A rat uterine perfusion model and a method for constructing the same and applications thereof

Abstract

The application provides a rat uterine perfusion model and a construction method and application thereof, and belongs to the technical field of animal models. The construction method comprises the following steps: ligating blood vessels at a left kidney of a dead pregnant rat, blood vessels at a bifurcation of the upper end of the left kidney, bifurcation of the upper side and both sides of the internal iliac artery and the internal iliac vein, the right kidney and the bifurcation of the abdominal aorta and the posterior vena cava, and then inserting a tube into the abdominal aorta and the posterior vena cava, and pumping perfusion liquid from the abdominal aorta to obtain the model. The model forms a reproductive artery-placenta-fetus-placenta-reproductive vein circulation, maximally guarantees placental activity, and retains complete placental tissue. The model is simple to prepare and easy to obtain, can more truly and accurately simulate the circulation process of maternal body temperature and complete nutrients in the placenta supplied to the fetus through the mother and the placenta in the whole permeability experiment, and thus more objectively studies the permeability of a compound in the placenta, and is more reliable than a human placental perfusion model.

Description

A rat uterine perfusion model, its construction method and application Technical Field

[0001] This invention belongs to the field of animal model technology, and in particular relates to a rat uterine perfusion model and its construction method and application. Background Technology

[0002] The fetal period is a critical period for human growth and development, during which mothers and infants are most sensitive to chemical exposure and are a high-risk group for environmental exposure. Epidemiological evidence shows that the fetus is directly exposed to various chemicals (such as pesticides, pharmaceuticals, and environmental pollutants) through the placenta during pregnancy. However, current research on mother-to-child transmission of chemicals lacks technological support, and knowledge about the interaction between chemicals and placental transport proteins remains limited to a few substances and a very small number of transport proteins. Furthermore, pregnant women may develop various acute and chronic diseases during pregnancy, requiring medication treatment. According to a WHO survey of 14,778 pregnant women in 22 countries across 4 continents, 86% of women took medication during pregnancy, with an average of 2.9 medications per woman, making the safety of medication use during pregnancy a growing concern. The extent of drug transplacental transport, i.e., drug exposure, is a key factor in fetal risk assessment. Therefore, conducting placental permeability studies of drugs is an important basis for evaluating the safety of medication use during pregnancy.

[0003] The placenta, derived from fetal and maternal tissues, is considered the first fetal organ exposed to exogenous substances. It is composed of the fetal chorionic villi and amnion, and the maternal decidua basalis. The placenta separates the maternal and fetal circulations, providing the fetus with a relatively stable environment less susceptible to external influences; hence, it is also known as the placental barrier. The placental barrier facilitates the exchange of substances between mother and fetus, providing nutrients to the fetus while expelling fetal metabolic waste. The placenta's transfer and barrier functions are crucial for fetal nutrition and protection. Researchers have focused on establishing simple, readily available, and reliable technical systems to explore the patterns and mechanisms of mother-to-child transmission of chemical substances and to further clarify the health risks posed by chemical substances to infants.

[0004] Studies on the placental permeability of compounds in humans are ethically problematic. Currently used in vitro models, such as cell culture, are limited by their inability to preserve intact placental tissue, high cost, long culture periods, demanding equipment, and inability to accurately simulate the dynamic changes in the human placental barrier structure during pregnancy. In contrast, the human placental in vitro circulation perfusion model is currently the only in vitro experimental model that preserves intact placental tissue for studying drug placental permeability. Its physiological state most closely resembles the in vivo environment, making it the most valuable research method. However, it suffers from drawbacks such as complex model preparation, short maintenance time, and inapplicability to early pregnancy. Placental tissue preparation models are simple to operate, and placental tissue from all stages of pregnancy can be cultured, but they cannot be used for studies on transplacental transport.

[0005] Rats have stable estrous cycles, rapid growth, and good reproductive performance, making them widely used in toxicology and pathophysiology research. Rats' genes are highly similar to human genes, and the placenta of rodents, including rats, is often used as a model of the human placenta due to its structural similarity. Therefore, establishing a rat uterine perfusion model to provide a new research subject for studying the placental permeability of compounds is particularly necessary. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a rat uterine perfusion model and its construction method and application. The rat uterine perfusion model retains complete placental tissue, maximizes placental activity, and is simple and easy to prepare. In the entire permeability experiment, it can more realistically and accurately simulate the circulation process of the pregnant woman's body temperature and the complete nutrients in the placenta being supplied to the fetus through the mother and placenta, thereby more objectively studying the permeability of compounds in the placenta.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a method for constructing a rat uterine perfusion model, comprising the following steps:

[0009] The blood vessels in the left kidney of the dead pregnant rats were ligated, as were the blood vessels at the upper bifurcation of the left kidney, the upper side of the bifurcation of the internal iliac artery and internal iliac vein at the lower end, the iliopsoas artery and iliopsoas vein on both sides, and the bifurcation of the right kidney with the abdominal aorta and posterior vena cava. Then, the abdominal aorta and posterior vena cava were cannulated, and perfusion fluid was pumped in through the abdominal aorta after cannulation to obtain the rat uterine perfusion model.

[0010] The perfusion solution comprises the following components at the following concentrations: 120–130 mM NaCl, 2.5–3.5 mM KCl, 1.0–1.5 mM KH₂PO₄, 1.0–1.5 mM MgSO₄, 2.0–2.8 mM CaCl₂, 8–12 mM glucose, 25–30 mM NaHCO₃, 2.0%–3.0% dextran by volume, and 2.5%–3.5% dextran 70 by volume.

[0011] Preferably, the body temperature of the deceased pregnant rat is maintained at 37°C, and the abdominal tissue of the deceased pregnant rat is moistened with phosphate buffer at 37°C.

[0012] Preferably, the abdominal aortic cannulation uses an indwelling needle with a diameter of 0.65 mm; the posterior vena cava cannulation uses an indwelling needle with a diameter of 1.2 mm.

[0013] Preferably, the pumped flow rate is 1.5 to 3.5 mL / min.

[0014] Preferably, the perfusion fluid is filled with 95% O2 and 5% CO2.

[0015] The present invention also provides a rat uterine perfusion model obtained by the above construction method.

[0016] The present invention also provides a perfusion fluid for constructing a rat uterine perfusion model, the perfusion fluid comprising the following components at the following concentrations: 120–130 mM NaCl, 2.5–3.5 mM KCl, 1.0–1.5 mM KH₂PO₄, 1.0–1.5 mM MgSO₄, 2.0–2.8 mM CaCl₂, 8–12 mM glucose, 25–30 mM NaHCO₃, 2.0%–3.0% dextran by volume, and 2.5%–3.5% dextran 70 by volume.

[0017] Preferably, the perfusion fluid is filled with 95% O2 and 5% CO2.

[0018] The present invention also provides an application of the above-mentioned perfusion fluid or rat uterine perfusion model in the preparation of compound placental permeability research products.

[0019] The present invention also provides an application of the above-mentioned perfusion fluid in constructing a rat uterine perfusion model.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] This invention provides a rat uterine perfusion model, its construction method, and its applications. This rat uterine perfusion model involves cannulation of the abdominal aorta and posterior vena cava to establish a gynecological artery-placenta-fetus-placenta-gynecological vein return flow. The uterus is immediately perfused with perfusion fluid to maximize placental viability while preserving intact placental tissue. This model is simple to prepare and readily available. Throughout the permeability experiment, it can more realistically and accurately simulate the circulation process of intact nutrients from the placenta through the mother and fetus, thereby providing a more objective study of compound permeability within the placenta. Compared to human placental in vitro perfusion models, this model is more reliable and provides technical support for elucidating and revealing the characteristics and related mechanisms of chemical substances (such as pesticides, pharmaceuticals, and environmental pollutants) migrating from the mother to offspring, and the impact of maternal exposure on offspring. This invention has wide applications and can be used to study the laws governing transplacental chemical transfer and transplacental transport mechanisms. Attached Figure Description

[0022] Figure 1 is a general diagram of the rat uterine perfusion model;

[0023] Figure 2 shows the permeation of different pollutants in the placenta, amnion, and fetal rats after rat perfusion.

[0024] Figure 3 shows the relationship between the permeation of different pollutants and their physicochemical properties, where (A) represents the concentration of 18 LCMs in fetal rats and their octanol-water partition coefficient (logK). ow (A) shows the correlation between the concentrations of 18 LCMs in fetal rats and their molecular weights (MW), and (B) shows the correlation between the concentrations of 18 LCMs in the placenta and their molecular weights (MW). (C) shows the correlation between the concentrations of 18 LCMs in the placenta and their molecular weights (MW) and their octanol-water partition coefficients (log K). ow (D) shows the correlation between the concentrations of 18 LCMs in the placenta and their molecular weights (MW).

[0025] Figure 4 shows the relative expression of transporters in the placenta after LCMs were perfused and in LCMs without perfusion. Detailed Implementation

[0026] This invention provides a method for constructing a rat uterine perfusion model, comprising the following steps:

[0027] The blood vessels in the left kidney of the dead pregnant rats, the blood vessels at the upper bifurcation of the left kidney, the upper side of the bifurcation of the internal iliac artery and internal iliac vein at the lower end, the bilateral iliopsoas arteries and iliopsoas veins, and the bifurcation of the right kidney with the abdominal aorta and posterior vena cava were ligated. Then, the abdominal aorta and posterior vena cava were cannulated, and perfusion fluid was pumped in through the abdominal aorta to obtain the rat uterine perfusion model.

[0028] The perfusion solution comprises the following components at the following concentrations: 120–130 mM NaCl, 2.5–3.5 mM KCl, 1.0–1.5 mM KH₂PO₄, 1.0–1.5 mM MgSO₄, 2.0–2.8 mM CaCl₂, 8–12 mM glucose, 25–30 mM NaHCO₃, 2.0%–3.0% dextran by volume, and 2.5%–3.5% dextran 70 by volume.

[0029] In this invention, the pregnant rat is preferably a rat pregnant for 18.5 days. The rat is preferably an SD rat, which has a stable estrous cycle, rapid growth, good reproductive performance, and its genes are highly similar to human genes. Rodent placentas, including rats, are used as models of the human placenta due to their similar structure to the human placenta. Before the pregnant rat dies, 1-2 mL of heparin sodium is injected to achieve systemic heparinization and anticoagulation. The heparin sodium content is preferably 95-105 IU / mL, and the solvent is physiological saline at 37°C. The body temperature of the deceased pregnant rat is maintained at 37°C. This can be achieved by placing the deceased pregnant rat on a temperature-controlled pad, maintaining the rat's body temperature at 37°C during the experiment and minimizing the loss of placental activity due to rat death. The abdominal tissue of the deceased pregnant rat is preferably moistened with phosphate buffer at 37°C. This invention does not have a specific limitation on the source of the phosphate buffer; it can be prepared using commercially available products or known preparation methods.

[0030] In this invention, the blood vessels at the left kidney of a deceased pregnant rat, the blood vessels at the upper bifurcation of the left kidney, the upper side of the bifurcation of the internal iliac artery and internal iliac vein at the lower end, the bilateral iliopsoas arteries and veins, and the bifurcation of the right kidney with the abdominal aorta and posterior vena cava are ligated. Then, the abdominal aorta and posterior vena cava are cannulated. The rat uterine perfusion model constructed by this invention establishes a reproductive artery-placenta-fetus-placenta-reproductive vein return flow through abdominal aortic and posterior vena cava cannulation, accurately forming a "placental barrier system." This provides technical support for elucidating and revealing the characteristics and related mechanisms of chemical migration from the mother to the offspring, and the impact of maternal exposure on offspring. The abdominal aortic cannulation uses a 0.65 mm diameter indwelling needle, such as a 26G indwelling needle; the posterior vena cava cannulation uses a 1.2 mm diameter indwelling needle, such as an 18G indwelling needle. In this invention, the pumped flow rate is preferably 1.5–3.5 mL / min, more preferably 1.8–3.2 mL / min.

[0031] This invention also provides a rat uterine perfusion model obtained by the above-described construction method. This rat uterine perfusion model is an experimental model used to study the mother-to-child transmission of chemical substances. The uterus of the rat is perfused immediately after death. Throughout the permeability experiment, it can more realistically and accurately simulate the circulation process of the pregnant woman's body temperature and the complete supply of nutrients from the placenta to the fetus. This allows for a more objective study of the permeability of compounds within the placenta, enabling researchers to intuitively understand whether and to what extent the chemical substances that pregnant women are exposed to daily affect the fetus. Furthermore, this rat uterine perfusion model can simultaneously perfuse both uteruses.

[0032] The present invention also provides a perfusion fluid for constructing a rat uterine perfusion model, the perfusion fluid comprising the following components at the following concentrations: 120–130 mM NaCl, 2.5–3.5 mM KCl, 1.0–1.5 mM KH₂PO₄, 1.0–1.5 mM MgSO₄, 2.0–2.8 mM CaCl₂, 8–12 mM glucose, 25–30 mM NaHCO₃, 2.0%–3.0% dextran by volume, and 2.5%–3.5% dextran 70 by volume.

[0033] In this invention, as a preferred embodiment, the perfusion solution comprises the following components at the following concentrations: 122–128 mM NaCl, 2.7–3.3 mM KCl, 1.1–1.3 mM KH₂PO₄, 1.1–1.4 mM MgSO₄, 2.2–2.6 mM CaCl₂, 9–11 mM glucose, 26–28 mM NaHCO₃, 2.2%–2.8% dextran by volume, and 2.7%–3.2% dextran 70 by volume. The volume percentages of dextran and dextran 70 are calculated based on the volume of the perfusion solution. Preferably, the perfusion solution is filled with 95% O₂ and 5% CO₂ to ensure the activity of placental barrier cells and realistically simulate the in vivo environment. The pH value of the perfusion solution is preferably 7.2–7.6. After preparation, the perfusion solution is preheated in a 37°C water bath and maintained at this temperature during the perfusion process. The Krebs-Ringer's perfusion solution used in this invention (122–128 mM NaCl, 2.7–3.3 mM KCl, 1.1–1.3 mM KH₂PO₄, 1.1–1.4 mM MgSO₄, 2.2–2.6 mM CaCl₂, 9–11 mM glucose, 26–28 mM NaHCO₃) is suitable for various mammalian tissues, such as nerve tissue, isolated heart perfusion, blood vessel or intestinal perfusion, etc. It can be used to clean tissues and maintain the normal physiological functions of isolated tissues, providing water and inorganic salts to meet normal cell metabolism for cell growth and proliferation, providing nutrients such as amino acids to meet the needs of cell growth and proliferation metabolism, adding carbohydrates (glucose) and metabolic intermediates to meet the energy needs of cells, and providing a suitable osmotic pressure environment for cells. The addition of dextran and dextran 70 to the perfusion solution of this invention can prevent thrombosis, increase plasma volume, maintain blood pressure, and more closely resemble the effect of real blood, thus more realistically reflecting the material transport process of the placental barrier in vivo. In this invention, there are no special limitations on the sources of NaCl, KCl, KH2PO4, MgSO4, CaCl2, glucose, and NaHCO3; commercially available products known in the art can be used. The dextran used in this invention was purchased from Maclean's, catalog number D888362, and dextran 70 was purchased from Maclean's, catalog number D806715.

[0034] The present invention also provides an application of the above-mentioned perfusion fluid or rat uterine perfusion model in the preparation of compound placental permeability research products.

[0035] In this invention, when studying the placental permeability of a compound using a perfusion fluid or a rat uterine perfusion model, the blood is first flushed with a blank perfusion fluid containing no target substance for 5 minutes before the formal experiment begins. During perfusion, the perfusion fluid pumped into the abdominal aorta passes through the following pathways: uterine artery—placenta—fetus—placenta—uterine vein, and drips from a drainage tube inserted into the posterior vena cava. The perfusion fluid is collected every 5 minutes.

[0036] The present invention also provides an application of the above-mentioned perfusion fluid in constructing a rat uterine perfusion model.

[0037] The rat uterine perfusion model has significant scientific value and research significance. It accurately simulates the circulation process of nutrients from the placenta to the fetus in pregnant women, allowing observation of the permeability of foreign substances such as pesticides, medicines, and environmental pollutants within the human placenta. It provides a direct understanding of whether substances that pregnant women are exposed to daily will affect the fetus and the extent of that effect. It also guides pregnant women on how to ensure the healthy and rapid growth and development of the fetus, improves the fetus's health level, and effectively avoids the risks of in vivo experiments.

[0038] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1

[0040] A method for constructing a rat uterine perfusion model, comprising the following steps:

[0041] (1) Pregnant rats (Sprague-Dawley, SD) with a gestation period of 18.5 days were fed with standard diet at room temperature. They were fasted for 12 hours before treatment and had free access to water.

[0042] (2) The rats were euthanized by intravenous injection of 2 mL of 100 IU / mL heparin sodium preheated to 37°C to achieve systemic heparinization and anticoagulation. Ten minutes later, the rats were euthanized by cervical dislocation. The abdomen was immediately opened to expose the uterus, abdominal aorta, posterior vena cava, gonadal artery, gonadal vein, renal artery, renal vein, iliopsoas artery, iliopsoas vein, internal iliac artery, and internal iliac vein. The animals' body temperature was maintained at 37°C using a temperature-controlled pad, and all exposed tissues were kept moist with phosphate-buffered saline preheated to 37°C.

[0043] (3) After opening the rat's abdomen, the blood vessels were ligated at the following five locations: ① left kidney; ② bifurcation at the upper end of the left kidney; ③ upper side of the internal iliac artery and internal iliac vein at the lower end; ④ bilateral iliopsoas arteries and iliopsoas veins; ⑤ bifurcation of the right kidney with the abdominal aorta and posterior vena cava.

[0044] (4) After ligating all blood vessels except the genital artery and vein, insert a cannula:

[0045] ① A 26G indwelling needle was used to insert a cannula into the abdominal aorta;

[0046] ②Use an 18G indwelling needle to insert a cannula into the posterior vena cava;

[0047] ③ Secure the indwelling needle tightly to the blood vessel to prevent it from slipping out.

[0048] (5) Pump the preheated perfusion fluid to 37°C into the abdominal aorta at a flow rate of 2 mL / min, and fill the perfusion fluid with 95% O2 and 5% CO2.

[0049] (6) After pre-perfusion rinsing the blood with blank perfusion fluid that does not contain the target substance for 5 minutes, the formal experiment can begin. During perfusion, the perfusion fluid pumped into the abdominal aorta passes through: uterine artery—placenta—fetus—placenta—uterine vein, and drips from the drainage tube inserted into the posterior vena cava. The perfusion fluid is collected every 5 minutes. After the perfusion experiment, the fetal mice are euthanized, and tissue samples such as amniotic fluid, placenta, fetal mice, and amniotic fluid can be collected.

[0050] The perfusion solution consists of the following components: 126 mM NaCl, 3 mM KCl, 1.2 mM KH2PO4, 1.3 mM MgSO4, 2.4 mM CaCl2, 10 mM glucose, 26 mM NaHCO3, 2.5% dextran, 3% dextran 70, and the balance being ultrapure water.

[0051] A summary diagram of the specific rat uterine perfusion model is shown in Figure 1.

[0052] Example 2

[0053] The difference between this embodiment and Embodiment 1 is that the perfusion solution is composed of the following components: 120mM NaCl, 3.5mM KCl, 1.0mM KH2PO4, 1.5mM MgSO4, 2.0mM CaCl2, 12mM glucose, 30mM NaHCO3, 2.0% dextran, and 3.5% dextran 70. The other steps are the same as in Embodiment 1.

[0054] Example 3

[0055] The difference between this embodiment and Embodiment 1 is that the perfusion solution is composed of the following components: 130mM NaCl, 2.5mM KCl, 1.5mM KH2PO4, 1.0mM MgSO4, 2.8mM CaCl2, 8mM glucose, 25mM NaHCO3, 3.0% dextran, and 2.5% dextran 70. The other steps are the same as in Embodiment 1.

[0056] Example 4 was used to study interfetal differences in mammalian multiple pregnancies.

[0057] Liquid crystal monomers (LCMs) are a class of artificially synthesized organic chemicals specifically used in the production of liquid crystal displays. Their production volume and ecological and health risks are increasing, making them a "new pollutant." Currently, liquid crystal monomers are being used as model compounds in laboratory experiments.

[0058] 4.1 A method for constructing a rat uterine perfusion model, the steps of which are as follows:

[0059] (1) Pregnant rats (Sprague-Dawley, SD) with a gestation period of 18.5 days were fed with standard diet at room temperature. They were fasted for 12 hours before treatment and had free access to water.

[0060] (2) The rats were euthanized by intravenous injection of 2 mL of 100 IU / mL heparin sodium preheated to 37°C to achieve systemic heparinization and anticoagulation. Ten minutes later, the rats were euthanized by cervical dislocation. The abdomen was immediately opened to expose the uterus, abdominal aorta, posterior vena cava, gonadal artery, gonadal vein, renal artery, renal vein, iliopsoas artery, iliopsoas vein, internal iliac artery, and internal iliac vein. The animals' body temperature was maintained at 37°C using a temperature-controlled pad, and all exposed tissues were kept moist with phosphate-buffered saline preheated to 37°C.

[0061] (3) After opening the rat's abdomen, the blood vessels were ligated at the following five locations: ① left kidney; ② bifurcation at the upper end of the left kidney; ③ upper side of the internal iliac artery and internal iliac vein at the lower end; ④ bilateral iliopsoas arteries and iliopsoas veins; ⑤ bifurcation of the right kidney with the abdominal aorta and posterior vena cava.

[0062] (4) After ligating all blood vessels except the genital artery and vein, insert a cannula:

[0063] ① A 26G indwelling needle was used to insert a cannula into the abdominal aorta;

[0064] ②Use an 18G indwelling needle to insert a cannula into the posterior vena cava;

[0065] ③ Secure the indwelling needle tightly to the blood vessel to prevent it from slipping out.

[0066] (5) Pump the preheated perfusion fluid to 37°C into the abdominal aorta at a flow rate of 2 mL / min, and fill the perfusion fluid with 95% O2 and 5% CO2.

[0067] (6) First, use a perfusion fluid without LCMs to pre-perfuse and rinse the blood for 5 minutes before starting the formal experiment.

[0068] (7) The perfusion fluid contained 17 LCMs at a concentration of 30 ng / mL (specific compound information is shown in Table 1). During perfusion, the perfusion fluid pumped into the abdominal aorta passed through the following pathway: uterine artery—placenta—fetus—placenta—uterine vein, and dripped from the drainage tube inserted into the posterior vena cava. The perfusion fluid was collected every 5 minutes, and the experiment lasted for 40 minutes. A total of eight groups of samples were tested. After the perfusion experiment, the fetal mice were euthanized, and tissue samples such as amniotic fluid, placenta, fetal mice, and amniotic fluid were collected.

[0069] The perfusion solution is composed of the following components: 126 mM NaCl, 3 mM KCl, 1.2 mM KH2PO4, 1.3 mM MgSO4, 2.4 mM CaCl2, 10 mM glucose, 26 mM NaHCO3, 2.5% dextran, and 3% dextran 70.

[0070] 4.217 detection methods for LCMs:

[0071] Regarding placental and amniotic tissues:

[0072] 1) Place it in a 2mL screw tube and add 20ng of internal standard (the structure of d4-DFPrB is listed in Table 1);

[0073] 2) Add 1 mL of extraction solution—hexane:ethyl acetate = 1:1 (v:v), 1 pickle bead, and homogenize using a tissue homogenizer;

[0074] 3) Transfer the sample to a 15 mL centrifuge tube, rinse the original screw-top tube with 1 mL of extraction buffer, add another 6 mL of extraction buffer, shake at 2400 rpm for 10 min, sonicate for 10 min, centrifuge at 2809 g for 10 min, and collect the extraction buffer. Repeat the extraction once with 5 mL of extraction buffer.

[0075] 4) Add 10 mg Envi-carb and 100 mg C18, shake for 10 min, centrifuge at 2809 g for 10 min, and collect the supernatant;

[0076] 5) Blow the extract to dryness with nitrogen, add 1 mL of n-hexane to reconstitute, filter through a membrane, and wait for injection.

[0077] For fetal mouse tissue:

[0078] 1) Add 20 ng of internal standard (the structure of d4-DFPrB is listed in Table 1);

[0079] 2) Add 4 mL of extract—hexane:ethyl acetate = 1:1 (v:v) and homogenize using a hand homogenizer;

[0080] 3) Add 2 mL of extraction solution—hexane:ethyl acetate = 1:1 (v:v);

[0081] 4) Shake for 10 min, sonicate for 10 min, centrifuge at 2809g for 10 min, collect the extract, add 5 mL of extract and repeat the extraction once;

[0082] 5) Add 10 mg Envi-carb and 100 mg C18, shake for 10 min, centrifuge at 2809 g for 10 min, and collect the supernatant;

[0083] 6) Blow the extract to dryness with nitrogen, add 1 mL of n-hexane to reconstitute, filter through a membrane, and wait for injection.

[0084] Quantitative detection and analysis of LCMs in samples were performed using an Agilent 7890A gas chromatography system (Agilent Technologies, Santa Clara, CA). This system was equipped with a 7693 autosampler (CTC Analytics, Zwingen, Switzerland) and connected in tandem with an XEVO TQ-S quadrupole mass spectrometer equipped with an atmospheric pressure gas chromatograph source (APGC, Milford, MA, USA), employing multiple reaction monitoring (MRM) mode. API positive ion mode and sensitivity mode were selected. A DB-5MS gas chromatography column (Agilent Technologies) with an inner diameter of 30 μm × 0.250 mm and a film thickness of 0.25 μm was used. The column oven temperature program was as follows: initial temperature 60 °C, held for 0.52 min, ramped up to 130 °C at 30 °C / min, then ramped up to 250 °C at 7 °C / min, and then ramped up to 300 °C at 10 °C / min and held for 5 min, for a total run time of 30 min. The corona voltage was 2.2 kV, the conical gas flow rate was set to 150 L / h, and the ion source temperature was 150 °C. Helium was used as the carrier gas at a flow rate of 1.2 mL / min. The injection volume was set to 5.0 μL, and splitless injection was used.

[0085] Table 1. Material information of the 17 LCMs in Example 4

[0086]

[0087]

[0088] The results in Figure 2 show that 5 out of 17 LCMs (LCM-73, LCM-39, LCM-69, LCM-57 and LCM-17) crossed the placental barrier. The transplacental transport of LCMs is selective, and the penetration of LCMs varies in different placental units. The chemical substances differ between mammalian multiple fetuses.

[0089] Example 5 was used to study the transmission patterns of different chemical substances between mother and child.

[0090] The difference between the rat uterine perfusion model construction method in this embodiment and part 4.1 of Example 4 is that in this embodiment, step (7) involves a perfusion fluid containing 18 LCMs at a concentration of 1 μg / mL (specific compound information is shown in Table 2). During perfusion, the perfusion fluid pumped into the abdominal aorta passes through the following pathway: uterine artery—placenta—fetus—placenta—uterine vein, and drips from a drainage tube inserted into the posterior vena cava. The perfusion fluid is collected every 5 minutes, and the experiment lasts for 40 minutes. A total of 5 groups of samples were tested. After the perfusion experiment, the fetal rats were euthanized, and placental and fetal rat tissue samples were collected.

[0091] For the detection method of LCMs, please refer to section 4.2 of Example 4.

[0092] Table 2. Material information of the 18 LCMs in Example 5

[0093]

[0094]

[0095] The results in Figure 3 show that the concentration of LCMs in fetal mice and placenta is strongly negatively correlated with the physicochemical properties of LCMs (octanol-water partition coefficient (log Kow) and molecular weight (MW)), indicating that substances with stronger lipophilicity and larger molecular weight are more difficult to penetrate the placental barrier.

[0096] Example 6

[0097] The difference between the rat uterine perfusion model construction method in this embodiment and part 4.1 of Example 4 is that in this embodiment, step (7) involves a perfusion fluid containing 22 LCMs at a concentration of 1 μg / mL (specific compound information is shown in Table 3). During perfusion, the perfusion fluid pumped into the abdominal aorta passes through the following pathway: uterine artery—placenta—fetus—placenta—uterine vein, and drips from a drainage tube inserted into the posterior vena cava. The perfusion fluid is collected every 5 minutes, and the experiment lasts for 50 minutes. Three groups of samples were tested. After the perfusion experiment, the fetal rats were euthanized, and placental and fetal rat tissue samples were collected. A control group was prepared using a perfusion fluid without LCMs.

[0098] Table 3. Material information of the 22 LCMs in Example 3

[0099]

[0100]

[0101]

[0102] Real-time PCR experimental steps:

[0103] 1) Extract total RNA:

[0104] a. Add 1 / 4 of a mouse placenta (approximately 100 mg) to 1 mL of lysis buffer RZ and homogenize.

[0105] b. Let the homogenized sample stand at room temperature for 5 minutes;

[0106] c. Centrifuge at 4℃ and 12000rpm for 5 minutes, collect the supernatant and transfer it to a new centrifuge tube;

[0107] d. Add 200 μL of chloroform, cap the tube, shake for 15 seconds, and let stand at room temperature for 3 minutes;

[0108] e. Centrifuge at 4℃, 12000rpm for 10min, take the upper aqueous phase and transfer it to a new tube;

[0109] f. Slowly add 0.5 times the volume of anhydrous ethanol and mix well;

[0110] g. Transfer all the liquid into the CR3 adsorption column, centrifuge at 12000 rpm for 1 min at 4℃, and discard the waste liquid in the collection tube.

[0111] h. Add 500 μL of protein removal solution RD to CR3, centrifuge at 4℃ and 12000 rpm for 1 min, discard the waste liquid, and put CR3 into a collection tube.

[0112] i. Add 500 μL of rinsing buffer RW to CR3, let stand at room temperature for 2 min, centrifuge at 12000 rpm for 1 min at 4℃, and discard the waste liquid.

[0113] j. Repeat step (i);

[0114] k. Place the adsorption column CR3 into a 2mL collection tube, centrifuge at 4℃ and 12000rpm for 2min to remove residual liquid.

[0115] 1. Place on a clean bench and air dry for 30 minutes;

[0116] m. Transfer CR3 to a new 1.5mL centrifuge tube, add 60μL of ultrapure water, incubate at room temperature for 2min, then centrifuge at 12000rpm for 2min at 4℃.

[0117] n. Repeat step (m) (add 40 μL of ultrapure water) and combine the two liquids;

[0118] o. Measure RNA concentration, or freeze at -80℃;

[0119] 2) Reverse transcription: RNA was reverse transcribed into cDNA using the FastKing gDNA Dispelling RT SuperMix (TIANGEN) reverse transcription kit, as shown in Table 4. The reaction mixture was prepared on ice, gently vortexed to mix, and placed in a PCR instrument. The temperature program was set as follows: 42℃ for 15 min → 95℃ for 3 min.

[0120] Table 4 RNA reverse transcription system

[0121]

[0122] 3) Real-time quantitative PCR: The reverse-transcribed cDNA was used as a template for PCR detection using the Hieff UNICON qPCR SYBR Green Master Mix kit. The qRT-PCR reaction system was prepared on ice, as shown in Table 5. The reaction temperature program was: 95℃ 30s → 95℃ 10s → 60℃ 20s → 72℃ 20s (40 cycles). The melting curve was set using the instrument's default settings. Primer sequences are shown in Table 6.

[0123] Table 5 cDNA amplification system

[0124]

[0125] Table 6. Transporter primer sequences

[0126]

[0127] As shown in Figure 4, the expression levels of organic anion transporters (oat10), organic cation transporters (oct3), folate receptor (folr1), serotonin transporters (sert), sodium / bile acid cotransporters (soat), balanced nucleotide transporters (ent1), multidrug resistance transporters (mdr1a), and neurotransmitter transporters (net) were upregulated compared to the blank perfusion group without LCMs. These transporters may be involved in the transplacental transport of LCMs.

[0128] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing a rat uterine perfusion model, characterized in that, Includes the following steps: The blood vessels in the left kidney of deceased pregnant rats were ligated, including the vessels at the upper bifurcation of the left kidney, the upper side of the bifurcation of the internal iliac artery and internal iliac vein at the lower end, the bilateral iliopsoas arteries and veins, and the bifurcation of the right kidney with the abdominal aorta and posterior vena cava. Cannulation was then performed on the abdominal aorta and posterior vena cava, and perfusion fluid was pumped in through the abdominal aorta to establish a rat uterine perfusion model. The perfusion fluid contained the following concentrations: 120-130 mM NaCl, 2.5-3.5 mM KCl, 1.0-1.5 mM KH2PO4, 1.0-1.5 mM MgSO4, 2.0-2.8 mM CaCl2, 8-12 mM glucose, and 25-30 mM... NaHCO3, 2.0%~3.0% dextran by volume and 2.5%~3.5% dextran 70 by volume; during perfusion, the perfusion fluid pumped into the abdominal aorta passes through: uterine artery—placenta—fetus—placenta—uterine vein, and drips from the drainage tube inserted into the posterior vena cava, and the perfusion fluid is collected every 5 minutes.

2. The construction method according to claim 1, characterized in that, The body temperature of the deceased pregnant rats was maintained at 37°C, and the abdominal tissues of the deceased pregnant rats were moistened with phosphate buffer at 37°C.

3. The construction method according to claim 1, characterized in that, The abdominal aortic cannulation uses an indwelling needle with a diameter of 0.65 mm; the posterior vena cava cannulation uses an indwelling needle with a diameter of 1.2 mm.

4. The construction method according to claim 1, characterized in that, The pumped flow rate is 1.5~3.5 mL / min.

5. The construction method according to claim 1, characterized in that, The perfusion fluid is filled with 95% O2 and 5% CO2.

6. A rat uterine perfusion model obtained by any one of the construction methods according to claims 1 to 5.

7. The application of the rat uterine perfusion model of claim 6 in the preparation of compound placental permeability research products.

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