following steps:

[0047] Using human villous tissue to construct trophoblast organoids;

[0048] After culturing and passaged, trophoblast organoids are then treated with L-NAME at a final concentration of 15-60 mM to induce injury, thus obtaining a trophoblast organoid injury model. Preferably, the final concentration of L-NAME is 30 mM.

[0049] The successful construction of the trophoblast organoid injury model was verified by detecting the expression level of the proliferation-related protein KI67 in the trophoblast organoids.

[0050] In this embodiment of the invention, L-NAME is an endothelial nitric oxide synthase inhibitor, commonly used in the construction of animal models of hypertension, including preeclampsia mice and rat models. L-NAME induces hypertension by reducing nitric oxide production, leading to sustained vasoconstriction and increased peripheral resistance. L-NAME also promotes the generation of reactive oxygen species, resulting in elevated levels of inflammatory factors and apoptosis. The L-NAME-induced trophoblast organoid injury model provided in this embodiment of the invention can be used to simulate the pathological state of the placenta in preeclampsia. This embodiment of the invention induces damage to trophoblast organoids by adding L-NAME at varying concentrations, and uses immunofluorescence to detect changes in the expression levels of trophoblast cell proliferation-related proteins KI67, EPCAM, and differentiation-related protein HLA-G, screening for suitable modeling concentrations and detection indicators. This model is then applied to the screening of therapeutic drugs (such as isorhizine), providing a more suitable in vitro screening model for the clinical treatment of preeclampsia.

[0051] In practical applications, the method for constructing the above-mentioned trophoblast organoid injury model may specifically include the following steps:

[0052] S1. Human chorionic villus tissue was sequentially digested with trypsin and collagenase V to obtain cells, as detailed below:

[0053] Human chorionic villus tissue was transferred to a solution containing 0.25 wt% EDTA-trypsin for enzymatic digestion for 3-5 minutes. The digested solution and tissue were filtered through a 70-100 μm cell sieve, and the filtrate was retained and labeled as filtrate #1. The tissue remaining on the cell sieve was then subjected to a second digestion with 0.8-1.2 mg / mL collagenase V for 3-5 minutes, and filtered again through a 70-100 μm cell sieve. The filtrate was retained and labeled as filtrate #2. Filtrates #1 and #2 were centrifuged separately, the supernatant was discarded, the precipitates were combined, and the mixture was resuspended and washed in DMEM / F12 medium, then centrifuged again, the supernatant was discarded, and the cells were obtained. The human chorionic villus tissue referred to was chorionic villus tissue from individuals at 6-9 weeks of gestation. It should be noted that the acquisition of human chorionic villus tissue samples must strictly adhere to ethical guidelines and procedures.

[0054] S2. Mix the cells with matrix gel and culture them using a special culture medium for trophoblast organoids to obtain trophoblast organoids, as detailed below:

[0055] The cells were mixed with pre-thawed matrix gel and the cell clumps were broken up to obtain a cell-matrix mixture. The cell-matrix mixture was added to a culture plate that had been preheated to 37°C. The culture plate was placed in an incubator and incubated for 10-20 minutes to allow the matrix gel to solidify. Then, a special culture medium for trophoblast organoids was added for culture to obtain trophoblast organoids.

[0056] S3. Culture and passage the trophoblast organoids. The specific methods include the following steps:

[0057] The trophoblast organoids were cultured in a dedicated trophoblast organoid medium, with medium changes every 2-3 days. Morphological changes in the trophoblast organoids were observed. When 50% of the trophoblast organoids in each well reached a diameter of 200-300 μm, they were passaged. During passage, the medium was not removed; instead, a low-absorption pipette tip was used to scrape the growth surface of each well to separate the matrix gel into the medium, resulting in a matrix gel-medium medium suspension. The matrix gel-medium medium suspension was centrifuged, the supernatant was discarded, and DMEM / F12 medium was added to the precipitate. The mixture was then agitated to separate the trophoblast organoids from the matrix gel. DMEM / F12 medium was added again, and the mixture was centrifuged, discarding the supernatant. Preheated Accutase enzyme (37°C) was then added for digestion. After digestion, DMEM / F12 medium was added again, and the mixture was centrifuged multiple times, discarding the supernatant. Matrix gel was added at a 1:2 ratio, and the mixture was thoroughly mixed to obtain the trophoblast organoid-Matrix gel suspension, which was then inoculated into new culture plates for further culture.

[0058] S4. Inoculate 5-10 μL of the trophoblast organoid-Matrix gel suspension into a black transparent bottom 96-well plate for high content detection. On the second day after passage, add 15-60 mM L-NAME to induce damage, and the trophoblast organoid injury model can be obtained.

[0059] In a preferred embodiment of the present invention, a special culture medium for trophoblast organoids is crucial for successfully culturing trophoblast organoids and maintaining their long-term viability, and its composition can be optimized according to actual experimental needs. In this embodiment of the present invention, the main components of the special culture medium for trophoblast organoids include:

[0060] Basic culture medium; preferably Advanced DMEM / F12, which provides the essential nutrients required for cell growth;

[0061] Serum substitutes: B27 supplement (preferably diluted 1:50) and N-2 supplement (preferably diluted 1:100) provide lipids, vitamins, antioxidants and other trace elements to support long-term cell survival and proliferation, while avoiding the batch differences and unknown components that may be present in serum.

[0062] Antibiotic: Primocin, at a concentration of 100 μg / mL, to prevent bacterial contamination;

[0063] Antioxidant: N-acetyl-L-cysteine, concentration 1.25mM;

[0064] Amino acids: L-glutamine, at a concentration of 2 mM, provides essential amino acids for cell growth;

[0065] Key growth factors:

[0066] Epidermal growth factor (EGF), concentration 50 ng / mL;

[0067] Fibroblast growth factor 2 (FGF-2), at a concentration of 100 ng / mL;

[0068] R-spondin 1 (R-spondin 1 conditioned medium or recombinant protein), at a concentration of 80 ng / mL;

[0069] HGF (Hepatocyte Growth Factor), concentration 50 ng / mL.

[0070] Small molecule compounds:

[0071] A83-01 (TGFβ and SMAD inhibitor), at a concentration of 500 nM.

[0072] CHIR99021 (WNT activator), concentration 1.5-5 μM; preferred concentration 5 μM.

[0073] prostaglandin E2, at a concentration of 2.5-10 μM; preferably 10 μM.

[0074] Y-27632 (ROCK inhibitor), with a concentration of 2-10 μM; the preferred concentration is 10 μM, enhances cell viability and promotes cell growth.

[0075] All the above-mentioned culture media should be of cell culture grade and should be prepared and filtered under strict aseptic conditions (0.22μm filter membrane) in a laminar flow hood to ensure the sterility of the culture environment and prevent any microbial contamination from adversely affecting organoid growth.

[0076] In another embodiment of the present invention, a method for screening drugs for the prevention and / or treatment of preeclampsia is also provided, specifically including the following steps:

[0077] Different concentration gradients of the drugs to be screened were added to the trophoblast organoid injury model to obtain the drug-treated trophoblast organoid injury model.

[0078] Immunofluorescence staining was performed on the trophoblast organoid injury model after drug treatment to detect the expression level of KI67 protein. Fluorescence images and data were acquired using a high-content imaging system to obtain the detection data of KI67 protein expression level.

[0079] Based on the detection data of KI67 protein expression level, the therapeutic effect of the drugs to be screened on preeclampsia was evaluated by comparing them with an untreated trophoblast organoid injury model.

[0080] The trophoblast organoid injury model constructed in this invention is an in vitro model suitable for long-term drug development, reducing difficulties in sample acquisition and ethical restrictions. Furthermore, it allows for accurate initial screening results using simple modeling methods and clearly defined detection indicators. This invention has been preliminarily applied to the pharmacodynamic study of isorhizine, an important monomer from Uncaria rhynchophylla, in the treatment of preeclampsia, showing promising results.

[0081] Example 1: This example provides a method for constructing trophoblast organoids, specifically including the following steps:

[0082] (1) Acquisition and Cell Preparation of Human Villus Tissue Samples: First, the villus tissue samples used in this embodiment of the invention were obtained from individuals whose consent was fully informed and approved by the Ethics Committee of Sir Run Run Shaw Hospital, affiliated with Zhejiang University School of Medicine. The human villus tissue was obtained from abortion samples taken during 6-9 weeks of pregnancy via negative pressure aspiration. After acquisition, the samples should be immediately placed in ice-cold tissue preservation solution and transported to the laboratory as soon as possible for further processing to maximize cell viability and prevent contamination. The tissue preservation solution can maintain tissue cell viability for up to 24 hours. In a biosafety cabinet, the acquired villus tissue was carefully pretreated and enzymatically digested. First, using a sterile scalpel or scissors, the tissue samples with blood clots were carefully removed to reduce potential subsequent contamination. Then, the villi were scraped off from the chorionic villi membrane using a No. 22 scalpel. The scraped human villus tissue was transferred to a solution containing 0.25% EDTA-trypsin for enzymatic digestion for 3-5 minutes. The digested solution and tissue were filtered through a 100μm cell sieve, and the filtrate was retained and labeled as filtrate #1. Tissue from the cell sieve was digested a second time for 4 minutes using 1 mg / mL collagenase V, and then filtered through a 100 μm cell sieve. The filtrate was retained and labeled as filtrate #2. Filtrates #1 and #2 were centrifuged at 400 g for 5 minutes, and the supernatant was discarded. The two cell pellets were combined, resuspended and washed in Advanced DMEM / F12 medium, and transferred to 1.5 mL centrifuge tubes. The mixture was centrifuged at 600 g for 6 minutes, and the supernatant was discarded. Approximately 200 μL of pre-thawed matrix gel was added at 10 times the volume of the cell pellet, and cell clumps were dispersed to obtain a cell-matrix gel mixture. The cell-matrix gel mixture was added to 25 μL of each well in a preheated 48-well plate at 37 °C. The plate was incubated for 15 minutes to allow the matrix gel to solidify, and then cultured in Trophoblast Organoids Medium (TOM) to obtain trophoblast organoids. The trophoblast organoid culture medium contains the following components: B27 supplement (1:50 dilution), N-2 supplement (1:100 dilution), Primocin antibiotic 100 μg / mL, N-acetyl-L-cysteine ​​1.25 mM, L-glutamine 2 mM, EGF 50 ng / mL, FGF-2 100 ng / mL, R-spondin 1 80 ng / mL, HGF 50 ng / mL, A83-01 500 nM, CHIR99021 5 μM, prostaglandin E2 10 mM, Y-27632 10 μM, and the remainder is Advanced DMEM / F12 basal culture medium.

[0083] (2) Culture and passage of trophoblast organoids: Trophoblast organoids require medium change every 2-3 days, and morphological changes should be observed. When the diameter reaches approximately 200 μm, passage should be performed (7-8 days). Without removing the culture medium, use a 1 mL low-absorption pipette tip to scrape back and forth on the growth surface of each well to separate the matrix gel into the culture medium, obtaining a matrix gel-culture medium suspension. Transfer the matrix gel-culture medium suspension from each of 3-4 wells to a 1.5 mL centrifuge tube, centrifuge at 600 g for 6 minutes, and discard the supernatant. Add 150 μL of Advanced DMEM / F12 culture medium to the cell pellet, and use a 200 μL low-absorption pipette tip and an electric pipette to pipette about 400 times to separate the organoids from the matrix gel. Add 1 mL of Advanced DMEM / F12 culture medium, centrifuge at 600 g for 6 minutes, and discard the supernatant. Add 500 μL of Accutase digestive enzyme preheated in a 37°C water bath, and digest at 37°C and 60 rpm for 5 minutes with shaking. Add 1 mL of Advanced DMEM / F12 medium, centrifuge at 600 g for 6 minutes, and discard the supernatant. Add 150 μL of Advanced DMEM / F12 medium and manually pipette 80 times. Add 1 mL of Advanced DMEM / F12 medium, centrifuge at 600 g for 6 minutes, and discard the supernatant. Subculture at a 1:2 ratio, add an appropriate volume of matrix gel, mix well to obtain a trophoblast organoid-Matrix gel suspension, and seed it into a new 48-well plate for culture. Alternatively, seed it into a confocal dish for immunofluorescence staining and confocal imaging. For trophoblast organoids passaged >3 times, on day 8 of growth, remove the matrix gel using Cell Recovery reagent, fix with paraformaldehyde, embed in agarose, and use for subsequent section staining.

[0084] Results: The success rate of the trophoblast organoids constructed in this embodiment of the invention is approximately 75%, with a maximum passage length of 20 generations and a culture period of up to 162 days (March 20, 2025 to August 20, 2025). Trophoblast organoids (cells) isolated from human villous tissue formed spherical structures with a diameter of approximately 50 μm on day 5 of in vitro culture, and reached a diameter of 200-300 μm on day 8 (e.g., Figure 1 (As shown). Furthermore, trophoblast organoids express trophoblast cell-specific proteins KRT7, proliferation-related proteins KI67 and EPCAM, and cell cycle-related protein P63, consistent with villous tissue characterization (e.g., ...). Figure 2 (As shown). The above results demonstrate that the embodiments of the present invention successfully constructed a trophoblast organoid model capable of long-term culture and passage.

[0085] Example 2: This example provides a method for constructing an L-NAME-induced trophoblast organoid injury model, as detailed below:

[0086] The trophoblast organoids obtained in Example 1 were cultured for 7-8 days, and then passaged. The trophoblast organoid-Matrix gel suspension was seeded at 10 μL per well in a black transparent-bottomed 96-well plate specifically designed for high-content assays. On the second day after passage, 15, 30, and 60 mM of L-NAME were added to induce injury, respectively, to obtain a trophoblast organoid injury model. Previous studies found that the half-maximal inhibitory concentration (IC50) of L-NAME in the trophoblast cell line (HTR-8 / SVneo) was 15.27 mM; therefore, the concentration gradient for inducing trophoblast organoid injury was set starting at 15 mM. If further pharmacodynamic studies were to be conducted on the second day after adding L-NAME, the therapeutic drug was added until day 6 for immunofluorescence staining. If only the injury-inducing effect of L-NAME was to be assessed, immunofluorescence staining was performed on day 4 (e.g., ...). Figure 3 (As shown).

[0087] Results: Microscopic observation revealed that 60 mM L-NAME caused irreversible damage to trophoblast organoids, rendering them unable to maintain their three-dimensional structure. Immunofluorescence staining of L-NAME-induced damaged trophoblast organoids showed that the expression level of the proliferation-related protein KI67 was significantly reduced under both 15 mM and 30 mM L-NAME treatment, with a reduction of >50% at 30 mM (e.g., ...). Figure 4 (As shown). The expression level of the proliferation-related protein EPCAM did not change significantly after L-NAME treatment. The expression level of the differentiation-related protein HLA-G was significantly decreased after treatment with 30 mM L-NAME, while the expression level of the cytoskeletal protein F-actin did not change significantly after L-NAME treatment (as shown). Figure 5 (As shown). The above results indicate that 30 mM L-NAME significantly affects the proliferation and differentiation process of trophoblast organoids and can be used to model the pathological state of placenta in preeclampsia. Among them, KI67 can be used as a damage indicator and an indicator for subsequent drug screening.

[0088] Example 3: This example provides a method for detecting drug sensitivity in an L-NAME-induced trophoblast organoid injury model, as detailed below:

[0089] Uncaria rhynchophylla is a commonly used traditional Chinese medicine for treating hypertension. Its main active ingredients are alkaloids, including isorhynchophylline, which is present in high amounts. To verify the sensitivity of the L-NAME-induced trophoblast organoid injury model provided in this invention to drug treatment, the efficacy results of isorhynchophylline on in vitro trophoblast cell lines were compared with those on the new model as an example.

[0090] 1. Safety evaluation of isorhizine in HTR-8 / SVneo cells: After passage, HTR-8 / SVneo cells were seeded into 96-well plates. 24 hours later, 25, 50, and 100 μM isorhizine were added. Cell viability was detected by CCK8 assay after 48 hours.

[0091] 2. Pharmacodynamic study of isorhizine in HTR-8 / SVneo cells: After passage, HTR-8 / SVneo cells were seeded into 96-well plates. After 24 hours, 15 mM L-NAME was added to induce damage. After 48 hours, the treatment was changed to 25, 50, and 100 μM isorhizine. Cell viability was detected by CCK8 assay after 72 hours.

[0092] 3. Pharmacodynamic study of isorhodopsin in L-NAME-induced trophoblast organoid injury model: After passage, the trophoblast organoids constructed in Example 1 were seeded into high-content 96-well plates. On the second day, 30 mM L-NAME was added to induce injury. On the fourth day, the treatment was changed to 15, 30, and 60 μM isorhodopsin. On the sixth day, immunofluorescence staining was performed, and images were taken and analyzed using a high-content imaging system to detect changes in KI67 expression levels.

[0093] Results: Isorhizine inhibited the proliferation of HTR-8 / SVneo cells at concentrations above 100 μM. Therefore, in subsequent efficacy studies, the concentration was set below 100 μM. Figure 6 As shown in A; the pharmacodynamic study results showed that isorhizine significantly reversed the inhibition of L-NAME-induced HTR-8 / SVneo cell proliferation at both 50 μM and 100 μM, but the reversal effect was not significant at 25 μM, as detailed below. Figure 6 As shown in B. The above results indicate that isorhizine promotes the proliferation of trophoblast cells in trophoblast cell lines, but the effect is not significant at low concentrations.

[0094] The efficacy of isorhynchine was further investigated using three different trophoblastic organoid injury models derived from human villous tissue. Results showed that 30 and 60 μM isorhynchine may have an inhibitory effect on trophoblastic organoid growth, with their average diameter being significantly lower than that of other treatment groups. Specifically, [details omitted]. Figure 7 As shown in A. Isorhodopsin showed the most significant KI67 expression reversion in L-NAME-induced trophoblast organoid injury at 15 μM, specifically as follows... Figure 7 As shown in B, C, and D. These results indicate that the L-NAME-induced trophoblast organoid injury model is more sensitive to drug treatment, and significant changes in indicators can be detected even at low concentrations.

[0095] In summary, the embodiments of this invention successfully constructed a trophoblast organoid model capable of long-term culture and passage, and simultaneously induced trophoblast organoid damage using 30 mM L-NAME, thus successfully constructing a trophoblast organoid damage model to simulate the pathological state of the placenta in preeclampsia. Furthermore, this invention can use the fluorescence detection of KI67 as an indicator for drug screening, combined with a high-content imaging system and analysis module for high-throughput drug screening. Compared with existing technologies, the embodiments of this invention have the following significant differences in technical means:

[0096] (1) Compared with the prior art, which uses placental samples from gestational age greater than 20 weeks to construct trophoblast organoids, the present invention uses samples from gestational age of 6-9 weeks to construct trophoblast organoids. The constructed trophoblast organoids make up for the shortcomings of the prior art in that they cannot be cultured and passaged for a long time, reduce the difficulty of obtaining clinical samples, and the early pregnancy trophoblast organoids are more suitable for early prevention and treatment research of preeclampsia.

[0097] (2) Compared with the existing technology that uses hypoxia to induce damage to trophoblast organoids, the present invention uses the inhibitor L-NAME to induce damage, which can reduce the limitations of instruments and equipment, and the method is convenient and effective.

[0098] (3) Compared with the existing technology which does not have clear indicators for subsequent drug screening studies, the embodiments of the present invention can use fluorescence detection KI67 combined with high content detection technology to screen drugs, which can conduct drug safety and efficacy studies more sensitively and efficiently.

[0099] The above embodiments merely illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this patent should be determined by the appended claims.

Claims

1. A method for constructing an L-NAME-induced trophoblast organoid injury model, characterized by, The method comprises the following steps: constructing a trophoblast organ using human chorionic tissue; inducing damage by adding L-NAME at a final concentration of 15-60 mM to the trophoblast organ after culturing and subculturing the trophoblast organ, to obtain a trophoblast organ damage model.

2. The method for constructing an L-NAME-induced trophoblast spheroid model according to claim 1, wherein The final concentration of the L-NAME is 30 mM.

3. The method for constructing an L-NAME-induced trophoblast spheroid model according to claim 1, wherein The step of constructing a trophoblast organ using human chorionic tissue specifically comprises: digesting the human chorionic tissue with trypsin and collagenase V in sequence to obtain cells; the human chorionic tissue is human chorionic tissue of 6-9 weeks of pregnancy; mixing the cells with Matrigel and culturing the cells using a trophoblast organ special medium to obtain the trophoblast organ.

4. The method for constructing an L-NAME-induced trophoblast spheroid model according to claim 3, wherein The step of digesting the human chorionic tissue with trypsin and collagenase V in sequence to obtain cells specifically comprises: transferring the human chorionic tissue to a solution containing 0.25 wt% EDTA-trypsin for enzymolysis for 3-5 minutes; filtering the solution and the tissue after enzymolysis through a 70-100 μm cell screen, retaining the filtrate and marking it as filtrate #1; digesting the tissue on the cell screen with 0.8-1.2 mg / mL collagenase V for 3-5 minutes, filtering it through a 70-100 μm cell screen again, retaining the filtrate and marking it as filtrate #2; centrifuging the filtrate #1 and the filtrate #2 respectively, discarding the supernatant, combining the precipitates, resuspending and washing them in DMEM / F12 medium, centrifuging them again, discarding the supernatant, and obtaining the cells.

5. The method for constructing an L-NAME-induced trophoblast spheroid model according to claim 3, wherein The step of mixing the cells with Matrigel and culturing the cells using a trophoblast organ special medium to obtain the trophoblast organ specifically comprises: mixing the cells with Matrigel that has been thawed in advance, and blowing the cell clumps to obtain a cell-Matrigel mixture; adding the cell-Matrigel mixture to a culture plate that has been preheated at 37°C in advance; placing the culture plate in an incubator for 10-20 minutes to solidify the Matrigel, and then adding a trophoblast organ special medium to culture the trophoblast organ; the trophoblast organ special medium comprises a WNT activator at a concentration of 1.5-5 μM, a prostaglandin E2 at a concentration of 2.5-10 μM, and a ROCK inhibitor at a concentration of 2-10 μM.

6. The method for constructing an L-NAME-induced trophoblast spheroid model according to claim 1, wherein The method for culturing and subculturing the trophoblast organ comprises the following steps: culturing the trophoblast organ in a trophoblast organ special medium, changing the medium every 2-3 days, and observing the morphological changes of the trophoblast organ; when the diameter of 50% of the trophoblast organ in each well reaches 200-300 μm, subculturing the trophoblast organ; when subculturing, not removing the medium, using a low-adsorption gun head to scrape back and forth on the growth surface of each well to separate the Matrigel into the medium, to obtain a Matrigel-medium suspension; The Matrigel-culture medium suspension is centrifuged, the supernatant is discarded, DMEM / F12 culture medium is added to the precipitate for blowing to separate the trophoblast organoids and Matrigel; then DMEM / F12 culture medium is continuously added for centrifugation, and the supernatant is discarded; then preheated Accutase digestion enzyme at 37℃ is added for digestion; after the digestion is completed, DMEM / F12 culture medium is added for multiple centrifugation, the supernatant is discarded, Matrigel is added at a ratio of 1:2 for subculture, and the trophoblast organoids-Matrigel suspension is obtained after mixing, and is inoculated into a new culture plate for culture.

7. The method for constructing an L-NAME-induced trophoblast spheroid model according to claim 1, wherein Further comprising the following steps: The success of the construction of the trophoblast organoid injury model is verified by detecting the expression level of the proliferation-related protein KI67 in the trophoblast organoids.

8. An L-NAME-induced trophoblast organoid injury model obtained by the construction method of any one of claims 1-7.

9. Use of the L-NAME-induced trophoblast organoid injury model of claim 8 in screening drugs for preventing and / or treating preeclampsia.

10. Use according to claim 9, characterized in that, The screening index of the drug is that the proliferation-related protein KI67 is detected by fluorescence; and the screening method of the drug comprises the following steps: Different concentrations of the drug to be screened are added to the trophoblast organoid injury model to obtain a trophoblast organoid injury model treated with the drug; The trophoblast organoid injury model treated with the drug is subjected to immunofluorescence staining to detect the expression level of the KI67 protein, and a high-content imaging system is used to obtain fluorescence images and data to obtain detection data of the expression level of the KI67 protein; Based on the detection data of the expression level of the KI67 protein, the trophoblast organoid injury model without drug treatment is compared to evaluate the therapeutic effect of the drug to be screened on preeclampsia.

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