CN113413457A - Application of TAX2 polypeptide in preparation of medicine for preventing and/or treating abdominal aortic aneurysm formation and/or rupture - Google Patents

Abstract

The invention provides an application of TAX2 polypeptide in preparing a medicament for preventing and/or treating abdominal aortic aneurysm formation and/or rupture. The invention discloses that by supplementing TAX2 polypeptide, macrophage infiltration of abdominal aorta vessel wall, activation of matrix metalloproteinase system and apoptosis of vascular smooth muscle cell are obviously inhibited, and the risk of forming and rupturing abdominal aortic aneurysm is finally reduced.

Description

Application of TAX2 polypeptide in preparation of medicine for preventing and/or treating abdominal aortic aneurysm formation and/or rupture

Technical Field

The invention belongs to the field of medicines, relates to an application of a medicine for preventing and/or treating abdominal aortic aneurysm formation and/or rupture, and particularly relates to an application of a TAX2 polypeptide in preparation of a medicine for preventing and/or treating abdominal aortic aneurysm formation and/or rupture.

Background

Abdominal Aortic Aneurysm (AAA) is a relatively common disease of abdominal aortic aneurysm with tumor-like distensibility, and AAA is defined as a general disease with a diameter expansion of more than 50%. AAA is a chronic degenerative disease that seriously threatens national health and can cause focal dilatation of the layers of the abdominal aortic wall. AAA is a common cause of aortic disease and the tenth leading cause of death in men over 65 years of age, often with no obvious symptoms during its early development and progression, and with a total mortality rate of over 80% in the event of rupture, is a serious life-threatening safety.

With the aging and popularization of surgical treatment and the continuous development of perioperative care and monitoring in recent years, the incidence and mortality of AAA postoperative complications have become a downward trend. Currently, the main operation modes of AAA include traditional open operation, compound operation and full-cavity inner covering film stent cavity repair operation. However, not all AAA are suitable for surgical treatment. For AAA with larger diameters (male diameter ≥ 55mm, female diameter ≥ 50 mm) or those with overt symptoms, the guidelines recommend active surgical intervention. For patients with small diameters or no symptoms, no surgical intervention is recommended, and AAA surgical treatments are more traumatic and at higher risk. Therefore, the search for effective drug intervention for this group of patients has been a research focus in recent years. In the process of numerous drug screening and development, metformin is considered to be the most valuable AAA therapeutic drug at present, and multiple studies find that metformin has the potential to limit AAA formation and rupture. Correlation entityExperimental studies also demonstrated that metformin significantly reduces AngII-induced ApoE^-/-Mouse AAA formation, whose protective effect depends on activation of the AMPK signaling pathway. However, the protective role of metformin in AAA remains controversial, and a large-scale clinical study found that chronic use of metformin was not effective in reducing the risk of rupture and mortality in patients with AAA. In addition, hypertension is an important risk factor for AAA formation and rupture, but studies have shown that intensive hypotensive treatment only results in a small reduction in the AAA rupture rate in patients, and still does not prevent most AAA rupture events.

Therefore, multidimensionally elucidating the potential molecular mechanism of the disease, searching for a new therapeutic target, and developing effective drugs for inhibiting the generation and development of AAA are still the bottleneck and core scientific problems to be broken through by AAA.

Disclosure of Invention

In order to solve the above problems, the present invention aims to provide an application of a TAX2 polypeptide in preparing a medicament for preventing and/or treating abdominal aortic aneurysm formation and/or rupture, thereby breaking through the bottleneck that no effective drug treatment means is available for abdominal aortic aneurysm nowadays.

In order to achieve the above object, the present invention provides a use of a TAX2 polypeptide in the preparation of a medicament for the prevention and/or treatment of abdominal aortic aneurysm formation and/or rupture.

The invention also provides application of the TAX2 polypeptide in preparing a medicament for preventing and/or treating the inflammation reaction of a blood vessel wall in the process of forming and/or rupturing an abdominal aortic aneurysm.

The invention also provides application of the TAX2 polypeptide in preparing a medicament for inhibiting activation of a vascular wall matrix metalloproteinase system.

The invention also provides application of the TAX2 polypeptide in preparing a medicament for inhibiting apoptosis of smooth muscle cells of a blood vessel wall.

The amino acid sequence of the TAX2 polypeptide is shown as Seq ID No. 1: CEVSQLLKGDAC are provided.

Wherein, the medicine also comprises a pharmaceutically acceptable carrier.

Preferably, the pharmaceutically acceptable carrier comprises any one or more of diluents, excipients, disintegrants, fillers, binders, lubricants, flavoring agents, surface active agents, and stabilizers.

Wherein the dosage form of the medicine is any one of injection, tablet, electuary, granule, pill and capsule.

The present invention proposes the polypeptide TAX2 for preventing and treating abdominal aortic aneurysm formation and rupture.

The invention has the beneficial effects that:

the invention provides an application of TAX2 polypeptide in preparing a medicament for preventing and treating abdominal aortic aneurysm formation and rupture, which remarkably inhibits macrophage infiltration of abdominal aortic vessel wall, activation of matrix metalloproteinase system and apoptosis of vascular smooth muscle cell by supplementing TAX2 polypeptide, and finally reduces the risk of abdominal aortic aneurysm formation and rupture.

Drawings

FIG. 1A is a typical view of aorta observed under a microscope in each group of mouse abdominal aortic aneurysm models.

FIG. 1B is a statistical chart of aneurysm formation rate and rupture rate of each group of mouse abdominal aortic aneurysm models.

Fig. 2A is abdominal ultrasound images of various groups of mouse abdominal aortic aneurysm models.

FIG. 2B is a statistical chart of the end-diastolic inner diameter of abdominal aorta of each group of mouse abdominal aortic aneurysm models.

FIG. 2C is a statistical chart of pulse wave velocity of each group of mouse abdominal aortic aneurysm models.

FIG. 2D is a statistical chart of the dilatability of blood vessels of each group of mouse abdominal aortic aneurysm models.

FIG. 3A is a Western bolt representative picture of LH1 in each group of mouse abdominal aortic aneurysm models.

FIG. 3B is a gray scale histogram of FIG. 3A.

FIG. 3C is a representative graph of immunofluorescence of LH1/Elastin of various groups of blood vessel walls in mouse abdominal aortic aneurysm model.

FIG. 4 is a 14-day systolic pressure statistical chart of each model of mouse abdominal aortic aneurysm model.

FIG. 5 is a flowchart of RNA library construction and transcriptome sequencing used in the present invention.

FIG. 6 is a flow chart of transcriptome sequencing data analysis used in the present invention.

FIG. 7A is a graph of RNA sequencing cluster analysis of abdominal aorta vessels of various groups of mouse abdominal aortic aneurysm models.

FIG. 7B is the analysis diagram of the differential gene interaction method of abdominal aorta blood vessel of each group of mouse abdominal aortic aneurysm models.

FIG. 7C shows wild-type mice administered to an abdominal aortic aneurysm modelPlod1 ^-/-Disease enrichment annotation graph after Human photonic Ontology analysis of mouse abdominal aorta vascular differential genes.

FIG. 7D shows wild-type mice administered to an abdominal aortic aneurysm modelPlod1 ^-/-Functions of the mouse abdominal aorta vascular differential Gene annotated map after Gene Ontology analysis.

FIG. 8A is a representative graph of immunofluorescence of vascular wall macrophages/Elastin in each group of mouse abdominal aortic aneurysm models.

FIG. 8B shows the vascular walls of each group of mice abdominal aortic aneurysm modelccl2Genes (upper) andil6quantitative PCR statistical map of genes (bottom).

FIG. 8C is a statistical chart of the ELISA method for detecting IL-6 (upper) and TNF-alpha (lower) levels in various groups of mice abdominal aortic aneurysm models.

FIG. 9 is a representation of gelatinase spectra of various groups of vascular walls reflecting MMP-9 and MMP-2 activities in a mouse abdominal aortic aneurysm model.

FIG. 10A is a representative graph of immunofluorescence of vascular wall TUNEL/Elastin in various groups of mouse abdominal aortic aneurysm models.

FIG. 10B is a graph of the apoptosis statistical analysis of FIG. 10A.

FIG. 10C is a representative graph of immunofluorescence of the vascular wall α -SMA/Elastin of each group of mouse abdominal aortic aneurysm models.

Fig. 10D is a statistical analysis chart of fig. 10C.

FIG. 11 shows wild-type mice administered to an abdominal aortic aneurysm modelPlod1 ^-/-Volcano Plot of differential genes in abdominal aortic vessels in mice.

FIG. 12A is a Western bolt representative of each group of thrombospondin-1 in mouse abdominal aortic aneurysm model.

FIG. 12B is an immunofluorescence representation of the vascular wall thrombospondin-1/Elastin of each group in a mouse abdominal aortic aneurysm model.

FIG. 13 is a scheme showing the biosynthesis process of TAX2 polypeptide and Scramble peptide.

FIG. 14A shows an abdominal aortic aneurysm after moldingPlod1 ^-/-Mice were administered either TAX2 polypeptide or scarmblepeptide for intervention and the aorta representative images obtained were photographed under a stereomicroscope.

FIG. 14B shows the abdominal aortic aneurysm after moldingPlod1 ^-/-Mice were administered either TAX2 polypeptide or Scamble peptide for intervention, and two groups of statistical plots of aneurysm formation and rupture rates.

FIG. 15A shows an abdominal aortic aneurysm after moldingPlod1 ^-/-Mice were given either a TAX2 polypeptide or a script peptide for intervention and two groups were sonicated to show a representative image of the abdominal aorta.

FIG. 15B shows the abdominal aortic aneurysm after moldingPlod1 ^-/-Mice were administered either TAX2 polypeptide or Scamble peptide for intervention, and two sets of abdominal aorta end diastolic inner diameter statistical plots.

FIG. 15C shows the abdominal aortic aneurysm after moldingPlod1 ^-/-Mice were administered either TAX2 polypeptide or Scamble peptide for intervention, and two abdominal pulse wave velocity statistics.

FIG. 15D shows a view of an abdominal aortic aneurysm after moldingPlod1 ^-/-Mice were administered either TAX2 polypeptide or Scamble peptide for intervention, two sets of statistical plots of vasodilation.

FIG. 16 shows the abdominal aortic aneurysm after moldingPlod1 ^-/-Mice were administered either TAX2 polypeptide or Scamble peptide for intervention, and two groups of vascular walls were subjected to H&E. VVG, Masson staining typical map.

FIG. 17 shows the abdominal aortic aneurysm after moldingPlod1 ^-/-Representative picture of immunofluorescence of two groups of vascular wall macrophages/elastins following mouse administration of either TAX2 polypeptide or Scamble peptide for intervention.

FIG. 18 shows an abdominal aortic aneurysm after moldingPlod1 ^-/-Mice were administered either TAX2 polypeptide or Scamble peptide for intervention, with two groups of vascular walls reflecting MMP-9 and MMP-2The active gelatin zymogram represents the graph.

FIG. 19A shows an abdominal aortic aneurysm after moldingPlod1 ^-/-Representative picture of immunofluorescence of two groups of vascular wall TUNEL/Elastin in mice administered either TAX2 polypeptide or Scamble peptide for intervention.

FIG. 19B shows the abdominal aortic aneurysm after moldingPlod1 ^-/-TAX2 polypeptide or Scamble peptide is given to mice for intervention, and two groups of representative graphs and statistical analysis graphs of immunofluorescence of vascular wall alpha-SMA/Elastin are shown.

FIG. 20A is a representative graph of immunofluorescence of LH1/Elastin in abdominal aortic aneurysm patients and normal control population.

FIG. 20B is a histogram of the two sets of fluorescence intensities of FIG. 20A.

FIG. 20C is a representative graph of immunofluorescence of abdominal aortic vessel wall thrombosporin-1/Elastin in patients with abdominal aortic aneurysm and in normal control populations.

FIG. 20D is a two-panel fluorescence intensity histogram of FIG. 20C.

Detailed Description

The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and not to limit the scope of the invention. The experimental methods in the following examples, which are not specified under specific conditions, are generally carried out under conventional conditions.

The TAX2 polypeptide is a synthetic polypeptide consisting of 12 amino acids (CEVSQLLKGDAC) as shown in Seq ID No.1, developed and synthesized by Dedieu S research team, university of Lance, France, 2015, and is capable of binding with thrombospondin-1 (thrombospondin-1) highly selectively and specifically blocking transduction of thrombospondin-1 signaling pathway. The TAX2 polypeptide has been proved to have antithrombotic and antitumor effects in recent studies, but the research on the aspect of maintaining the vascular structure and function, particularly the abdominal aorta blood vessel, is quite limited, and no direct evidence indicates the relationship between the TAX2 polypeptide and the onset of abdominal aortic aneurysm.

Materials and instruments

1. The TAX2 polypeptide is synthesized by Beijing Zhongke Asia photobiology science and technology Limited, and the sequence is shown as Seq ID No. 1: CEVSQLLKGDAC are provided.

2. Scaramble peptide (scrambled peptide) was synthesized by Beijing Zhongke Asia photoBiotech, Inc., and the sequence is shown in Seq ID No. 2: LSVDESKAQGIL are provided.

3.4 months old C57BL/6 backgroundPlod1 ^−/−Mice and their littermate control C57BL/6 wild-type mice were purchased from biotechnology limited, baioose, beijing, and housed in an IVC facility in an SPF-grade barrier environment.

AngII (angiotensin II) was purchased from Sigma, and was specified: 50 mg.

5. Isoflurane (Huoning) was purchased from Shanghai Yapeh pharmaceuticals, Inc., and was specified: 100 mL.

6. anti-LH 1 antibody was purchased from Abcam, product model: ab 262947.

7. Anti-thrombospondin-1 antibody was purchased from Abcam, product model: ab 1823.

8. Physiological Saline (Saline) was purchased from Shijiazhuang four drugs, Inc., and specified: 500 mL.

9.10% Paraformaldehyde was purchased from Shanghai song Van Biotech limited, Specification: 500 mL.

10. Total RNA extraction kit purchased from qiagen, model: RNeasy Kit 50.

11. The RNA detection kit is purchased from Agilent company, and has the model number: 2100 RNA Nano 6000.

12. PBS was purchased from GE, specification: 500 mL.

13. ALZET micro osmotic pumps were purchased from DURECT corporation, model: 2006.

14. small animal gas anaesthesia machines were purchased from veteiqp corporation, specifications: RC2 single channel.

15. The small animal high resolution ultrasound imaging system was purchased from fuji film company, model: vevo 2100.

16. The small animal in vivo blood vessel function analysis software is purchased from fuji film company, model: vevo VASC.

17. The small animal non-invasive sphygmomanometer was purchased from Softron corporation, model: 2010A.

18. Body style microscopes were purchased from Carl Zeiss, model: and V8.

19. Peristaltic pumps were purchased from lange constant flow pump ltd, model: the MU 100.

20. Tissue homogenizers were purchased from dunnes, type: the special purpose is for mice.

21. Centrifuge was purchased from Thermo corporation, model: fresco 17.

22. The library-building sequencer was purchased from Illumina corporation, model: hiseq 2500.

23. The spectrophotometer was purchased from keo corporation, model: K5500.

the experimental design of the invention conforms to the animal management implementation rule of medical experiment, is approved by the animal experiment ethics committee of the Fuweisan hospital of the Chinese medical science institute, and is implemented according to the animal experiment guideline.

Example 1: angiotensin II inductionPlod1 ^−/−Mice developed abdominal aortic aneurysm

1. Experiment grouping

A first group: wild type mouse + physiological Saline (WT + Saline) (n = 20)

Second group: wild type mouse + angiotensin II (WT + AngII) (n = 20)

Third group:Plod1 ^−/−mouse + physiological saline (Plod1 ^−/−+Saline）（n=20）

And a fourth group:Plod1 ^−/−mouse + angiotensin II (Plod1 ^−/−+AngII）（n=20）

2 mouse abdominal aortic aneurysm model establishment

Mice were anesthetized with isoflurane and the neck was dehaired, the skin of the neck was cut and the skin and subcutaneous muscle tissue of the mice were separated with forceps, an osmotic pump (ALZET) containing AngII solution (1000 ng/kg BW/min) or PBS was implanted subcutaneously along the incision, and the position of the pump on the back of the mice was adjusted to reduce the effect of the pump on the daily behavior of the mice. Following the micro-osmotic pump implantation, the wound was sutured and disinfected. The mice were evaluated for abdominal aortic aneurysm formation by taking the material 4 weeks later.

3 mouse abdominal aorta ultrasonic detection

The abdomen of the mice was actively examined ultrasonically using a Vevo2100 sonicator. Mice were induced for anesthesia with 3% isoflurane at an air flow rate of 1L/min, mounted in a supine position on a hot plate, and maintained under anesthesia with 1% isoflurane by wearing a nose mask. Depilatory cream was applied to the mouse breast. The end-diastolic abdominal aorta internal diameter was measured using the MS550D probe, placing the probe on a mouse abdominal vernier probe to find the abdominal aorta suprarenal segment. The pulse wave velocity and the blood vessel expansibility of the mouse abdominal aorta are measured and calculated by using a Vevo VASC blood vessel function module.

4 mouse noninvasive blood pressure monitoring

And a noninvasive tail artery pressure meter is adopted to dynamically monitor the conscious blood pressure of the mouse. The mouse is horizontally placed in a fixed cage, placed in a heating cover, the exposed tail part is placed in an inductor, and monitoring is started after the mouse is quiet and motionless. After the waveform was stabilized, the pressure was applied and the systolic pressure was measured. The average of three consecutive measurements was taken each time. Before the experiment, the operation steps are repeated at the same time for 3 days respectively until the mouse is completely adapted to the measurement environment, and after the measurement data are stable and consistent, formal experiment and data acquisition can be carried out.

5-dimensional microscope observation of mouse aneurysm formation and rupture

Mice were euthanized on day 28 after abdominal aortic aneurysm molding. The thorax is cut off quickly, then the heart is fully exposed, about 10ml of ice physiological saline is slowly irrigated to the left ventricle by a peristaltic pump, and the blood in the blood vessel is fully irrigated. The aorta of the mice was then completely isolated under a Zeiss V8 stereomicroscope, and the aneurysm formation and rupture of each treatment group were observed in the brightfield channel and image acquisition was performed.

Fig. 1A is a typical aorta view under a microscope for each group of mice, and fig. 1B is a statistical diagram of aneurysm formation rate and rupture rate. As can be seen from FIGS. 1A and 1B, only 1 of the WT mice had an abdominal aortic aneurysm after 4 weeks of AngII molding, whereas only 1 had an abdominal aortic aneurysm after the administration of AngIIPlod1 ^−/−In the + AngII group, 18 mice were probed for abdominal aortic aneurysms, 8 of which developed aneurysm rupture bleeding,Plod1 ^−/−the tumor formation rate and the rupture rate of the mouse are obviously higher than those of a WT mouse.In addition to this, the present invention is,Plod1 ^−/−+ Saline group mice did not develop abdominal aortic aneurysm, suggesting 4 months of agePlod1 ^−/−Mice did not spontaneously develop abdominal aortic aneurysm, and 4 weeks of continuous AngII stimulation was essential for aneurysm formation and rupture.

In addition to significant damage to vessel wall structure, long-term vascular remodeling can have a severe impact on vascular function. The structure and function of the abdominal aorta were further evaluated by mouse high-resolution abdominal ultrasound, and the results are shown in fig. 2A. As can be seen from fig. 2A, similar to the observation result of the stereomicroscope,Plod1 ^−/−the degree of abdominal aortic dilation was significantly increased in the + AngII group mice compared to WT + AngII, as shown in fig. 2B. In addition, the important parameters of pulse wave velocity shown in FIG. 2C and blood vessel dilatation shown in FIG. 2D, which reflect the blood vessel functions, arePlod1 ^−/−The + AngII group changes more significantly, indicating that after an abdominal aortic aneurysm is modeled,Plod1 ^−/−the degree of deterioration of vascular function was more severe in the mice than in the WT mice.

Plod1The gene codes for lysine hydroxylase 1 (LH 1) protein, which is a key enzyme for post-transcriptional modification of collagen, and can strengthen the arrangement and connection between collagens, and is essential for the collagen to maintain extracellular matrix homeostasis. Elastin is the most important protein for maintaining the wall structure of a large blood vessel, and the large blood vessel structure can be displayed by fluorescent staining of the Elastin. After the WT mice are stimulated by AngII, Western felt test is performed, the result is shown in fig. 3A, and gray scale statistics is performed on fig. 3A, and the result is shown in fig. 3B, it can be seen that the expression of the blood vessel wall LH1 stimulated by the AngII of the WT mice is significantly increased compared with that of a control group (salt), and the increase of the expression of LH1 induced by the AngII is an important compensation mechanism for the anti-pathological stimulation of the blood vessel, which can make the arrangement of extracellular collagen more compact, thereby resisting the effect of the AngII and avoiding the formation of aneurysm due to the expansion of the blood vessel wall, as shown in fig. 3C. However,Plod1 ^−/−mice are lack of LH1 in the body per se, and cannot express increased LH1 after being stimulated by AngIIFor compensatory protection, the Elastin structure is incomplete, and thusPlod1 ^−/−Mice were more susceptible to the formation of abdominal aortic aneurysms than WT mice, as shown in fig. 3A, 3B and 3C.

Notably, hypertension is a significant risk factor for the formation of abdominal aortic aneurysms. The results of measuring the systolic blood pressure of each group of mice given AngII molding for 2 weeks are shown in FIG. 4. As can be seen from FIG. 4, both WT mice and WT mice were usedPlod1 ^−/−Mice, all had significant increases in systolic blood pressure after AngII treatment, but there was no significant difference in the magnitude of the increase in blood pressure between the two groups. This means thatPlod1 ^−/−The significant increase in the tumor formation and rupture rates of abdominal aortic aneurysms in mice was not due to further elevated blood pressure.

Through the results, the invention successfully constructs a stable mouse abdominal aortic aneurysm model, namely, the invention is used for solving the problems that the existing method is not suitable for the existing methodPlod1 ^−/−AngII stimulation was given for 4 weeks in mice with up to 90% incidence of abdominal aortic aneurysms (18/20). The model compares the conventional ApoE^-/-The abdominal aortic aneurysm model (aneurysm incidence rate is about 60%) caused by the stimulation of the mouse by AngII is simpler and more convenient, the modeling time is shorter and the model success rate is higher.

Example 2: the preliminary locking of the thrombospondin-1 by transcriptome sequencing may bePlod1 ^−/−Key regulatory protein for mice to form abdominal aortic aneurysm

1 mouse abdominal aorta blood vessel extraction of Total RNA

The total RNA extraction kit is used for extraction, and the related pipes and reagents are all from the kit.

1) The pellet was dissolved well with 350. mu.L of RLT lysate.

2) The liquid was transferred to a 1.5mL collection tube and centrifuged at 12000rpm for 3 min.

3) The supernatant was transferred to a new 1.5mL collection tube (the pellet was not aspirated) and an equal volume of 70% ethanol (anhydrous ethanol and RNase free water for RNA) was added. The mixture was quickly mixed with a tip without centrifugation.

4) Remove 700. mu.L of the mixture quickly to the Rneasy collection column, cover it, centrifuge at 12000rpm at 4 ℃ for 15 seconds, and pour off the liquid in the collection tube.

5) mu.L of Buffer RW1 was added to the column, carefully capped, centrifuged at 12000rpm at 4 ℃ for 15 seconds, and the liquid in the collection tube was decanted.

6) Preparation of DNase 1 mix: 10 μ L DNase 1 (first time using RNase free water preparation) + 70 μ L Buffer RDD, reverse mixing, low speed simple centrifugation.

7) 80 μ L of DNase 1 mix was added directly to the membrane in the column and allowed to stand at ambient temperature for 15 min.

8) mu.L of Buffer RW1 was added to the column, carefully capped, centrifuged at 12000rpm at 4 ℃ for 15 seconds, and the liquid in the collection tube was decanted.

9) Add 500. mu.L Buffer RPE to the column, carefully cover it, centrifuge at 12000rpm at 4 ℃ for 15 seconds, and pour off the liquid in the collection tube.

10) Add 500. mu.L Buffer RPE to the column and carefully cover it and centrifuge at 12000rpm for 2min at 4 ℃.

11) The collection tube was discarded along with the collected liquid, and the column was moved into a new 2mL collection tube, capped, and centrifuged at full speed for 1 min.

12) The column was transferred into a new 1.5mL collection tube (supplied), 30-50. mu.L of RNase-free water was added to the column (which was spread over the whole membrane), the cover was carefully closed, and after standing for one minute, the column was centrifuged at 12000rpm at 4 ℃ for 1min to elute the RNA.

13) RNA quantification was performed using a Nano drop, and the abdominal aorta vessels of the mice were approximately 100ug/mL and the total amount was approximately 5ug, as measured after zeroing with RNase-free water. The A260/280 is controlled to be between 1.8 and 2.0 as much as possible.

2 vascular RNA library construction and transcriptome sequencing

A flow chart of vascular RNA library construction and transcriptome sequencing is shown in FIG. 5.

1) Transcriptome library preparation

A transcriptome sequencing library was constructed using 3. mu.g of the total RNA that was qualified as starting material. Eukaryotic mRNA was first enriched using magnetic beads with oligo (dT) and after enrichment mRNA was fragmented using fragmentation buffer. Using these fragmented RNAs as templates, single-stranded cDNA was synthesized using random primers. Then, a double-stranded cDNA was synthesized using buffer, dNTPs, RNase H and DNA polymerase I. After synthesis of the double-stranded cDNA, the double-stranded cDNA was purified using the AMPureXP beads kit. And (3) carrying out end repair, such as poly A tail adding, connection sequencing joint reaction and the like on the purified double-stranded cDNA. AMPure XP beads were used for fragment size selection and finally cDNA libraries were obtained by PCR enrichment.

2) Cluster and on-machine sequencing

The cDNAs were clustered on cBot using HiSeq PE Cluster Kit v4-cBot-HS (Illumia) reagent and the paired-end sequencing program (PE 125/PE 150) was run on the HiSeq2500 sequencing platform, resulting in 125bp paired-end sequencing Reads.

3) Filtering, alignment and visualization of transcriptome sequencing data

Because the Raw off-line sequences (Raw Reads) contain information such as low quality sequences and linker contamination, the Raw data is filtered to obtain high quality sequences (Clean Reads). Subsequent analyses were performed using Clean Reads.

The filtering of the raw data mainly involves: (1) reads to remove the joint contamination; (2) removing low quality Reads; (3) the Reads with an N content of more than 5% are removed. After removing these Reads, the sequencing quality was evaluated by data filtering statistics, sequencing quality distribution, base distribution, and the like. After the raw data is filtered, alignment analysis is performed, i.e., the sequencing sequence is aligned with the reference gene to locate it to the genome. TopHat is special software for comparison of transcriptome data, and has the advantage that the sequences which are not compared in the early stage can be cut and compared for the second time, so that the purpose of identifying the Exon-Exon splice sites is achieved. After the alignment is completed, the alignment result is visualized using an Integrated Genomics Viewer.

4) Analysis of expression level and analysis of differential Gene in transcriptome sequencing

FPKM (fragments per Kilobase per Millon Mapped fragments) is a very effective parameter commonly used in RNA-Seq technology for quantitative estimation of gene expression values. And performing gene differential expression analysis by using DESeq, and selecting genes with the Fold change of more than or equal to 1.5 and the P of less than 0.05 as up-regulated genes and selecting genes with the Fold change of less than 0.67 and the P of less than 0.05 as down-regulated genes when comparing the treated group with the control group.

3 analysis of vascular transcriptome sequencing data

The flow chart for analyzing the sequencing data of the vascular transcriptome is shown in FIG. 6.

1) GO analysis of transcriptome sequencing results

The differential gene obtained by screening is analyzed by GO and KEGG on a DAVID website (website: http:// DAVID. abcc. ncifcrf. gov /), and the analysis process is as follows: and opening the website and entering a main interface of the DAVID Database. Pasting the screened expression difference GENEs into a Paste a List tool bar, selecting 'office GENE SYMBOL' in a Select Identifier pull-down option, selecting 'GENE LIST' in a List type option, and then clicking to submit. After the click submission, the gene species was selected, and this study analyzed the mouse brain vascular tissue, so the species was "Mus musculus". Click "Start Analysis" after selecting the species. Selecting 'Function association Clustering' on an incoming 'Analysis Wizard' interface, clearing all default options, selecting 'GoTERM-BP-DIRECT', 'GoTERM-CC-DIRECT' and 'GoTERM-MF-DIRECT' in a GENE ONTOLOGY pull-down menu, then clicking 'Function association chart' to obtain an Analysis result and downloading the obtained result in a document form. And opening the downloaded document by using EXCEL, drawing by using 'GO TERM' as a vertical coordinate and using-log p value as a horizontal coordinate, and visualizing the GO and KEGG analysis results.

4 ELISA

The ELISA reaction process is as follows: TNF-alpha and IL-6 standards diluted in concentration gradients were added to a 96-well plate pre-coated with antibody, 100. mu.L per well. Blank control was added to the same volume of sample dilution. Diluting a serum protein sample by 5 times by using a sample diluent, adding the diluted serum protein sample into a 96-well plate, reacting at 37 ℃ for 90 min, wherein each well is 100 mu L; after the reaction is finished, discarding the liquid in the 96-well plate, and washing the plate for 3 times for 5min by using TBST; after washing, adding biotinylated TNF-alpha and IL-6 antibodies, reacting for 90 min at 37 ℃ in 100 mu L per well; washing with TBST for 3 times after the reaction is finished; after washing, 100 mu L of ABC liquid is added into each hole, and reaction is carried out at 37 ℃ for 30 min; after the reaction is finished, the plate is washed 3 times by using 0.01M TBS, TMB color developing agent is added, each hole is 90 mu L, the reaction is carried out for 15-20 min in a dark place at 37 ℃, and stop solution is added when the plate holes corresponding to the first four concentration gradients of the standard are blue, and each hole is 100 mu L. After the stop solution is added, the absorbance value at 450 nm is detected on a microplate reader, and the contents of TNF-alpha and IL-6 in the sample are calculated according to a standard curve.

5 gelatin zymogram

Gelatinase assay was performed according to the conventional method, since proteins of the same protein amount in mouse abdominal aorta blood vessel samples were loaded and separated on 10% Tris-glycine gel with 0.1% gelatin as a base. Then, washed and renatured with 2.5% Triton X-100 buffer. After incubation of the buffer at 37 ℃ for 24 hours, the gel was incubated with 0.05% Coomassie R-250 dye (Sigma-Aldrich, MO, USA) for 30 minutes. Gelatinase standards include MMP-9 and MMP-2 (Sino Biological Inc., Beijing, China). Finally, MMP-9 and MMP-2 activities were evaluated based on optical density.

6 immunofluorescence staining of abdominal aorta section

After the endoscopic image acquisition, the abdominal aorta of the mouse is fixed in 4% paraformaldehyde (W/V% in PBS) for 24 hours, and then is placed in 20% and 30% sucrose solutions (W/V% in PBS) in turn for 16 hours to be fully dehydrated and balanced. Then wrapped in OCT embedding medium (Tissue-Tek) and frozen in a refrigerator at-80 ℃. Before immunofluorescent staining, the sections were allowed to re-incubate at room temperature for 2 hours. The slice tissues were sealed in a 37 ℃ incubator for 1 hour using 10% donkey serum, and the serum was discarded before adding a prepared primary antibody (Anti-F4/80 antibody (1:500, Abcam); Anti-SMA antibody (1:1000, Abcam)) to the slices. DAPI nuclear staining was performed after addition of fluorescent secondary antibody and statistical analysis was performed on a Leica Q550 workstation (Leica Microsystems Imaging Solutions, Cambridge, UK).

For further clarificationPlod1 ^−/−A detailed molecular mechanism for forming the mouse abdominal aortic aneurysm is that the abdominal aortic blood vessels of each treatment group of mice are separated on the 14 th day of model building, RNA is extracted, and then transcriptome sequencing is carried out. FIG. 7A shows the transcript of the day 14 differential gene in mice model of each treatment groupThis cluster heatmap. Wherein, the color blocks with orange red represent the expression amount larger than the average value of all samples, the color blocks with blue represent the expression amount smaller than the average value, and the dendrogram shows the clustering condition of the transcript (left side). The number of genes that differed between the groups was further visualized as shown in fig. 7B. In thatPlod1 ^−/−In mice, a total of 503 genes (209 upregulated genes and 294 downregulated genes) were differentially expressed in the AngII-treated group and the Saline-treated group (| logFC |>1 and q-value <0.05); however, only 31 genes (18 up-regulated and 13 down-regulated genes) were expressed in WT mice altered following AngII administration. More importantly, in the case of the same AngII treatment,Plod1 ^−/−the expression of 108 genes was significantly changed in mice compared to WT mice, and in view of the large difference between the abdominal aortic aneurysm formation rates between the two groups of mice, it was suggested that the 108 genes are the key gene set responsible for the phenotypic difference between the two groups, i.e., explanationPlod1 ^−/−Mice form an important clue to abdominal aortic aneurysms.

The 108 genes are firstly analyzed in a Human genotype database, and the enrichment display of Human diseases related to the genes is attempted. As shown in FIG. 7C, it was suggested that these genes are mainly involved in a series of large vessel diseases such as arterial dilatation, aneurysm formation and vascular rupture. This result again strongly suggests that the 108 genes are associated withPlod1 ^−/−The pathogenesis of abdominal aortic aneurysms in mice is closely related. Further, Gene Ontology enrichment analysis was performed on these differential genes in order to identify the biological functions exerted by these genes. Fig. 7D shows functional annotation diagrams of GO corresponding to 108 different genes. Wherein, the sequence from top to bottom is from small to large according to q-value. The results suggest that these differential genes are closely related to inflammatory processes, Matrix Metalloproteinase (MMP) activation, and vascular smooth muscle cell apoptosis, and that the above-mentioned biological functions are precisely the most important mechanisms known for the formation and rupture of abdominal aortic aneurysms.

Furthermore, the key biological processes obtained by the analysis and enrichment of GO are verified. Visualization of large vessel structures using Elastin immunofluorescence staining, by immunofluorescenceAfter the AngII treatment was found by staining, it can be seen from FIG. 8APlod1 ^−/−The infiltration of macrophages in the vascular wall is more severe in mice than in WT mice, and it can be seen from FIG. 8B that proinflammatory cytokines are associated with the vascular wallccl2Andil6the expression of (a) also increased significantly. In agreement, it can be seen from FIG. 8C that the levels of IL-6 and TNF- α are in the plasmaPlod1 ^−/−Also significantly increased in mice. Furthermore, it can be seen from FIG. 9 that the activities of MMP-2 and MMP-9, which are key regulatory proteins for mediating extracellular matrix remodeling, are also shown in the experiments of gelatinasePlod1 ^−/−Is significantly elevated in mice. In addition, large vessel structures were visualized using Elastin immunofluorescence staining, which was found by TUNEL stainingPlod1 ^−/−The mouse vascular wall apoptotic cell number was significantly higher than that of the WT mouse, and the results are shown in fig. 10A and 10B, and the abdominal aorta smooth muscle layer was significantly thinned, and the results are shown in fig. 10C and 10D.

For further clarificationPlod1 ^−/−A key regulatory target for mouse abdominal aortic aneurysm formation, the 108 differential genes were displayed in the form of Volcano Plot, as shown in fig. 11. As can be seen from FIG. 11, thrombospondin-1 is ranked first among these differential genes, whether ranked by p-value or LogFC. Thrombospondin-1 belongs to a member of the stromal cell Thrombospondin family. Interestingly, thrombospondin-1 has been shown to be effective in promoting macrophage infiltration and activating pro-inflammatory processes, and furthermore, thrombospondin-1 has been reported to be involved in regulating MMP activity in macrophages and promoting vascular smooth muscle cell apoptosis, and it is precisely these thrombospondin-1's biological functions that have been reportedPlod1 ^−/−It is found in the blood vessels of mice developing abdominal aortic aneurysms. Therefore, LH1 (fromPlod1Encoded) is probably by up-regulating the expression of the key protein of thrombospondin-1, thereby leading to the enhancement of the inflammatory response of the abdominal aorta vessel wall, the continuous activation of MMP and the increase of the apoptosis level of vascular smooth muscle cells, further leading to the thinning of the vascular smooth muscle layer and finally leading to the formation and the rupture of abdominal aortic aneurysm.

Immunofluorescence Using ElastinThe light staining revealed large vessel structures, confirmed by Western blot and immunofluorescence staining as shown in fig. 12A and 12B, after administration of AngII treatment,Plod1 ^−/−the expression level of the mouse vascular wall thrombospondin-1 is obviously improved compared with that of the WT mouse, which indicates that the abdominal aortic aneurysm formation is further inhibited by blocking the biological function of the thrombospondin-1.

Example 3: the specific blocking of the thrombospondin-1 signal pathway by the TAX2 polypeptide can be obviously reducedPlod1 ^−/−Mouse abdominal aortic aneurysm formation and risk of rupture

Biosynthesis of TAX2 polypeptide and Scamble peptide (Fmoc solid phase Synthesis)

FIG. 13 shows a scheme for the biosynthesis of TAX2 polypeptide and Scramble peptide.

The basic principle of synthesis:

on the high molecular resin, amino acids are connected into specific polypeptide molecules in sequence from the carboxyl terminal according to the amino acid sequence of the polypeptide molecules.

Repeating (condensation → washing → deprotection → neutralization and washing → next round of condensation) operation to reach the length of the peptide chain to be synthesized, finally cracking the peptide chain from the resin, and obtaining the required polypeptide after purification and other treatments.

1) Deprotection: fmoc protected monomers must have the amino protecting group removed with a basic solvent (piperidine).

2) Activation and crosslinking: the carboxyl group of the next amino acid is activated by an activator. The activated monomer reacts with the free amino group to crosslink, forming a peptide bond.

3) And (3) circulation: the first two reactions are repeatedly circulated until the synthesis is completed.

4) Elution and deprotection: the polypeptide is eluted from the resin, and the protecting groups are eluted and deprotected with a deprotecting agent (TFA).

5) Analysis and purification of the polypeptide: separating different polypeptide molecules according to the hydrophobicity difference by using a reversed phase HPLC chromatography method, so that the purity of the TAX2 polypeptide and the Scamble peptide is improved to more than 98%.

The TAX2 polypeptide and the script peptide were synthesized by Beijing Zhongke Asia photoBiotech Co., Ltd in accordance with the above-mentioned principles. Scramblepeptide, as a negative control polypeptide for the TAX2 polypeptide, was arranged to exclude non-specific effects of the TAX2 polypeptide, to ensure that the therapeutic effect exerted by the TAX2 polypeptide was true, not non-specific off-target effects of the TAX2 polypeptide, keeping the number of amino acids the same, but the ordering different.

2 Abdominal aortic section H & E staining, VVG staining and Masson staining

After the endoscopic image acquisition, the abdominal aorta of the mouse is fixed in 4% paraformaldehyde (W/V% in PBS) for 24 hours, and then is placed in 20% and 30% sucrose solutions (W/V% in PBS) in turn for 16 hours to be fully dehydrated and balanced. Then wrapped in OCT embedding medium (Tissue-Tek) and frozen in a refrigerator at-80 ℃. Sections were allowed to re-incubate at room temperature for 2 hours prior to H & E staining. The sections were inserted into hematoxylin stain for 8 minutes, placed in a running water tank, and rinsed with running water for 10 minutes. Sections were stained for 1 minute by inserting them into eosin staining solution. Then putting the slices into 70% ethanol, 95% ethanol I, 95% ethanol II, absolute ethanol I and absolute ethanol II in sequence. Then placing in xylene I and xylene II. Then, the sheet was sealed with a neutral resin. The collagen fibers in the arterial tissue were stained using a modified Masson trichrome staining solution kit (ThermoFisher Scientific) to give blue color of collagen fibers and red color of muscle fibers and cytoplasm. The sections were viewed photographed using a Leica Q550 workstation (Leica Microsystems Imaging Solutions, Cambridge, UK).

3 the rest of the methods for modeling and model evaluation of abdominal aortic aneurysm were the same as in example 1.

4. Results

To further determine whether in vivo prevention of thrombospondin-1 (blocking its signal transduction pathway) is effective in reducing the incidence and rupture rate of abdominal aortic aneurysm, the following method is providedPlod1 ^−/−The mice were modelled with AngII and were given TAX2 polypeptide (dose: 10 mg/kg body weight/day, 100. mu.L each time, i.e. intraperitoneal injection, daily until the end of the experiment). The TAX2 polypeptide has been described in various documentsIt was confirmed that it can bind to thrombospondin-1 (thrombospondin-1) with high selectivity and specifically block transduction of the thrombospondin-1/CD47 signaling pathway. To exclude the nonspecific effect of the TAX2 polypeptide, a script peptide (control) was used as a negative control for the TAX2 polypeptide-treated group, and the dose and manner of administration of the script peptide group (abbreviated as Scrb) were identical to those of the TAX2 polypeptide group, and the results are shown in fig. 14A and 14B.

As can be seen from fig. 14A and 14B, after 28 days of AngII modeling, compared with the scarmble group, the group treated with TAX2 polypeptide had a significantly decreased abdominal aortic aneurysm tumor formation rate and rupture rate, from 90% to 40% and from 35% to 20%. Further, the structure and function of the abdominal aorta were further evaluated by mouse high-resolution abdominal ultrasound, and as a result, as shown in fig. 15A, 15B, 15C and 15D, it was found that, similarly to the observation result of the stereomicroscope, as shown in fig. 15A and 15B,Plod1 ^−/−the abdominal aorta of the mice in the AngII + TAX2 group is expanded to a greater degreePlod1 ^−/−The AngII + scarmble ratio was significantly reduced; as shown in FIGS. 15C and 15D, the pulse wave velocity and the vascular dilation, which are important parameters reflecting the vascular function, were also significantly improved, indicating that the TAX2 polypeptide was significantly improvedPlod1 ^−/−Mouse abdominal aortic vascular function. Furthermore, as shown in fig. 16, at the pathological level by H&E staining shows that the TAX2 polypeptide is obviously improvedPlod1 ^−/−Abdominal aorta dilation of the mice; it was found by VVG staining that the TAX2 polypeptide significantly reduced the effect of AngIIPlod1 ^−/−The mouse vascular elastic plate is broken and arranged disorderly; the TAX2 polypeptide was found to significantly reduce AngII-mediated collagen deposition in the vessel wall by Masson staining.

Elastin immunofluorescence staining is used for displaying a large blood vessel structure, and further the methods such as immunofluorescence staining and gelatin zymogram prove that the TAX2 polypeptide shown in figure 17 can effectively relieve AngII-mediated diseasesPlod1 ^−/−Inflammatory reaction of vascular wall of mice, TAX2 polypeptide shown in figure 18 can effectively reduce activation of MMP2 and MMP9 of vascular wall, TAX2 polypeptide shown in figures 19A and 19B can obviously reduce apoptosis of vascular wall cells and inhibit vascular wall cells from apoptosisThe anti-smooth muscle layer becomes thin.

The results suggest that in vivo administration of TAX polypeptide successfully blocks the thrombospondin-1 signaling pathway and significantly alleviates AngII-mediated eventsPlod1 ^−/−Mouse abdominal aortic aneurysm formation and risk of rupture.

Example 4: the expression of LH1 in affected blood vessel of patients with abdominal aortic aneurysm is remarkably reduced, while the expression of thrombospondin-1 is remarkably increased

1. Collecting human blood vessel samples and preparing slices:

blood vessel samples of 6 human-derived patients with abdominal aortic aneurysm were collected from 6 patients at the time of performing an aneurysm thoracotomy operation in the hospital extrafuge, academy of medicine, china. These 6 patients were confirmed to be abdominal aortic aneurysms by ultrasound examination or CT angiography. Blood vessel samples of 6 non-abdominal aortic aneurysm control populations 6 were collected at the time of heart transplant open chest surgery for diagnosis of hypertrophic cardiomyopathy or dilated cardiomyopathy.

Fresh samples were collected and immediately preserved in formalin, paraffin embedded and subsequently cut into 7 μm thick pathological sections for immunofluorescence experiments.

The study was approved by the ethical committee of clinical research of the academy of medical sciences of china (2014 CB 541601), and informed consent was filled in all the groups of people who were included in the study.

2. The clinical information of the human blood vessel sample patient is shown in the table 1.

TABLE 1 clinical information of human-derived vascular samples

3. Immunofluorescence experiment of human blood vessel sample: the method is the same as the mouse blood vessel immunofluorescence experiment method.

It has been demonstrated by animal level studies that under sustained high blood pressure stimulation,Plod1the LH1 expression defect caused by deletion can obviously increase the morbidity risk of abdominal aortic aneurysm, and the mechanism is to induce the expression of thrombospondin-1, the latter further induces vascular inflammatory reaction, activates MMP2 and MMP9 of blood vessel walls and induces vascular smoothingApoptosis of the smooth muscle cells eventually leads to thinning and dilation of the vessel wall, resulting in the formation of abdominal aortic aneurysms. And in vivo administration of TAX polypeptide can successfully block the thrombospondin-1 signal channel and remarkably relieve AngII mediatedPlod1 ^−/−Mouse abdominal aortic aneurysm formation and risk of rupture.

To further enhance the clinical significance and potential clinical transformation value of this study, the abdominal aortic vessels of 6 patients with abdominal aortic aneurysm and 6 control populations were collected and subjected to immunofluorescence experiments, and the structures of large vessels were shown using Elastin immunofluorescent staining, with the results shown in fig. 20A to 20D. From fig. 20A and 20B, it can be seen that the expression of the blood vessel wall LH1 in the abdominal aortic aneurysm patients is significantly reduced compared to the blood vessel wall in the normal control population, while from fig. 20C and 20D, it can be seen that the expression of thrombospondin-1 is significantly enhanced compared to the blood vessel wall in the normal control population. The result is highly consistent with the expression level of animal blood vessel wall LH1/thrombospondin-1, which indicates that LH1 is commonly existed in the pathogenesis of abdominal aortic aneurysm due to the down-regulation of blood vessel wall expression, and that the activation of thrombospondin-1 signal pathway caused by the down-regulation of LH1 is a key initiating link in the pathogenesis of abdominal aortic aneurysm of human, thus indicating that the thrombospondin-1 can be a new treatment target for abdominal aortic aneurysm diseases. The antagonist of the tetramospondin-1 and the downstream signal path thereof can be used for clinically preventing and treating the abdominal aortic aneurysm disease through the TAX2 polypeptide specificity in the early stage, so that the bottleneck that no effective drug treatment means exists in the abdominal aortic aneurysm at present can be broken through.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

SEQUENCE LISTING

<110> Fuweisan Hospital, Chinese academy of science

Application of <120> TAX2 polypeptide in preparation of medicine for preventing and/or treating abdominal aortic aneurysm formation and/or rupture

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 12

<212> PRT

<213> Artificial sequence

<400> 1

Cys Glu Val Ser Gln Leu Leu Lys Gly Asp Ala Cys

1 5 10

<210> 2

<211> 12

<212> PRT

<213> Artificial sequence

<400> 2

Leu Ser Val Asp Glu Ser Lys Ala Gln Gly Ile Leu

1 5 10

Claims (8)

1. Use of a TAX2 polypeptide in the manufacture of a medicament for the prevention and/or treatment of abdominal aortic aneurysm formation and/or rupture.
2. Use of a TAX2 polypeptide in the manufacture of a medicament for the prevention and/or treatment of an inflammatory response in a blood vessel wall during the formation and/or rupture of an abdominal aortic aneurysm.
3. Application of TAX2 polypeptide in preparing medicine for inhibiting activation of vascular wall matrix metalloproteinase system.
4. Application of TAX2 polypeptide in preparing medicine for inhibiting apoptosis of smooth muscle cells in blood vessel wall.
5. 5. The use according to any one of claims 1 to 4, wherein the amino acid sequence of the TAX2 polypeptide is as set forth in Seq ID No. 1.
6. 6. The use of any one of claims 1 to 4, wherein the medicament further comprises a pharmaceutically acceptable carrier.
7. 7. The use of claim 6, wherein the pharmaceutically acceptable carrier comprises any one or more of diluents, excipients, disintegrants, fillers, binders, lubricants, flavoring agents, surfactants, stabilizers.
8. 8. The use of claim 7, wherein the medicament is in the form of any one of injection, tablet, granule, pill and capsule.

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