Application of CTHRC1 protein as aortic aneurysm and / or aortic dissection marker

Abstract

The invention discloses application of a CTHRC1 protein as an aortic aneurysm and / or aortic dissection marker, belongs to the technical field of gene diagnosis and treatment of molecular biology, and aims to solve the problems that aortic aneurysm and / or aortic dissection related surgical treatment has a high death risk, and the long-term curative effect and prognosis of a patient are poor. According to the present invention, the recombinant human CTHRC1 protein is adopted as the immunogen so as to obtain the mouse anti-CTHRC1 monoclonal antibody specifically bound to the human CTHRC1 antigen; the monoclonal antibody can neutralize and effectively block the specific biological function activity of a CTHRC1 immunogen through high binding affinity, can be applied to a new treatment target of aortic aneurysm and / or aortic dissection diseases, reduces the death rate of the diseases, and improves the prognosis of patients.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene diagnosis and treatment of molecular biology, and in particular relates to the application of CTHRC1 protein as a marker of aortic aneurysm and / or aortic dissection. Background Art

[0002] Aortic aneurysm and / or aortic dissection is one of the cardiovascular diseases that seriously endangers the life and health of patients. It has an acute onset and is a serious condition. It can cause aortic rupture and death in a short period of time. Since surgical treatment related to aortic disease has a high risk of death, the long-term efficacy and prognosis of patients are poor. Although some drugs now have a therapeutic effect on aortic dissection, such as antibiotics such as doxycycline and roxithromycin, as well as statins, beta-blockers, angiotensin-converting enzyme inhibitors, etc., these drugs have not been widely used due to their large side effects or insignificant clinical effects.

[0003] Aortic aneurysm is the result of pathological dilatation of the aorta, which can exceed the normal aortic vessel diameter by about 50%. Aortic dissection is caused by a tear in the aortic intima. Driven by arterial pressure, blood enters the middle layer through the tear in the intima, forming a dissection hematoma and extending along the aortic wall to form a true and false cavity. Aortic dissection without intervention is a fatal emergency, with a mortality rate of about 40% at the first visit, and this rate increases at a rate of 1% per hour, and can even be as high as 90% annual mortality. Based on population epidemiological research data, the incidence of aortic dissection is about 3-5 / 100,000 cases per year, and it is increasing year by year. In addition, there is a lack of effective and safe drug treatment methods for the treatment of aortic aneurysm and / or aortic dissection in clinical practice, and the main means are concentrated in surgical treatment and endovascular interventional repair. It is worth noting that due to the high risk of death associated with surgical treatment of aortic aneurysm and / or aortic dissection, the long-term efficacy and prognosis of patients are poor.

[0004] Recent studies have found that adventitial fibroblasts of the aortic wall can be activated into myofibroblasts under pathological conditions such as mechanical stress or injury. At the same time, adventitial fibroblasts can participate in pathological remodeling through autocrine pathways or complex paracrine interactions with vascular smooth muscle cells, providing a theoretical basis for new diagnostic and therapeutic strategies targeting fibroblast populations for aortic aneurysms and / or aortic dissections.

[0005] Collagen Triple HelixRepeat Containing 1 (CTHRC1) is a highly expressed gene that was first screened out by Pyagay et al. from differential gene expression analysis of rat arterial injury models. Subsequently, researchers such as Tang also detected the expression of CTHRC1 in 19 solid tumors and found that the expression of CTHRC1 was increased in 16 of them. Currently, most studies focus on the role of CTHRC1 in the occurrence and development of various tumors, but there are few reports on the study of CTHRC1 in aortic aneurysms and / or aortic dissections, and its biological function and specific mechanism of action for the vascular adventitial fibroblast population are still unclear. Summary of the invention

[0006] In response to the above problems, the present application provides an application of CTHRC1 protein as a marker for aortic aneurysm and / or aortic dissection, including a diagnostic kit capable of accurately diagnosing selected aortic aneurysm and / or aortic dissection, the use of a diagnostic kit for the CTHRC1 gene, and a monoclonal antibody targeting the biological function of CTHRC1, and provides an application of the above monoclonal antibody, which can bind with high affinity and neutralize and block the specific biological functional activity of the CTHRC1 immunogen, and can be applied to new diagnostic and therapeutic targets for aortic aneurysm and / or aortic dissection.

[0007] In a first aspect, the present invention provides use of CTHRC1 protein in preparing a diagnostic reagent for aortic aneurysm and / or aortic dissection.

[0008] The amino acid sequence of the CTHRC1 protein is as follows:

[0009] SEIPKGKQKAQLRQREVVDLYNGMCLQGPAGVPGRDGSPGANGIPGTPGIPGRDGFKG

[0010] EKGECLRESFEESWTPNYKQCSWSSLNYGIDLGKIAECTFTKMRSNSALRVLFSGSLRLKCR

[0011] NACCQRWYFTFNGAECSGPLPIEAIIYLDQGSPEMNSTINIHRTSSVEGLCEGIGAGLVDVAIW

[0012] VGTCSDYPKGDASTGWNSVSRIIIEELPK;

[0013] In a preferred example of this aspect, the diagnostic agent for aortic aneurysm and / or aortic dissection is a monoclonal antibody against CTHRC1 protein.

[0014] CTHRC1 protein monoclonal antibodies, as follows:

[0015] Light chain variable region amino acid sequence

[0016] QLILTQSSSASFSLGASAKLTCTLSSQHSTYTIEWYQQQPLKPPKYVMEVKKDGHSTGDGIP DRFSGSSSGADRYLSISNIQPEDEAIYICGVGDTIKEQFVYVFGGGTRVTVL;

[0017] Heavy chain variable region amino acid sequence

[0018] EVQLVETGGGLVQPKGSMKLSCAASGFTFNINAMNWVRQAPGKGLEWVARIRSKSNNYAT YYADSVKDRFTISRDDSQRMLYLQMNNLKTEDTAKYYCVSNWDWYFDVWGAGTTVTVSS;

[0019] In a second aspect, the present invention provides use of CTHRC1 protein in preparing a diagnostic kit for aortic aneurysm and / or aortic dissection.

[0020] In this aspect, an enzyme-linked immunosorbent assay kit for detecting the content of CTHRC1 protein in a sample to be tested is provided, comprising a CTHRC1 protein monoclonal antibody.

[0021] In a third aspect, the present invention provides the use of CTHRC1 protein as a drug target in screening drugs for treating aneurysms and / or aortic dissection, and provides monoclonal antibodies against the biological functions of CTHRC1 that target vascular adventitial fibroblast populations, which can bind with high affinity and neutralize and block the specific biological functional activity of CTHRC1 immunogens.

[0022] In a preferred example of this aspect, the drug includes an anti-CTHRC1 monoclonal antibody, which can bind with high affinity and neutralize and block the specific biological functional activity of the CTHRC1 immunogen, and can be applied as a new targeted therapeutic target for aortic aneurysm and / or aortic dissection.

[0023] In a fourth aspect, the present invention provides use of an inhibitor of CTHRC1 protein in preparing a medicament for treating aortic aneurysm and / or aortic dissection, wherein the inhibitor of CTHRC1 protein is a monoclonal antibody against CTHRC1 protein.

[0024] In a preferred example of this aspect, the drug comprises a pharmaceutically acceptable carrier and an effective amount of an active ingredient, wherein the active ingredient is an inhibitor of CTHRC1 protein.

[0025] In a fifth aspect, the present invention provides a drug for treating aortic aneurysm and / or aortic dissection, comprising a pharmaceutically acceptable carrier and an effective amount of the following active ingredient, wherein the active ingredient is a monoclonal antibody against CTHRC1 protein.

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] The present invention uses recombinant human CTHRC1 protein as an immunogen to obtain a mouse anti-CTHRC1 monoclonal antibody that specifically binds to the human CTHRC1 antigen. The monoclonal antibody can neutralize and effectively block the specific biological functional activity of the CTHRC1 immunogen through high binding affinity, and can be applied to a new therapeutic target for aortic aneurysm and / or aortic dissection, reduce disease mortality, and improve patient prognosis.

[0028] In the present invention, in the in vitro CTHRC1 protein stimulation culture of vascular smooth muscle cells interacting with vascular adventitial fibroblasts, the present invention has biological activities including directly inhibiting the phenotype conversion of vascular smooth muscle cells by inhibiting the ERK signaling pathway, and the amino acid sequence of the antibody with the most ideal biological activity is sequenced and humanized to prepare an anti-CTHRC1 monoclonal antibody that can be used as a therapeutic agent. By using an anti-CTHRC1 monoclonal antibody targeting the vascular adventitial fibroblast population, the present invention has the advantages of high efficiency, low trauma, high safety, etc., and can effectively reduce the risk of death from aortic aneurysm and / or aortic dissection and improve the prognosis of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a diagram of the CTHRC1 protein structure.

[0030] Figure 2 The diagram shows the amino acid sequence of the variable region of the CTHRC1 protein monoclonal antibody; (a) light chain; (b) heavy chain.

[0031] Figure 3 This is the SDS-PAGE detection result of CTHRC1 protein.

[0032] Figure 4 is the CTHRC1 content in the serum of patients with human aortic dissection.

[0033] Figure 5 The immunohistochemical method was used to detect the expression level of CTHRC1 in the vascular tissue of patients with aortic dissection.

[0034] Figure 6 The ELISA method was used to detect the content of CTHRC1 in the serum of mice in the model group.

[0035] Figure 7The expression level of CTHRC1 in the aortic tissue of mice in the model group was detected by immunoblotting.

[0036] Figure 8 The immunohistochemistry method was used to detect the expression level of CTHRC1 in the aortic tissue of mice in the model group.

[0037] Fig. 9 This is a general picture of aortic lesions in wild-type mice and CTHRC1 knockout mice.

[0038] Fig.10 The incidence of aortic dissection in wild-type mice and CTHRC1 knockout mice.

[0039] Fig.11 Figure 2 shows the mortality rate of aortic dissection in wild-type mice and CTHRC1 knockout mice.

[0040] Fig.12 This is a gross image of aortic lesions in wild-type mice after treatment with anti-CTHRC1 neutralizing antibody.

[0041] Fig.13 The incidence of aortic lesions in wild-type mice treated with an anti-CTHRC1 neutralizing antibody.

[0042] Fig.14 Mortality from aortic dissection in wild-type mice treated with an anti-CTHRC1 neutralizing antibody.

[0043] Fig.15 These are the in vitro functional blocking results of CHTRC1 neutralizing antibodies. DETAILED DESCRIPTION

[0044] The preferred embodiments of the present invention are described in detail below in conjunction with the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0045] Example 1: Preparation of monoclonal antibodies against CTHRC1 protein

[0046] 1. Preparation of CTHRC1 Immunization Antigen for Sensitizing Mice

[0047] By using whole gene synthesis to protein expression and purification (31-243aa), eukaryotic protein expression of CTHRC1 protein with His was performed, and SDS-PAGE detection was shown in Figure 3 .

[0048] Amino acid series of CTHRC1 protein:

[0049] SEIPKGKQKAQLRQREVVDLYNGMCLQGPAGVPGRDGSPGANGIPGTPGIPGRDGFKGEKGECLRESFEESWTPNYKQCSWSSLNYGIDLGKIAECTFTKMRSNSALRVLFS GSLRLKCRNACCQRWYFTFNGAECSGPLPIEAIIYLDQGSPEMNSTINIHRTSSVEGLCEGIGAGLVDVAIWVGTCSDYPKGDASTGWNSVSRIIIEELPK; CTHRC1 protein structure is as follows Figure 1 shown.

[0050] 2. Immunization of Mice and Serum Evaluation

[0051] Use 7-week to 11-week-old Balb / c female mice as the animals to be immunized, weighing about 18-22g, and about 7-10 weeks old. Select healthy animals with shiny fur and free movement. Take the CTHRC1 antigen out of the -20℃ refrigerator and thaw at room temperature to avoid repeated freezing and thawing. Mark the syringe with the project number and animal number. Mix the antigen thoroughly. The concentration of the first immunization antigen is 0.5mg / mL, and the mouse immunization dose is 0.1mL / mouse; the concentration of the second-fourth immunization antigen is 0.5mg / mL, and the mouse immunization dose is 0.1mL / mouse. The main method of mouse immunization is subcutaneous and intraperitoneal multi-point injection. Immunization cycle: The second immunization is performed on the 21st day after the first immunization, and the interval between the second immunization to the third immunization and the third immunization to the fourth immunization is 14 days. Orbital blood sampling is performed on the 7th day after the fourth immunization of mice. Finally, the mouse serum titer is tested. Antigen was diluted to 1 μg / mL with coating buffer, and 50 μL / well was added to the ELISA plate, and coated overnight at 4°C. The liquid in the wells was shaken dry, and 1% BSA was added to the ELISA plate at 100 μL / well, and incubated in a 37°C electric constant temperature incubator for 1 hour. The liquid in the wells was shaken dry. The mouse serum after immunization was diluted with PBS at 1:300, 1:900, 1:2700, 1:8100, 1:24300, 1:72900, and 1:218700. 50 μL / well was added to the first to seventh wells in the same column of the ELISA plate in descending order of dilution multiples, and BSA was added to the eighth well as a negative control. Each dilution gradient was repeated. Incubate at 37°C for 30 minutes. Washing solution (1X TBST) was added to the ELISA plate at 180 μL / well, and the ELISA plate was washed twice. 1% BSA was used to dilute GAM-HRP to a working concentration (1:5000), and 50 μL / well was added to the ELISA plate, and incubated at 37°C for 30 minutes. Color development, termination and reading: the liquid in the well was discarded, and washing solution was added to the ELISA plate at a volume of 180 μL / well, and the ELISA plate was washed 3 times; 100 μL of freshly prepared TMB color substrate was added to each reaction well, and incubated at 37°C for 5 minutes; then 90 μL / well of stop solution was added to terminate the reaction, and the OD value at 450nm was measured on the ELISA reader. The specific measurement results are shown in Table 1.

[0052] Table 1: Serum titer test results of immunized mice

[0053] Dilution multiple YM246-10 YM246-12 Negative mice 1：300 3.072 2.987 0.017 1：900 1.884 1.837 0.013 1：2700 0.79 0.712 0.002 1：8100 0.304 0.248 0.006 1：24300 0.117 0.089 0.007 1：72900 0.054 0.041 0.008 1：218700 0.028 0.023 0.006 PBS 0.016 0.015 0.01

[0054] 3. Mouse Fusion Rescreening

[0055] Take mice on the fourth day after booster immunization. Check the mouse number. Grab the mouse tail, tighten the skin of the mouse neck to keep its head from turning freely, and make its eyeball protrude outward. Use elbow tweezers to remove the protruding eyeball and collect orbital blood. After the blood has flowed out, kill the mouse by breaking the neck and put it in a beaker containing 75% alcohol for disinfection. Bring the beaker, mouse and alcohol into the sterile room together. Use large tweezers to remove the mouse from the alcohol, drain the alcohol, and put it in a kidney-shaped dish on the clean bench. Use large tweezers to lift the mouse's abdominal fur layer, cut an inverted triangle incision with sterile ophthalmic scissors, and tear open its abdominal fur layer with hemostats to expose the abdominal cavity. Use sterile small tweezers to lift the mouse peritoneal membrane, and use another sterile ophthalmic scissors to cut the peritoneal membrane until the entire abdominal cavity is exposed. Find the spleen in the upper right part of the abdominal cavity, carefully remove it, and put it in a glass plate containing preheated IMDM culture medium, change three plates, and wash three times. Use forceps to make a small hole at one end of the spleen, then use two forceps to squeeze out the spleen cells, and then use a 1mL gun to blow the spleen cells apart, collect them in a 50mL centrifuge tube, and centrifuge at 1500rpm for 3min. Discard the supernatant, knock the precipitate apart, add 30mL IMDM culture medium, and centrifuge at 1500rpm for 3min. At the same time, blow down the SP2 / 0 cells in good growth state, and centrifuge at 1500rpm for 3min. Discard the supernatant and resuspend the cells with 30mL preheated IMDM culture medium. Centrifuge at 1500rpm for 3min again. Discard the supernatant and resuspend the cells with 30mL preheated IMDM culture medium. Take the corresponding amount of cells according to the ratio of mouse spleen cells to SP2 / 0 cells of 10:1, mix them evenly in the imported 50mL centrifuge tube, and centrifuge at 1500rpm for 3min. Use a liquid pump to drain the supernatant. Slowly add 1mL preheated PEG (1500) along the wall at the bottom of the centrifuge tube, and add it within 60s. Place in a 37°C water bath for 1 minute. Slowly drip 5 mL of preheated IMDM culture medium along the tube wall, then gradually increase the speed, add 15 mL of preheated IMDM culture medium, and finally add to 40 mL. Centrifuge the above fused cell suspension at 1200 rpm for 5 minutes. Discard the supernatant, add 25 mL of preheated SP2 / 0 myeloma cell culture medium, and carefully resuspend the cells with a 10 mL pipette. Spread 100 μL / well of the fused hybridoma cell suspension onto a 96-well cell culture plate. After 4-6 hours, add 2×HAT culture medium, 100 μL / well.

[0056] 4. Monocloning of positive hybridomas

[0057] After fusion, the cells are grown for 7 days and tested positive. Positive cells are picked. After blowing the cells in the positive cell wells, 200 cells are spread on 96-well plates for sub-screening and monoclonal antibodies are selected. The sub-plate cells are generally grown for 8-10 days and the supernatant is tested to select positive cell lines. After selecting the monoclonal cell line, expand the culture, collect the cell supernatant and freeze the cells. Dilute the antigen to 1μg / mL with coating buffer, add 50μL to the 96-well plate of the ELSIA plate, and incubate at 4℃ overnight. The next day, discard the solution in the well, wash once with 1xTBST washing buffer with 180μL per well, and pat dry. Add 100μL of 1% BSA to each well for blocking and incubate at 37℃ for 1h. Then discard the blocking solution. Add 50μL of cell supernatant to the above-mentioned blocked reaction wells. At the same time, set up positive (1:500 dilution of mouse serum after immunization) and negative control wells (add 1% BSA). Incubate at 37°C for 30 min, pat dry, wash twice with 180 μL per well of 1xTBST washing buffer, and pat dry. Add diluted goat anti-mouse secondary antibody-HRP (1:5000 dilution, diluted with 1% BSA), add 50 μL / well to the ELISA plate wells, incubate at 37°C for 30 min, then discard the liquid, wash three times with 180 μL per well of 1xTBST washing buffer, and pat dry. Add 100 μL of the prepared TMB substrate solution to each reaction well (A solution and B solution are prepared at 1:1, prepared and used immediately), and react at 37°C for 5 min. Add 50 μL of 2M sulfuric acid to each reaction well to terminate the reaction. And measure the OD value at 450nm on the ELISA reader. The specific measurement results are shown in Table 2.

[0058] Table 2: Positive hybridoma supernatant titer test results

[0059]

[0060] Count the positive hybridoma cells. Add 6.5mL of sub culture medium to the sample tank, add positive hybridoma cells to the culture medium to make the cell concentration 125 / 200μL; add cells to the 96-well cell culture plate at 200μL / well, add 3 columns of 24 wells; then add 5mL of fresh sub culture medium, plate 3 columns of 24 wells at the same amount; then repeat the operation in sequence. Finally, four cell gradients are formed, namely 125 / well, 25 / well, 5 / well and 1 / well. The cell culture plate is placed at 37℃ and 5% CO2 for culture. Dilute the antigen to 1μg / mL with coating buffer, add 50μL to the 96-well plate of the ELSIA plate, and incubate at 4℃ overnight. The next day, discard the solution in the wells, wash once with 1xTBST washing buffer at 180μL per well, and pat dry. Add 50μL of cell supernatant to the above-mentioned blocked reaction wells. Incubate at 37°C for 30 min, pat dry, wash twice with 180 μL per well of 1xTBST washing buffer, and pat dry. Incubate with secondary antibodies for typing: Add various secondary antibodies for typing (1:1000 dilution, diluted with 1% BSA), add 50 μL / well to the wells of the ELISA plate, incubate at 37°C for 30 min, then discard the solution, wash three times with 180 μL per well of 1xTBST washing buffer, and pat dry. Add 100 μL of the prepared TMB substrate solution to each reaction well (A solution and B solution are prepared at 1:1, and are prepared and used immediately), and react at 37°C for 5 min. Add 100 μL of 2M sulfuric acid to each reaction well to terminate the reaction. And measure the OD value at 450nm on the ELISA reader. The specific measurement results are shown in Table 3.

[0061] Table 3: Monoclonal cell supernatant titer detection

[0062]

[0063] 5. Preparation of Purified Antibodies

[0064] One week in advance, 0.5 mL of liquid paraffin was injected into the peritoneal cavity of mice used to prepare ascites to sensitize the mice. Generally, ascites is formed in mice in 7-10 days. A drainage needle is inserted into the abdominal cavity of the mouse, and the needle is carefully rotated to allow the ascites to flow out slowly; after collecting the ascites, centrifuge at 10000 rpm for 5 minutes, collect the supernatant, determine the titer and purify. Take out the semi-finished ascites from the raw material warehouse, thaw at room temperature or 2-8 ° C, and observe the sample. The appearance of normal ascites under natural light is as follows: light yellow liquid, relatively clear, with some oil and tissue. Add 10 mL of mouse ascites to a clean glass beaker, record the sample number, volume and other information, use a measuring cylinder to measure 20 mL of acetate buffer (pH 4.0) and add it to the beaker, and place it on a magnetic stirrer for stirring. Add 330 μL of n-octanoic acid according to the ascites volume ratio of 3.33%, and the addition speed is 2-3s / drop. Start timing for 30 minutes after the addition. After stirring, put the sample into a round-bottom centrifuge tube and centrifuge at 4°C 10000rpm for 30min. After centrifugation, filter with 8 layers of gauze and funnel, put the supernatant into a clean beaker, and discard the precipitate. Dialyze the filtered supernatant into 1×PBS, dialyze with a dialysate volume ratio of 30 times or more, stir at room temperature during the day, change the liquid every 3h, and stir and dialyze overnight in a 4C° display cabinet at night, and change the liquid 3 times in total. Record the supernatant number obtained in the previous step, and the volume is 20mL. Set the peristaltic pump flow rate to 10rpm, and use a peristaltic pump to add 20mL of saturated ammonium sulfate solution to the beaker. Stir while adding, and stir thoroughly on the stirrer for 30min. After stirring, centrifuge at 4C°, 10000rpm for 10min, discard the supernatant, take the antibody precipitate and dilute and dissolve it with an appropriate amount of 1×PBS. Dialyze the antibody dissolved in the previous step into 1×PBS, and the dialysis ratio is greater than 50 times. During the day, place it at room temperature and stir it. Change the solution every 3 hours. At night, place it in a 4C° display cabinet and stir it overnight. Change the solution 4 times in total. After dialysis, centrifuge the antibody at 4C°, 10000rpm for 10 minutes and filter it with a 0.22um filter. Dilute the dialyzed sample to an appropriate multiple and measure the OD value at 280nm in a nucleic acid protein detector. The OD range is 0.2-0.8.

[0065] CTHRC1 protein monoclonal antibodies are as follows:

[0066] Light chain variable region amino acid sequence

[0067] QLILTQSSSASFSLGASAKLTCTLSSQHSTYTIEWYQQQPLKPPKYVMEVKKDGHSTG DGIPDRFSGSSSGADRYLSISNIQPEDEAIYICGVGDTIKEQFVYVFGGGTRVTVL;

[0068] Among them, CDR1:TLSSQHSTYTIE; CDR2:VKKDGSHSTGD; CDR3:GVGDTIKEQFVYV, such as Figure 2 As shown in (a).

[0069] Heavy chain variable region amino acid sequence

[0070] EVQLVETGGGLVQPKGSMKLSCAASGFTFNINAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQRMLYLQMNNLKTEDTAKYYCVSNWDWYFDVWGAGTTVTVSS;

[0071] Among them, CDR1: INAMN; CDR2: RIRSKSNNYATYYADSVKD; CDR3: NWDWYFDV, such as Figure 2 As shown in (b).

[0072] Example 2: CTHRC1 content in serum of patients with aortic aneurysm and / or aortic dissection

[0073] 1. Main Reagents

[0074] Human CTHRC1 ELSIA Kit. Serum from patients with aortic aneurysm and / or aortic dissection and healthy controls.

[0075] 2. Experimental Procedure

[0076] 2.1 Collect serum samples from patients with aortic aneurysm and / or aortic dissection and healthy controls. Collect whole blood into a test tube without anticoagulant and place it at room temperature for 1 hour. After the whole blood naturally coagulates and serum precipitates, centrifuge it at about 1500g for 10 minutes at 4℃, and take the yellow supernatant to obtain serum.

[0077] 2.2 Dilute the sample five times and add 100 μL / well to the corresponding wells as the sample group, standard group, and blank group, and incubate at 37 degrees for 2 hours.

[0078] 2.3 Add 300-400 μL of washing solution to each well and wash the plate three times. After the last wash, place the plate on thick absorbent paper and pat dry.

[0079] 2.4 Add biotin-conjugated anti-human CTHRC1 detection antibody to all wells at 100 μL / well, cover with sealing film, and incubate at 37 degrees in the dark for 1 hour.

[0080] 2.5 Add 300-400 μL of washing solution to each well and wash the plate five times. After the last wash, place the plate on thick absorbent paper and pat dry.

[0081] 2.6 Add 100 μL of diluted streptavidin-HRP to all wells, cover with sealing film, and incubate at room temperature in the dark for 30 minutes.

[0082] 2.7 Add 100 μL TMB colorimetric solution to all wells, cover with sealing film, and incubate at room temperature in the dark for 15 minutes.

[0083] 2.8 Add 50 μL of stop solution to all wells, mix well and measure the A450 value immediately.

[0084] 3. ELISA results Figure 4 As shown, it can be found that the CTHRC1 content is significantly increased in the serum of human patients with aortic aneurysm and / or aortic dissection.

[0085] Example 3: CTHRC1 expression level in vascular tissues of patients with aortic aneurysm and / or aortic dissection

[0086] 1. Materials

[0087] Monoclonal antibody against CTHRC1 protein; vascular tissues from patients with aortic aneurysm and / or aortic dissection and healthy controls.

[0088] 2. Experimental Procedure

[0089] 2.1 Dewaxing of paraffin sections to water: Vascular sections of patients with aortic aneurysm and / or aortic dissection and blood vessels of healthy controls were sequentially placed in xylene I for 15 minutes - xylene II for 15 minutes - xylene III for 15 minutes - anhydrous ethanol I for 5 minutes - anhydrous ethanol II for 5 minutes - 85% alcohol for 5 minutes - 75% alcohol for 5 minutes - and washed with distilled water.

[0090] 2.2 Antigen repair: Place the tissue sections in a repair box filled with EDTA antigen repair buffer (PH9.0) in a microwave oven for antigen repair, heat for 10 minutes until boiling, turn off the heat for 10 minutes to keep warm, and then turn to medium-low heat for 7 minutes. During this process, prevent excessive evaporation of the buffer and do not dry the slides. After natural cooling, place the slides in PBS (PH7.4) and shake on a decolorizing shaker to wash 3 times, 5 minutes each time.

[0091] 2.3 Blocking endogenous peroxidase: Put the slices into 3% hydrogen peroxide solution (hydrogen peroxide: pure water = 1:9), incubate at room temperature in the dark for 25 minutes, put the slides in PBS (PH7.4) and wash them 3 times on a decolorizing shaker, each time for 5 minutes.

[0092] 2.4 Treat with triton-x100 (0.3%) for 10 minutes and wash with PBS three times, 5 minutes each time.

[0093] 2.5 Serum blocking: Add 10% donkey serum in the histochemical circle to evenly cover the tissue and block at room temperature for 30 minutes.

[0094] 2.6 Add primary antibody: Gently shake off the blocking solution, add CTHRC1 monoclonal antibody prepared in a certain proportion with PBS on the slices, and incubate the slices flat in a humidified box at 4°C overnight (add a small amount of water in the humidified box to prevent the antibody from evaporating).

[0095] 2.7 Add secondary antibody: Place the slide in PBS (PH7.4) and shake on a decolorizing shaker for 3 times, 5 minutes each time. After the slices are slightly dried, add the secondary antibody (HRP labeled) of the corresponding species in the circle to cover the tissue and incubate at room temperature for 50 minutes.

[0096] 2.8 DAB color development: Place the slide in PBS (PH7.4) and shake on a decolorizing shaker for 3 times, 5 minutes each time. After the slices are slightly dried, add freshly prepared DAB color development solution in the circle, and control the color development time under a microscope. The positive color is brown-yellow, and the slices are rinsed with tap water to stop the color development.

[0097] 2.9 Counterstaining of cell nuclei: Counterstain with Harris hematoxylin for about 3 minutes, wash with tap water, differentiate with 1% hydrochloric acid alcohol for a few seconds, rinse with tap water, turn blue with ammonia water, and rinse with running water.

[0098] 2.10 Dehydration and sealing: Put the slices into 75% alcohol for 5 minutes - 85% alcohol for 5 minutes - anhydrous ethanol I for 5 minutes - anhydrous ethanol II for 5 minutes - xylene I for 5 minutes to dehydrate and make them transparent. Take the slices out of xylene, dry them slightly, and seal them with neutral gum.

[0099] 2.11 Microscope examination, image acquisition and analysis.

[0100] 3. Immunohistochemistry results Figure 5 As shown, it can be found that the expression level of CTHRC1 is increased in the vascular tissues of human patients with aortic aneurysm and / or aortic dissection.

[0101] Example 4: Combination of Angiotensin II (Ang II) and β-aminopropionitrile (BAPN) to induce aortic aneurysm and / or aortic dissection mouse model

[0102] 1. Experiment on animal aortic aneurysm and / or aortic dissection model induced by combined use of Ang II and BAPN

[0103] SPF grade C57BL / 6 mice and CTHRC1 knockout mice were purchased from Saiye Biotechnology. Ang II and BAPN combined to induce aortic aneurysm and / or aortic dissection mouse model: 6-week-old male C57BL / 6 wild-type and CTHRC1- / - mice in the same cage were selected and given subcutaneous micro-osmotic sustained-release pumps filled with Ang II and BAPN, which were continuously infused at a rate of 2500ng / (kg·min) and 150mg / (kg·day), respectively, for 14 days. The survival of the four groups of mice was recorded within 14 days during the experiment, and all mice were killed after 2 weeks, and the aorta and serum were collected for experiments.

[0104] Animal experiment of CTHRC1 neutralizing antibody against aortic structural disorder and phenotypic conversion of middle vascular smooth muscle: The induced modeling experiment was the same as above. The experimental animals were 6-week-old male C57BL / 6 wild-type mice, divided into CTHRC1 neutralizing antibody treatment group and IgG treatment group. The CTHRC1 treatment group and the IgG treatment group were given neutralizing antibody or IgG 200ug / mouse / time, intraperitoneally, three times a week, for a total of 2 weeks. All mice were killed 48h after the last injection, and the aorta and serum were collected for subsequent experiments.

[0105] 2. Experimental steps:

[0106] 2.1 ELISA method to detect the level of CTHRC1 in mouse serum

[0107] 2.1.1 Collect serum samples from mice in the model group and the control group. Collect whole blood into a test tube without anticoagulant and place it at room temperature for 1 hour. After the whole blood coagulates naturally and the serum is precipitated, centrifuge it at about 1500g for 10 minutes at 4℃, and take the yellow supernatant to obtain the serum.

[0108] 2.1.2 Dilute the sample five times and add 100 μL / well to the corresponding wells as the sample group, standard group, and blank group, and incubate at 37 degrees for 2 hours.

[0109] 2.1.3 Add 300-400 μL of washing solution to each well and wash the plate three times. After the last wash, place the plate on thick absorbent paper and pat dry.

[0110] 2.1.4 Add biotin-conjugated anti-human CTHRC1 detection antibody to all wells at 100 μL / well, cover with sealing film, and incubate at 37 degrees in the dark for 1 hour.

[0111] 2.1.5 Add 300-400 μL of washing solution to each well and wash the plate five times. After the last wash, place the plate on thick absorbent paper and pat dry.

[0112] 2.1.6 Add 100 μL of diluted streptavidin-HRP to all wells, cover with sealing film, and incubate at room temperature in the dark for 30 minutes.

[0113] 2.1.7 Add 100 μL of TMB colorimetric solution to all wells, cover with a sealing film, and incubate at room temperature in the dark for 15 minutes.

[0114] 2.1.8 Add 50 μL of stop solution to all wells, mix well and immediately measure the A450 value.

[0115] 2.2 Immunohistochemical detection of CTHRC1 expression levels in vascular tissues of model and control mice

[0116] 2.2.1 Dewaxing of paraffin sections to water: The blood vessel sections of the mice in the model group and the control group were sequentially placed in xylene I for 15 minutes - xylene II for 15 minutes - xylene III for 15 minutes - anhydrous ethanol I for 5 minutes - anhydrous ethanol II for 5 minutes - 85% alcohol for 5 minutes - 75% alcohol for 5 minutes - and washed with distilled water.

[0117] 2.2.2 Antigen repair: Place the tissue sections in a repair box filled with EDTA antigen repair buffer (PH9.0) in a microwave oven for antigen repair, heat for 10 minutes until boiling, turn off the heat for 10 minutes to keep warm, and then turn to medium-low heat for 7 minutes. During this process, prevent the buffer from evaporating excessively and do not dry the slides. After natural cooling, place the slides in PBS (PH7.4) and shake on a decolorizing shaker to wash 3 times, 5 minutes each time.

[0118] 2.2.3 Blocking endogenous peroxidase: Put the sections into 3% hydrogen peroxide solution (hydrogen peroxide: pure water = 1:9), incubate at room temperature in the dark for 25 minutes, place the slides in PBS (PH7.4) and wash them 3 times on a decolorizing shaker, each time for 5 minutes.

[0119] 2.2.4 Treat with triton-x100 (0.3%) for 10 minutes and wash with PBS three times, 5 minutes each time.

[0120] 2.2.5 Serum blocking: Add 10% donkey serum in the histochemical circle to evenly cover the tissue and block at room temperature for 30 minutes.

[0121] 2.2.6 Add primary antibody: Gently shake off the blocking solution, add CTHRC1 monoclonal antibody prepared in a certain proportion with PBS on the slices, and incubate the slices flat in a humidified box at 4°C overnight (add a small amount of water in the humidified box to prevent the antibody from evaporating).

[0122] 2.2.7 Add secondary antibody: Place the slide in PBS (PH7.4) and shake on a decolorizing shaker for 3 times, 5 minutes each time. After the slices are slightly dried, add the secondary antibody (HRP labeled) of the corresponding species in the circle to cover the tissue and incubate at room temperature for 50 minutes.

[0123] 2.2.8 DAB color development: Place the slide in PBS (PH7.4) and shake on a decolorizing shaker for 3 times, 5 minutes each time. After the slices are slightly dried, add freshly prepared DAB color development solution in the circle, and control the color development time under a microscope. The positive color is brown-yellow, and the slices are rinsed with tap water to stop the color development.

[0124] 2.2.9 Counterstaining of cell nuclei: Counterstain with Harris hematoxylin for about 3 minutes, wash with tap water, differentiate with 1% hydrochloric acid alcohol for a few seconds, rinse with tap water, turn blue with ammonia water, and rinse with running water.

[0125] 2.2.10 Dehydration and sealing: Place the slices in 75% alcohol for 5 minutes - 85% alcohol for 5 minutes - anhydrous ethanol I for 5 minutes - anhydrous ethanol II for 5 minutes - xylene I for 5 minutes to dehydrate and make them transparent. Take the slices out of xylene, dry them slightly, and seal them with neutral gum.

[0126] 2.2.11 Microscope examination, image acquisition and analysis.

[0127] 2.3 Immunoblotting to detect the expression level of CTHRC1 in vascular tissues of mice in the model group and the control group

[0128] 2.3.1 Use pre-cooled tools to separate the vascular tissues of the corresponding parts of the model group and control group mice, and try to keep them on ice to prevent protease hydrolysis;

[0129] 2.3.2 Place the tissue block in a round-bottom microcentrifuge tube or EP tube, add liquid nitrogen to freeze the tissue, homogenize and grind on ice, add about 200 μl of pre-cooled lysis buffer (add protease and phosphatase inhibitors before use) for every about 10 mg of tissue, homogenize in an ice bath, and shake at 4°C for 2 hours.

[0130] 2.3.3 Centrifuge at 12000rpm for 20min at 4℃. Gently aspirate the supernatant and transfer it to a newly pre-cooled microcentrifuge tube and place it on ice. This is the protein sample and discard the precipitate.

[0131] 2.3.4 Protein quantification: According to the number of samples, prepare an appropriate amount of BCA working solution by adding 50 volumes of BCA reagent A to 1 volume of BCA reagent B (50:1), and mix thoroughly; place the ELISA plate on an oscillator for 30 seconds, place it at 37°C for 30 minutes, and then perform colorimetric determination at 562nm. According to the absorbance value of the measured sample, the corresponding protein concentration can be found on the standard curve.

[0132] 2.3.5 Denaturation and reduction of protein samples: Protein denaturation is generally performed using a loading buffer containing a cationic denaturing detergent such as SDS and boiled at 95-100°C for 5 minutes.

[0133] 2.3.6 Electrophoresis: The amount of vascular tissue protein antigen sample is 30ug. Take the treated sample solution, use a microinjector to absorb an appropriate amount of sample solution, and slowly add the sample solution to the concave part of the gel plate (the sample point entry point). During electrophoresis, use low voltage constant voltage electrophoresis for the upper gel, turn on the power and adjust the voltage to 80v (usually about 15min), and use high voltage constant voltage electrophoresis when bromophenol blue enters the lower gel, adjust the voltage to 120v until bromophenol blue reaches the bottom of the gel, then stop electrophoresis.

[0134] 2.3.7 Transfer: After SDS-PAGE electrophoresis is completed, use a blade or a thin plate to gently pry open the two glass plates of the gel plate, so that the gel is tilted on one of the glass plates. Use a blade to cut the separation gel along the junction of the separation gel and the concentrated gel on the gel, and cut off a small corner at the upper left corner of the separation gel to mark the sample order. Then carefully transfer the gel into the transfer buffer. Cut a 0.45um PVDF membrane of the same size as the separation gel and soak it in methanol for 5s. Cut 6 pieces of filter paper of the same size and balance them with the PVDF membrane and gel with transfer buffer for 15 minutes. Place sponge gasket, filter paper, gel, membrane, filter paper, and sponge gasket (from bottom to top) on the transfer device from the negative electrode (black bottom) to the positive electrode. Set the transfer current to constant current, 200mA, and the time is about 40min (the time required varies depending on the size of the protein, generally about 1min for 1KD). After transfer, take out the membrane and carefully cut off a corner at the same position as the gel to mark the electrophoresis direction and the membrane surface where the protein is adsorbed.

[0135] 2.3.8 Blocking: Incubate with 5% skimmed milk powder or BSA solution at room temperature or 37°C with slow shaking for 1-2 hours.

[0136] 2.3.9 Add primary antibody: dilute CTHRC1 monoclonal antibody at 1:1000 and incubate overnight at 4°C with slow shaking on a shaker.

[0137] 2.3.10 After incubation, absorb the primary antibody, add TBST to a square fresh-keeping box, place the membrane in it, make sure that the TBST covers the PVDF membrane, shake at low speed on a shaker for 10 minutes, and repeat washing 3 times.

[0138] 2.3.11 Dilute the secondary antibody of the corresponding species at 1:10000 and incubate on a shaker at room temperature for 1 hour. After incubation, aspirate the secondary antibody, add TBST to a square fresh-keeping box, place the membrane in it, make the TBST cover the PVDF membrane, shake at low speed on a shaker for 10 minutes, and repeat washing 3-5 times.

[0139] 2.3.12 Chemiluminescence color development and band gray value analysis.

[0140] 3. ELISA, immunohistochemistry, and immunoblotting methods found that CTHRC1 expression was significantly upregulated in the model group mice. At the same time, combined with the single-cell sequencing data of diseased blood vessels, it was found that CTHRC1 was mainly highly expressed in a group of heterogeneous fibroblasts. The aortic lesions of the model group mice were more severe, and the middle smooth muscle cells showed a transformation to a proliferative phenotype. The experimental results are as follows Figure 6 , 7 , as shown in Figure 8.

[0141] 4. Compared with CTHRC1 knockout mice, wild-type mice had a higher incidence of aortic aneurysms and / or aortic dissections, a higher mortality rate, and more severe lesions. Fig. 9 , 10 , as shown in 11.

[0142] 5. In wild-type mice, CTHRC1 neutralizing antibodies can reduce the incidence and mortality of aortic aneurysms and / or aortic dissection induced by the combination of angiotensin II (Ang II) and β-aminopropionitrile (BPAN). It can also reduce the degree of aortic lesions, improve vascular structural disorders, and maintain normal function. The experimental results are as follows Fig.12 , 13 , as shown in 14.

[0143] Example 5: Vascular smooth muscle cell phenotype conversion experiment

[0144] 1. Materials

[0145] CTHRC1 recombinant protein, CTHRC1 monoclonal antibody, vascular aortic smooth muscle cells, α-SMA, SM22α, p-ERK, and ERK primary antibodies were purchased from Abcam.

[0146] 2. The expression plasmid of CTHRC1 was constructed and transfected into 293T cells, and the recombinant CTHRC1 protein was purified. Through immunoblotting experiments, it was observed that the recombinant CTHRC1 protein can promote the migration of vascular smooth muscle cells by activating the ERK signaling pathway and aggravate the loss of the contractile phenotype of vascular smooth muscle cells. The use of CTHRC1 neutralizing antibodies can reverse this effect.

[0147] 3. Experimental steps: Immunoblotting method to detect the protein levels of vascular smooth muscle cell contractile markers

[0148] 3.1 Wash the vascular smooth muscle cells in the six-well plate with pre-cooled PBS, remove the supernatant, and keep it on ice as much as possible to prevent protease hydrolysis;

[0149] 3.2 Add about 250 μl of pre-cooled lysis buffer (add protease and phosphatase inhibitors before use) for every approximately 10^6 cells, scrape the cells with a cell scraper, and place them at 4°C for 15 minutes to lyse.

[0150] 3.3 Centrifuge at 12000 rpm for 20 min at 4°C. Gently aspirate the supernatant and transfer it to a newly pre-cooled microcentrifuge tube and place it on ice. This is the protein sample and discard the precipitate.

[0151] 3.4 Protein quantification: According to the number of samples, prepare an appropriate amount of BCA working solution by adding 50 volumes of BCA reagent A to 1 volume of BCA reagent B (50:1), and mix thoroughly; place the ELISA plate on an oscillator for 30 seconds, place it at 37°C for 30 minutes, and then perform colorimetric determination at 562nm. According to the absorbance value of the measured sample, the corresponding protein concentration can be found on the standard curve.

[0152] 3.5 Denaturation and reduction of protein samples: Protein denaturation is generally performed using a loading buffer containing a cationic denaturing detergent such as SDS and boiled at 95-100°C for 5 minutes.

[0153] 3.6 Electrophoresis: The amount of vascular tissue protein antigen sample is 30ug. Take the treated sample solution, use a microinjector to absorb an appropriate amount of sample solution, and slowly add the sample solution to the concave part of the gel plate (the sample point entry point). During electrophoresis, use low voltage constant voltage electrophoresis for the upper gel, turn on the power and adjust the voltage to 80v (usually about 15min), and use high voltage constant voltage electrophoresis when bromophenol blue enters the lower gel, adjust the voltage to 120v until bromophenol blue reaches the bottom of the gel and stop electrophoresis.

[0154] 3.7 Transfer: After SDS-PAGE electrophoresis, use a blade or a thin plate to gently pry open the two glass plates of the gel plate, so that the gel is overturned on one of the glass plates. Use a blade to cut the separation gel along the junction of the separation gel and the concentrated gel on the gel, and cut off a small corner at the upper left corner of the separation gel to mark the sample order. Then carefully transfer the gel into the transfer buffer. Cut a 0.45um PVDF membrane of the same size as the separation gel and soak it in methanol for 5s. Cut 6 pieces of filter paper of the same size and balance them with the PVDF membrane and gel with transfer buffer for 15 minutes. Place sponge gasket, filter paper, gel, membrane, filter paper, sponge gasket (from bottom to top) on the transfer device from the negative electrode (black bottom) to the positive electrode. Set the transfer current to constant current, 200mA, and the time is about 40min (the time required varies depending on the size of the protein, generally about 1min for 1KD). After transfer, take out the membrane and carefully cut off a corner at the same position as the gel to mark the electrophoresis direction and the membrane surface where the protein is adsorbed.

[0155] 3.8 Blocking: Incubate with 5% skimmed milk powder or BSA solution at room temperature or 37°C with slow shaking for 1-2 hours.

[0156] 3.9 Add primary antibodies: dilute α-SMA, SM22α, p-ERK1 / 2, ERK1 / 2 at 1:1000 and incubate overnight at 4°C with slow shaking on a shaker.

[0157] 3.10 After incubation, absorb the primary antibody, add TBST to a square fresh-keeping box, place the membrane in it, make sure that the TBST covers the PVDF membrane, shake at low speed on a shaker for 10 minutes, and repeat washing 3 times.

[0158] 3.11 Dilute the secondary antibody of the corresponding species at 1:10000 and incubate on a shaker at room temperature for 1 hour. After incubation, aspirate the secondary antibody, add TBST to a square fresh-keeping box, place the membrane in it, make the TBST cover the PVDF membrane, shake at low speed on a shaker for 10 minutes, and repeat washing 3-5 times.

[0159] 3.12 Chemiluminescence color development and band gray value analysis.

[0160] 4. The experimental results show that CTHRC1 recombinant protein can aggravate the loss of contractile phenotype of vascular smooth muscle cells by activating the ERK signaling pathway, while CHTRC1 neutralizing antibody can reverse this effect. Fig.15 shown.

[0161] The above experiments confirmed that CTHRC1 is involved in the occurrence and development of aortic aneurysm and / or aortic dissection, and worsens the course of the disease. CTHRC1 can aggravate the progression of aortic aneurysm and / or aortic dissection in animals, promote the disorder of the adventitial structure of the blood vessels, and aggravate the loss of the contractile phenotype of smooth muscle cells in the tunica media. In vitro, CTHRC1 can accelerate the migration of vascular smooth muscle cells and accelerate the loss of their contractile phenotype. The use of CTHRC1 neutralizing antibodies can block its effects. After using CTHRC1 monoclonal antibodies in animals with their high affinity to neutralize and block, the incidence and mortality of aortic aneurysm and / or aortic dissection can be greatly reduced, and the progression of the lesions can be slowed down. The above results show that the gene can be accurately used for the diagnosis kit selected from aortic aneurysm and / or aortic dissection, the use of the diagnostic kit for providing the CTHRC1 gene, and the new target for new targeted therapy for aortic aneurysm and / or aortic dissection.

[0162] The above are only a few embodiments of the present application and do not constitute any form of limitation to the present application. Although the present application is disclosed as above with preferred embodiments, it is not intended to limit the present application. Any technician familiar with the profession, without departing from the scope of the technical solution of the present application, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. Application of CTHRC1 protein in the preparation of diagnostic reagents for aortic aneurysm and / or aortic dissection.

2. The use according to claim 1, characterized in that: The diagnostic reagent comprises a CTHRC1 protein monoclonal antibody.

3. Application of CTHRC1 protein in the preparation of a diagnostic kit for aortic aneurysm and / or aortic dissection.

4. The use according to claim 3, characterized in that: The kit comprises CTHRC1 protein monoclonal antibody.

5. Application of CTHRC1 protein as a drug target in screening drugs for the treatment of aneurysm and / or aortic dissection.

6. The use according to claim 5, wherein the drug comprises a monoclonal antibody against CTHRC1 protein, which can bind with high affinity and neutralize and block the specific biological function activity of the CTHRC1 immunogen.

7. Use of an inhibitor of CTHRC1 protein in the preparation of a drug for treating aortic aneurysm and / or aortic dissection, wherein the inhibitor of CTHRC1 protein is a monoclonal antibody against CTHRC1 protein.

8. The use according to claim 7, characterized in that: The medicine comprises a pharmaceutically acceptable carrier and an effective amount of active ingredients, wherein the active ingredients are inhibitors of CTHRC1 protein.

9. A drug for treating aortic aneurysm and / or aortic dissection, characterized in that: The invention comprises a pharmaceutically acceptable carrier and an effective amount of the following active ingredients, wherein the active ingredient is a monoclonal antibody against CTHRC1 protein.

Images