Application of inhibiting mutual combination of endothelial cells PHACTR1 and HSP90 in treatment of abdominal aortic aneurysm

Abstract

The invention relates to the technical field of biological medicine, particularly discloses application of inhibiting mutual combination of endothelial cells PHACTR1 and HSP90 in treatment of abdominal aortic aneurysm, and is technically characterized by providing application of a substance for blocking or inhibiting combination of PHACTR1 protein and HSP90 protein in preparation of an endothelial function protective product. The invention relates to an application of a substance for blocking or inhibiting combination of a PHACTR1 protein and an HSP90 protein in preparation of a product for inhibiting animal or human abdominal aortic aneurysm. The substance is a PHACTR1 or HSP90 small-molecule interfering RNA (Ribonucleic Acid), or a polypeptide or a small-molecule substance for inhibiting the combination of a PHACTR1 protein and an HSP90 protein. In the scheme of the invention, the mutual combination of the PHACTR1 protein and the HSP90 protein is a new drug target of the abdominal aortic aneurysm, a new treatment means is provided for the treatment of the abdominal aortic aneurysm, and the scheme of the invention has important application value in the development of drugs for treating the abdominal aortic aneurysm.

Description

Technical Field

[0001] The patent of this invention relates to the field of biomedicine technology, specifically, to the application of inhibiting the mutual binding between endothelial cell PHACTR1 and HSP90 in the treatment of abdominal aortic aneurysm. Background Art

[0002] Abdominal aortic aneurysm (AAA) refers to the long-term irreversible local expansion of the abdominal aorta to form a tumor-like expansion. It is a high-fatality disease with great harm. Its risk factors include old age (≥65 years old), smoking, hypertension, hyperlipidemia, atherosclerosis, male sex, congenital weak aortic wall vessels, vasculitis (inflammation of the large arteries, etc.) and other aneurysmal lesions. The disease is insidious, with a low rate of early diagnosis, and often no obvious symptoms in the early stages. As the disease progresses, the diameter of the tumor increases, leading to tumor rupture, distal organ embolism and compression of adjacent organs, and symptoms such as abdominal and back pain and hypotension will appear, which will attract the attention of patients. Once the tumor ruptures, the mortality rate is as high as 90%.

[0003] At present, the treatment of AAA rupture is mainly surgical, including endovascular repair or open repair surgery. However, the surgical risk is high and the prognosis is poor. Therefore, early screening and detection of the disease are crucial. With the gradual promotion of AAA screening programs, more and more early asymptomatic AAA patients are discovered and closely monitored. Unfortunately, the current non-surgical treatment mainly targets the risk factors of AAA, such as smoking cessation and blood pressure control, and there is no direct drug treatment for AAA. Therefore, in-depth exploration of the pathogenesis of abdominal aortic aneurysm and the development of targeted drugs are key technologies that need to be addressed in this disease field.

[0004] The pathogenesis of abdominal aortic aneurysm is complex. The currently known mechanisms include degradation of elastic fibers, degeneration of the vascular media, and local vascular inflammation, including: (1) Elastic fiber rupture is mainly related to the remodeling of extracellular matrix proteins caused by changes in FBN1, MFAP5, and LOX genes. (2) Changes in smooth muscle cell function are the main cause of vascular media degeneration, including changes in smooth muscle cell contractile function mediated by ACTA2, MYH11, MYLK, PRKG1, etc., smooth muscle metabolic dysfunction mediated by the MAT2A gene, and abnormal smooth muscle viability regulated by genes such as FOXE3. (3) Local vascular inflammation is mainly due to the recruitment of inflammatory cells, mainly macrophages, to the injured site after local vascular injury or local vasculitis, which further promotes the release and aggregation of inflammatory factors such as TNF-α, IL-6, IL-12, and chemokines, further aggravating vascular injury.

[0005] In recent years, the important role of endothelial cell function in the pathogenesis of abdominal aortic aneurysm has received increasing attention. Endothelial cells are widely distributed in the body and are arranged inside all blood vessels and lymphatic vessels of the body. Vascular endothelial cells are an important line of defense for the body and play a key role in transporting oxygen and nutrients, regulating the transport of immune cells, and maintaining tissue homeostasis. Under normal circumstances, endothelial cells sense the surrounding environment and release vasoactive substances such as nitric oxide (NO) to cause vasodilation, or secrete mediators such as endothelin-1 to cause vasoconstriction, thereby regulating local blood flow. Under pathological conditions, inflammatory factors, abnormal shear force and other stimuli lead to endothelial dysfunction, which is mainly manifested as reduced synthesis and release of nitric oxide (NO) in vascular endothelial cells, impaired vasodilation function, and upregulated expression of inflammation-related molecules. It is an early pathological event in the formation of AAA, inducing oxidative stress and inflammation, thereby leading to further degradation of the arterial wall. Improving endothelial barrier function can delay the early progression of abdominal aortic aneurysm and is expected to become a new target for the treatment of abdominal aortic aneurysm, with important potential application value. However, there are currently no treatments related to endothelial function.

[0006] NO is mainly synthesized by vascular endothelial cells and catalyzed by endothelial NO synthase (eNOS). Under normal circumstances, after activation, eNOS uses nicotinamide adenine dinucleotide phosphate (NADPH) and molecular oxygen as substrates to oxidize the terminal guanidine nitrogen atom of L-arginine to generate NO, while producing L-citrulline as a byproduct. Studies have reported that abnormal expression or function of eNOS can cause or aggravate AAA. Proteins related to regulating the expression and function of endothelial eNOS can provide new targets for the treatment of AAA.

[0007] Phosphatase and actin regulatory factor-1 (PHACTR1) is a member of the phosphatase and actin regulatory protein family, which contains an N-terminal nuclear localization signal, four RPEL repeat sequences and a C-terminal protein phosphatase 1 (PP1) binding domain. The applicant's research found that PHACTR1 is upregulated in aortic aneurysms, and PHACTR1 in vascular endothelial cells can bind to the molecular chaperone protein HSP90 of eNOS, thereby inhibiting the expression and activity of eNOS and promoting the occurrence and development of abdominal aortic aneurysms.

[0008] To this end, the present invention aims to provide an application of inhibiting the binding between endothelial cell PHACTR1 and HSP90 in the treatment of abdominal aortic aneurysm to solve the above problems. Summary of the invention

[0009] The purpose of the present invention is to solve the technical problems raised in the above-mentioned background technology, and to provide the purpose of the present invention is to provide the use of PHACTR1 protein and HSP90 protein binding to each other, and the use of inhibiting the binding to each other in preparing endothelial function protective products or in preparing products for inhibiting the occurrence and development of abdominal aortic aneurysms in animals or humans.

[0010] The above-mentioned purpose of the present invention is achieved like this:

[0011] The present invention provides application of a substance for inhibiting endothelial cell PHACTR1 protein in preparing a product for protecting endothelial function.

[0012] The present invention also provides the use of the substance inhibiting PHACTR1 protein in preparing a product for inhibiting abdominal aortic aneurysm in animals or humans.

[0013] Furthermore, the substance is a PHACTR1 antibody.

[0014] Furthermore, the above-mentioned products are medicines or health products.

[0015] The present invention also provides the use of a substance for blocking or inhibiting the binding of PHACTR1 protein with HSP90 protein in the preparation of an endothelial function protective product.

[0016] The present invention also provides the use of a substance for blocking or inhibiting the binding between PHACTR1 protein and HSP90 protein in the preparation of a product for inhibiting abdominal aortic aneurysm in animals or humans.

[0017] Furthermore, the above-mentioned substance is a PHACTR1 or HSP90 small molecule interfering RNA, or a polypeptide or a small molecule substance that inhibits the binding between the PHACTR1 protein and the HSP90 protein.

[0018] Furthermore, the above-mentioned product is a medicine.

[0019] The difficulty and significance of the technical problem solved by the present invention are:

[0020] Treatments for abdominal aortic aneurysms include surgical treatment and non-surgical treatment. The main treatment for ruptured abdominal aortic aneurysms is surgical treatment: for patients with good systemic conditions who can tolerate surgery, classic open repair is used for treatment, but this operation is difficult and has many complications, and is not suitable for high-risk patients; while endovascular repair of abdominal aortic aneurysm is less invasive and has reduced risks, it has complications such as endoleak, graft displacement or occlusion, and a high rate of re-intervention after surgery. Therefore, early screening and early intervention of abdominal aortic aneurysms are particularly important. However, current non-surgical treatments are mainly based on reducing risk factors, such as smoking cessation and blood pressure control, and there is still a lack of direct targeted drug treatment. Existing research evidence shows that eNOS-mediated endothelial dysfunction is an early pathological event in the formation of abdominal aortic aneurysm, inducing oxidative stress and inflammation, thereby leading to further degeneration of the arterial wall. Therefore, improving endothelial barrier function can delay the early progression of abdominal aortic aneurysm, and is expected to become a new target for the treatment of abdominal aortic aneurysm, with important potential application value. However, there are currently no treatments related to endothelial function. To this end, the inventors of the present application have conducted in-depth research on the pathogenesis of abdominal aortic aneurysm and provided the use of the mutual binding of PHACTR1 protein and HSP90 protein (molecular chaperone of eNOS), as well as the use of inhibiting the mutual binding in the preparation of endothelial function protective products or in the preparation of products for inhibiting the occurrence and development of abdominal aortic aneurysm in animals or humans, which has important application value for the development of new targeted drugs for the treatment of abdominal aortic aneurysm.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The scheme of the present invention provides a new use of the mutual binding of PHACTR1 protein and HSP90 protein, and the use of inhibiting the mutual binding in preparing endothelial function protective products or in preparing products for inhibiting the occurrence and development of abdominal aortic aneurysms in animals or humans. The mutual binding of PHACTR1 protein and HSP90 protein is a new drug target for abdominal aortic aneurysm, providing a new treatment method for the treatment of abdominal aortic aneurysm. The scheme of the present invention has important application value in the development of drugs for treating abdominal aortic aneurysm. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the immunohistochemical staining result of PHACTR1 in normal human aortic tissue and aortic aneurysm tissue in the embodiment of the present invention;

[0024] Figure 2 PHACTR1 knockout mouse gene identification diagram in the embodiment of the present invention (AB: PCR and agarose electrophoresis were performed to identify the mouse genotypes for gene editing to construct systemic (A) and endothelial cell-specific PHACTR1 knockout mice (B);

[0025] Figure 3The figures are the tumor formation conditions of abdominal aortic aneurysm model in mice at different time points in the embodiments of the present invention (AB: Abdominal aortic aneurysm model was induced in wild-type male mice using elastase, and the abdominal aorta of the mice was observed on days 0, 7, 14 and 21 after modeling (A), and the maximum outer diameter of the blood vessel was statistically analyzed (B). *P<0.05. Data are expressed as mean ± SEM, and analyzed using unpaired two-tailed T test and Welch's correction);

[0026] Figure 4 In the present invention, systemic knockout of PHACTR1 can significantly inhibit the progression of abdominal aortic aneurysm in mice (AB: systemic PHACTR1 knockout male mice (Phactr1 - / - ) and wild-type control mice (WT) were used to establish abdominal aortic aneurysm models. The morphology of the abdominal aorta was observed 14 days later (A) and the maximum outer diameter of the blood vessels was statistically analyzed (B). C: Systemic PHACTR1 knockout mice (Phactr1 - / - HE staining of abdominal aortic aneurysms of ) and control wild-type mice (WT). *P<0.05. Data are expressed as mean ± SEM and analyzed using unpaired two-tailed T test and Welch's correction);

[0027] Figure 5 In the embodiment of the present invention, endothelial cell-specific knockout of PHACTR1 can significantly inhibit the progression of abdominal aortic aneurysm in mice (AB: endothelial cell-specific PHACTR1 knockout male mice (Phactr1 ECKO ) and littermate control mice (Phactr1 f / f ) were used to establish abdominal aortic aneurysm models. The morphology of the abdominal aorta was observed 14 days later (A) and the maximum outer diameter of the blood vessels was counted (B). CD: Endothelial cell-specific PHACTR1 knockout mice (Phactr1 ECKO ) and littermate control mice (Phactr1 f / f ) HE staining of abdominal aortic aneurysm (C) and statistical analysis of vascular wall thickness (D). *P<0.05. Data are expressed as mean ± SEM and analyzed using unpaired two-tailed T test and Welch's correction);

[0028] Figure 6 RNA-seq and bioinformatics analysis in the present invention suggest that PHACTR1 is involved in regulating NO synthesis (in 8-week-old endothelial cell-specific PHACTR1 knockout mice (Phactr1 ECKO ) and littermate control mice (Phactr1 f / f), lungs were harvested and digested, and CD31+CD45- endothelial cells were obtained by magnetic bead sorting for RNA-seq. KEGG pathway enrichment analysis (A) and GO function enrichment analysis (B) were performed on the up-regulated differentially expressed genes. The red highlighted texts are NO synthesis-related processes);

[0029] Figure 7 It is that knockdown of PHACTR1 in the embodiment of the present invention can upregulate eNOS expression and NO release (AC: after HUVEC was transfected with PHACTR1-specific siRNA (siPHACTR1) or negative control siRNA (siCtrl), WB was used to detect eNOS protein expression and quantification (AB); then the cell supernatant was collected, and the NO content in the supernatant was detected by a kit to evaluate NO release (C). n=3. DE: PHACTR1 was overexpressed in HUVEC by adenovirus, and eNOS protein expression was detected by WB and quantification. n=3. LF: laminar flow. *P<0.05, **P<0.01);

[0030] Figure 8 In the embodiment of the present invention, HSP90 is a PHACTR1 interacting protein (A: PHACTR1 (HA tag) was overexpressed in 293FT cells, the cell lysate was collected, and immunoprecipitation (IP) was performed with HA tag antibody, and the IP complex was subjected to SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining; BC: protein spectrum detection was performed on the protein band of about 90 kDa obtained by IP). DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0032] The implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0033] With reference to the accompanying drawings of the specification, the following are specific embodiments of the solutions provided by the present invention.

[0034] Example: The present invention provides an embodiment of the invention for use of a substance that inhibits endothelial cell PHACTR1 protein in the preparation of a product that protects endothelial function, or for use of a substance that inhibits PHACTR1 protein in the preparation of a product that inhibits abdominal aortic aneurysm in animals or humans. The substance is a PHACTR1 antibody. The product can be a medicine.

[0035] The present invention also provides the use of a substance that blocks or inhibits the binding between PHACTR1 protein and HSP90 protein in the preparation of an endothelial function protective product.

[0036] The present invention also provides the use of a substance that blocks or inhibits the binding of PHACTR1 protein to HSP90 protein in the preparation of a product for inhibiting abdominal aortic aneurysm in animals or humans. The substance is a PHACTR1 or HSP90 small molecule interfering RNA, or a polypeptide or small molecule substance that inhibits the binding of PHACTR1 protein to HSP90 protein.

[0037] In the above-mentioned embodiments of the present invention, the mechanism of action of the present invention is studied as follows:

[0038] 1. Up-regulation of PHACTR1 expression in human aortic aneurysm tissue: In order to clarify the relationship between PHACTR1 and the pathogenesis of abdominal aortic aneurysm, the inventors of the present application used aortic tissue and aortic aneurysm tissue of healthy subjects to perform PHACTR1 immunohistochemical staining and observed that in the histochemical sections of patients with aortic aneurysm, the expression of PHACTR1 was significantly increased (e.g. Figure 1 shown).

[0039] 2. Endothelial cell-specific knockout of PHACTR1 can inhibit the occurrence and development of abdominal aortic aneurysms in mice: In order to successfully construct an abdominal aortic aneurysm model, the inventors of the present application successfully established an abdominal aortic aneurysm model induced by elastase using male wild-type mice by consulting literature and experiments, and detected the abdominal aortic tumor formation in mice at 0, 7, 14, and 21 days after modeling (such as Figure 2 As shown in the figure, as time goes by, the degree of bulging in the abdominal aorta of mice gradually increases. These results indicate that the inventors of the present application have successfully established an abdominal aortic aneurysm model in mice.

[0040] To explore the role of endothelial PHACTR1 in the formation of abdominal aortic aneurysm, the inventors of the present application used CRISPR / Cas9 technology to construct systemic knockout (Phactr1 - / - ) and endothelial cell-specific knockout of PHACTR1 (Phactr1 ECKO ) of mice (e.g. Figure 3 The abdominal aortic aneurysm model was established in knockout mice and control mice according to the above method.

[0041] For systemic knockout mice, samples were taken 21 days after modeling and it was found that compared with wild-type mice (WT), the expression of Phactr1 - / - The outer diameter of the aorta in mice decreased significantly (e.g. Figure 4 HE staining of vascular tissue showed that Phactr1 - / - The lumen diameter of the mouse was significantly smaller than that of the control wild-type mice (e.g. Figure 4 as shown in C in the figure).

[0042] Phactr1 ECKOSimilar results were also found in mice. After 21 days of abdominal aortic aneurysm model creation in knockout mice and control mice, the abdominal aorta was isolated and observed to have significantly reduced aneurysm expansion after endothelial-specific knockout of PHACTR1, as evidenced by a reduction in the outer diameter of the blood vessels (e.g. Figure 5 HE staining further showed that Phactr1 ECKO The inner diameter and vascular wall thickness of the abdominal aorta of mice were significantly reduced (e.g. Figure 5 The above results indicate that PHACTR1, especially PHACTR1 in endothelial cells, is involved in regulating the occurrence and progression of abdominal aortic aneurysms induced by elastase.

[0043] 3. Endothelial cell PHACTR1 inhibits the expression and activity of eNOS: As mentioned above, endothelial dysfunction caused by endothelial cell eNOS deficiency is an important factor in the development of abdominal aortic aneurysm. The inventors of this application used transcriptome sequencing (RNA-seq) to analyze the potential function of PHACTR1 in endothelial cells. The lungs contain a large number of endothelial cells, so the inventors of this application took Phactr1 ECKO and littermate control mice (Phactr1 f / f ) lung tissue, digested and prepared single cell suspension, magnetic bead sorting was used to obtain CD31+CD45- endothelial cells, and RNA-seq was performed. Functional enrichment of up-regulated differentially expressed genes was performed, and GO analysis showed that the functions of differentially expressed genes were related to NO biosynthesis and metabolic processes (such as Figure 6 KEGG analysis showed that differentially expressed genes were concentrated in arginine biosynthesis and metabolism (as shown in A in Figure 2). Figure 6 This indicates that PHACTR1 can regulate NO synthesis and mediate endothelial dysfunction.

[0044] The inventors of the present application further analyzed the regulatory effect of PHACTR1 on eNOS using human umbilical vein endothelial cells (HUVEC). PHACTR1 was knocked down by siRNA in HUVEC, and WB results showed that eNOS expression was significantly increased (e.g. Figure 7 AB), the NO content in the cell culture supernatant also increased significantly (as shown in Figure 7 After adenovirus overexpressed PHACTR1, the expression of eNOS protein decreased significantly (as shown in C). Figure 7 The above results indicate that PHACTR1 can negatively regulate eNOS expression and NO release.

[0045] 4. PHACTR1 can bind to HSP90, a molecular chaperone of eNOS: To clarify the molecular basis of PHACTR1 regulating eNOS function, the inventors of the present application performed immunoprecipitation (IP) combined with protein spectrum analysis to search for potential interacting proteins of PHACTR1. PHACTR1 with HA tag was overexpressed in 293FT cells. After IP was performed using HA tag antibody, the IP complex was separated by SDS-PAGE electrophoresis and compared with the control antibody IP group and the IP group without overexpression of PHACTR1 to obtain differential bands (such as Figure 8 A differential band at about 90 kDa was taken and mass spectrometry was performed on the band. It was found to be HSP90 protein (as shown in Figure 1). Figure 8 BC in the figure). HSP90 is an important molecular chaperone that plays an important biological function by directly binding to and regulating the stability, protein folding and activation of substrate / client proteins. eNOS is one of the substrates of HSP90, and its phosphorylation activation depends on the binding of HSP90. Therefore, the above results suggest that PHACTR1 binds to HSP90, thereby inhibiting the activity of eNOS and promoting the occurrence and development of abdominal aortic aneurysm.

[0046] The following is a specific experiment implemented by the present invention:

[0047] 1. Establishment of PHACTR1 whole-body and endothelial-specific knockout mice and genotype identification methods:

[0048] (1) Transgenic mice were constructed by Biocytogen using CRISPR / Cas9 technology to obtain PHACTR1 systemic knockout mice in the C57BL / 6J background (Phactr1 - / - ) and conditional knockout mice (Phactr1 f / f Tie2-Cre mice (B6.Cg-Tg[Tek-cre]1Ywa / J) were purchased from Jackson Laboratory. f / f The mice were crossed with Tie2-Cre mice to generate endothelial-specific PHACTR1 knockout mice (Phactr1 ECKO Only male mice were used in this study, and all mice were bred in the Tongji University Laboratory Animal Room with a 12-hour light / dark cycle and free access to food and water. All animal procedures were approved by the Animal Care and Use Committee of the Shanghai Tenth People's Hospital (SHDSYY-0050 and SHDSYY-202-T0009). The experiments outlined in this invention were in accordance with the Guide for the Care and Use of Laboratory Animals (8th edition, 2011) published by the National Institutes of Health.

[0049] (2) Genotype identification method of transgenic mice:

[0050] ① The mice's tails were docked 9-14 days after birth, and the tail length was about 0.2-0.5 cm.

[0051] ② Lysis of rat tail tissue: After cutting the rat tail tissue into small pieces, add lysis buffer and proteinase K, incubate at 55°C for 20 minutes, and then heat at 95°C for 5 minutes to inactivate proteinase K.

[0052] ③ Centrifugation: Centrifuge the sample and take the supernatant as the template DNA for the PCR reaction.

[0053] ④ Configure the PCR reaction system: According to the amount of primers and template DNA, prepare an appropriate amount of PCR reaction mixture.

[0054] ⑤Perform PCR reaction: Place the prepared PCR mixture into the PCR instrument and set appropriate conditions for amplification.

[0055] ⑥Prepare 2% agarose gel.

[0056] ⑦ Load the PCR product and DNA marker together and perform electrophoresis separation at 120V for 36 minutes.

[0057] ⑧Observe the electrophoresis results in the gel imaging system and determine the genotype based on the position of the bands.

[0058] 2. Establishment and evaluation methods of mouse abdominal aortic aneurysm model:

[0059] (1) Male C57BL / 6 mice aged 8 to 10 weeks were selected and given drinking water containing 0.2% β-aminopropionitrile (BAPN, A3134, Sigma-Aldrich). Two days later, abdominal aortic aneurysm models were established.

[0060] (2) After the mouse was anesthetized by isoflurane inhalation, a midline laparotomy was performed and the intestines were moved to the upper side of the peritoneal cavity; the infrarenal abdominal aorta was exposed from the level of the left renal artery to the iliac bifurcation, and the aorta was carefully separated from the vena cava and surrounding tissues to expose the pretreatment site.

[0061] (3) A small piece of sterile gelatin sponge was soaked with 47 U / mL purified porcine elastase (E7885, Sigma-Aldrich), placed around the abdominal aorta, and incubated for 15 minutes. In sham-operated mice, normal saline was used instead of elastase.

[0062] (4) Remove and discard the sponge, carefully wipe the aorta with a clean cotton swab to absorb excess fluid, place the organ in place, suture the abdominal opening in layers, and then place the mouse in an incubator to recover.

[0063] (5) The experimental mice were continuously given drinking water containing 0.2% BAPN (β-aminopropionitrile) until the end of the experiment.

[0064] (6) Evaluation method: At the specified time point, mice were anesthetized and euthanized, and the abdominal aorta was exposed and the surrounding connective tissue was cleaned as described above. A needle with a diameter of 0.45 mm was placed next to the blood vessel as a ruler, and photos were taken under a microscope. The outer diameter of the aorta was measured using ImageJ software.

[0065] 3. HUVEC culture and subculture method:

[0066] (1) When the cell density reaches about 80%, aspirate the medium in the original culture flask and rinse the cells twice with PBS buffer.

[0067] (2) Add an appropriate amount of 0.05% trypsin for digestion. The digestion time can be adjusted according to the actual situation until the cell edges are reduced and the cell wall is loosened.

[0068] (3) After digestion is complete, remove the trypsin, add fresh culture medium to neutralize the trypsin, and gently blow the cell layer to make the cells fall off and form a cell suspension. Collect the cell suspension in a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, and retain the cell pellet. Add appropriate culture medium to it, mix the cell suspension, evenly add it to a 10 cm dish, and then place it in an incubator for culture.

[0069] (4) Change the medium regularly (2-3 times a week) and repeat the above steps when the cell density reaches 80% again.

[0070] 4. siRNA transfection method for HUVEC:

[0071] (1) HUVECs were digested from T75 culture flasks and inoculated into 5-6 6 cm dishes.

[0072] (2) On the second day, preheat Opti-MEM medium. Mix solution A and solution B according to the following ingredients.

[0073] A: 250 μL Opti-MEM medium + SiRNA (100 nM);

[0074] B: 250 μL Opti-MEM medium + 6 μL Lipofectamine 2000;

[0075] Mix liquid A and liquid B and let it stand at room temperature for 20 minutes.

[0076] (3) Wash HUVECs twice with preheated Opti-MEM medium, using 3 mL each time.

[0077] (4) Add 1.5 mL of pre-warmed Opti-MEM medium to the culture dish.

[0078] (5) Add 0.5 mL of the mixed AB solution to each dish and shake gently to mix well.

[0079] (6) Incubate at 37°C for 2 hours.

[0080] (7) Discard the culture medium and replace with complete HUVEC culture medium to continue culturing.

[0081] (8) After 48 hours, collect the plates for testing.

[0082] 5. Adenovirus packaging method:

[0083] (1) Cell transfection (transfection 6-well plate):

[0084] ① Prepare Mix A with 250 μL DMEM and 9 μL PEI and let it stand for more than 5 minutes.

[0085] ②250μL DMEM, 1μg shuttle plasmid, and 2μg packaging plasmid (i.e., adenovirus backbone plasmid) were prepared into MixB.

[0086] ③ Mix MixA and MixB, vortex to mix, centrifuge for a moment, and let stand for more than 30 minutes to form a DNA-PEI complex.

[0087] ④ During the static period, HEK293A cells were plated in a 6-well plate, with a cell number of about 0.3-0.5×10 6 / hole (the plates can also be laid in advance).

[0088] ⑤ Add the mixture after standing to the 6-well plate drop by drop. Mix well and place in a CO2 incubator for culture.

[0089] (2) Amplification and virus collection steps:

[0090] ① About 14 days after transfection, blow down the cells with CPE in the six-well plate together with the culture medium and add them to the 10 cm dish. After mixing, place them in a CO2 incubator for culture.

[0091] ② Collect the 10 cm dish cells with CPE, transfer them to a 15 mL centrifuge tube, and mark them accordingly;

[0092] ③ Centrifuge at 3000rpm for 10min. After centrifugation, remove the centrifuge tube, retain the cell pellet, and save the supernatant in another centrifuge tube for later use.

[0093] ④ Add adenovirus freezing solution to the cell pellet, continuously blow up the pellet with a pipette to ensure complete dissolution, and then transfer to a 1.5mL EP tube.

[0094] ⑤ Freeze and thaw repeatedly at -80℃ and 37℃ for 4 times.

[0095] ⑥After freeze-thawing is completed, centrifuge at 12000g for 2 minutes.

[0096] ⑦ Transfer the supernatant to a tube using a 1 mL pipette and store in a -80°C refrigerator.

[0097] (3) Large-scale amplification and virus collection steps:

[0098] ① Add the collected virus supernatant evenly to 10 10cm dishes, mix well and place in a CO2 incubator for culture, and collect the virus after 2-3 days;

[0099] ② Collect 10 10 cm dishes of cells with CPE, transfer them into 50 ml centrifuge tubes using an electric pipette, and mark them accordingly;

[0100] ③ Centrifuge at 3500 rpm for 7 min, and collect the supernatant and cell pellet respectively;

[0101] ④ Add PEG8000 and NaCl to the viral supernatant, mix well and place upright at 4°C overnight; store the cell pellet at -80°C for later use.

[0102] (4) Treatment before purification:

[0103] ① Virus supernatant treatment:

[0104] a. The next day, centrifuge the supernatant that has been kept at 4°C overnight at 3500g, 4°C, for 30 min;

[0105] b. After centrifugation, discard the supernatant and collect the virus precipitate;

[0106] c. Resuspend the virus pellet in 2 mL of freezing buffer and collect it in a 15 mL tube for later use.

[0107] ②Cell precipitation treatment:

[0108] a. Resuspend the cell pellet with 6 mL of adenovirus freezing solution, mix well, and freeze-thaw four times;

[0109] b. After freeze-thaw, centrifuge at 3500g, 4°C for 30 min, and collect the supernatant and precipitate (cell debris);

[0110] c. Mix the supernatant with the resuspended virus pellet; resuspend the cell fragments in 2 mL of freezing buffer;

[0111] ③Ultrasonic cell disruption:

[0112] a. Ultrasonic disruption of resuspended cell fragments until the liquid is no longer viscous;

[0113] b. After the ultrasound is completed, the liquid is divided into two EP tubes and centrifuged at 12000g and 4℃ for 10min;

[0114] c. After centrifugation, mix with the virus supernatant and cell pellet treated samples.

[0115] (4) Purification and concentration:

[0116] ①Purification——Iodixanol density gradient centrifugation:

[0117] a. Prepare different concentrations of iodixanol;

[0118] b. Take a supernatant tube and slowly add different concentrations of iodixanol layer by layer using an electric pipette;

[0119] c. Add the treated virus solution to the top layer;

[0120] d. Ultracentrifugation, 48000rpm, 150min. Before centrifugation, the corresponding ultracentrifuge tube should be balanced and the error should be controlled within 0.1g.

[0121] ② Concentration:

[0122] a. After centrifugation, puncture the bottom of the supernatant tube with a needle and collect the adenovirus layer into a 15 mL tube;

[0123] b. Dilute the collected virus solution to a volume of 15 mL with freezing buffer and then filter with a 0.2 μm filter membrane;

[0124] c. Place the filtered liquid in a 15 mL ultrafiltration tube and centrifuge at 3500 g for 50 min to concentrate. Generally, the predetermined volume is reached after 2-3 centrifugations.

[0125] d. Repeatedly blow and aspirate the remaining liquid in the ultrafiltration tube into a virus storage tube and mark it with the name and date.

[0126] e. Vortex the collected viruses and centrifuge them, then aspirate 10 μL of the virus solution for titer detection.

[0127] (5) Virus titer detection:

[0128] Real-time quantitative PCR was used to determine the number of adenovirus particles in crude virus lysate and purified virus samples, and quantitative analysis was performed based on the standard curve.

[0129] 6. Adenovirus overexpression method for HUVEC:

[0130] (1) Cell preparation: Inoculate cells into the desired cell culture dish and culture overnight to ensure that the cell density is between 30% and 50% when virus infection is performed the next day.

[0131] (2) Virus infection (1 / 2 small volume infection method) and medium replacement: Before infection, take out the virus from the refrigerator and slowly thaw it on ice, remove the original cell culture medium, add 1 / 2 volume of fresh culture medium, add the appropriate volume of virus according to the MOI value found, mix gently, and infect. After 4 hours, add the volume to the complete culture volume. 10-16 hours after infection, discard the virus-containing culture medium, replace with fresh complete culture medium, and continue to culture at 37°C.

[0132] (3) Detection results: 24-48 hours after infection, the target gene was detected by qPCR, WB, immunofluorescence, immunohistochemistry and other methods.

[0133] 7. Protein extraction and WB experimental methods of HUVEC:

[0134] (1) Cell washing: Rinse the cell culture dish repeatedly with ice-cold PBS twice to ensure that there is no residual culture medium on the bottom of the dish.

[0135] (2) Cell protein extraction: Add protein lysis buffer (containing 1x PMSF, added immediately before use) to the dish, collect the cells using a cell scraper, and incubate on ice for 10-15 minutes.

[0136] (3) Removal of cell debris: Centrifuge the liquid in step 2 at 4°C, 12,000 rpm, for 10 minutes. Keep the supernatant, which is the cell protein.

[0137] (4) BCA protein quantification: Use an ELISA reader to measure the absorbance at 562 nm, create a standard curve, and calculate the sample protein concentration.

[0138] (5) Protein denaturation: Add the corresponding volume of 5×SDS-PAGE loading buffer to each protein sample (the volume ratio of 5×SDS-PAGE loading buffer to protein sample is 1:4), vortex mix, and heat and boil in a 95℃ metal water bath for 5 min to fully denature it. After cooling on ice, centrifuge and vortex before loading. The remaining sample is stored in a -80℃ refrigerator.

[0139] (6) Protein loading and polyacrylamide gel electrophoresis: After loading, start electrophoresis at a voltage of 60 V. After the protein molecular weight maker is separated, adjust the voltage to 80 V. After half an hour of electrophoresis, adjust the voltage to 90 V and continue electrophoresis. Stop electrophoresis when bromophenol blue reaches the bottom of the glass plate.

[0140] (7) Transfer: After using methanol to activate the membrane, place the black plastic plate (cathode), sponge, 3 layers of filter paper, separation gel, PVDF membrane, 3 layers of filter paper, sponge, and white plastic plate (anode) in this order. Place the plywood and ice plate in the transfer tank, cover the transfer cover, and place the entire transfer tank in crushed ice to maintain a low temperature environment. Start electrotransfer, transfer conditions: 200 mA, 120 min.

[0141] (8) Blocking and antibody incubation: After the transfer, take out the membrane and block it with 5% skim milk for 1.5-2 hours; after the blocking, shake it with 1×TBS solution on a shaker at 180rpm for 3 minutes and wash it three times; antibody incubation: incubate it on a shaker in a 4℃ refrigerator overnight. According to the specificity and titer of the antibody, the incubation time can be appropriately extended; incubate the secondary antibody: wash it with 1×TBST solution, each time for 8 minutes, wash it 3 times. After washing, incubate it with the diluted secondary antibody on a shaker at room temperature for 1 hour; wash the membrane: wash it with 1×TBST solution, each time for 8 minutes, wash it 3 times.

[0142] (9) Chemiluminescence result detection: The ultrasensitive ECL colorimetric solution A and B components were mixed in a ratio of 1:1 in the dark, and the colorimetric solution was added dropwise onto the PVDF membrane to evenly cover it, and a Western blot imaging system was used to develop the image.

[0143] 8. Experimental method of co-immunoprecipitation:

[0144] (1) Sample preparation: Wash the cells in a 10 cm culture dish three times with PBS, then add 1 mL of lysis buffer, collect the cells with a cell scraper, incubate on ice for 15 min, fully lyse the cells, and obtain the cell supernatant after centrifugation.

[0145] (2) Immunoprecipitation: Take 1 mg of protein sample and dilute it to 1 mg / mL with lysis buffer. Take 500 μL of lysis buffer and add the target antibody (1:100) and IgG of the corresponding species, respectively. Rotate at 4°C overnight.

[0146] (3) Binding to protein A / G agarose beads: Add 30 μL of washed protein A / G agarose beads to each of the above mixtures and incubate on a shaker at 4°C for 3-4 hours.

[0147] (4) Washing: After centrifugation at 2000 rpm at 4°C, discard the supernatant and retain the precipitate. Wash the precipitate with ice-cold 1xPBS and centrifuge at 2000 rpm at 4°C. Repeat 5 times, 3 minutes each time, to ensure that the beads are thoroughly washed.

[0148] (5) Add 20-40 μl of 1× SDS-PAGE electrophoresis loading buffer to each tube, mix well to resuspend the precipitate, and boil in a boiling water bath for 5 minutes. Take part or all of the sample for SDS-PAGE electrophoresis. Samples that are not used temporarily can be stored at -20°C.

[0149] In summary, the embodiments of the present invention provide the use of the mutual binding of PHACTR1 protein and HSP90 protein, and the use of inhibiting the mutual binding in preparing endothelial function protective products or in preparing products for inhibiting the occurrence and development of abdominal aortic aneurysms in animals or humans.

[0150] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of substances that inhibit endothelial cell PHACTR1 protein in the preparation of products that protect endothelial function.

2. Use of substances inhibiting PHACTR1 protein in the preparation of products for inhibiting abdominal aortic aneurysms in animals or humans.

3. The use according to claim 1 or 2, characterized in that: The substance is PHACTR1 antibody.

4. The use according to claim 3, characterized in that: The product is a medicine or a health product.

5. Use of substances that block or inhibit the binding of PHACTR1 protein to HSP90 protein in the preparation of endothelial function protective products.

6. Use of a substance that blocks or inhibits the binding of PHACTR1 protein to HSP90 protein in the preparation of a product for inhibiting abdominal aortic aneurysm in animals or humans.

7. The use according to claim 5 or 6, characterized in that: The substance is a PHACTR1 or HSP90 small molecule interfering RNA, or a polypeptide or a small molecule substance that inhibits the binding of PHACTR1 protein to HSP90 protein.

8. The use according to claim 7, characterized in that: The product described is a pharmaceutical product.

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