Preparation method of placental trophoblast-derived transporter and application of placental trophoblast-derived transporter in medicine for treating spontaneous abortion

Abstract

The invention relates to a preparation method of a placenta trophoblast-derived transporter and application of the placenta trophoblast-derived transporter in a medicine for treating spontaneous abortion, which comprises the following steps of: taking out a placenta of a suckling mouse, cutting the placenta into fine tissue blocks, resuspending by using digestive juice, digesting in a water bath kettle at 37 DEG C for 20 minutes, and then stopping digestion by using 1640 culture containing high-temperature heat-inactivated fetal calf serum; diluting and neutralizing collagenase digestive juice according to 5 times of volume; and centrifuging the collagenase digestive juice for multiple times to obtain a precipitate, namely the exosome. And carrying out density gradient centrifugation on the coarse migration body by using Optiprep as a density medium, washing, precipitating and centrifuging by using PBS on an ultra-high-speed centrifuge by using a horizontal rotor to obtain the placental trophoblast source migration body. The invention finds that the generation of the placental trophoblast cell source migration body is related to the physiological process of pregnancy for the first time, and the placental trophoblast cell source migration body can enter the circulatory system and reach the placenta through in-vitro supplementation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of placental trophoblast-derived migratory organisms as biomarkers in the preparation of drugs for predicting and treating placental trophoblast damage during pregnancy. Background Technology

[0002] Abnormal placental trophoblast cell function is associated with various adverse pregnancy outcomes, including fetal growth restriction, eclampsia, and spontaneous abortion. Related studies have reported that vesicles released by placental trophoblasts (EVTs) mediate communication between maternal and fetal tissues and organs, maintaining maternal-fetal homeostasis. However, there are no reports on cellular vesicles as biomarkers for detecting structural and functional damage to placental trophoblast cells during pregnancy.

[0003] In recent years, a novel organelle has been discovered in migratory cells, named the migratory body because its formation depends on cell migration. Migratory bodies are widely distributed in various cell types. As cells move forward, numerous contractile fibers form at the tail, from which migratory bodies grow. When the cell leaves its original position and moves forward, the contractile fibers lengthen and break, releasing the migratory body into the extracellular environment. Migratory bodies contain proteins and nucleic acids, enabling them to be taken up by surrounding cells and release their contents into the microenvironment after rupture, thus transmitting biological information. Furthermore, migratory bodies can remain on the cell's migration trail, providing spatial location information. Therefore, they play a unique role in intercellular communication and possess important biological functions.

[0004] However, research on the production of migratory bodies in the placental trophoblast is currently lacking, and research on the role of migratory bodies in disease is still in its early stages. The placenta, an organ that transports maternal-fetal information, has the ability to produce abundant cellular vesicles; however, given the difficulty in determining the origin of these vesicles at the maternal-fetal interface, the importance of placental cellular vesicles, including migratory bodies, in the detection of placental trophoblast damage is greatly diminished.

[0005] The present invention aims to provide a trophoblast-derived migration and to accurately analyze it, which will help to provide new directions and clues for the treatment of pregnancy-related diseases. Summary of the Invention

[0006] Given the limited understanding of the mechanisms underlying placental trophoblast structural and functional damage, this invention explores the role of placental trophoblast migration bodies in maintaining pregnancy by examining the role of trophoblasts in intercellular communication within the placenta and utilizing placental villous trophoblast migration bodies as a novel communication medium.

[0007] A method for preparing placental trophoblast-derived migratory organisms includes the following steps: Step (1) On the 6.5, 8.5 and 10.5 days of pregnancy in pregnant mice, the placenta of the suckling mice was removed by sacrificing mice. The placenta was cut into small tissue pieces and resuspended in digestive solution. It was then placed in a 37°C water bath for 20 minutes to digest. Digestion was stopped by culture with 1640 containing heat-inactivated fetal bovine serum. The collagenase digestive solution was diluted 5 times by volume. Step (2) Centrifuge the collagenase digestion solution from step (1) at 1000 rpm for 5 minutes to obtain cell pellet; centrifuge the supernatant at 1000 g for 10 minutes to remove larger fragments, and centrifuge the supernatant at 4000 g for 20 minutes to remove cell fragments; centrifuge the supernatant at 20,000 g for 30-60 minutes to obtain a pellet of coarse migratory bodies; transfer the supernatant to a new centrifuge tube and centrifuge at 160,000 g for 2-3 hours to obtain a pellet of exosomes. All the above centrifugation operations are performed at 4°C.

[0008] Step (3) The coarse migratory bodies obtained in step (2) were subjected to density gradient centrifugation using Optiprep as the density medium to establish density gradients of 40% (1 ml), 35% (1 ml), 30% (1 ml), 25% (1 ml), 20% (1 ml), 15% (1 ml), 10% (1 ml), and 5% (1 ml) and the sample (5%, 1.5 ml). The samples were centrifuged at 4°C with a horizontal rotor at 150,000 g for 4 hours at 4°C. The purified migratory body samples were enriched between 10% and 25%. The precipitate was washed with PBS and centrifuged again at 20,000 g for 30 minutes to obtain placental trophoblast-derived migratory bodies.

[0009] Furthermore, the digestive solution in step (1) of the present invention is a mixture of type I and type IV collagenase.

[0010] The present invention provides an extracellular secretory vesicle, which is a migratory body derived from placental trophoblast cells of pregnant rats.

[0011] The application of a placental trophoblast-derived migratory organism in the treatment of spontaneous abortion, specifically the following process: Abortion model mice were constructed by intraperitoneal injection of lipopolysaccharide (LPS) into wild-type healthy C57 / BL6 female mice with a gestation period of 6.5 days. The LPS-induced abortion mice were divided into two groups, and on days 6.5, 8.5, and 10.5 of gestation, they were injected via tail vein with 100 μl (0.2 μg / μl) of placental migratory body suspension from wild-type pregnant mice of the corresponding gestation period or an equivalent volume of physiological saline.

[0012] The present invention adopts the above technical solution and has the following beneficial effects compared with the prior art: 1. This invention is the first to discover that the generation of placental trophoblast-derived migratory bodies is related to the physiological process of pregnancy, and that placental trophoblast-derived migratory bodies can be supplemented in vitro, enter the circulatory system, and reach the placenta.

[0013] 2. LPS-induced abortion mice supplemented with migration cells at regular intervals during pregnancy help reduce the abortion rate and increase the weight of the placenta and suckling mice, thus helping to maintain pregnancy. Attached Figure Description

[0014] Figure 1 Immunofluorescence image of WGA-labeled migratory cells on the placenta of aborted pregnant mice after injection into the mice.

[0015] Figure 2 Flowchart of the experiment for creating a miscarriage model mouse model using LPS and rescuing the miscarriage rate using migration bodies.

[0016] Figure 3 A schematic diagram and statistical analysis of the miscarriage rate in a mouse model of miscarriage after injection of migratory bodies. Figure 4 This is a statistical analysis graph showing the changes in placental weight and neonatal weight in aborted mice. Detailed Implementation

[0017] The technical solution of the present invention will be described in detail below: The first objective of this invention is to provide a migratory body derived from placental trophoblast cells; A second objective of this invention is to provide placental trophoblast-derived migratory bodies as biomarkers for the preparation or auxiliary prediction of trophoblast damage and products; A third objective of this invention is to provide a protective effect in maintaining pregnancy in migratory bodies and its application in the preparation of drugs for the diagnosis and prevention of spontaneous abortion.

[0018] To achieve the above objectives, the following technical solutions can be used: The present invention provides an extracellular secretory vesicle, which is a migratory body derived from placental trophoblast cells of pregnant rats.

[0019] This invention proposes that the labeled migratory organisms can be detected on the placenta of pregnant mice by intravenous injection of the labeled migratory organisms.

[0020] This invention proposes that placental trophoblast cell-derived migratory organisms can effectively reduce the miscarriage rate in LPS-induced abortion model mice.

[0021] This invention proposes that placental trophoblast cell-derived migratory organisms can effectively alleviate the reduction in placental weight and suckling weight in LPS-induced abortion model mice.

[0022] A method for preparing placental trophoblast-derived migratory organisms includes the following steps: Step (1) On the 6.5, 8.5 and 10.5 days of pregnancy in pregnant mice, the placenta of the suckling mice was removed by sacrificing mice. The placenta was cut into small tissue pieces and resuspended in digestive solution. It was then placed in a 37°C water bath for 20 minutes to digest. Digestion was stopped by culture with 1640 containing heat-inactivated fetal bovine serum. The collagenase digestive solution was diluted 5 times by volume. Step (2) Centrifuge the collagenase digestion solution from step (1) at 1000 rpm for 5 minutes to obtain cell pellet; centrifuge the supernatant at 1000 g for 10 minutes to remove larger fragments, and centrifuge the supernatant at 4000 g for 20 minutes to remove cell fragments; centrifuge the supernatant at 20,000 g for 30-60 minutes to obtain a pellet consisting of coarse migratory bodies; transfer the supernatant to a new centrifuge tube and centrifuge at 160,000 g for 2-3 hours to obtain a pellet consisting of exosomes constituting coarse migratory bodies. All the above centrifugation operations are performed at 4°C.

[0023] Step (3) The coarse migratory bodies obtained in step (2) were subjected to density gradient centrifugation using Optiprep as the density medium to establish density gradients of 40% (1 ml), 35% (1 ml), 30% (1 ml), 25% (1 ml), 20% (1 ml), 15% (1 ml), 10% (1 ml), and 5% (1 ml) and the sample (5%, 1.5 ml). The samples were centrifuged at 4°C with a horizontal rotor at 150,000 g for 4 hours at 4°C. The purified migratory body samples were enriched between 10% and 25%. The precipitate was washed with PBS and centrifuged again at 20,000 g for 30 minutes to obtain placental trophoblast-derived migratory bodies.

[0024] The specific steps for detecting migratory bodies on the placenta are as follows: (1) The purified migration bodies obtained in Example 1 were injected into pregnant mice. On days 6.5, 8.5 and 10.5 of gestation, WGA-labeled migration bodies or WGA dye of the same volume from the placenta were injected into the pregnant mice via the tail vein. The mice were sacrificed on day 11.5 of gestation, and the placenta was removed and sectioned.

[0025] (2) After fixing the placenta overnight with pre-cooled paraformaldehyde, the placenta was soaked in 10% sucrose solution and 30% sucrose solution respectively. After the placental tissue settled to the bottom of the sucrose solution, the placental tissue was embedded and frozen with OCT and then cut into 10 μm thick frozen sections.

[0026] (3) After the sections are dried, stain the cell nuclei in the dark, fix and mount them.

[0027] (4) Use a confocal microscope to image placental tissue to check for WGA signals. From Figure 1 The results showed that there was no green signal of WGA on the placenta of pregnant mice injected with WGA dye, while there was a green signal of the migratory organism on the placenta of pregnant mice injected with WGA-labeled migratory organisms, indicating that the migratory organisms can reach the placenta.

[0028] like Figure 2 As shown, the specific steps to reduce the miscarriage rate by supplementing mice with placental migration bodies derived from normal pregnant mice are as follows: (1) Abortion model mice were constructed by intraperitoneal injection of lipopolysaccharide (LPS) into wild-type healthy C57 / BL6 female mice with a gestation period of 6.5 days. The LPS-induced abortion mice were divided into two groups. At 6.5 days, 8.5 days, and 10.5 days of gestation, 100 μl (0.2 μg / μl) of placental migratory body suspension from wild-type pregnant mice of the corresponding gestation period or an equal volume of physiological saline was injected via the tail vein. (2) At 11.5 days of gestation, neonatal mice and placentas were removed from the uterus of euthanized mice, weighed, and the abortion rates of the two groups were statistically analyzed. Figure 2 The results show that the abortion rate in mice injected with the migration body was significantly lower than that in the saline injection group. Figure 3 A, 3B), and the weight of the suckling mice and the weight of the placenta were significantly higher than those of the group injected with saline (A, 3B). Figure 4 (A, 4B) indicates that placental migration bodies can improve miscarriage in mice, and placental migration bodies are associated with pregnancy maintenance.

[0029] The scope of protection of this invention is not limited to the above embodiments. Any variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. A method for preparing placental trophoblast-derived migratory organisms, characterized in that... Includes the following steps: Step (1) On the 6.5, 8.5 and 10.5 days of pregnancy in pregnant mice, the placenta of the suckling mice was removed by sacrificing mice. The placenta was cut into small tissue pieces and resuspended in digestive solution. It was then placed in a 37°C water bath for 20 minutes to digest. Digestion was stopped by culture with 1640 containing heat-inactivated fetal bovine serum. The collagenase digestive solution was diluted 5 times by volume. Step (2) Centrifuge the collagenase digestion solution from step (1) at 1000 rpm for 5 minutes to obtain cell pellet; centrifuge the supernatant at 1000g for 10 minutes to remove larger fragments, and centrifuge the supernatant at 4000g for 20 minutes to remove cell fragments. Centrifuge at 20,000 g for 30–60 minutes to obtain coarse migratory bodies as the precipitate; transfer the supernatant to a new centrifuge tube and continue centrifuging at 160,000 g for 2–3 hours to obtain exosomes as the precipitate. All centrifugation operations were performed at 4°C. Step (3) The coarse migratory bodies obtained in step (2) were subjected to density gradient centrifugation using Optiprep as the density medium to establish density gradients of 40% (1 ml), 35% (1 ml), 30% (1 ml), 25% (1 ml), 20% (1 ml), 15% (1 ml), 10% (1 ml), and 5% (1 ml) and the sample (5%, 1.5 ml). The samples were centrifuged at 4°C with a horizontal rotor at 150,000 g for 4 hours at 4°C. The purified migratory body samples were enriched between 10% and 25%. The precipitate was washed with PBS and centrifuged again at 20,000 g for 30 minutes to obtain placental trophoblast-derived migratory bodies.

2. The preparation method according to claim 1, characterized in that... The digestive solution in step (1) above is a mixture of type I and type IV collagenase.

3. An extracellular secretory vesicle obtained as an extracellular vesicle from a placental trophoblast-derived migratory body prepared by the method of claim 1.

4. The application of a placental trophoblast-derived migratory organism as a biomarker in the preparation or auxiliary prediction of trophoblast damage and products.

5. The application of a placental trophoblast-derived migration body in the preparation of drugs for the diagnosis and prevention of spontaneous abortion.

6. The application according to claim 5, characterized in that... The specific application process is as follows: Abortion model mice were constructed by intraperitoneal injection of lipopolysaccharide (LPS) into wild-type healthy C57 / BL6 female mice with a gestation period of 6.5 days. The LPS-induced abortion mice were divided into two groups, and at 6.5 days, 8.5 days, and 10.5 days of gestation, respectively, they were injected via tail vein with 100 μl (0.2 μg / μl) of placental-derived migratory bodies from wild-type pregnant mice of the corresponding gestation period or an equivalent volume of physiological saline.

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