A method for constructing a preeclampsia rat model with pregnancy complicated with thrombocytopenia

Abstract

The application provides a construction method of a preeclampsia rat model with pregnancy and concurrent thrombocytopenia, relates to the technical field of disease animal model construction, and comprises the following steps: obtaining a pregnancy model by mating a pregnant female mouse with a male mouse; injecting an adenovirus into the uterus of the model group pregnant female mouse in the pregnancy cycle of the pregnancy model, and injecting a carrier virus into the control group; and observing and measuring typical clinical characteristics of preeclampsia of the rat. After being verified by a systematic preeclampsia animal model evaluation method, the preeclampsia-like mouse constructed by the method has typical clinical characteristics of preeclampsia such as thrombocytopenia, is highly similar to the performance of a preeclampsia patient, has good clinical symptom simulation, and is of great significance for in-depth study of the cause, occurrence and development mechanism and treatment scheme of PE.

Description

Technical Field

[0001] This invention relates to the field of animal model construction technology, specifically to a method for constructing a rat model of preeclampsia complicated by thrombocytopenia during pregnancy. Background Technology

[0002] Preeclampsia (PE) is a serious pregnancy complication with an incidence of approximately 3% to 5%. It is characterized by hypertension, proteinuria, and fetal growth restriction. Severe PE can lead to hemolysis, elevated liver enzymes, and thrombocytopenia, causing damage to multiple organ systems and is a major cause of maternal mortality. PE also increases the risk of offspring abnormalities, such as stillbirth, neonatal death, perinatal mortality, congenital abnormalities, fetal growth restriction (FGR), small for gestational age (SGA), low birth weight (SP), very low birth weight (VLS), and premature birth. Statistics show that the risk of offspring mortality increases by 29% in women with PE.

[0003] In recent years, significant progress has been made in the study of placental endovascular encephalopathy (PE). Studies have shown that dysregulation of multiple placental signaling pathways is closely related to PE. Differential expression of these pathways can impair the proliferation, migration, and infiltration of trophoblasts, leading to shallow placental implantation and failure of spiral artery remodeling. Reduced uteroplacental blood flow perfusion can cause placental ischemia and hypoxia, hindering normal fetal development. On the other hand, cytokines released from the placenta can enter the maternal bloodstream, excessively activating the inflammatory response, causing vascular endothelial damage, and resulting in various clinical manifestations of PE.

[0004] Currently, there is a lack of effective treatments for premature ejaculation (PE), often leading to termination of pregnancy to save the lives of both mother and fetus. This is a fundamental cause of high maternal and infant mortality and disability rates. Therefore, elucidating the exact pathogenesis of PE, identifying accurate predictive indicators, and exploring effective treatments are urgent tasks. However, the pathogenesis of PE is complex, involving multiple pathways, factors, and mechanisms, and a definitive answer remains elusive.

[0005] An ideal animal model should exhibit the clinical characteristics of the disease. Preeclampsia (PE) specifically occurs in the mid-to-late stages of pregnancy, manifesting as insufficient trophoblastic cell infiltration, impaired spiral artery remodeling, accompanied by hypertension, diffuse vascular endothelial dysfunction, and further leading to kidney damage, proteinuria, and other end-organ dysfunction. Thrombocytopenia is one of the important symptoms of preeclampsia, but current models primarily focus on hypertension, with little attention paid to platelet count.

[0006] Because the causative factors are unclear and the systemic symptoms are widespread, current known models cannot fully represent all the symptoms of preeclampsia (PE). In particular, PE complicated by thrombocytopenia can increase the risk of thrombosis and postpartum hemorrhage, leading to serious complications such as HELLP syndrome and DIC. Existing methods for constructing preeclampsia-like animal models are relatively complex and lack screening before model construction and a systematic evaluation system for platelet parameters associated with PE. This limits further in-depth research on PE complicated by thrombocytopenia. Therefore, establishing animal models with typical clinical characteristics of PE, including those complicated by thrombocytopenia, and improving the evaluation system related to platelet parameters are of great significance for in-depth research on the etiology, pathogenesis, and treatment of PE complicated by thrombocytopenia. Summary of the Invention

[0007] In view of this, the purpose of this invention is to propose a method for constructing a rat model of preeclampsia (PE) complicated by thrombocytopenia during pregnancy, based on the current state of the technology, and in particular, to provide a rat model with typical clinical features of preeclampsia complicated by thrombocytopenia during pregnancy based on the pathogenesis of preeclampsia (PE). This invention provides a method for constructing a rat model of preeclampsia (PE), and a rat animal model of preeclampsia (PE) with typical clinical features in early, mid, and late pregnancy (including indicators of hypertension, proteinuria, thrombocytopenia, and fetal growth restriction).

[0008] To achieve the above objectives, in a first aspect, the present invention provides a method for constructing a rat model of preeclampsia complicated by thrombocytopenia during pregnancy, the method comprising the following steps: Step 1: Establishing a pregnancy model: A pregnancy model is obtained by mating pregnant female mice with male mice; Step 2, Adeno-associated virus injection: During the gestation period of the pregnancy model, adeno-associated virus was injected into the uterus of gestational-age female mice in the model group, while the control group was injected with the vector virus. Step 3: Observation and Measurement: Observe and measure the typical clinical features of preeclampsia in rats.

[0009] As a further aspect of the present invention, when establishing a pregnancy model, gestational-age female mice and male mice are mated at a ratio of 2:1.

[0010] As a further aspect of the present invention, the species of the gestational age female and male mice are selected from SD rats, Wistar rats, or Lewis rats.

[0011] As a further aspect of the present invention, the gestational age female rats are 7-8 week old SD rats that have not mated. 18 gestational age female SD rats with normal estrous cycles are caged with mature male rats of the same strain at a ratio of 2:1. The presence of vaginal plugs the next morning is considered as 0.5 days of pregnancy.

[0012] As a further aspect of the present invention, pregnant female mice were randomly divided into a normal control group (control) and a preeclampsia model group (sh_Tagln2).

[0013] As a further aspect of the present invention, during the gestation period of the pregnancy model, adeno-associated virus was injected into the uterus of pregnant female mice on days 7.5 and 12.5, respectively.

[0014] As a further aspect of the present invention, the mTagln2 [shRNA] adeno-associated virus was injected into the uterus of pregnant female mice.

[0015] As a further aspect of the present invention, mTagln2 [shRNA] adeno-associated virus is injected twice into the uterus, with each injection dose being 1×10⁻⁶. 9 PFU, 100μL.

[0016] As a further aspect of the present invention, the adeno-associated virus and the vector virus were purchased from Shanghai Jikai Gene Technology Co., Ltd., wherein: Model group virus code: AV-U6>mTagln2 [shRNA#2]-PGK>EGFP; Control group virus catalog number: AV-U6>Scramble-shRNA-PGK>EGFP.

[0017] As a further aspect of the present invention, the typical clinical features of preeclampsia include hypertension, proteinuria, thrombocytopenia, and indicators of fetal growth restriction in gestational-age female mice.

[0018] The method for constructing a preeclampsia rat model involved in this invention is simple and feasible. After verification by a systematic evaluation method for preeclampsia-like animal models, the preeclampsia-like rats constructed by this method have typical clinical features of preeclampsia, including hypertension, proteinuria, thrombocytopenia, and fetal growth restriction. They are highly similar to the manifestations of preeclampsia patients and have good clinical symptom simulation. This is of great significance for in-depth research on the etiology, pathogenesis, and treatment of PE combined with thrombocytopenia.

[0019] Compared with existing technologies, the method for constructing a rat model of preeclampsia with thrombocytopenia during pregnancy proposed in this invention has the following beneficial effects: The present invention discloses a method for constructing a rat model of preeclampsia in pregnancy complicated with thrombocytopenia. The method involves injecting adeno-associated virus into the uterus of pregnant female rats. The construction process is simple and feasible. After verification by a systematic evaluation method for preeclampsia-like animal models, the preeclampsia-like rats constructed by this method have typical clinical features of preeclampsia and are highly similar to the manifestations of preeclampsia patients. They have good clinical symptom simulation, which is of great significance for in-depth research on the etiology, pathogenesis and treatment of PE.

[0020] These or other aspects of this application will become more apparent from the following description of embodiments. It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only, and are not intended to limit the application. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other embodiments can be obtained based on these drawings without creative effort.

[0022] In the diagram: Figure 1 In the figure, A represents the statistical graph of systolic blood pressure results of the Control group and the sh_Tagln2 group on days 7.5, 11.5, 14.5, and 17.5 of gestation in the method for constructing a rat model of preeclampsia with thrombocytopenia during pregnancy according to an embodiment of the present invention.

[0023] Figure 1 In the figure, B represents the statistical results of urinary protein (mg / mL) in the Control group and sh_Tagln2 group on days 11.5 and 17.5 of pregnancy in the method for constructing a rat model of preeclampsia with thrombocytopenia during pregnancy according to an embodiment of the present invention.

[0024] Figure 1 C in the figure represents a statistical comparison of platelet count (PLT) between the Control group and the sh_Tagln2 group in the method for constructing a rat model of preeclampsia with thrombocytopenia during pregnancy according to an embodiment of the present invention.

[0025] Figure 1 In the figure, D represents the weight statistics of fetal rats in the Control group and the sh_Tagln2 group in the method for constructing the preeclampsia rat model of pregnancy complicated with thrombocytopenia in this embodiment of the invention.

[0026] Figure 2In Figure A, the comparison chart of plateletcrit (PCT) between the Control group and the sh_Tagln2 group in the method for constructing the preeclampsia rat model of pregnancy complicated with thrombocytopenia in this embodiment of the invention is shown.

[0027] Figure 2 B in the figure is a statistical comparison of platelet distribution width (PDW) between the Control group and the sh_Tagln2 group in the method for constructing a rat model of preeclampsia with thrombocytopenia during pregnancy according to an embodiment of the present invention.

[0028] Figure 2 C in the figure represents a statistical comparison of mean platelet volume (MPV) between the Control group and the sh_Tagln2 group in the method for constructing a rat model of preeclampsia with thrombocytopenia during pregnancy according to an embodiment of the present invention.

[0029] Figure 2 In the figure, D represents a statistical comparison of the duration of tail vein bleeding between the Control group and the sh_Tagln2 group in the method for constructing a rat model of preeclampsia with thrombocytopenia during pregnancy according to an embodiment of the present invention.

[0030] Figure 3 The image shows the placental weight statistics of the Control group and the sh_Tagln2 group in the method for constructing a rat model of preeclampsia with thrombocytopenia during pregnancy, as described in this embodiment of the invention.

[0031] Figure 4 In the figure, A represents the actual images of fetal mice and placentas in the Control group and sh_Tagln2 group in the method for constructing the preeclampsia rat model of pregnancy complicated with thrombocytopenia in this embodiment of the invention.

[0032] Figure 4 In the figure, B is a photograph of the uterus and placenta of the fetal rat in the sh_Tagln2 group in the method for constructing a rat model of preeclampsia with thrombocytopenia during pregnancy in an embodiment of the present invention.

[0033] Figure 4 In the figure, C represents the immunoblot images of TAGLN2 protein expression in the placenta of pregnant rats in the Control group and sh_Tagln2 group in the method for constructing a rat model of preeclampsia with thrombocytopenia during pregnancy in this embodiment of the invention.

[0034] Figure 4 In the diagram, D represents the statistical difference in the relative protein expression of TAGLN2 in the placenta of pregnant rats in the sh_Tagln2 group compared to that in the Control group during the construction of the rat model of preeclampsia with thrombocytopenia in pregnancy according to the embodiment of the present invention. Detailed Implementation

[0035] The present application will now be further described in conjunction with the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0036] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be further described in detail below with reference to specific examples and the accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative and are not intended to limit the scope of this application.

[0037] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0038] The flowchart shown in the attached diagram is for illustrative purposes only and does not necessarily include all content and operations / steps, nor does it necessarily have to be performed in the order described. For example, some operations / steps can be broken down, combined, or partially merged, so the actual execution order may change depending on the actual situation.

[0039] The adeno-associated virus used in the following examples was purchased from Shanghai Jikai Gene Technology Co., Ltd.

[0040] The following detailed description of some embodiments of this application is provided in conjunction with the accompanying drawings. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0041] This invention provides a method for constructing a rat model of preeclampsia with thrombocytopenia during pregnancy. The method involves the screening of female rats before model construction. Specifically, 7-8 week old SD rats that have not mated are used as the research subjects. The rats are kept at a temperature of 21-23°C with free access to food. After one week of adaptive feeding, 18 SD rats with normal estrous cycles are paired with mature male rats of the same strain (2:1). The presence of vaginal plugs the following morning is considered 0.5 days of gestation. The female rats are randomly divided into a normal control group (control) and a preeclampsia model group (sh_Tagln2).

[0042] During the adeno-associated virus (AAV) injection phase, SD female rats on day 7.5 of gestation were randomly divided into a control group (n=9) and a shTagln2-RNA group (n=9). Pregnant rats in the shTagln2-RNA group received uterine injections of AAV on days 7.5 and 12.5 of gestation: AV-U6>mTagln2[shRNA#2]-PGK>EGFP (1×10⁻⁶). 9 PFU, 100 μL); control group pregnant rats were injected uterinely with control virus at corresponding time points: AV-U6>Scramble-shRNA-PGK>EGFP (1×10⁻⁶ PFU, 100 μL); 9 PFU (100 μL); From days 6.5, 9.5, 12.5, and 15.5 of gestation to days 7.5, 10.5, 13.5, and 16.5 of gestation, experimental rats were placed in metabolic cages to collect urine for 24 hours; and on days 7.5, 10.5, 13.5, and 16.5, the tail artery blood pressure of the experimental rats was monitored by an animal blood pressure measurement system before they were sacrificed.

[0043] Blood pressure measurement method: Turn on the intelligent non-invasive blood pressure monitor (ZS-Z), set the temperature of the insulated container to 37℃, and preheat for 10 minutes; place the female mouse in the mouse bag, leaving the tail exposed, and place the head in the mouse net. Place the wrapped female mouse in the 37℃ insulated container in a comfortable position, soothe her emotions, and keep her as quiet as possible; place the sensor at the base of the mouse's tail to monitor the blood flow in the female mouse's tail; start the measurement when the pulse is stable and complete the measurement within 10 minutes. During the measurement, pay attention to soothing the female mouse and you can gently stroke the rat's tail; record the measured systolic blood pressure. Record more than 5 values ​​for each female mouse. Take the systolic blood pressure values ​​of 3 consecutive groups with a difference of less than 6 mmHg within each group under the female mouse's resting state, and calculate their mean as the blood pressure value measured for that mouse on that day.

[0044] Observation and detection indicators: Peripheral anticoagulated blood was collected by enucleation and platelet parameters (including platelet count, plateletcrit, platelet distribution width, and mean platelet volume) were detected. Urine collection and urinary protein quantification in animals: Experimental rats were placed in metabolic cages to collect urine over 24 hours. The collected urine was centrifuged at 2000 rpm for 15 minutes at room temperature. The supernatant was collected, aliquoted, and stored at -80°C for later analysis of urinary protein and creatinine levels. The ratio of urinary microalbumin to creatinine was used as the final determination of urinary protein levels (excluding errors caused by varying muscle mass in different rats).

[0045] Abdominal aortic blood collection: Experimental rats were treated with cervical dislocation and immersed in 75% alcohol for 3-5 minutes. The abdominal cavity was opened using sterilized surgical instruments. A needle was inserted anterior to a branch of the common iliac artery and slowly moved centripetally along the abdominal aorta to collect approximately 5 mL of anticoagulated blood from pregnant rats. Platelet parameters were measured using a whole blood analyzer. Purified platelets were then obtained after gradient centrifugation for transcriptomics sequencing.

[0046] Embryo and placental tissue specimen collection: After blood collection, the rats were dissected, the uterus of the pregnant rats was completely removed, the decidua basalis, placenta and fetus were separated, and the rats were washed with pre-cooled PBS to remove blood, blood clots, etc. The number of surviving embryos and absorbed embryos, as well as the size, shape and wet weight of the placenta, were observed and recorded.

[0047] In rats, blood pressure and urinary protein levels increased during pregnancy, but blood pressure returned to normal postpartum. See also... Figure 1 As shown in Figure A, rat blood pressure increased during pregnancy. Statistical graph of systolic blood pressure results in the Control group and sh_Tagln2 group on days 7.5, 11.5, 14.5, and 17.5 of gestation; Systolic Blood pressure (mmHg): represents rat systolic blood pressure; ns: P > 0.05 compared to the control group; *: P < 0.05 compared to the control group; See also Figure 1 As shown in B, urinary protein levels increased in rats during pregnancy. The statistical results of urinary protein (mg / mL) in the Control group and the sh_Tagln2 group on days 11.5 and 17.5 of gestation are shown in the figure; *: P < 0.05 compared to the control group, where P represents a statistically significant difference.

[0048] like Figure 1 and Figure 2 As shown, pregnant mice injected with mTagln2 [shRNA] adeno-associated virus showed a significant increase in systolic blood pressure during pregnancy, while the control vector virus group showed no significant difference in systolic blood pressure. This indicates that exogenous downregulation of Tagln2 can lead to increased blood pressure in pregnant mice. Pregnant mice also showed a significant increase in urinary protein during pregnancy compared to the control group, while the control group showed no change. The results indicate that downregulation of Tagln2 can lead to typical eclampsia-related symptoms in pregnant mice, such as increased blood pressure and increased urinary protein.

[0049] In this embodiment, the control group was injected with the vector virus, while the injection of recombinant shTagln2-RNA induced a reduction in the volume of the placenta and fetus in pregnant rats, and a decrease in the weight of the rat fetus and placenta. See [link to relevant documentation]. Figure 3 As shown, the fetal weight of rats decreased. Fetal weight statistics of the Control group and the sh_Tagln2 group; ***: P < 0.05 compared with the control group; see also Figure 3 As shown, the placental weight of rats decreased. Placental weight statistics of the Control group and the sh_Tagln2 group; ***: P < 0.05 compared with the control group. During observation and measurement, female rats in each group were sacrificed at 16.5 days of gestation after the aforementioned treatment steps, and the uterus was dissected, and the fetuses and placentas were separated; Figure 3 As shown, the number of implantations and fetal weight in pregnant mice differed significantly between the shTagln2-RNA injection group and the control group. Figures 1 to 3As shown, the size of fetal mice and placentas in the shTagln2-RNA injection group was significantly smaller than that in the control group, suggesting that Tagln2 downregulation leads to restricted embryonic growth and development and poor placental development. Combined with... Figure 1 , Figure 2 and Figure 3 The results showed that exogenous knockdown of Tagln2 can lead to typical symptoms of preeclampsia in pregnant mice, including elevated blood pressure, increased proteinuria, decreased platelet count, restricted embryonic growth, and placental dysplasia.

[0050] Among them, see Figure 1 As shown in C, platelet count decreased in rats. A statistical chart comparing platelet (PLT) counts in the Control group and the sh_Tagln2 group is provided; **: P < 0.01 compared to the control group; see [link / reference]. Figure 2 As shown, plateletcrit decreased in rats. A statistical chart comparing plateletcrit (PCT) between the Control group and the sh_Tagln2 group is presented; *: P < 0.05 compared to the control group; see also... Figure 2 As shown, platelet distribution width (PDW) decreased in rats. A statistical comparison of platelet distribution width (PDW) between the Control group and the sh_Tagln2 group is presented; *: P < 0.05 compared to the control group; see also... Figure 2 As shown, platelet volume decreased in rats. A statistical comparison of mean platelet volume (MPV) between the Control group and the sh_Tagln2 group is presented; **: P < 0.01 compared to the control group.

[0051] In this embodiment, the experimental results of low expression of TAGLN2 in placental samples from pregnant mice in the sh_Tagln2 group model group are shown in [reference needed]. Figure 4 C and Figure 4 As shown in D, the relative protein expression level of TAGLN2 in the placenta of pregnant mice in the sh_Tagln2 group was significantly lower than that in the Control group. *: P < 0.05 compared with the control group.

[0052] In summary, the method for constructing a rat model of preeclampsia in pregnancy complicated with thrombocytopenia of the present invention involves injecting adeno-associated virus into the uterus of pregnant female rats. The construction process is simple and feasible. After verification by a systematic evaluation method for preeclampsia-like animal models, the preeclampsia-like rats constructed by this method have typical clinical features of preeclampsia and are highly similar to the manifestations of preeclampsia patients, with good clinical symptom simulation. This is of great significance for in-depth research on the etiology, pathogenesis, and treatment of PE.

[0053] The above are exemplary embodiments disclosed in this invention. However, it should be noted that various changes and modifications can be made without departing from the scope of the embodiments of this invention as defined by the claims. The functions, steps, and / or actions of the methods according to the disclosed embodiments described herein do not need to be performed in any particular order. Furthermore, although the elements disclosed in the embodiments of this invention may be described or claimed individually, they may be understood as multiple unless explicitly limited to a singular number.

[0054] It should be understood that, as used herein, the singular form "a" is intended to include the plural form as well, unless the context clearly supports an exception. It should also be understood that, as used herein, "and / or" refers to any and all possible combinations of one or more of the associatedly listed items. The embodiment numbers disclosed above are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0055] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of the invention (including the claims) is limited to these examples. Within the framework of the invention, technical features of the above embodiments or different embodiments can be combined, and many other variations of different aspects of the invention exist, which are not provided in the details for the sake of brevity. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the invention should be included within the protection scope of the invention.

Claims

1. A method for constructing a rat model of preeclampsia complicated by thrombocytopenia during pregnancy, characterized in that, The method includes the following steps: Step 1: Establishing a pregnancy model: A pregnancy model is obtained by mating pregnant female mice with male mice; Step 2, Adeno-Associated Virus Injection: During the gestation period of the pregnancy model, the uterus of pregnant female mice in the model group was injected with mTagln2 [shRNA] adeno-associated virus on days 7.5 and 12.5, respectively, while the control group was injected with the vector virus; the uterus was injected with mTagln2 [shRNA] adeno-associated virus twice, with each injection dose being 1×10 9 PFU, 100μL; Step 3: Observation and Measurement: Observe and measure the typical clinical features of preeclampsia in rats.

2. The method for constructing a rat model of preeclampsia with thrombocytopenia during pregnancy according to claim 1, characterized in that, When establishing the pregnancy model, pregnant female mice were mated with male mice at a ratio of 2:

1.

3. The method for constructing a rat model of preeclampsia complicated with thrombocytopenia during pregnancy according to claim 2, characterized in that, The gestational age of the female and male rats was selected from SD rats, Wistar rats, or Lewis rats.

4. The method for constructing a rat model of preeclampsia complicated with thrombocytopenia during pregnancy according to claim 3, characterized in that, The gestational age female rats were 7-8 week old SD rats that had not mated. SD rats with normal estrous cycles were gestational age female rats and mature male rats of the same strain were caged together at a ratio of 2:

1. The presence of vaginal plugs the next morning was considered as 0.5 days of gestation.

5. The method for constructing a rat model of preeclampsia with thrombocytopenia during pregnancy according to claim 4, characterized in that, Pregnant female mice were randomly divided into a normal control group and a preeclampsia model group.

6. The method for constructing a rat model of preeclampsia complicated by thrombocytopenia during pregnancy according to claim 1, characterized in that, The typical clinical features of preeclampsia include hypertension, proteinuria, thrombocytopenia, and indicators of fetal growth restriction in gestational-age female mice.

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