Construction method and application of severe preeclampsia rat model based on TET2

Abstract

The invention discloses a construction method and application of a severe preeclampsia rat model based on TET2, and belongs to the technical field of biomedicine.Ad-shTET2 is injected into a pregnant rat in the gestation period, the blood pressure and the 24-h urine protein level of the pregnant rat are monitored, the intrauterine development condition of a fetal rat and histomorphological changes of the placenta, the liver and the kidney of the pregnant rat are observed, and the severe preeclampsia rat model is established. According to the method, the model of the rats with severe preeclampsia is constructed, the model has characteristic symptoms and sign changes of severe preeclampsia, the severity of the disease can be reflected, the operation controllability is good, the stability is good, and the method has important significance on screening of candidate drugs for treating severe preeclampsia and research on pathogenesis of severe preeclampsia.

Description

Technical Field

[0001] The present invention is a method for constructing a severe preeclampsia rat model based on TET2 and its application, specifically relates to a method for constructing a severe preeclampsia rat model using TET2 regulatory protein and related applications of this method or model in the treatment of severe preeclampsia, belonging to the field of biomedical technology. Background Art

[0002] Preeclampsia is a multifactorial, multi-pathway, and multi-organ syndrome of pregnancy with new-onset hypertension (≥140 / 90 mmHg) as the main clinical feature after 20 weeks of gestation. It can be divided into preeclampsia and severe preeclampsia according to its severity. The pathological basis is shallow trophoblast infiltration and spiral artery remodeling disorder, but the specific pathogenesis is still unclear.

[0003] Due to the medical ethics restrictions on human experiments, animal models have become an important tool for studying the occurrence and development of preeclampsia. However, there is currently no animal model that can well reflect the severity of severe preeclampsia. The way of placental formation and the degree of trophoblast invasion in rodents are similar to those in humans. The mother has uterine spiral artery remodeling. After successful modeling, the typical phenotype of preeclampsia may appear. The rat model of preeclampsia used in the past was constructed by injecting sFlt1 adenovirus into the rat tail vein, but this model generally does not cause damage to multiple organs and cannot well reflect severe preeclampsia.

[0004] In order to better simulate the clinical characteristics of patients with preeclampsia, in the prior art, Chinese patent CN115843748A constructs an animal model of preeclampsia by administering N-nitro-L-arginine methyl ester or its salt during female rat pregnancy, which can make the constructed rat model of preeclampsia have typical preeclampsia-like phenotypes such as hypertension, proteinuria, liver and kidney dysfunction, angiogenesis-related factor disorder, placental angiogenesis disorder, and fetal development restriction. In addition, Chinese patent CN117797182A constructs a pregnant rat model of preeclampsia by injecting adenovirus overexpressing TPBG gene during rat pregnancy, which can induce clinical symptoms of preeclampsia such as increased blood pressure, proteinuria, kidney damage, and abnormal expression levels of sFlt-1 and PlGF in pregnant rats.

[0005] TET2 is a demethylase for DNA methylation and RNA methylation in epigenetic regulation and is an important regulatory protein. Studies have reported that TET2 affects the biological behaviors of tumor cells such as hepatocellular carcinoma, such as proliferation, migration, invasion, and cell cycle. Based on the similar biological behaviors of trophoblasts and tumor cells, it is speculated that abnormal expression of TET2 may be involved in regulating the biological behaviors of trophoblasts and is a central link in the occurrence and development of preeclampsia. However, existing studies have only reported that TET2 is related to the proliferation, invasion, migration, and cell cycle of tumor cells. There are currently no studies on the effects of TET2 on the biological behaviors of trophoblasts and related reports on its relationship with severe preeclampsia. Summary of the invention

[0006] The purpose of the present invention is to provide a method for constructing a severe preeclampsia rat model based on TET2 and its application. By injecting Ad-shTET2 into pregnant mice, a model with characteristic symptoms and signs of severe preeclampsia can be constructed, which can reflect the severity of the disease, has good controllability and stability, and is of great significance for the screening of candidate drugs for the treatment of severe preeclampsia and the study of the pathogenesis of severe preeclampsia.

[0007] The present invention is achieved through the following technical scheme: a method for constructing a severe preeclampsia rat model based on TET2, injecting Ad-shTET2 into pregnant mice during gestation, monitoring the blood pressure and 24-hour urine protein level of pregnant mice, observing the intrauterine development of fetal mice, and the tissue morphological changes of the placenta, liver and kidney of pregnant mice, to construct a severe preeclampsia rat model.

[0008] The pregnant mice are SPF grade SD rats, and the age is 10 weeks.

[0009] The pregnant mice were injected with Ad-shTET2 via the tail vein on day 9.5 of pregnancy.

[0010] The dose of Ad-shTET2 injected into the pregnant mice was 2×10 9 PFU.

[0011] The application of the severe preeclampsia rat model obtained by the above-mentioned construction method in screening candidate drugs for the treatment of severe preeclampsia.

[0012] The severe preeclampsia rat model obtained by the above-mentioned construction method is used in the study of the pathogenesis of severe preeclampsia.

[0013] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) The present invention obtains a pregnant mouse model with decreased TET2 expression by injecting Ad-shTET2 (recombinant adenovirus interfering with TET2) into pregnant mice. This model can reflect the relevant characteristic symptoms and physical changes of severe preeclampsia, and is of great value for exploring the pathogenesis of severe preeclampsia.

[0014] (2) The severe preeclampsia rat model established by the present invention not only has typical symptoms and signs of preeclampsia such as increased blood pressure and proteinuria, but also has structural and functional damage to the placenta, liver and kidneys, and intrauterine growth restriction of the fetus. These all reflect the systemic multi-systemic nature and severity of severe preeclampsia, which is more representative.

[0015] (3) The severe preeclampsia rat model constructed based on TET2 in the present invention has typical preeclampsia-like symptoms such as increased blood pressure and 24-hour proteinuria, which is consistent with the pathological process of human preeclampsia. In addition, structural and functional damage to the placenta, liver and kidneys was detected, and intrauterine growth restriction of fetal mice was observed. These can well reflect the systemic multi-system dysfunction and severity of severe preeclampsia, and can be used to study the mechanism of occurrence and development of severe preeclampsia.

[0016] In summary, the present invention provides a standardized, safe, efficient, stable and reliable severe preeclampsia animal model, which provides a new animal model for screening candidate drugs for the treatment of severe preeclampsia and exploring the pathogenesis of severe preeclampsia. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Blood pressure changes in pregnant SD mice.

[0018] Figure 2 The changes of 24h urine protein level in SD pregnant mice.

[0019] Figure 3 This is the intrauterine development of fetal mice.

[0020] Figure 4 The figure shows the changes in placenta weight and tissue morphology of pregnant SD mice.

[0021] Figure 5 The figure shows the histomorphological changes of liver and kidney in pregnant SD mice.

[0022] Figure 6 Serum PlGF and sFlt-1 levels in pregnant SD mice.

[0023] Figure 7 The expression of TET2 protein in the placenta of pregnant SD mice. DETAILED DESCRIPTION

[0024] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0025] The main reagents and materials involved in the embodiments of the present invention are as follows: SPF SD rats (Chengdu Dashuo Experimental Animal Co., Ltd.), recombinant adenovirus vector (Shanghai Hanheng Biotechnology Co., Ltd.), BCA kit (Yishen Biotechnology Co., Ltd.), rat PlGF and sFlt-1 ELISA research kit (Jiangsu ELISA Industrial Co., Ltd.).

[0026] The main instruments involved in the embodiments of the present invention are as follows: Medlab blood pressure measurement system (Nanjing Calvin Biotechnology Co., Ltd.), rat metabolic cage (Shanghai Yuyan Scientific Instrument Co., Ltd.), scanning electron microscope (Olympus Corporation, Japan).

[0027] Example 1: Establishment and identification of severe preeclampsia rat model Ten-week-old SPF SD rats were selected and the ratio of male to female was 2:1. After the pregnancy was confirmed, the female rats were randomly divided into three groups, with 6 female rats in each group. On the 9.5th day of pregnancy, 2×10 9 PFU of Ad-shTET2 (experimental group), Ad-GFP (GFP negative control group, i.e., GFP negative control group under injection of adenoviral vector) and normal saline (NS, blank control group).

[0028] Then, the blood pressure and 24-hour urine protein level of the pregnant mice were monitored to observe the intrauterine development of the fetuses, as well as the histomorphological changes of the placenta, liver and kidneys of the pregnant mice, as follows: (A) Non-invasive blood pressure (systolic blood pressure and mean arterial pressure) was measured on gestational days 6, 9, 12, 15, and 17 of pregnant SD mice, and invasive blood pressure (systolic blood pressure) was measured on gestational day 19 of SD mice.

[0029] Among them, the non-invasive systolic blood pressure and mean arterial pressure measurement method is to use the tail artery pressure measurement system, fix the pregnant mouse on the heated test table with a fixture (temperature 34℃, lasting about 10 minutes), put the pressurized tail cuff and pulse transducer on the appropriate position of the mouse tail in turn, inflate and pressurize with a rubber ball, increase the pressure in the tail cuff until the pulse disappears completely, and then continue to pressurize by about 20mmHg, and then slowly deflate and decompress until the pulse signal returns to the starting level. Read the systolic blood pressure and mean arterial pressure data from the recording system. Measure three times in a row, and take the average value as the measurement value; the invasive systolic blood pressure measurement method is to anesthetize the pregnant mouse and fix it on the experimental table, separate one side of the common carotid artery, insert a catheter filled with heparin saline, connect the pressure transducer, record the blood pressure waveform, and read the systolic blood pressure data from the recording system. Measure three times in a row, and take the average value as the measurement value.

[0030] Figure 1 The following are the changes in blood pressure of SD pregnant mice (*: P<0.05, **: P<0.01). In the figure, A is systolic blood pressure, B is mean arterial pressure, and C is the invasive blood pressure (systolic blood pressure) of SD pregnant mice on the 19th day of pregnancy. Figure 1 As shown in the figure, compared with the Ad-GFP group and the NS group, the blood pressure of SD pregnant mice in the Ad-shTET2 group was significantly increased on days 15, 17, and 19 of pregnancy. This indicates that knocking down TET2 can cause symptoms of increased blood pressure in pregnant mice in the late pregnancy.

[0031] (ii) Urine was collected from pregnant SD mice on gestational days 9, 15, 17, and 19 for 24-hour urine protein testing.

[0032] The 24-hour urine protein detection method is to collect rat urine for 24 hours using a metabolic cage, record the urine volume, collect the supernatant after urine centrifugation, and use the BCA kit to measure the urine protein concentration in the supernatant. The product of the concentration and the urine volume is the 24-hour urine protein amount.

[0033] Figure 2 The changes of 24-hour urine protein levels in pregnant SD mice (**: P<0.01). Figure 2 As shown in the figure, compared with the Ad-GFP group and the NS group, the 24-hour urine protein of SD pregnant mice in the Ad-shTET2 group increased significantly. This shows that knocking down TET2 can cause the symptoms of increased 24-hour urine protein in pregnant mice in the late pregnancy.

[0034] (III) On the 19th day of pregnancy, pregnant SD mice were killed and fetuses were removed by cesarean section. The fetuses in each litter were counted, weighed, and measured in length to observe their intrauterine development. The placenta was weighed, and the placenta, liver, and kidney were collected for tissue morphology staining to observe organ damage.

[0035] Furthermore, the specific steps of Masson's trichrome staining of SD pregnant mouse placenta include: a. Dehydrate the fixed placenta tissue in a gradient manner and embed it in paraffin, then slice, bake, dewax and hydrate, and rinse with ddH2O for a few seconds; b. Stain with hematoxylin solution for 5 minutes; c. Differentiate with 1% hydrochloric acid alcohol for a few seconds, and wash thoroughly with ddH2O to remove acidic substances; d. Return to blue with Masson's blue solution for 3-5 minutes, and wash thoroughly with ddH2O for 1 minute; e. Stain with Ponceau fuchsin solution for 5-10 minutes; f. Wash with the prepared weak acid working solution (weak acid solution and ddH2O are mixed in a ratio of 1:2) for 1 minute; g. Wash with phosphomolybdic acid solution for 1-2 minutes, and wash with weak acid working solution for 1 minute; h. Stain with aniline blue solution for 1-2 minutes, and wash with weak acid working solution for 1 minute; i. Dehydrate, make it transparent, place it in a fume hood to dry naturally, then seal the slide, and observe and take pictures under a microscope.

[0036] The specific steps of hematoxylin-eosin staining of SD pregnant mouse liver include: a. Dehydrate the fixed liver tissue in a gradient manner and embed it in paraffin, then slice, bake, dewax and hydrate, and rinse with ddH2O for a few seconds; b. Soak the slices in a hematoxylin staining jar for 7-10 min, and rinse with ddH2O to remove excess dye on the slices; c. Differentiate the slices with 1% hydrochloric acid ethanol for a few seconds, and wash them thoroughly with ddH2O to remove acidic substances; d. Soak the slices in 1% ammonia water for a few seconds, and rinse with ddH2O for 8 min; e. Stain with 91% eosin for 2-5 min; f. Dehydrate, make it transparent, place it in a fume hood to dry naturally, then seal the slices, observe and take pictures under a microscope.

[0037] The specific steps of glycogen staining of SD pregnant mouse kidneys include: a. Dehydrate the fixed kidney tissues in a gradient manner and embed them in paraffin, then slice, bake, dewax and hydrate, and rinse with ddH2O for a few seconds; b. Stain with 0.5% periodic acid aqueous solution for 5 min, and wash thoroughly with ddH2O; c. React the slices in Schiff reagent for 30 min; d. Treat the slices with 0.5% sodium bisulfite 3 times, 2 min each time, and rinse with ddH2O for 5-10 minutes; e. Counterstain the cell nuclei with hematoxylin staining solution for 3 min, differentiate with 1% hydrochloric acid alcohol for a few seconds, and wash thoroughly with ddH2O to remove acidic substances; f. Dehydrate, make transparent, place in a fume hood to dry naturally, then seal the slices, observe and take pictures under a microscope.

[0038] Figure 3 The intrauterine development of the fetus (*: P<0.05, **: P<0.01). Figure A shows the size of the fetus, placenta and uterus, and Figure B shows the changes in fetal weight, body length and number of fetuses from left to right. Figure 3 It can be seen that compared with the Ad-GFP group and the NS group, the fetal weight and body length of the Ad-shTET2 group were significantly reduced, and there was no statistically significant difference in the number of fetal mice per litter.

[0039] Figure 4 Figure 1 shows the changes in placental weight and tissue morphology of SD pregnant mice (*: P<0.05). Figure A shows the changes in placental weight of SD pregnant mice. It can be seen that the placental weight of SD pregnant mice in the Ad-shTET2 group was significantly reduced compared with the Ad-GFP group and the NS group; B shows the placenta of SD pregnant mice stained with Masson's trichrome (200×). It can be seen that compared with the Ad-GFP group and the NS group, there was collagen matrix deposition in the labyrinth area of ​​the Ad-shTET2 group (see the area indicated by the yellow arrow and enlarged in the lower right corner); C shows the changes in the impaired development of the placenta of SD pregnant mice. It can be seen that compared with the Ad-GFP group and the NS group, the placental development of the Ad-shTET2 group was impaired, the area of ​​the labyrinth area was reduced, and the area of ​​the connection area was increased. This shows that knocking down TET2 can lead to impaired placental development in pregnant mice.

[0040] Figure 5 Figure 1 shows the morphological changes in the liver and kidney tissues of SD pregnant mice. Figure A shows the hematoxylin-eosin staining of the liver of SD pregnant mice (100×). It can be seen that compared with the Ad-GFP group and the NS group, the Ad-shTET2 group has mild necrosis of the portal vein between the liver lobules of pregnant mice, and the surrounding hepatocytes are arranged in disorder (see the area indicated by the yellow arrow); Figure B shows the glycogen staining of the kidney of SD pregnant mice (400x). Compared with the Ad-GFP group and the NS group, the Ad-shTET2 group has enlarged glomerular capsules and mild mesangial hyperplasia (see the area indicated by the yellow arrow). This shows that knocking down TET2 can cause liver and kidney damage in pregnant mice.

[0041] (IV) On the 19th day of pregnancy, serum samples of SD pregnant mice were collected for detection of sFlt-1 and PlGF.

[0042] Among them, sFlt-1 and PlGF detection methods both use ELISA method.

[0043] Figure 6 Figure 1 shows the levels of serum sFlt-1 and PlGF in SD pregnant mice (*: P<0.05). Figure 1 shows the changes in serum sFlt-1 levels in SD pregnant mice, Figure 1 shows the changes in serum PlGF levels in SD pregnant mice, and Figure 1 shows the changes in serum sFlt-1 / PlGF in SD pregnant mice. Figure 6 It can be seen that compared with the Ad-GFP group and the NS group, the serum sFlt1 level of SD pregnant mice in the Ad-shTET2 group was significantly increased, the PlGF level was significantly decreased, and the sFlt1 / PlGF ratio was significantly increased. This shows that knocking down TET2 can lead to an imbalance between angiogenesis and anti-angiogenesis in pregnant mice.

[0044] (V) The placentas of SD pregnant mice were collected on gestational day 19 and the expression of TET2 protein in the placenta was detected.

[0045] Total protein of placental tissue was extracted, and the protein expression of TET2 was detected by western blotting.

[0046] Figure 7 Figure 2 is the change of TET2 protein expression in the placenta of SD pregnant mice (**: P<0.01). Figure A is the protein immunoblot of TET2 in the placenta of pregnant mice in the NS group, Ad-GFP group and Ad-shTET2 group, and Figure B is the statistical analysis of TET2 protein expression in the placenta of pregnant mice in the NS group, Ad-GFP group and Ad-shTET2 group. Figure 7It can be seen that compared with the Ad-GFP group and the NS group, the protein expression of TET2 in the placenta of the Ad-shTET2 group was significantly reduced. This shows that the expression of TET2 in the placenta of pregnant mice was successfully knocked down by tail vein injection of shTET2 adenovirus.

[0047] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification or equivalent change made to the above embodiment based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for constructing a severe preeclampsia rat model based on TET2, characterized in that: Ad-shTET2 was injected into pregnant mice during pregnancy, and a severe preeclampsia rat model was established by monitoring the blood pressure and 24-hour urine protein level of the pregnant mice, observing the intrauterine development of the fetuses, and the histomorphological changes of the placenta, liver, and kidneys of the pregnant mice.

2. The construction method according to claim 1, characterized in that: The pregnant mice were SPF grade SD rats, and were 10 weeks old.

3. The construction method according to claim 1, characterized in that: The pregnant mice were injected with Ad-shTET2 via the tail vein on day 9.5 of pregnancy.

4. The construction method according to claim 1, characterized in that: The dose of Ad-shTET2 injected into the pregnant mice was 2×10 9 PFU.

5. Use of the severe preeclampsia rat model obtained by the construction method as described in claim 1 in screening candidate drugs for the treatment of severe preeclampsia.

6. Application of the severe preeclampsia rat model obtained by the construction method as described in claim 1 in the study of the pathogenesis of severe preeclampsia.

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