Application of BMAL1 overexpressed adenovirus in pulmonary arterial hypertension treatment

Abstract

The invention discloses an application of BMAL1 overexpressed adenovirus in pulmonary arterial hypertension treatment, the application is that the BMAL1 overexpressed adenovirus can relieve proliferation and migration of pulmonary arterial smooth muscle in pulmonary arterial hypertension, and specifically, the overexpressed adenovirus is constructed, and a rat pulmonary arterial smooth muscle cell BMAL1 overexpressed model is constructed by using a transfection technology. By constructing a mouse pulmonary vessel BMAL1 overexpression model, it is elaborated that BMAL1 can effectively inhibit vascular remodeling of pulmonary arteries under the hypoxia condition by adjusting related signal channels, pulmonary arterial pressure can be relieved, right heart pressure can be reduced by explaining BMAL1 overexpression of pulmonary vessels, pulmonary vessel injury can be relieved, and therefore clinical symptoms of pulmonary arterial hypertension are improved; the invention discloses an important role of the BMAL1 overexpressed adenovirus in pulmonary arterial hypertension, and provides a potential target for developing a novel treatment method aiming at the disease.

Description

Technical Field

[0001] The present invention relates to the technical field of pulmonary hypertension treatment, and in particular to application of a BMAL1 overexpressing adenovirus in the treatment of pulmonary hypertension. Background Art

[0002] Pulmonary hypertension (PH) is a serious circulatory disease, which is defined as changes in pulmonary vascular structure and / or function caused by a variety of heterogeneous disease causes and different pathogenic mechanisms. These changes lead to increased pulmonary vascular resistance and elevated pulmonary artery pressure, forming a complex clinical and pathophysiological syndrome. The etiology of PH is extremely complex and can be primary or secondary. The etiology of primary PH is still unclear and may be related to genetic factors, drug or toxin exposure, and certain inflammatory diseases, while secondary PH may be related to a variety of underlying diseases, such as left heart disease, metabolic diseases, etc. Different etiologies and pathogenesis lead to It causes abnormalities in the structure and function of the pulmonary blood vessels, such as spasm and remodeling of the pulmonary arterioles. These changes reduce the compliance of the pulmonary blood vessels, increase blood flow resistance, and ultimately lead to increased pulmonary artery pressure. As the disease progresses, the continuously elevated pulmonary artery pressure increases the burden on the right ventricle, because the right ventricle is the pump responsible for pumping blood into the lungs. Long-term high load causes the right ventricle to gradually thicken and dilate, and may eventually develop into right heart failure. Right heart failure is a serious heart disease characterized by a decrease in the heart's pumping function, which cannot meet the body's needs for blood and oxygen. Patients will experience symptoms such as fatigue and edema. The diagnosis of PH usually requires a combination of clinical symptoms, echocardiogram and other examination results.

[0003] In recent years, the role of the biological clock gene BMAL1 in the cardiovascular system has gradually attracted attention. As a core component of the biological clock, BMAL1 is involved in regulating a variety of physiological processes, including cell proliferation, migration and metabolism. Studies have shown that abnormal expression of BMAL1 in pulmonary artery smooth muscle cells is closely related to the occurrence and development of pulmonary hypertension. However, it is not yet fully understood how BMAL1 specifically affects the proliferation and migration of PASMCs, and how to treat pulmonary hypertension by regulating BMAL1. Summary of the invention

[0004] The purpose of the present invention is to provide a use of a BMAL1 overexpression adenovirus in the treatment of pulmonary hypertension, which has the advantages of high efficiency, accuracy and high sensitivity, and solves the problems mentioned in the background technology.

[0005] To achieve the above object, the present invention provides the following technical solution: an application of a BMAL1 overexpressing adenovirus in the treatment of pulmonary hypertension, the application comprising:

[0006] BMAL1 overexpression adenovirus can reduce the proliferation and migration of pulmonary artery smooth muscle in pulmonary hypertension. Specifically, a rat pulmonary artery smooth muscle cell BMAL1 overexpression model is constructed by adenovirus transfection technology, which includes the following steps:

[0007] Step 1: Perform cck8 experiment, which shows that after hypoxia, the proliferation of PASMCs transfected with empty adenovirus and overexpressing BMAL1 is increased compared with PASMCs in normoxia, while after hypoxia, the proliferation of PASMCs transfected with overexpressing BMAL1 is reduced compared with PASMCs transfected with empty adenovirus;

[0008] Step 2: Three experimental groups were set up, namely, empty adenovirus under normoxic condition, empty adenovirus under hypoxia condition, and BMAL1 overexpression adenovirus under hypoxia condition. The migration ability of rat pulmonary artery smooth muscle cells after hypoxia treatment with BMAL1 overexpression adenovirus was reduced compared with that of the control group after hypoxia.

[0009] Step 3: Extract the protein of rat pulmonary artery smooth muscle cells for Western Blot experiment: the BMAL1 protein level in pulmonary artery smooth muscle cells with BMAL1 overexpression was significantly upregulated;

[0010] BMAL1 overexpression adenovirus can alleviate pulmonary vascular remodeling in pulmonary hypertension, specifically: constructing a mouse pulmonary hypertension disease model by combining hypoxia with SU5416, and constructing a mouse pulmonary vascular BMAL1 overexpression model by nasal drops, which includes the following steps:

[0011] Step 1: Three experimental groups were set up, namely, empty adenovirus nasal drops under normoxic conditions, empty adenovirus nasal drops under hypoxia conditions, and BMAL1 overexpression adenovirus nasal drops under hypoxia conditions. The right heart catheter pressure measurement showed that the right heart pressure of mice in the pulmonary hypertension disease group was higher than that in the control group, and the right heart pressure of mice in the BMAL1 overexpression treatment group after hypoxic administration was lower than that in the pulmonary hypertension group;

[0012] Step 2: Perform pulmonary angiography on the mice in step 1; the pulmonary vascular damage in the mice in the pulmonary hypertension group was more severe than that in the control group, and the pulmonary vascular damage in the mice in the BMAL1 overexpression treatment group was less severe than that in the pulmonary hypertension group;

[0013] Step 3: Extract lung tissue proteins from mice for Western Blot experiment to verify that the expression of BMAL1 protein in the lung tissue of mice in the pulmonary hypertension group was downregulated compared with that in the control group, and the expression of BMAL1 protein in the lung tissue of mice in the BMAL1 overexpression treatment group was significantly upregulated.

[0014] Preferably, the preparation process of the BMAL1 overexpression adenovirus is as follows:

[0015] Step 1: Construction of recombinant plasmid

[0016] According to the CDS sequence of the BMAL1 coding gene published in GenBank, the BMAL1 coding sequence was codon optimized considering codon preference, mRNA secondary structure, repeat sequence, GC content, etc. EcoRI and BamHI restriction sites were added at the 5' and 3' ends, respectively, the chemically synthesized BMAL1 fragment and shuttle plasmid pCADV were digested with EcoRI and BamHI, respectively, connected with Solution I and transformed into Escherichia coli DH5α strain (E.coliDH5α), and screened on ampicillin resistance plates. Transformants were selected to extract plasmids, and recombinant plasmids (pcADV-BMAL1) were identified by double digestion with Mlul (located upstream of the EF1 promoter) and EcoRI and DNA sequencing.

[0017] Step 2: Expression detection of recombinant plasmid

[0018] Mix 0.8ug of pcADV-BMAL1 in 50μL serum-free medium; add 2μL transfection reagent Lip2000 to 50μL serum-free medium and incubate at room temperature for 5min; mix the above solution and incubate at room temperature for 20min, add HEK293T cells with a confluency of 60%-80%. Observe under a fluorescence microscope 48h after transfection. Lyse cells on ice with RIPA cell lysis buffer and collect cell proteins by centrifugation. After BCA quantification and 10% SDS-PAGE electrophoresis, transfer to PVDF membrane, incubate anti-FLAG (1:5000) and anti-GAPDH (1:5000) antibodies respectively after blocking, and then incubate with HRP-IgG (1:5000), and detect the expression of target protein by chemiluminescence.

[0019] Step 3: Packaging of Ad-BMAL1

[0020] According to the AdMax adenovirus packaging kit, the specific steps are as follows: dissolve 2μg of recombinant shuttle plasmid pcADV-BMAL1 and 2μg of backbone plasmid in 250mL Opti-MEM medium to obtain plasmid dilution solution: add 10μL transfection reagent Lip2000 to 250μL Opti-MEM medium to obtain transfection reagent dilution solution. Add the transfection reagent dilution solution dropwise to the plasmid dilution solution, incubate at room temperature for 20 minutes, and then add HEK293T for adenovirus packaging. Observe under a fluorescence microscope. When most cells show typical cytopathic effects and more than 50% of the cells are detached from the wall, collect the cell supernatant to obtain Ad-BMAL1.

[0021] Step 4. Amplification and purification of Ad-BMAL1

[0022] HEK293T cells were infected with Ad-BMAL1 and cultured for 2-3 days. Then, 10% NP40 was added to lyse the cells and the supernatant was collected by centrifugation at 12000r / min for 10min. 50mL of virus precipitate (20% PEG8000, 2.5M NaCl) was added to each 100mL supernatant, ice-bathed for 1h, and the precipitate was collected by centrifugation at 12000r / min for 20min and resuspended in 10mL of CsCI solution with a density of 1.10g / mL. The virus was purified by density gradient centrifugation. The virus was collected, dialyzed at 4℃ overnight, and stored at -80℃.

[0023] Step 5: Ad-BMAL1 titer determination

[0024] 100 μL of 10 -4 -10 -7 Diluted Ad-BMAL1 was added to 293T cells and cultured for 48 hours. After pre-cooled formaldehyde fixation, 1% BSA was used for blocking, and anti-adenovirus capsid protein Hexon antibody and HRP-labeled goat anti-mouse IgG (1:5000) were incubated in sequence. Diaminobenzidine (DAB) was used for staining, and the number of positive cells was counted under the bright field of the microscope. Virus titer (PFU / mL) = (number of infected cells / number of fields of view) x (number of fields of view per well) x (dilution factor) / (0.1mL).

[0025] Step 6: Identification of target protein expressed by Ad-BMAL1

[0026] 293T cells were infected with Ad and rAd-BMAL1, respectively. Total cell protein was prepared 48 hours after infection. The expression of BMAL1 was detected by Western blot. The positive control was purified BMAL1 recombinant protein with His tag. The primary antibody was BMAL1 monoclonal antibody (1:1000)1, and the secondary antibody was goat anti-mouse HRP-IgG (1:5000). At the same time, 293T cells were infected with Ad and Ad-BMAL1, and BMAL1 expression was detected by immunofluorescence 24 hours after infection. The primary antibody was BMAL1 monoclonal antibody (1:500) and the secondary antibody was goat anti-mouse IgG labeled with red fluorescent Cy3 (1:200). The cell nucleus was stained with DAPI and observed by fluorescence.

[0027] Preferably, the adenovirus transfection method comprises the following steps:

[0028] Step 1: On the first day, inoculate the target cells in good condition into the culture dish. Ensure that the cell confluence is between 50% and 70% when infected on the second day;

[0029] Step 2: On the second day, preheat the culture medium, take out the virus, put it on ice to melt, and centrifuge it in a desktop centrifuge to make the virus liquid accumulate at the bottom of the tube; discard the culture medium, add the prepared virus to the serum-free culture medium, mix well, and add it to the culture dish;

[0030] Step 3: After 2 hours (shake evenly every 0.5 hours to increase infection efficiency), add the same amount of normal culture medium containing 10% FBS as in step 3;

[0031] Step 4: Replace with fresh normal culture medium after 12-24 hours;

[0032] Step 5: Observe fluorescence expression and perform related tests 48-72 hours after infection.

[0033] Preferably, the bmal1 overexpression adenovirus is ordered from Heyuan Biotechnology, gene name: Bmal1, gene ID: 11865, transcript: NM_001368412.1, species: Rat, gene size: 1899.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The present invention constructs a mouse model and illustrates that BMAL1 overexpression adenovirus can effectively inhibit the proliferation and migration of pulmonary artery smooth muscle cells under hypoxic conditions by regulating related signal pathways, which helps to alleviate pulmonary vascular remodeling. The present invention also constructs a mouse model to explain that BMAL1 overexpression adenovirus can reduce right heart pressure and alleviate pulmonary vascular damage, thereby improving the clinical symptoms of pulmonary hypertension and providing a new target for the treatment of pulmonary hypertension: The present invention reveals the important role of BMAL1 overexpression adenovirus in pulmonary hypertension and provides a potential target for the development of new treatment methods for the disease. By deeply exploring the mechanism of action of BMAL1, it is helpful to further understand the pathophysiological process of pulmonary hypertension and promote the in-depth development of research in this field. The application of BMAL1 overexpression adenovirus in the treatment of pulmonary hypertension has the advantages of high efficiency, accuracy and high sensitivity, and solves the problems mentioned in the background technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a schematic diagram of the cck8 experimental results of the present invention;

[0037] Figure 2 It is a diagram illustrating the migration ability of rat pulmonary artery smooth muscle cells after hypoxia treatment with empty adenovirus in normoxic state, empty adenovirus in hypoxic state, BMAL1 overexpressing adenovirus in hypoxic state, and BMAL1 overexpressing adenovirus in hypoxic state according to the present invention;

[0038] Figure 3 This is a schematic diagram of the Westen Blot experimental results of the present invention;

[0039] Figure 4 This is a schematic diagram of the pressure measurement result of the right cardiac catheter of the present invention;

[0040] Figure 5 Schematic diagram of pulmonary angiography of the present invention. DETAILED DESCRIPTION

[0041] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] See also Figure 1-5 The present invention provides a technical solution: an application of a BMAL1 overexpression adenovirus in the treatment of pulmonary hypertension, the application comprising:

[0043] BMAL1 overexpression adenovirus can reduce the proliferation and migration of pulmonary artery smooth muscle in pulmonary hypertension. Specifically, a rat pulmonary artery smooth muscle cell BMAL1 overexpression model is constructed by adenovirus transfection technology, which includes the following steps:

[0044] Step 1: Perform cck8 experiment, which shows that after hypoxia, the proliferation of PASMCs transfected with empty adenovirus and overexpressing BMAL1 is increased compared with PASMCs in normoxia, while after hypoxia, the proliferation of PASMCs transfected with overexpressing BMAL1 is reduced compared with PASMCs transfected with empty adenovirus;

[0045] Step 2: Three control groups were set up, including empty adenovirus under normoxic conditions, empty adenovirus under hypoxic conditions, and BMAL1 overexpression adenovirus under hypoxic conditions. The migration ability of rat pulmonary artery smooth muscle cells treated with BMAL1 overexpression adenovirus after hypoxia was reduced compared with that of the control group;

[0046] Step 3: Extract the protein of rat pulmonary artery smooth muscle cells for Western Blot experiment: the BMAL1 protein level of BMAL1 overexpressed pulmonary artery smooth muscle cells was significantly upregulated;

[0047] BMAL1 overexpression adenovirus can alleviate pulmonary vascular remodeling in pulmonary hypertension, specifically: constructing a mouse pulmonary hypertension disease model by combining hypoxia with SU5416, and constructing a mouse pulmonary vascular BMAL1 overexpression model by nasal drops, which includes the following steps:

[0048] Step 1: Three control groups were set up, including empty adenovirus under normoxic conditions, empty adenovirus under hypoxic conditions, and BMAL1 overexpression adenovirus under hypoxic conditions. The results showed that the right heart pressure of mice in the pulmonary hypertension disease group was higher than that in the control group, and the right heart pressure of mice in the BMAL1 overexpression treatment group after hypoxic administration was lower than that in the pulmonary hypertension group.

[0049] Step 2: Perform pulmonary angiography on the mice in step 1; the pulmonary vascular damage in the mice in the pulmonary hypertension group was more severe than that in the control group, and the pulmonary vascular damage in the mice in the BMAL1 overexpression treatment group was less severe than that in the pulmonary hypertension group;

[0050] Step 3: Extract lung tissue proteins from mice for Western Blot experiment to verify that the expression of BMAL1 protein in the lung tissue of mice in the pulmonary hypertension group was downregulated compared with that in the control group, and the expression of BMAL1 protein in the lung tissue of mice in the BMAL1 overexpression treatment group was significantly upregulated.

[0051] In the present invention, the preparation process of BMAL1 overexpression adenovirus is as follows:

[0052] Preferably, the preparation process of the BMAL1 overexpression adenovirus is as follows:

[0053] Step 1: Construction of recombinant plasmid

[0054] According to the CDS sequence of the BMAL1 coding gene published in GenBank, the BMAL1 coding sequence was codon optimized considering codon preference, mRNA secondary structure, repeat sequence, GC content, etc. EcoRI and BamHI restriction sites were added at the 5' and 3' ends, respectively, the chemically synthesized BMAL1 fragment and shuttle plasmid pCADV were digested with EcoRI and BamHI, respectively, connected with Solution I and transformed into E.coliDH5α, and screened on ampicillin resistance plates. Transformants were selected to extract plasmids, and recombinant plasmids were identified by double digestion with Mlul (located upstream of the EF1 promoter) and EcoRI and DNA sequencing.

[0055] Step 2: Expression detection of recombinant plasmid

[0056] Mix 0.8ug of pcADV-BMAL1 in 50μL serum-free medium; add 2μL transfection reagent Lip2000 to 50μL serum-free medium and incubate at room temperature for 5min; mix the above solution and incubate at room temperature for 20min, add HEK293T cells with a confluency of 60%-80%. Observe under a fluorescence microscope 48h after transfection. Lyse cells on ice with RIPA cell lysis buffer and collect cell proteins by centrifugation. After BCA quantification and 10% SDS-PAGE electrophoresis, transfer to PVDF membrane, incubate anti-FLAG (1:5000) and anti-GAPDH (1:5000) antibodies respectively after blocking, and then incubate with HRP-IgG (1:5000), and detect the expression of target protein by chemiluminescence.

[0057] Step 3: Packaging of Ad-BMAL1

[0058] According to the AdMax adenovirus packaging kit, the specific steps are as follows: dissolve 2μg of recombinant shuttle plasmid pcADV-BMAL1 and 2μg of backbone plasmid in 250ML Opti-MEM medium to obtain plasmid dilution solution: add 10μL transfection reagent Lip2000 to 250μL Opti-MEM medium to obtain transfection reagent dilution solution. Add the transfection reagent dilution solution dropwise to the plasmid dilution solution, incubate at room temperature for 20 minutes, and then add HEK293T for adenovirus packaging. Observe under a fluorescence microscope. When most cells show typical cytopathic effects and more than 50% of the cells are detached from the wall, collect the cell supernatant to obtain Ad-BMAL1.

[0059] Step 4. Amplification and purification of Ad-BMAL1

[0060] HEK293T cells were infected with Ad-BMAL1 and cultured for 2-3 days. Then, 10% NP40 was added to lyse the cells and the supernatant was collected by centrifugation at 12000r / min for 10min. 50mL of virus precipitate (20% PEG8000, 2.5M NaCl) was added to each 100mL supernatant, ice-bathed for 1h, and the precipitate was collected by centrifugation at 12000r / min for 20min and resuspended in 10mL of CsCI solution with a density of 1.10g / mL. The virus was purified by density gradient centrifugation. The virus was collected, dialyzed at 4℃ overnight, and stored at -80℃.

[0061] Step 5: Ad-BMAL1 titer determination

[0062] 100 μL of 10 -4 -10- 7Diluted Ad-BMAL1 was added to 293T cells and cultured for 48 hours. After pre-cooled formaldehyde fixation, 1% BSA was used for blocking, and anti-adenovirus capsid protein Hexon antibody and HRP-labeled goat anti-mouse IgG (1:5000) were incubated in sequence. Diaminobenzidine (DAB) was used for staining, and the number of positive cells was counted under the bright field of the microscope. Virus titer (PFU / mL) = (number of infected cells / number of fields of view) x (number of fields of view per well) x (dilution factor) / (0.1mL).

[0063] Step 6: Identification of target protein expressed by Ad-BMAL1

[0064] 293T cells were infected with Ad and rAd-BMAL1, respectively. Total cell protein was prepared 48 hours after infection. The expression of BMAL1 was detected by Western blot. The positive control was purified BMAL1 recombinant protein with His tag. The primary antibody was BMAL1 monoclonal antibody (1:1000)1, and the secondary antibody was goat anti-mouse HRP-IgG (1:5000). At the same time, 293T cells were infected with Ad and Ad-BMAL1, and BMAL1 expression was detected by immunofluorescence 24 hours after infection. The primary antibody was BMAL1 monoclonal antibody (1:500) and the secondary antibody was goat anti-mouse IgG labeled with red fluorescent Cy3 (1:200). The cell nucleus was stained with DAPI, and fluorescence observation was performed.

[0065] In the present invention, the adenovirus transfection method comprises the following steps:

[0066] Step 1: On the first day, inoculate the target cells in good condition into the culture dish. Ensure that the cell confluence is between 50% and 70% when infected on the second day;

[0067] Step 2: On the second day, preheat the culture medium, take out the virus, put it on ice to melt, and centrifuge it in a desktop centrifuge to make the virus liquid accumulate at the bottom of the tube; discard the culture medium, add the prepared virus to the serum-free culture medium, mix well, and add it to the culture dish;

[0068] Step 3: After 2 hours (shake evenly every 0.5 hours to increase infection efficiency), add the same amount of normal culture medium containing 10% FBS as in step 3;

[0069] Step 4: Replace with fresh normal culture medium after 12-24 hours;

[0070] Step 5: Observe fluorescence expression and perform related tests 48-72 hours after infection

[0071] In the present invention, the experimental steps and methods for verifying BMAL1 overexpression adenovirus are as follows:

[0072] Step 1: Lung tissue / smooth muscle cell protein extraction

[0073] 1) Take out the frozen lung tissue (lung tissue contains a lot of blood, so it needs to be fully irrigated with PBS and stored at -80℃), thaw on ice, weigh about 20 mg of lung tissue and put it into an enzyme-free EP tube / smooth muscle cells are taken out of the incubator and gently washed three times with PBS.

[0074] 2) Add 200uL of lysis buffer to the EP tube, add protease inhibitors and phosphatase inhibitors in proportion, cut the cells with ophthalmic scissors on ice, and then homogenize them on ice with a tissue homogenizer / add 100uL of lysis buffer to the culture dish, scrape the cells to one side of the culture dish on ice with a scraper, transfer them to the EP tube, and break them with an ultrasonic machine on ice.

[0075] 3) Precool the centrifuge to 4°C, and then centrifuge the sample at 12,000 rpm for 40-50 min.

[0076] 4) Transfer the supernatant to a new 1.5 mL EP tube, mark it, and remove the precipitate.

[0077] 5) Quantify the BCA protein concentration of the supernatant. After the concentration is measured, the prepared lysis buffer is used as a supplement to adjust the concentration of each group of samples to the same concentration. Then, 1 / 4 of the volume of 5x loading buffer is added to each sample according to its volume. After mixing with a vortex oscillator, place it in a 100°C metal bath for 10 minutes.

[0078] 6) After heating, take out the sample and place it in ice to cool down, then divide it into portions and store it in a -80℃ refrigerator.

[0079] Step 2: Tissue protein quantification

[0080] 1) First, prepare the protein standard according to the instructions: pipette 2mL PBS into an EP tube containing 20mg BSA, vortex thoroughly to dissolve and prepare a 10mg / mL protein stock solution. Then dilute the stock solution with PBS to prepare protein standards with final concentrations of (0, 0.025, 0.125, 0.25, 0.5, 0.75, 1.0, 1.5mg / mL).

[0081] 2) Prepare 200 L AB working solution (A:B=50:1) per well according to the number of samples and labeled product.

[0082] 3) Take a 96-well plate, pipette 5uL of standard into each well according to the concentration, and then fill it up to 20p with PBS solution.

[0083] 4) Similarly, take 5uL of the sample to be tested into a new well, set up multiple wells for quantification and take the average value as the concentration result. After all samples are added, add 20L of PBS solution to each sample well.

[0084] 5) Pipette 200L of mixed BCA working solution and add it to each well, avoiding bubbles as much as possible, and then place it in a 37℃ incubator for 30 minutes.

[0085] 6) Finally, use an ELISA reader to measure the absorbance at 560 nm. Draw a standard curve equation based on the known concentration of the standard and the corresponding absorbance, and input the absorbance of the sample to be tested to calculate the sample concentration.

[0086] Step 3: Western Blot

[0087] According to the known molecular weight of the protein, the required gel concentration can be selected by referring to the table below.

[0088] Protein molecular weight (kDa) Gel concentration (%) 12-45 15 10-70 12 15-100 10 25-200 8 45-250 6

[0089] According to the required electrophoresis gel concentration, prepare acrylamide separation and concentration gel according to the ratio in the table below.

[0090] Prepare different concentrations of Tris-glycine SDS for acrylamide electrophoresis separation gel

[0091] Concentration (total volume 20mL) 6％ 8％ 10％ 12％ 15％ dHO 10.6 9.3 7.9 6.6 4.6 30% acrylamide mixed solution 4.0 5.3 6.7 8.0 10.0 1.5MTris(pH8.8) 5.0 5.0 5.0 5.0 5.0 10% SDS 0.2 0.2 0.2 0.2 0.2 10% ammonium persulfate 0.2 0.2 0.2 0.2 0.2 TEMED 0.016 0.012 0.008 0.008 0.008

[0092] Preparation of Tris-Glycine SDs without Acrylamide Electrophoresis 5% Stacking Gel

[0093] Total 6mL 15mL ddH20 4.1L 10.2mL 30% acrylamide mixed solution 1mL 2.53mL 1.0MTris-HCl(pH6.8) 0.75mL 1.88mL 10% SDS 0.06mL 0.15mL 10% ammonium persulfate 0.06mL 0.15mL TEMED 0.006mL 0.015mL

[0094] 1) Clean and dry the glass plate, assemble the glue-making glass plate and perform leak detection.

[0095] 2) Prepare separation gel of corresponding concentration according to the formula in the table above and the size of the protein to be tested.

[0096] 3) Use a 1 mL pipette to carefully pour the prepared separation gel between the two glass plates, and stop pouring the gel about 1.5 cm away from the upper edge of the short glass.

[0097] 4) Use the gun tip to absorb anhydrous ethanol or isopropanol, and slowly add it to the sealing liquid in the glass plate. After it solidifies at room temperature for 30 minutes, a clear dividing line can be seen between the separation glue and the upper sealing liquid. Then pour in the sealing layer and absorb the excess sealing liquid with filter paper.

[0098] 5) Prepare concentrated gel according to the formula in the table above, pipette the corresponding components into a clean 50 mL tube in order, mix well, add TEMED and mix, and immediately pour the upper layer until it is full.

[0099] 6) Quickly insert the sample comb rinsed with deionized water into the glass plate from one side, making sure there are no bubbles, and let it stand at room temperature for 30 minutes.

[0100] 7) Take out the glass plate with the prepared gel from the frame, assemble the electrophoresis frame with it, put it into the electrophoresis tank, and pour the electrophoresis buffer into the inner and outer tanks. Slowly pull out the sample comb vertically, and gently blow each hole with a needle to remove the residual gel.

[0101] 8) Loading: Pipette a sample with a total protein content of 40-60ug and add it to the bottom of the loading well according to the experimental design grouping. Add protein markers as indicators to the leftmost and rightmost wells.

[0102] 9) Electrophoresis: Turn on the electrophoresis power supply and start electrophoresis at a constant voltage of 60V until the sample bromophenol blue enters the separation gel interface, at which time the protein marker also has different color layers. Then adjust the voltage to a constant voltage of 110V and continue electrophoresis until the bromophenol blue runs to the bottom.

[0103] 10) Transfer: Cut the PVDF membrane into pieces of about 6.5 cm*8.5 cm, and then soak the membrane in methanol for 1 min to activate the membrane. Then immerse it in pre-cooled transfer buffer for equilibrium.

[0104] 11) Turn off electrophoresis, open the electrophoresis tank, and remove the glass plate after running the gel. Insert the glued plastic sheet between the two glued glass plates, gently pry open the short glass plate from one side, use the plastic sheet to cut off the upper glue, keep the separation gel, and then carefully transfer it to the filter paper soaked in the pre-cooled transfer buffer.

[0105] 12) Immerse the transfer clip in pre-cooled transfer buffer, open it, and assemble it into a "sandwich" structure in the order of black clip - filter screen - filter paper - filter paper - separation gel - PVDF membrane - filter paper - filter screen - white clip, and carefully remove the bubbles between the separation gel and PVDF membrane.

[0106] 13) After assembling the transfer splint, place it in the transfer tank, pay attention to "black to black, white to red", add pre-cooled transfer buffer to the tank, put it in the freezer, turn on the power, and rotate the membrane at a constant current of 400mA for 2h. The entire transfer tank is buried in an ice-water mixture to keep it low temperature.

[0107] 14) After the transfer is completed, quickly take out the PVDF membrane, soak it in TBST solution, and gently shake it on a horizontal shaker to wash it for 5 minutes.

[0108] 15) Prepare 5% milk blocking solution with TBST and skimmed milk powder, and soak the PVDF membrane in a shaker at room temperature for 2 hours.

[0109] 16) After the blocking is completed, the milk blocking solution is recovered, and the PVDF membrane is rinsed on a shaker for 3 times with TBST solution, each time for 10 minutes, and the target band is cut according to the molecular weight indicated in the Marker manual. Each band is rolled into a centrifuge tube with the corresponding primary antibody solution diluted with 5% BSA.

[0110] 17) Place the centrifuge tube on a rotator and incubate at 4°C overnight.

[0111] 18) The next day, place the membrane incubated with primary antibody in TBST solution and wash it three times on a horizontal shaker, each time for 20 minutes.

[0112] 19) Then add the membrane to the prepared secondary antibody solution to achieve the same incubation formula and incubate at room temperature for 2 hours.

[0113] 20) Place the membrane incubated with secondary antibody in TBST and shake on a horizontal shaker 3 times for 20 minutes each time.

[0114] 21) Move the cleaned membrane to a dark room and avoid light. Use ECL colorimetry to prepare a colorimetric solution with a ratio of 1:1 between solution A and solution B, soak the strips, and then expose, develop, and fix. Finally, scan the film with a scanner and perform statistical analysis of the grayscale value using ImageJ software.

[0115] In the present invention, the steps of pulmonary angiography are as follows:

[0116] After the mice were anesthetized, they were fixed and roughly disinfected with 75% alcohol. The skin was cut upside down from the lower abdomen with a "T" shape to avoid bleeding as much as possible. The same method was to cut the peritoneum and chest cavity to fully expose the heart, lungs, liver and other organs. The inferior vena cava below the liver was removed, and the mouse's liver was carefully removed to separate the lungs and heart from the liver. 0.1mL of heparin was injected into the heart from the toe, and a live knot was made around the root of the pulmonary artery with a thread. 10mL of PBS solution preheated at 37°C was drawn with a syringe and fixed on the injection pump. Adjust the speed of the injection pump, make a small incision at the entrance of the pulmonary artery into the right atrium, carefully place the perfusion fluid tube into the pulmonary artery through this incision, and then kill and fix the small black silk thread before so that the tube mouth cannot move at will. Then make a small incision at the apex of the heart to ensure that all the perfusion fluid is pumped into the pulmonary artery instead of flowing into the heart. Turn on the injection pump, PBS begins to slowly pump into the pulmonary artery, and the lungs begin to slowly turn white. Prepare perfusion contrast solution: add MV coating agent (67.5) to the premixed perfusion solution compound (yellow, 600L) + diluent (750JL) shaker, and then use a 10mL syringe to draw 1mL of perfusion solution. Carefully replace the syringe: separate the 10mL syringe originally filled with PBS from the perfusion needle, and then connect the 10mL syringe filled with 0.7mL contrast solution. Re-adjust the speed of the syringe pump and empty the bubbles generated by the replacement syringe. Accurately record the total perfusion volume of the perfusion contrast solution: under the microscope, the yellow perfusion solution immediately begins to reach the pulmonary artery, and start recording the time. When the terminal pulmonary blood vessels are fully stained, stop pumping immediately, record the time to stop, centrifuge the total perfusion volume according to the infusion pump speed, add a certain amount of PBS to rinse the skin of the lungs, heart and other organs, mark the mouse number, and store in a 4C refrigerator for 2 hours. After 2 hours, separate the heart, left lung and right lung, and place the lung tissue in a 15mL tube.

[0117] 10 mL of PBS was added to each tube, and then mixed on a shaker in a refrigerator at 4°C overnight. The next day, after gradual dehydration with different concentrations of alcohol, 10 mL of methyl salicylate was added to each tube, and the lungs were photographed after gently shaking at room temperature overnight.

[0118] In the present invention, the CCK8 experimental steps are as follows:

[0119] I) PASMC cells transfected with adenovirus were inoculated into 96-well plates (3 samples*6 replicate wells) and cultured overnight with 200ul culture medium.

[0120] 2) When the cells are 70%-80% confluent, place them in a normoxic / hypoxic incubator.

[0121] 3) Aspirate the culture medium and wash with PBS three times.

[0122] 4) Add 100ul of 10% CCK8 working solution prepared with basal culture medium to each well, and keep it away from light.

[0123] 5) The absorbance at a wavelength of 450 nm was measured every half hour using an ELISA reader, and statistical analysis and graphing were performed using Microsoft Excel 2010.

[0124] In the present invention, the Transwell experiment is as follows:

[0125] Before inoculation, cells were transfected with adenovirus, and the specific steps were as follows.

[0126] 1) Inoculation of cells: Resuspend PASMC in serum-free DMEM culture medium to a cell density of 5x10 5 / mL, evenly add 200L of cell suspension to each Transwell chamber, and add 500uL of DMEM culture medium containing 20% ​​FBS to the lower chamber, and pay attention to remove bubbles.

[0127] 2) After routine overnight culture, the cells were allowed to become 60%-70% confluent and incubated in an oxygen-deficient incubator at 37°C, 5% CO2, and 1% 0.04°C for 48 hours.

[0128] 3) Remove the liquid from the upper and lower chambers, wash twice with PBS solution, and gently wipe off the cells in the upper chamber with a cotton swab.

[0129] 4) Fix the cells with 4% paraformaldehyde solution for 30 min, stain with 0.1% crystal violet for 10 min-20 min at room temperature, rinse with ultrapure water, randomly select 1-5 high-power fields (×400) under a microscope to take pictures, count the migrated cells in each Transwell chamber, and perform statistical analysis.

[0130] In the present invention, the adenovirus transfection method comprises the following steps:

[0131] Step 1: On the first day, inoculate the target cells in good condition into the culture dish. Ensure that the cell confluence is between 50% and 70% when infected on the second day;

[0132] Step 2: On the second day, preheat the culture medium, take out the virus, put it on ice to melt, and centrifuge it in a desktop centrifuge to make the virus liquid accumulate at the bottom of the tube; discard the culture medium, add the prepared virus to the serum-free culture medium, mix well, and add it to the culture dish;

[0133] Step 3: After 2 hours (shake evenly every 0.5 hours to increase infection efficiency), add the same amount of normal culture medium containing 10% FBS as in step 3;

[0134] Step 4: Replace with fresh normal culture medium after 12-24 hours;

[0135] Step 5: Observe fluorescence expression and perform related tests 48-72 hours after infection.

[0136] In the present invention: bmal1 overexpression adenovirus was ordered from Heyuan Biotechnology, gene name: Bmal1, gene ID: 11865, transcript: NM_001368412.1, species: Rat, gene size: 1899.

[0137] In summary: The application of the BMAL1 overexpression adenovirus in the treatment of pulmonary arterial hypertension, through the construction of a mouse model, illustrates that the BMAL1 overexpression adenovirus can effectively inhibit the proliferation and migration of pulmonary arterial smooth muscle cells under hypoxic conditions by regulating related signaling pathways, which helps to alleviate pulmonary vascular remodeling, and through the construction of a mouse model, explains that the BMAL1 overexpression adenovirus can reduce right heart pressure and alleviate pulmonary vascular damage, thereby improving the clinical symptoms of pulmonary hypertension, providing a new target for the treatment of pulmonary arterial hypertension: The present invention reveals the important role of BMAL1 overexpression adenovirus in pulmonary arterial hypertension, provides a potential target for the development of new treatment methods for the disease, and through in-depth exploration of the mechanism of action of BMAL1, helps to further understand the pathophysiological process of pulmonary arterial hypertension and promotes the in-depth development of research in this field. The application of the BMAL1 overexpression adenovirus in the treatment of pulmonary arterial hypertension has the advantages of high efficiency, accuracy and high sensitivity, and solves the problems mentioned in the background technology.

[0138] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0139] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A use of a BMAL1 overexpression adenovirus in the treatment of pulmonary arterial hypertension, characterized in that: The applications include: BMAL1 overexpression adenovirus can reduce the proliferation and migration of pulmonary artery smooth muscle cells in pulmonary hypertension, specifically: construct a BMAL1 overexpression adenovirus, and use transfection technology to construct BMAL1 overexpression in rat pulmonary artery smooth muscle cells, which includes the following steps: Step 1: CCK8 experiment was performed, which showed that after hypoxia, the proliferation of PASMCs (rat pulmonary artery smooth muscle cells) transfected with empty adenovirus and overexpressing BMAL1 increased compared with normoxic PASMCs, while after hypoxia, the proliferation of PASMCs transfected with overexpressing BMAL1 decreased compared with PASMCs transfected with empty adenovirus; Step 2: Perform transwell chamber experiments and set up three experimental groups, namely, PASMC transfected with empty adenovirus and placed in a normoxic incubator, PASMC transfected with empty adenovirus and placed in a hypoxic incubator, and PASMC transfected with BMAL1 overexpressing adenovirus and placed in a hypoxic incubator. The migration ability of rat pulmonary artery smooth muscle cells in the BMAL1 overexpressing adenovirus after hypoxia treatment was reduced compared with that in the control group, while the migration ability of these two groups was increased compared with that in the normoxic empty load group; Step 3: Extract the protein of rat pulmonary artery smooth muscle cells for Western Blot experiment: BMAL1 was significantly downregulated after hypoxia treatment of pulmonary artery smooth muscle cells, and the BMAL1 protein level of pulmonary artery smooth muscle cells transfected with BMAL1 overexpression adenovirus was significantly upregulated; BMAL1 overexpression adenovirus can alleviate pulmonary vascular remodeling in mice with pulmonary hypertension, specifically: constructing a mouse pulmonary hypertension disease model by combining hypoxia with SU5416, and constructing a mouse pulmonary vascular BMAL1 overexpression model by nasal drops, which includes the following steps: Step 1: Select 6-8 week old c57 mice and randomly divide them into three groups, namely control group (no-load adenovirus nasal drops under normoxic condition), pulmonary hypertension disease group (no-load adenovirus nasal drops under hypoxia condition), BMAL1 overexpression treatment group (BMAL1 overexpression adenovirus nasal drops under hypoxia condition). The first two groups were given 20ul no-load adenovirus nasal drops every week, and the third group was given 20ul BMAL1 overexpression adenovirus nasal drops every week. The mice in the control group were raised in a normoxic environment, and the mice in the disease group and treatment group were raised in a 10% oxygen environment, and 10mg / kg of Sugen5416 was subcutaneously injected every week. Subsequent experimental studies were conducted after 4-5 weeks. Step 2: The mice in step 1 were subjected to right cardiac catheter pressure measurement, and the conclusion was that the right cardiac pressure of mice in the pulmonary hypertension disease group (nasal drops of empty adenovirus under hypoxia) was higher than that in the control group (nasal drops of empty adenovirus under normoxic condition), and the right cardiac pressure of mice in the BMAL1 overexpression treatment group (nasal drops of BMAL1 overexpression adenovirus under hypoxia) was lower than that in the pulmonary hypertension group; Step 3: Perform pulmonary angiography on the mice in step 1; the pulmonary vascular damage in the mice in the pulmonary hypertension group was more severe than that in the control group, and the pulmonary vascular damage in the mice in the BMAL1 overexpression treatment group was less severe than that in the pulmonary hypertension group; Step 4: Extract lung tissue proteins from the mice in step 1 for Western Blot experiments to verify that the expression of BMAL1 protein in the lung tissue of mice in the pulmonary hypertension group was downregulated compared with that in the control group, and the expression of BMAL1 protein in the lung tissue of mice in the BMAL1 overexpression treatment group was significantly upregulated.

2. The use of a BMAL1 overexpressing adenovirus in the treatment of pulmonary hypertension according to claim 1, characterized in that: The research process of BMAL1 overexpression in the treatment of pulmonary hypertension is as follows: Step 1: Transfect the constructed adenovirus into the target cells (rat pulmonary artery smooth muscle cells) to achieve the introduction of BMAL1 gene; Step 2: Gene expression detection: Western blot was used to detect the expression level of BMAL1 protein; adenovirus was added with a fluorescent tag, and the transfection efficiency of BMAL1 could be evaluated by fluorescence microscopy; Step 3: Cell function experiments: after confirming BMAL1 overexpression, biological processes such as cell proliferation, differentiation, and apoptosis were studied; CCK-8 assay was used to detect changes in cell proliferation; Transwell chamber assay was used to evaluate the effects on cell migration and invasion abilities; Step 4: Animal model experiment: The constructed adenovirus is applied to the animal model through nasal infection, and the in vivo function of the BMAL1 gene is studied through local delivery to the pulmonary vasculature. The physiological changes and changes in histopathological characteristics of the animal model are observed, and the effects of BMAL1 on the disease in vivo and possible mechanisms of influence are evaluated.

3. The use of a BMAL1 overexpression adenovirus in pulmonary artery smooth muscle cells according to claim 1, characterized in that: The adenovirus transfection method comprises the following steps: Step 1: On the first day, inoculate the target cells in good condition into the culture dish. Ensure that the cell confluence is between 50% and 70% when infected on the second day; Step 2: On the second day, preheat the culture medium, take out the virus, put it on ice to melt, and centrifuge it in a desktop centrifuge to make the virus liquid accumulate at the bottom of the tube; discard the culture medium, add the prepared virus to the serum-free culture medium, mix well, and add it to the culture dish; Step 3: After 2 hours (shake evenly every 0.5 hours to increase infection efficiency), add the same amount of normal culture medium containing 10% FBS as in step 2; Step 4: Replace with fresh normal culture medium after 12-24 hours; Step 5: Observe fluorescence expression and perform related tests 48-72 hours after infection.

4. The use of a BMAL1 overexpressing adenovirus in the treatment of pulmonary hypertension according to claim 1, characterized in that: The bmal1 overexpression adenovirus was ordered from Heyuan Biotechnology, gene name: Bmal1, gene ID: 11865, transcript: NM_001368412.1, species: Rat, gene size: 1899.

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