Umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2, nucleotide sequence, application, medicine and medicine composition

Abstract

The invention discloses an umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2, a nucleotide sequence, application, a medicine and a medicine composition, the amino acid sequence of the umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 is shown as SEQ ID No: 1, and the umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 is an endogenous polypeptide derived from a dynamic protein heavy chain 5 and has small toxic and side effects; the compound can be used for preparing drugs and corresponding pharmaceutical compositions for recovering and maintaining the integrity and function of vascular endothelium or relieving preeclampsia, and can effectively improve the angiogenesis ability of damaged endothelial cells and reduce the expression of vascular endothelial injury markers.

Description

Technical Field

[0001] The present invention relates to endogenous polypeptides, and in particular to an umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2, a nucleotide sequence, an application, a medicine and a pharmaceutical composition. Background Art

[0002] Pre-eclampsia (PE) is a common and unique disease during pregnancy, with clinical manifestations of hypertension and proteinuria. In severe cases, it can lead to damage to important maternal organs (such as stroke, liver and kidney damage, heart failure, etc.), placental abruption, fetal growth restriction, premature birth, etc., resulting in approximately 76,000 maternal and 500,000 perinatal deaths each year, seriously threatening the life safety of mothers and infants. In addition, pre-eclampsia significantly increases the risk of long-term cardiovascular disease, type 2 diabetes, dyslipidemia and premature death in mothers, and the risk of cardiovascular disease, diabetes and obesity in their offspring after adulthood is significantly increased, and the risk of death increases by 6 times, affecting the quality of the newborn population and the quality of life. Although pre-eclampsia is extremely harmful, its pathogenesis has not yet been fully clarified, and the existing clinical treatment is limited to symptomatic treatment, which has failed to effectively curb the development of the disease. Therefore, in-depth exploration of new treatment strategies is not only of great significance for the clinical treatment of pre-eclampsia mothers, but also crucial for the health management of their offspring.

[0003] Among the many pathogenic factors, vascular endothelial cell damage is widely considered to be the core link of preeclampsia. Placental vascular remodeling disorders, placental-derived factors, excessive release of toxic substances and inflammatory mediators lead to systemic vascular endothelial cell damage, triggering systemic small blood vessel spasmodic contraction, and then leading to a series of clinical symptoms such as hypertension, proteinuria, and edema. From local placental lesions to systemic multi-organ damage, vascular endothelial cells are not only directly damaged target cells, but also key carriers of the occurrence and development of preeclampsia. Therefore, the repair and protection of vascular endothelial cell damage has become an important direction of research in the treatment of preeclampsia.

[0004] Endogenous peptides are a class of peptide molecules that are widely present in the body and mainly come from broken fragments of proteins. Although they have long been regarded as intermediate products of protein processing or degradation, recent studies have found that endogenous peptides play an important regulatory role in immune regulation, cell proliferation and apoptosis, neurotransmitter regulation, etc. Mesenchymal stem cells are a type of multipotent stem cells with multiple differentiation potentials. Among them, umbilical cord-derived mesenchymal stem cells have the characteristics of multidirectional differentiation potential and low immunogenicity, and low tumorigenicity in vivo. These characteristics make them further become an important cell source for studying alternative therapies and isolating endogenous peptides. However, no studies have yet found endogenous peptides that can protect vascular endothelial cell function and relieve the symptoms of preeclampsia. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide an endogenous umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2, nucleotide sequence and application, and the second purpose is to provide a drug and a pharmaceutical composition containing the peptide.

[0006] Technical solution: The umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 of the present invention has an amino acid sequence as shown in SEQ ID NO: 1.

[0007] Preferably, the umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 is an endogenous polypeptide derived from dynamin heavy chain 5

[0008] The nucleotide sequence of the present invention encodes the aforementioned umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2.

[0009] Application of the umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 of the present invention in the preparation of a drug for alleviating preeclampsia.

[0010] The invention discloses an application of the umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 in preparing a drug for restoring and maintaining the integrity and function of vascular endothelium.

[0011] Preferably, the method is used in the preparation of a drug for improving the angiogenesis ability of damaged endothelial cells.

[0012] Preferably, the invention is used in the preparation of a drug for reducing the expression of vascular endothelial injury markers.

[0013] The medicine of the present invention comprises the aforementioned umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 and a pharmaceutically acceptable salt thereof.

[0014] The pharmaceutical composition of the present invention comprises the aforementioned umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2, a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier thereof.

[0015] Preferably, the aforementioned pharmaceutically acceptable carrier includes one or more of a diluent, a filler, an excipient, a binder, a wetting agent, a disintegrant, an effervescent agent, a surfactant, an absorption enhancer, a lubricant, an adsorption carrier, a sustained-release microsphere, an implant, an in situ microsphere, a liposome, a microemulsion, an in situ hydrogel, and a nanoparticle.

[0016] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. Umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 is an endogenous polypeptide with small toxic and side effects; 2. It can effectively restore and maintain the integrity and function of the vascular endothelium, such as reducing the expression of vascular endothelial injury markers and improving the angiogenesis ability of damaged endothelial cells; 3. It can effectively alleviate preeclampsia. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1This is a graph showing the viability of human umbilical vein endothelial cells treated with umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2;

[0018] Figure 2 This is a graph showing the results of umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 intervention to improve the angiogenesis ability of human umbilical vein endothelial cells, where A is a microscopic image, and B is the statistical results of the number of vascular grid intersections, the number of vascular nodes, and the number of vascular grids from left to right;

[0019] Figure 3 This is the electron microscopic result of damaged human umbilical vein endothelial cells treated with umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2;

[0020] Figure 4 This is the result diagram of the effect of umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 on the gene expression of endothelial cell injury markers;

[0021] Figure 5 This is the result diagram of the effect of umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 on the expression of endothelial cell injury marker proteins;

[0022] Figure 6 These are the results of umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 alleviating preeclampsia-like conditions in pregnant mice, where A is a schematic diagram of the treatment process, B is a graph showing the blood pressure results of pregnant mice at different time points, C is a graph showing the urine protein content of pregnant mice at different time points, D is a comparison of fetal mice and placenta, and E is a graph showing the weight of fetal mice.

[0023] Figure 7 This is an analysis of the reasons why umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 alleviates preeclampsia-like symptoms in pregnant mice, where A is the expression result of vascular endothelial injury markers in the serum of pregnant mice, B is the result of H&E staining of pregnant mouse kidneys, and C is the result of H&E staining of pregnant mouse placenta. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further described below.

[0025] Example 1: Synthesis of Umbilical Cord Mesenchymal Stem Cell Anti-preeclampsia Peptide 2 (APEP2)

[0026] Shanghai Kepeptide Biotechnology Co., Ltd. was commissioned to synthesize APEP2 (amino acid sequence as shown in SEQ ID NO: 1: AQTKRLVGDVLLATAFLSYSGP) by solid phase method

[0027] The synthesized anti-preeclampsia peptide 2 was prepared into a 1 mM solution with DMSO and stored at -40°C for future use.

[0028] Example 2: Effect of umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 on the activity of human umbilical vein endothelial cells

[0029] Cells were seeded at a density of 2000 cells per well in a 96-well plate, and the culture medium was discarded after the cells adhered to the wall; complete culture medium containing APEP2 was added, wherein the final concentration of APEP2 was 10 μM, and the culture was continued; after culturing for 24 hours, the cells were taken out, the culture medium was discarded, and complete culture medium containing 10% CCK-8 reagent was added, and the cells were placed in an incubator for incubation for 1 hour, and the absorbance at 450 nm was detected by an enzyme marker; the results are shown in FIG. Figure 1 As shown, APEP2 intervention had no significant effect on the viability of human umbilical vein endothelial cells.

[0030] Example 3: Effect of umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 on angiogenesis of damaged human umbilical vein endothelial cells

[0031] After adding matrix gel to the 96-well plate, the plate was placed in an incubator and allowed to stand for 45 minutes. After the gel solidified, 10,000 human umbilical vein endothelial cells were inoculated in each well and divided into NC group, TNF-α injury group, TNF-α+Scr group, and TNF-α+APEP2 treatment group. Except for the NC group, the culture medium in the other groups contained TNF-α with a final concentration of 30 ng / ml. The TNF-α+Scr group was supplemented with a final concentration of 10 μM of scrambled peptide (Scr), and the TNF-α+APEP2 treatment group was supplemented with a final concentration of 10 μM of APEP2. Images were collected at regular intervals, and the length of the tube, coverage area, number of rings, and number of nodes were measured and recorded using ImageJ software. The results are shown in the figure. Figure 2 As shown, APEP2 intervention can significantly improve the angiogenesis ability of damaged endothelial cells.

[0032] Example 4: Effects of umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 on the morphology of damaged human umbilical vein endothelial cells

[0033] 10,000 human umbilical vein endothelial cells were inoculated in each well of a 6-well plate and divided into NC group, TNF-α injury group, TNF-α+Scr group, and TNF-α+APEP2 treatment group. Except for the NC group, the culture medium in the other groups contained TNF-α with a final concentration of 30 ng / ml, the TNF-α+Scr group was supplemented with a scrambled peptide (Scr) with a final concentration of 10 μM, and the TNF-α+APEP2 treatment group was supplemented with APEP2 with a final concentration of 10 μM. After 24 hours of culture, the cells were digested, centrifuged, and the cell pellets were collected. Electron microscopy fixative was added to resuspend and mix at 4°C for 24 hours, and then handed over to Wuhan Savier Company for transmission electron microscopy. The results are shown in Figure 3As shown in the figure, in the TNF-α injury group and the TNF-α+Scr group, the degree of cell edema was relatively obvious, manifested as matrix edema, mitochondrial membrane damage, matrix dissolution, and reduced rough endoplasmic reticulum. After APEP2 treatment, the degree of cell edema was relatively alleviated, the cytoplasm was abundant, the mitochondrial membrane structure was relatively intact and relatively abundant, and the rough endoplasmic reticulum did not expand, indicating that APEP2 can significantly improve the morphology of damaged endothelial cells.

[0034] Example 5: Effects of umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 on endothelial cell injury markers

[0035] 1. Effect of umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 on the gene levels of endothelial cell injury markers

[0036] 10,000 human umbilical vein endothelial cells were inoculated in each well of a 6-well plate and divided into NC group, TNF-α injury group, TNF-α+Scr group, and TNF-α+APEP2 treatment group. Except for the NC group, the culture medium of the other groups contained TNF-α with a final concentration of 30 ng / ml, the TNF-α+Scr group was additionally supplemented with a final concentration of 10 μM of scrambled polypeptide (Scr), and the TNF-α+APEP2 treatment group was additionally supplemented with a final concentration of 10 μM of APEP2.

[0037] After 24 hours, the 6-well plate was removed and washed with PBS. Total RNA of the cells was extracted using an RNA extraction kit and reverse transcribed. RT-qPCR was performed using a ChamQ Universal SYBR qPCR Master mix kit and a real-time fluorescence quantitative PCR system. GAPDH was used as an internal reference to calculate the relative expression of each gene in the cell. Two replicate wells were set up for each experiment and repeated three times. The results are shown in Figure 4 As shown, it can be seen that APEP2 can significantly inhibit the expression of vascular endothelial injury markers VCAM-1, ET-1, and sFlt-1 genes.

[0038] 2. Effect of umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 on the protein levels of endothelial cell injury markers

[0039] 10,000 human umbilical vein endothelial cells were inoculated in each well of a 6-well plate and divided into NC group, TNF-α injury group, TNF-α+Scr group, and TNF-α+APEP2 treatment group. Except for the NC group, the culture medium of the other groups contained TNF-α with a final concentration of 30 ng / ml, the TNF-α+Scr group was additionally supplemented with a final concentration of 10 μM of scrambled polypeptide (Scr), and the TNF-α+APEP2 treatment group was additionally supplemented with a final concentration of 10 μM of APEP2.

[0040] After 24 hours, the 6-well plate was removed, washed with PBS, and 100 μL / well of RIPA lysis buffer was added, and 1% protease inhibitor and phosphorylase inhibitor were added to extract protein. After electrophoresis, the protein sample was transferred to PVDF membrane; after blocking with blocking solution, mouse ET-1 primary antibody and rabbit VCAM-1 primary antibody were used for incubation at 4°C for 16 hours; after washing after the primary antibody incubation, the corresponding secondary antibody conjugated with horseradish peroxidase was used for incubation at room temperature for 1 hour; after washing, enhanced chemiluminescence solution was added for development, and the obtained image was analyzed in grayscale.

[0041] The results are as follows Figure 5 As shown, APEP2 can significantly inhibit the expression of vascular endothelial injury markers VCAM-1 and ET-1 proteins in human umbilical vein endothelial cells.

[0042] Example 6: Effects of umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 on preeclampsia-like mice

[0043] 1. Scr and APEP2 were modified by acetate conversion and endotoxin removal;

[0044] 2. Forty-nine healthy BALB / c pregnant mice housed under specific pathogen-free conditions were randomly divided into four groups: NC group, LPS modeling group, LPS+Scr group, and LPS+APEP2 treatment group;

[0045] 3. The systolic blood pressure of each mouse was measured and urine was collected on 5.5, 12.5 and 18.5 days of pregnancy. Except for the NC group, the pregnant mice in the other groups were intraperitoneally injected with 10 ng / g lipopolysaccharide (LPS) from 10.5 days of pregnancy to establish the mouse model of preeclampsia. The pregnant mice in the LPS+Scr group were intraperitoneally injected with 10 mg / kg scrambled polypeptide (Scr) at the same time, and the pregnant mice in the LPS+APEP2 treatment group were intraperitoneally injected with 10 mg / kg APEP2 at the same time.

[0046] 4. On day 18.5, all mice were euthanized with CO2, eye blood was collected for subsequent detection of the concentration of vascular endothelial injury markers in the maternal circulation, and the fetal placenta was weighed and photographed;

[0047] The results are as follows Figure 6 , 7 As shown, APEP2 significantly alleviated the preeclampsia-like phenotype of mice, lowered the maternal blood pressure of mice, and alleviated the growth restriction of mouse offspring; H&E staining of mouse kidneys showed glomerular capillary occlusion and endothelial cell proliferation in LPS model group mice, while APEP2 treatment could improve kidney and placental damage and reduce the concentrations of vascular endothelial injury markers ET-1, sFlt-1 and TNF-α in maternal serum.

Claims

1. An umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2, characterized in that: The polypeptide has an amino acid sequence as shown in SEQ ID NO:

1.

2. The polypeptide according to claim 1, characterized in that The polypeptide is an endogenous polypeptide derived from dynein heavy chain 5.

3. A nucleotide sequence, characterized in that The nucleotide sequence encodes the umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 according to claim 1.

4. Use of the umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 according to claim 1 in the preparation of a drug for alleviating preeclampsia.

5. Use of the umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 according to claim 1 in the preparation of a drug for restoring and maintaining the integrity and function of vascular endothelium.

6. The use according to claim 5, characterized in that: The application is application in preparing medicine for improving the angiogenesis ability of damaged endothelial cells.

7. The use according to claim 5, characterized in that: The application is application in preparing a drug for reducing the expression of vascular endothelial injury markers.

8. A drug, characterized in that It comprises the umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 according to claim 1 and a pharmaceutically acceptable salt thereof.

9. A pharmaceutical composition, characterized in that It comprises the umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 according to claim 1, a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier thereof.

10. The pharmaceutical composition according to claim 9, characterized in that: The pharmaceutically acceptable carrier includes one or more of a diluent, a filler, an excipient, a binder, a wetting agent, a disintegrant, an effervescent agent, a surfactant, an absorption promoter, a lubricant, an adsorption carrier, a sustained-release microsphere, an implant, an in situ microsphere, a liposome, a microemulsion, an in situ hydrogel, and a nanoparticle.

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