Application of loop CH-type finger protein 8 as biomarker in evaluation or auxiliary evaluation of vascular endothelial cell senescence

Abstract

The invention discloses an application of a loop CH type finger protein 8 as a biomarker in evaluation or auxiliary evaluation of vascular endothelial cell senescence. The expression level of MARCHF8 in vascular endothelial cells and / or on cell membranes is in negative correlation with the senescence degree of the vascular endothelial cells. And detecting the expression level of the MARCHF8 in the vascular endothelial cells and / or on cell membranes so as to carry out evaluation or auxiliary evaluation on senescence of the vascular endothelial cells. According to the application disclosed by the invention, the effect of MARCHF8 in a vascular senescence process is defined, and the MARCHF8 is used as a vascular endothelial cell surface marker for early diagnosis and monitoring of vascular senescence and targeted therapy of related diseases. Along with gradual aggravation of vascular senescence and atherosclerosis, MARCHF8 as a new biomarker and target will provide important support for early prevention, clinical diagnosis and treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of membrane-associated ring-CH finger protein 8 (MARCHF8) as a biomarker in evaluating or assisting in evaluating the senescence of vascular endothelial cells. Background Art

[0002] The vascular system is an indispensable part of the function of human organs. The aging of blood vessels constitutes the basic physiological and pathological basis for age-related changes in multiple organ systems, and is also the common etiological mechanism of various chronic diseases in the elderly population. Vascular aging is particularly associated with endothelial dysfunction, which promotes the development of cardiovascular and metabolic diseases, including atherosclerosis. Endothelial aging leads to a series of dysfunctions, such as vascular dilation, angiogenesis, and barrier dysfunction, which are considered to be the core mechanisms of vascular aging and cardiovascular and metabolic disorders. Therefore, early assessment and early diagnosis of vascular aging are important measures for evaluating and preventing vascular aging and related diseases, and have important medical value and social significance.

[0003] However, there is still a blank stage for cell surface biomarkers for vascular aging. Although some molecules that may be related to vascular aging have been found in existing studies, the clinical application value and early diagnostic role of these molecules still need to be verified. Therefore, how to discover new and reliable cell surface biomarkers related to vascular aging, especially biomarkers that can reflect the senescence of vascular endothelial cells, has become an urgent technical problem to be solved.

[0004] In the study of the senescence of vascular endothelial cells, in recent years, more and more evidence has shown that the senescence of vascular endothelial cells not only directly affects the function of blood vessels, but also promotes the occurrence and development of diseases such as atherosclerosis through multiple mechanisms. The main characteristics of vascular endothelial cell senescence include cell cycle arrest, increased inflammatory response, and decreased cell function. These senescent changes are usually accompanied by changes in the expression of biological markers, which provides potential targets for the early diagnosis and treatment of vascular aging.

[0005] Therefore, finding biomarkers that can reflect the process of vascular endothelial cell senescence has important clinical significance. Summary of the Invention

[0006] The object of the present invention is to provide the use of membrane-associated ring-CH-type finger protein 8 (MARCHF8) as a biomarker in the evaluation or auxiliary evaluation of vascular endothelial cell senescence. The present invention discovers a new cell surface biomarker, namely membrane-associated ring-CH-type finger protein 8 (MARCHF8), which is closely related to vascular endothelial cell senescence and vascular aging-related diseases (such as atherosclerosis). Research findings show that with the senescence of vascular endothelial cells, the gene and protein levels as well as the membrane protein level of MARCHF8 significantly decrease, and its expression level is closely related to the senescence of vascular endothelial cells, the reduction of vascular endothelial cell functions (reduction of cell proliferation, migration, tube formation, and nitric oxide synthesis ability), and the development of atherosclerosis. Therefore, MARCHF8 can not only be used as a biomarker for vascular endothelial cell senescence and vascular aging, but also as a biological biomarker and intervention treatment target for atherosclerosis, providing new ideas for the early diagnosis and treatment of related diseases.

[0007] Compared with the current evaluation methods for vascular aging, the MARCHF8 biomarker provided by the present invention has higher sensitivity and specificity, and can effectively reflect the early changes of vascular endothelial cell senescence. In addition, the experimental results in a mouse atherosclerosis model show that the increased expression of MARCHF8 can significantly inhibit the occurrence and development of atherosclerosis, further verifying its potential as a diagnostic and treatment target for atherosclerosis. Therefore, MARCHF8 can not only be used as a biomarker for vascular aging, but also has broad application prospects in clinical practice, including early screening, prevention, and targeted therapy.

[0008] By clarifying the role of MARCHF8 in the process of vascular aging, the present invention proposes to use it as a biomarker on the surface of vascular endothelial cells for the early diagnosis, monitoring, and targeted therapy of vascular aging. With the gradual aggravation of vascular aging and atherosclerosis, MARCHF8, as a new biomarker and target, will provide important support for early prevention, clinical diagnosis, and treatment.

[0009] In order to achieve the above object, the present application adopts the following technical solutions:

[0010] In the first aspect, the present invention provides the use of membrane-associated ring-CH-type finger protein 8 (MARCHF8) as a biomarker in the evaluation or auxiliary evaluation of vascular endothelial cell senescence.

[0011] Among them, the Gene ID of MARCHF8 is: 312656 (updated on 5-Mar-2024). Diseases related to vascular endothelial cell senescence include atherosclerosis, coronary heart disease, aortic dissection, vascular calcification, hypertension, kidney diseases, cerebrovascular diseases, etc.

[0012] In the above technical solution, the expression level of MARCHF8 in and / or on vascular endothelial cells is negatively correlated with the degree of senescence of the vascular endothelial cells. When evaluating or assisting in evaluating the degree of senescence and cell function of different vascular endothelial cells, the degree of senescence of the vascular endothelial cells to be tested with a low MARCHF8 expression level is higher than that of the vascular endothelial cells to be tested with a high MARCHF8 expression level, and the functions of the vascular endothelial cells to be tested with a low MARCHF8 expression level (cell proliferation, migration, tube formation, nitric oxide synthesis ability) are lower than those of the vascular endothelial cells to be tested with a high MARCHF8 expression level.

[0013] In the above technical solution, the expression level of MARCHF8 in and / or on vascular endothelial cells is detected to evaluate or assist in evaluating the senescence of vascular endothelial cells.

[0014] In a second aspect, the present invention provides the use of MARCHF8 in the preparation of a product for evaluating or assisting in evaluating the senescence or function of vascular endothelial cells, or the use of a substance that specifically binds to MARCHF8 or its gene in the preparation of a product for evaluating or assisting in evaluating the senescence or function of vascular endothelial cells.

[0015] The product for evaluating or assisting in evaluating the senescence or function of vascular endothelial cells (cell proliferation, migration, tube formation, nitric oxide synthesis ability) may contain a substance that specifically binds to MARCHF8 or its gene. The substance that specifically binds to MARCHF8 may be a monoclonal antibody or a polyclonal antibody against MARCHF8, and the substance that specifically binds to the MARCHF8 gene may be a specific primer pair for PCR amplification of the MARCHF8 gene.

[0016] In a third aspect, the present invention provides the use of MARCHF8 in the preparation of a product for regulating the senescence or function of vascular endothelial cells, or the use of a substance that specifically binds to MARCHF8 or its gene in the preparation of a product for regulating the senescence or function of vascular endothelial cells.

[0017] The product for regulating the senescence or function of vascular endothelial cells (cell proliferation, migration, tube formation, nitric oxide synthesis ability) may contain a substance that specifically binds to MARCHF8 or its gene. The substance that specifically binds to MARCHF8 may be a monoclonal antibody or a polyclonal antibody against MARCHF8, and the substance that specifically binds to the MARCHF8 gene may be a specific primer pair for PCR amplification of the MARCHF8 gene.

[0018] Products that regulate the senescence or function of vascular endothelial cells (cell proliferation, migration, tube formation, nitric oxide synthesis ability) also include substances that inhibit the expression of MARCHF8, such as small interfering RNAs that interfere with the expression of MARCHF8. Specifically, the target sequence of the small interfering RNA that interferes with the expression of MARCHF8 is st-h-MARCHF8 (i.e., CCTTGTATGTGCTCATTGA); or substances that promote the expression of MARCHF8, such as exogenous MARCHF8 and its analogs, and / or pharmaceutical ingredients that enhance the stability of MARCHF8 protein, and / or nucleic acid molecules that can express MARCHF8 and their vectors.

[0019] In a fourth aspect, the present invention provides the use of a probe capable of detecting the expression level of MARCHF8 in vascular endothelial cells and / or on the cell membrane in the preparation of a kit for evaluating or assisting in the evaluation of vascular endothelial cell senescence.

[0020] In a fifth aspect, the present invention provides the use of a kit in the evaluation or assistance in the evaluation of vascular endothelial cell senescence. The kit includes a probe and a hybridization solution, and the probe is capable of detecting the expression level of MARCHF8 in vascular endothelial cells and / or on the cell membrane.

[0021] In a sixth aspect, the present invention provides the use of an active ingredient in the preparation of a drug for delaying vascular aging and / or preventing and treating diseases related to vascular aging. The active ingredient includes a reagent that can increase the expression level of MARCHF8.

[0022] In the above technical solutions, the reagent includes exogenous MARCHF8 or its analogs; and / or the reagent includes a pharmaceutical ingredient that enhances the stability of MARCHF8; and / or the reagent includes a nucleic acid molecule that can express MARCHF8 and its vector.

[0023] In a seventh aspect, the present invention provides a drug for delaying vascular aging and / or preventing and treating diseases related to vascular aging. The drug includes a reagent that can increase the expression level of MARCHF8, and a pharmaceutically acceptable carrier.

[0024] The present invention has studied the role of MARCHF8 in vascular endothelial senescence and atherosclerosis through means such as cell experiments and animal models, and has revealed its application as a potential biomarker and target.

[0025] Study on the Role of MARCHF8 in the Process of Vascular Endothelial Cell Senescence: By studying the expression and role of MARCHF8 in vascular endothelial cells, it was found that MARCHF8 plays a key role in the process of endothelial cell senescence. Quantitative real-time PCR (qPCR) and Western blot (WB) assays were performed on young and senescent endothelial cells using PCR primers and specific antibodies of MARCHF8. It was observed that the expression levels of MARCHF8 gene and protein were significantly decreased in senescent vascular endothelial cells compared with young endothelial cells.

[0026] Discovery of MARCHF8 in Atherosclerosis, a Vascular Aging-Related Disease: In this invention, a mouse model of atherosclerosis induced by a high-fat diet was established to study the role of MARCHF8 in atherosclerosis. An overexpression experiment of MARCHF8 was conducted on the mouse atherosclerosis model by injecting adeno-associated virus (AAV) via the tail vein. The WB results showed that the expression level of MARCHF8 in the overexpression group was significantly increased, and both immunofluorescence and WB results supported this conclusion. Meanwhile, Oil Red O staining of arteries showed that the atherosclerotic plaque area in mice overexpressing MARCHF8 was reduced and lipid deposition was inhibited.

[0027] Role of MARCHF8 in the Mechanisms of Vascular Aging and Atherosclerosis

[0028] Cell Model: Vascular endothelial cells (HUVEC) were infected with lentiviruses packaged with empty plasmid (Ctrl) and lentiviruses containing MARCHF8 overexpression plasmid (M8-OE). Control Ctrl cell lines and MARCHF8-OE cell lines were obtained after puromycin screening. At the same time, cell lines with knockdown of MARCHF8 (si-MARCHF8) and its siRNA control group (si-NC) were constructed using siRNA technology. The effects of MARCHF8 on the growth and proliferation of endothelial cells were detected by cell proliferation assay (Edu), cell migration assay (scratch wound healing assay), and functional detection of vascular endothelial cells (detection of nitric oxide (NO) secretion). The results showed that knockdown of MARCHF8 inhibited the growth and proliferation of young vascular endothelial cells, damaged the function of vascular endothelial cells, and accelerated the aging process; overexpression of MARCHF8 promoted the growth and proliferation of endothelial cells, restored the function of senescent endothelial cells, and delayed the aging process.

[0029] Mouse model: The empty plasmid (CD-CV) and adeno-associated virus containing the MARCHF8 overexpression plasmid (HFD-M8) were injected into the atherosclerotic mouse model via the tail vein, and the development of arteriosclerosis was recorded at regular intervals. Four weeks after implantation, ultrasonic vascular imaging technology was used to evaluate vascular stiffness and elasticity, and oil red staining was used to evaluate lipid deposition in the vascular wall. It was found that the mice in the HFD-M8 group had significant improvements in vascular stiffness and elasticity and lipid deposition compared with the HFD-CV group, suggesting that the increase in MARCHF8 expression could delay vascular aging and the occurrence of atherosclerosis.

[0030] The experimental results of the present invention show that:

[0031] MARCHF8 plays a key role in the process of vascular endothelial cell aging and regulates aging-related proteins;

[0032] Overexpression of MARCHF8 can significantly reduce the area of atherosclerotic plaques in mice and inhibit lipid deposition;

[0033] Overexpression of MARCHF8 can improve the stiffness and elasticity of mouse blood vessels and delay the occurrence of atherosclerosis;

[0034] MARCHF8 is involved in the occurrence and development of vascular aging and atherosclerosis by regulating the growth, proliferation and function of vascular endothelial cells.

[0035] The MARCHF8 protein of the present invention can be used as an early diagnostic marker for vascular aging and atherosclerosis, and can be used to prepare new vascular protection drugs targeting MARCHF8, especially for delaying the process of vascular aging and improving atherosclerotic symptoms.

[0036] The beneficial effects of the present invention are as follows: The biomarker MARCHF8 of the present invention plays an important role in the screening and identification of vascular endothelial cells, can evaluate vascular aging and vascular aging-related diseases (including atherosclerosis), can also detect human vascular function and evaluate the degree of human vascular aging, etc., providing important molecular indicators for the research and intervention of vascular aging-related diseases, especially in the evaluation of human cardiovascular function and disease diagnosis, and has important clinical application value. Description of the Drawings

[0037] Figure 1 : The level of MARCHF8 mRNA is down-regulated during the process of vascular endothelial cell aging, accompanied by an increase in the aging index P21mRNA.

[0038] Figure 2 : The expression of MARCHF8 protein levels in cells and on the cell membrane is down-regulated with the aging of vascular endothelial cells.

[0039] Figure 3 : Knockdown of MARCHF8 in vascular endothelial cells promotes cell senescence.

[0040] Figure 4 : Knockdown of MARCHF8 in vascular endothelial cells results in a decrease in MARCHF8 mRNA levels, accompanied by an increase in the mRNA levels of cell inflammation indicators including IL6 and IL8.

[0041] Figure 5 : Knockdown of MARCHF8 in vascular endothelial cells results in a decrease in MARCHF8 protein levels, accompanied by an increase in the senescence indicator P21 protein.

[0042] Figure 6 : Overexpression of MARCHF8 in vascular endothelial cells inhibits cell senescence.

[0043] Figure 7 : Overexpression of MARCHF8 in vascular endothelial cells leads to an increase in MARCHF8 mRNA levels, accompanied by a decrease in the mRNA levels of cell inflammation indicators including IL6 and IL8.

[0044] Figure 8 : Overexpression of MARCHF8 in vascular endothelial cells leads to an increase in MARCHF8 protein levels, accompanied by a decrease in the senescence indicator P21 protein.

[0045] Figure 9 : MARCHF8 can improve the proliferation, migration, tube formation and NO synthesis functions of endothelial cells. Young HUVECs were transfected with siRNA targeting human MARCHF8 (siM8) and its negative control siRNA (siNC), and senescent HUVECs were infected with a control lentiviral vector (Ctrl) or a lentivirus overexpressing MARCHF8 (M8-OE). (A) Cell proliferation was detected by Edu binding assay. (B) Cell migration was detected by wound healing assay. Pictures were taken at 0, 16 and 24 hours. Scale bar, 50 μm. (C) Cell tube formation was observed in 96-well plates coated with Matrigel medium. Representative images of network morphological changes are shown. The total length and number of branches of cell tubes were quantified using Image J. (D) Representative images and histograms of nitric oxide (NO) production in HUVEC cells. Scale bar: 100 μm (si) / 500 μm (OE). Quantitative data are expressed as the mean ± SEM of three independent experiments (n = 3). * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001.

[0046] Figure 10 : ApoE - / -Overexpression of MARCHF8 in mice led to a significant increase in the level of MARCHF8 protein in the aorta of mice in the HFD-M8 group. ApoE - / - The mice were randomly divided into three groups (10 mice in each group): 1) Ctrl: control diet (CD) + AAV empty control vector (Control, Ctrl) group; 2) HFD-Ctrl: high-fat diet (HFD) + Ctrl group; 3) HFD-M8: HFD + AAV MARCHF8 overexpression (M8) group. WB analysis of the level of MARCHF8 protein in the aortas of the three groups, with GAPDH as the internal reference control (n = 6).

[0047] Figure 11 : ApoE - / - Overexpression of MARCHF8 in mice could significantly delay atherosclerosis. Aortic Oil Red O analysis was used to evaluate Figure 10 the degree of atherosclerosis in the three groups of mice after 12 weeks of feeding (n = 10). (A) The whole aorta of the mice was used to analyze the lipid deposition of atherosclerosis in the mice. There were three representative aortas for each group of mice. Scale bar, 20 μm. Quantitative analysis of the relative area of atherosclerotic lesions, expressed as a percentage of the total aortic area) (n = 3). (B) Representative examples and quantitative comparison of atherosclerotic lesions stained with Oil Red O at the aortic root (n = 3). Scale bar, 50 μm.

[0048] Figure 12 : ApoE - / - Overexpression of MARCHF8 in mice could significantly reduce the plasma IL6 level in mice caused by HFD and reduce the systemic inflammation level in mice.

[0049] Figure 13 : ApoE - / - Overexpression of MARCHF8 in mice could reduce the pulse wave velocity (PWV) in the atherosclerotic mouse model caused by HFD and reduce the vascular stiffness in mice. Detailed implementation manners

[0050] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The present invention can be implemented in many different forms and should not be construed as limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the present invention to those skilled in the art. The present invention will be defined only by the claims. In the present invention, the abbreviations used are shown in Table 1 below.

[0051] Table 1 Abbreviation table

[0052]

[0053] The materials and equipment used in the experiments of the present invention are shown in Table 2 below.

[0054] Table 2 Main Reagents

[0055]

[0056]

[0057] Cell culture: The use of human umbilical cords was approved by the Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. As previously described, HUVECs were extracted from three different human umbilical cords and cultured in EGM2 medium (Lonza) and EGM-2 supplement kit at 37 °C and 5% CO2. HUVECs were passaged every 2 or 3 days with 0.05% trypsin-EDTA (Gibco, USA), and the population doubling level (PDL) of the cells was measured. Replicative senescence of HUVECs was induced continuously by young cells until their proliferation almost stopped. Young cells (PDL5-13) and senescent cells (PDL25-33) were used in the experiments. This study was approved by the Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, and written informed consent was obtained from the donor's father (protocol number: TJ-IRB20230711).

[0058] Experimental animals: Animal experiments were conducted in accordance with the regulations of the Institutional Animal Use and Care Committee of Huazhong University of Science and Technology (HUST) (IACUC no. 3141). Five-week-old male ApoE - / - mice were obtained from Beijing HFK Bioscience Co., Ltd. MARCHF8 was overexpressed by bGlobin-MCS-AAV-EGFP-3-FLAG-WPRE-hGHpolyA carrying the CMV promoter, purchased from GeneChem Co., Ltd. (Shanghai, China), and the specific sequence is shown in Table 2-2. A blank vector (bGlobin-MCS-EGFP-3FLAG-WPRE-hGH polyA) containing the CMV promoter and enhanced green fluorescent protein was used as the control vector. The mice were randomly divided into 3 groups (10 mice in each group): Group 1, control group (CD) fed with + AAV empty control vector group (CV); Group 2, high-fat diet (HFD) fed with + CV; Group 3, HFD + AAV MARCHF8 overexpression group (M8). Two weeks before HFD (Cat.#XT108C, Xitong, Jiangsu, China), ApoE - / - mice were intravenously injected with overexpressed AAV MARCHF8 or AAV vector, and then subjected to a 12-week high-fat diet (HFD). After 12 weeks of HFD treatment, the three groups of mice were fasted overnight and euthanized, and then blood and tissues were collected. The remaining tissues were collected and frozen for future use.

[0059] Example 1: The expression level of MARCHF8 is down-regulated with the senescence of endothelial cells

[0060] Detection of intracellular mRNA levels of MARCHF8 and p21: The total RNA of young endothelial cells and senescent endothelial cells was extracted using an RNA purification kit (Guangzhou Meiji Biotechnology Co., Ltd.), and reverse transcription was performed using a ReverTra RT kit (Takara, Japan). The mRNA levels of genes were determined using Green Realtime PCR Master Mix (TOYOBO, Japan) and primers on an ABI Step One Plus (Applied Biosystems, USA). GAPDH and β-TUBULIN were used as endogenous normalization controls. The 2 -ΔΔCt -method was used to calculate the change in relative mRNA levels. Each detection was performed at least three independent times. The primer sequences are shown in Table 3. The quantitative results of the intracellular mRNA levels of MARCHF8 and p21 are as Figure 1 shown, and the results show that compared with young endothelial cells, the mRNA expression level of MARCHF8 in senescent endothelial cells is significantly decreased, accompanied by a significant increase in the p21 mRNA expression level.

[0061] Detection of intracellular MARCHF8 protein level: The experiment was performed using the standard Western blot (WB) method. The cells were lysed with RIPA buffer (China BoaoTe Co., Ltd.), and a protease inhibitor mixture (China Doctor De Co., Ltd.) was added to the buffer. The protein lysates were separated by polyacrylamide-sodium dodecyl sulfate gel electrophoresis and then transferred to a polyvinylidene difluoride membrane (Millipore, USA). After electrophoresis, the membrane was blocked with 5% skim milk and then incubated with the primary antibody. The membrane was then washed and incubated with the corresponding secondary antibody, and finally developed using a chemiluminescence kit (China Beyotime Co., Ltd.). Membrane proteins and cytoplasmic proteins were separated using a membrane extraction kit (China Kiggen Biotechnology Co., Ltd.). The antibodies used in this study are listed in the main reagent table. The WB results and quantitative results of the intracellular MARCHF8 protein level are as Figure 2 shown, and the results show that compared with young endothelial cells, the total cellular protein level and membrane protein level of MARCHF8 in senescent endothelial cells are significantly decreased.

[0062] Example 2: MARCHF8 knockdown experiment, MARCHF8 overexpression experiment

[0063] MARCHF8 gene interference: Small interfering RNAs (siRNAs) targeting MARCHF8, ADAM10 and their negative control (NC) were designed and synthesized (RiboBio, China). HUVECs were seeded in complete medium and allowed to grow to 70 - 80% confluence, and then transfected with siRNAs premixed with Lipofectamine TM 3000 (Thermo Fisher Scientific, USA) at a transfection concentration of 50 nM. After 6 hours, the medium containing siRNAs and Lipofectamine TM 3000 was replaced with complete medium.

[0064] Table 3 MARCHF8 target information

[0065] ID Target sequence information st-h-MARCHF8 CCTTGTATGTGCTCATTGA(SEQ ID NO.1)

[0066] st-h-MARCHF8 is hereinafter named si-MARCHF8

[0067] Interference gene infection of HUVEC cells and verification

[0068] Overexpression of MARCHF8 virus packaging:

[0069] ① Resuscitate 293T cells for lentivirus packaging and culture them in DMEM complete medium. It is required to use cells with an earlier passage number.

[0070] ② Day1: One day before transfection, digest 293T cells with trypsin for 1 min, count them and seed them in a 10 cm dish, trying to ensure that there are 6.5×10 6 cells in each dish, and the medium volume is 8.5 mL for transfection.

[0071] ③ Day2: Take a 2 mL EP tube, add 1.5 mL OPTI-MEM, then add 7.5 μg of the lentivirus packaging helper plasmid psPAX2, 2.5 μg of pMD2.G, and 10 μg of the target plasmid, mix well, and then add 30 μL of HG-Trans293 TM transfection reagent, mix well, let it stand at room temperature for 30 min, and then evenly add it to the 293T cells seeded the previous day, taking care not to blow up the cells. Replace with DMEM complete medium 8 - 12 h later.

[0072] ④ Day4: 48 h after transfection, collect the supernatant into a 50 mL centrifuge tube, temporarily store it at 4 °C, and replenish 10 mL of DMEM complete medium. Take a photo with a fluorescence microscope to observe the transfection efficiency, and then incubate it at 37 °C and 5% CO2.

[0073] ⑤Day 5: Collect the supernatant at 72 h, combine it with the supernatant at 48 h, centrifuge at 1000 g / min for 10 min, aliquot according to the specific usage amount, and store at -80 °C.

[0074] Virus infection of HUVEC cells:

[0075] Two target plasmids were synthesized by GeneChem, among which VC was an empty vector and M8-OE was a plasmid overexpressing MARCHF8. Both vectors were packaged into lentiviruses.

[0076] Table 4 Information of LV-MARCHF8 target

[0077]

[0078] Seed HUVEC cells in a six-well plate one day in advance, with 2×10 6 cells per well, and a total of 9 wells were seeded. Infection: For the two viruses VC and M8-OE, add 500 μL and 1000 μL of lentivirus respectively, and supplement the remaining volume to 2 mL with McCoy's 5A complete medium. Add 2 μL of the co-infection reagent polybrene to each well, mix well, and culture in an incubator. After 8 h, replace the medium with McCoy's 5A complete medium to infect HUVEC cells. Observe the cell morphology and infection efficiency under a fluorescence microscope at 24 h, 48 h, 72 h, and 96 h.

[0079] For the verification of the RNA levels of MARCHF8 knockdown and MARCHF8 overexpression, the steps of cell RNA extraction and PCR are the same as above.

[0080] Verification of the protein levels of MARCHF8 knockdown and MARCHF8 overexpression:

[0081] Collect cells 96 h after lentivirus infection (after trypsin digestion, neutralize with McCoy's 5A complete medium, centrifuge at 300 g / min to remove the medium, wash with PBS, centrifuge, and try to remove PBS as much as possible), lyse with 2D-lysis buffer, lyse the cells on ice for half an hour, shake once every 5 min, then centrifuge at 4 °C and 12000 rpm for 10 min, take the supernatant, and the quantification steps are the same as above. Load 15 μg of protein for immunoblotting, and the WB experimental steps are the same as above.

[0082] Example 3 MARCHF8 affects the expression of cell senescence and cell inflammation indicators

[0083] Cell phenotypic identification of cellular senescence: The SA-β-gal activity was measured using a senescence β-galactosidase staining kit (Beyotime, China). First, the 1x fixation buffer was applied to the cells at room temperature for 6 - 7 minutes, and then the cells were stained overnight with the staining mixture under 3℃ without carbon dioxide. Subsequently, the blue-stained cells and the total number of cells were observed under an optical microscope and counted using ImageJ software (National Institutes of Health, USA). Compared with the control group, the proportion of blue-stained cells in the MARCHF8 knockdown group increased significantly as Figure 3 shown, while compared with the control group, the proportion of blue-stained cells in the MARCHF8 overexpression group decreased significantly as Figure 6 shown; the results indicated that MARCHF8 could delay cellular senescence.

[0084] mRNA and WB detection of cellular senescence and cellular inflammation indicators. The cellular inflammation indicators used in this invention were interleukin 6 (IL6) and interleukin 8 (IL8) respectively, and the senescence indicator was p21. The primer information is shown in Table 5, and the antibody information is listed in the main reagent table; the cell RNA extraction and PCR steps and the cell protein extraction and WB procedures were the same as above. Compared with the control group, the mRNA levels of IL6 and IL8 in the MARCHF8 knockdown group increased significantly, as Figure 4 shown; the protein level of p21 increased significantly, as Figure 5 shown; while compared with the control group, the mRNA levels of IL6 and IL8 in the MARCHF8 overexpression group decreased significantly, as Figure 7 shown, and the protein level of p21 decreased significantly, as Figure 8 shown; the results indicated that MARCHF8 could reduce cellular inflammation and delay cellular senescence at the RNA and protein levels.

[0085] Table 5 PCR primer sequence table

[0086]

[0087]

[0088] Example 4 Effects of MARCHF8 on cell functions

[0089] (1) Cell proliferation function - Edu proliferation assay

[0090] 1) Prepare the constructed HUVECs with knocked-out MRACHF8 (si-M8) and its control group HUVECs (si-NC), and HUVECs with overexpressed MRACHF8 (MARCHF8-OE) and its control group HUVECs (VC) respectively according to the above steps. After digestion, count and prepare them into 5×10 4Cell suspension at [X] / mL was evenly added to a 96-well plate, 100 μL per well, so that the number of cells was maintained at 5×10 3 / well. After complete adhesion, the culture medium was discarded and the wells were washed with PBS, and then 500 μL of fresh culture medium was added back to the wells. The Edu working solution was taken and diluted to 20 μM / L with cell culture medium. 500 μL of the diluted Edu working solution was added to each well of the plate, so that the final concentration of the Edu working solution in the plate became 10 μM / L. After addition, the plate was placed back in the incubator and cultured for 24 h.

[0091] 2) Cell fixation and permeabilization: Discard the culture medium and wash with PBS. Fix with 4% paraformaldehyde for 15 min at room temperature, then discard the paraformaldehyde. Wash with PBS containing 3% BSA, and then add 0.5% Triton X-100 dissolved in PBS to the wells for permeabilization treatment, and let it stand at room temperature for 20 min.

[0092] 3) Edu detection: In this experiment, a 100 μL iClick reaction solution system was used, and the components of the system are shown in Table 6 below.

[0093] Table 6 Components of the system

[0094] Added component Volume iClick EdU reaction buffer 86μL CuSO4 4μL Andy Fluor 555azide 0.3μL 1x iclickEdU buffer additive 10μL Total volume 100μL

[0095] Discard the cell permeabilization solution and wash again with PBS containing 3% BSA. After washing, add the prepared iClick reaction solution to each well of the plate. Note that it should be prepared and used immediately, and let it stand at room temperature for 30 min, paying attention to avoiding light. Discard the reaction solution and wash with PBS containing 3% BSA.

[0096] 4) Imaging: Add 200 μL of DAPI anti-quenching agent to the wells, and then observe under a microscope.

[0097] 5) Repeat the above experiment 3 times.

[0098] The results of Edu fluorescence detection and quantitative results are as Figure 9 shown in A. The results show that compared with the control group, the fluorescence of cells in the MARCHF8 knockdown group was significantly reduced, while compared with the control group, the fluorescence of cells in the MARCHF8 overexpression group was significantly increased; the results indicate that MARCHF8 can promote the proliferation function of endothelial cells.

[0099] (2) Cell migration function - Cell scratch assay

[0100] 1) The migration ability of HUVEC was evaluated using a wound healing assay. Cells were cultured in a 6-well plate until confluent, and a straight line was scratched on the cell monolayer. Photos were taken at 0 h, 16 h, and 24 h after scratching, respectively.

[0101] 2) Repeat the above experiment three times.

[0102] The results detected by the cell scratch assay and the quantitative results are as Figure 9 shown in Figure B. The results show that compared with the control group, the scratch repair rate of the MARCHF8 knockdown group was significantly reduced; while compared with the control group, the scratch repair rate of the MARCHF8 overexpression group was significantly increased; the results indicate that MARCHF8 can promote the migratory function of endothelial cells.

[0103] (3) Cell tube formation function - Tube formation assay

[0104] 1) The tube formation ability of HUVECs was evaluated using the Matrigel tube formation assay. HUVECs were seeded onto 96-well plates pre-coated with growth factor-reduced Matrigel (Corning, USA) at a density of 20,000 cells / well, and then cultured for 6 hours at 37°C and 5% CO2. The tube formation process was photographed using an inverted optical microscope and quantified using the Angiogenesis Analyzer plugin of ImageJ.

[0105] 2) Repeat the above experiment three times.

[0106] The results detected by the tube formation assay and the quantitative results are as Figure 9 shown in Figure C. The results show that compared with the control group, both the tube formation ability and tube length of the MARCHF8 knockdown group were significantly reduced; while compared with the control group, both the tube formation ability and tube length of the MARCHF8 overexpression group were significantly increased; the results indicate that MARCHF8 can promote the tube formation function of endothelial cells.

[0107] (4) Cell NO synthesis function - NO synthesis assay

[0108] 1) The production of NO in HUVEC cells was evaluated using the specific NO probe DAF-FM DA (Beyotime, China). After removing the medium, cells were stained with 2.5 μmol / L DAF-FM DA in Hank's solution (pH 7.4) for 20 minutes at 37°C. After washing three times with Hank's solution to completely remove the probe, cells were observed and photographed using a laser confocal microscope (Nikon C2+, Tokyo, Japan).

[0109] 2) Repeat the above experiment three times.

[0110] The results detected by the NO synthesis assay and the quantitative results are as Figure 9 shown in Figure D. The results show that compared with the control group, the NO synthesis function of the MARCHF8 knockdown group was significantly reduced; while compared with the control group, the NO synthesis function of the MARCHF8 overexpression group was significantly increased; the results indicate that MARCHF8 can promote the NO synthesis of endothelial cells.

[0111] Example 5: MARCHF8 can delay atherosclerosis

[0112] Establish a mouse model of atherosclerosis: A mouse model of atherosclerosis was established with a 12-week high-fat diet (HFD), and lipid deposition in the aorta was detected by Oil Red O staining to determine the successful establishment of the model; after the mice were euthanized, they were perfused with ice-cold PBS for 5 minutes, and then perfused with 4% buffered paraformaldehyde containing 2 mM EDTA and 2% glucose.

[0113] (1) Obtaining differential proteins of MARCHF8 and validation experiments in the animal atherosclerosis model

[0114] Protein immunoblotting (WB) experiments verified the upregulation of MARCHF8 expression in the mouse atherosclerosis model overexpressing MARCHF8: Western blot (WB) experiments were performed on the aortic tissue of mice in 1) CD-CV: control diet (CD) + AAV empty control vector (CV) group; 2) HFD-CV: high-fat diet (HFD) + CV group; 3) HFD-M8: HFD + AAV MARCHF8 overexpression (M8) group, with 6 samples in each group (n = 6). WB analysis of the MARCHF8 protein level in the aortas of the three groups, with GAPDH as the internal reference control, and Western blot (WB) was performed using the standard WB method. Cells were lysed using RIPA buffer (Boster, China) supplemented with protease inhibitors. Protein lysates were separated by electrophoresis on a polyacrylamide-sodium dodecyl sulfate (SDS-PAGE) gel and then transferred to a PVDF membrane (Millipore, USA). After electrophoresis, the cell membrane was blocked with 5% skim milk and incubated with the primary antibody. Then the cell membrane was washed and incubated with the appropriate secondary antibody, and then visualized using a chemiluminescence kit (Beyotime, China). MARCHF8 was upregulated in the aortic tissue of mice in the HFD-M8 group, as Figure 10 shown.

[0115] (2) MARCHF8 can reduce lipid deposition in the mouse atherosclerosis model. The entire aorta was carefully dissected, removing connective tissue and fat, and longitudinally sectioned on a paraffin-coated petri dish. The aorta was incubated with Oil Red O in propylene glycol at room temperature for 2 hours. After staining, the aorta was decolorized 3 times with 60% ethanol. Then the aorta soaked in PBS was photographed against a black background, and the Image J was used to analyze the images to evaluate the percentage of the Oil Red O staining area. As Figure 11 shown, compared with the control group, the Oil Red staining area of the aorta in the MARCHF8 overexpression group was significantly reduced, indicating that MARCHF8 can reduce lipid deposition in the mouse atherosclerosis model.

[0116] (3) MARCHF8 can reduce systemic inflammatory response. The index used to reflect systemic inflammatory response in this invention is the IL6 level in plasma. BD TM Mouse IL6 detection kit for flow cytometry bead array (CBA) (#558301, BD Biosciences, USA) was used to measure the IL6 level in mouse plasma. Data was acquired and analyzed on a FACS Canto II flow cytometer (BD Biosciences, USA) using BD FACSDiva software. As Figure 12 shown, compared with the control group, the plasma IL6 level in the MARCHF8 overexpression group was significantly reduced, indicating that MARCHF8 can reduce the systemic inflammatory response in the mouse atherosclerosis model.

[0117] (4) MARCHF8 can reduce the PWV in the mouse atherosclerosis model. Regarding the measurement of the pulse wave velocity (PWV) in mice, the mice were placed in a closed gas anesthesia chamber and inhaled 3% isoflurane for about 15 s. After detecting the anesthesia level by pinching the toes, the mice were placed on a sensing plate and fixed, and the isoflurane concentration was adjusted to the maintenance concentration, 1.0% - 1.5%. After hair removal, ultrasonic coupling agent was applied. First, observe the electrocardiogram of the mouse through the sensor. After the electrocardiogram was stable, the ultrasonic sensor probe was slowly slid upward along the sternum of the mouse until the waveform of the thoracic aorta was found and photographed, and the position was recorded at the same time. Then the probe was slowly slid upward along the umbilicus until the waveform of the abdominal aorta was found and photographed to record the position. Measure the distance between the thoracic aorta and the abdominal aorta waveforms, and analyze the conduction time based on the waveforms. The pulse wave velocity was calculated as the separation distance (millimeters, mm) between the two measurement points divided by the pulse transmission time (milliseconds, ms). As Figure 13 shown, compared with the control group, the plasma PWV in the MARCHF8 overexpression group was significantly reduced, indicating that MARCHF8 can reduce the PWV in the mouse atherosclerosis model.

[0118] In the examples of this invention, the obtained data were all analyzed using SPSS 22.0 statistical software. The experiments were repeated 3 times, and P < 0.05 was considered to have statistical significance.

[0119] Obviously, the above examples are only for clear illustration and not limitations on the implementation methods. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. And the obvious changes or modifications derived therefrom are still within the protection scope of this invention.

Claims

1. Use of ring CH-type finger protein 8 (MARCHF8) as a biomarker in evaluating or assisting in the evaluation of vascular endothelial cell senescence.

2. The application according to claim 1, characterized in that: The expression level of MARCHF8 in and / or on the cell membrane of vascular endothelial cells is negatively correlated with the degree of senescence of the vascular endothelial cells.

3. The application according to claim 1, characterized in that: Detect the expression level of MARCHF8 in and / or on the cell membrane of vascular endothelial cells to evaluate or assist in the evaluation of vascular endothelial cell senescence.

4. Use of MARCHF8 in the preparation of a product for evaluating or assisting in the evaluation of vascular endothelial cell senescence or function, or use of a substance that specifically binds to MARCHF8 or its gene in the preparation of a product for evaluating or assisting in the evaluation of vascular endothelial cell senescence or function.

5. Use of MARCHF8 in the preparation of a product for regulating vascular endothelial cell senescence or function, or use of a substance that specifically binds to MARCHF8 or its gene in the preparation of a product for regulating vascular endothelial cell senescence or function.

6. Use of a probe capable of detecting the expression level of MARCHF8 in and / or on the cell membrane of vascular endothelial cells in the preparation of a kit for evaluating or assisting in the evaluation of vascular endothelial cell senescence.

7. Use of a kit in evaluating or assisting in the evaluation of vascular endothelial cell senescence, characterized in that: The kit includes a probe and a hybridization solution, and the probe is capable of detecting the expression level of MARCHF8 in and / or on the cell membrane of vascular endothelial cells.

8. Use of an active ingredient in the preparation of a drug for delaying vascular aging and / or preventing and treating diseases related to vascular aging, characterized in that: The active ingredient includes a reagent that can increase the expression level of MARCHF8.

9. The application according to claim 8, wherein: The reagent includes exogenous MARCHF8 or its analog; and / or the reagent includes a pharmaceutical ingredient that enhances the stability of MARCHF8; and / or the reagent includes a nucleic acid molecule capable of expressing MARCHF8 and its vector.

10. A drug for delaying vascular aging and / or preventing and treating vascular aging-related diseases, characterized in that: The drug includes a reagent that can increase the expression level of MARCHF8, and a pharmaceutically acceptable carrier.

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