Application of reagents for increasing DJ-1 gene expression in the preparation of anti-vascular calcification drugs

Abstract

The present invention discloses the use of a reagent for increasing the expression of the DJ-1 gene in the preparation of an anti-vascular calcification drug, and relates to the field of biomedicine technology. The nucleotide sequence of the DJ-1 gene is shown in SEQ ID NO.1. The present invention has found that increasing the expression of the DJ-1 gene can inhibit the expression of the ALPL, Runx2 and BMP2 genes, and can effectively alleviate vascular calcification, thereby effectively reducing the incidence of vascular calcification. The present invention provides a new technical means for preventing and treating vascular calcification, and has important clinical application value. The present invention also illustrates the mechanism of action of preventing and treating vascular calcification with the DJ-1 gene as the target, which provides strong technical support for screening drugs for anti-vascular calcification.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the use of a reagent for increasing the expression level of DJ-1 gene in the preparation of an anti-vascular calcification drug. Background Art

[0002] Vascular calcification (VC) is a common pathological change in which calcium and phosphate mineral crystals are deposited ectopically on the blood vessel wall. Vascular calcification greatly increases the incidence and mortality of cardiovascular diseases. According to the different parts of the blood vessel wall where calcification occurs, it is mainly divided into intimal calcification and medial calcification. Intimal calcification mainly occurs in atherosclerosis, while medial calcification often occurs in the development of diseases such as chronic kidney disease, aging, and diabetes. Vascular calcification leads to plaque instability, increased vascular stiffness, and reduced compliance, which makes adverse cardiovascular events such as cardiovascular and cerebrovascular embolism, hypertension, and heart failure more likely to occur.

[0003] Previous studies believed that vascular calcification is a passive degenerative process, but in recent years, a large number of studies have revealed that vascular calcification is a highly regulated and active biological osteogenesis process. The most important link is the transformation of vascular smooth muscle cells from a contractile phenotype to an osteogenic / chondrogenic phenotype, accompanied by the upregulation of chondrogenic / osteogenic transcription factors (ALPL, Runx2, BMP2). The pathogenesis of vascular calcification is still unclear, and currently no effective treatment for vascular calcification has been found in clinical practice. Therefore, further clarifying the mechanism of vascular calcification and finding effective intervention measures are a major issue that needs to be addressed in the current field of cardiovascular disease. Summary of the invention

[0004] The purpose of the present invention is to provide an agent for increasing the expression of the DJ-1 gene for use in the preparation of an anti-vascular calcification drug to solve the problems of the above-mentioned prior art. The present invention has found that increasing the expression of the DJ-1 gene can inhibit the expression of the ALPL, Runx2 and BMP2 genes, thereby effectively alleviating vascular calcification, thereby effectively reducing the incidence of vascular calcification.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides use of a reagent for increasing the expression level of a DJ-1 gene in the preparation of an anti-vascular calcification drug, wherein the nucleotide sequence of the DJ-1 gene is shown in SEQ ID NO.1.

[0007] Furthermore, the reagent is an adeno-associated virus that overexpresses the DJ-1 gene.

[0008] Furthermore, the method for constructing an adeno-associated virus that overexpresses the DJ-1 gene includes constructing the DJ-1 gene into an adeno-associated virus overexpression vector to obtain a recombinant plasmid, and then packaging and purifying the recombinant plasmid with an adeno-associated virus.

[0009] Furthermore, the adeno-associated virus overexpression vector is a pAV-SM22A-P2A-GFP vector.

[0010] The present invention also provides an anti-vascular calcification drug, the active ingredients of which include an agent for increasing the expression level of DJ-1 gene;

[0011] The nucleotide sequence of the DJ-1 gene is shown in SEQ ID NO.1.

[0012] Furthermore, the reagent is an adeno-associated virus that overexpresses the DJ-1 gene.

[0013] Furthermore, the method for constructing an adeno-associated virus that overexpresses the DJ-1 gene includes constructing the DJ-1 gene into an adeno-associated virus overexpression vector to obtain a recombinant plasmid, and then packaging and purifying the recombinant plasmid with an adeno-associated virus.

[0014] Furthermore, the adeno-associated virus overexpression vector is a pAV-SM22A-P2A-GFP vector.

[0015] Furthermore, the anti-vascular calcification drug also includes pharmaceutically acceptable excipients.

[0016] Furthermore, the anti-vascular calcification drug is in the form of an injection.

[0017] The present invention discloses the following technical effects:

[0018] The present invention has found that increasing the expression of DJ-1 gene can inhibit the expression of ALPL, Runx2 and BMP2 genes, thereby effectively alleviating vascular calcification, thereby effectively reducing the incidence of vascular calcification.

[0019] The present invention provides a new technical means for preventing and treating vascular calcification and has important clinical application value.

[0020] The present invention also clarifies the mechanism of preventing and treating vascular calcification by taking DJ-1 gene as the target, which provides strong technical support for screening drugs against vascular calcification. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 Western blot detection results (A) and expression level statistics (B) of the adeno-associated overexpression vector of DJ-1 gene; the Control group represents the mouse tail vein injection of empty adeno-associated virus AAV-NC; the AAV-DJ-1 group represents the mouse tail vein injection of DJ-1 overexpression adeno-associated virus AAV-DJ-1 (n=6);

[0023] Figure 2 Alizarin red staining shows the calcification of mouse primary vascular smooth muscle cells after 6 days of high Pi stimulation (n=3);

[0024] Figure 3 The graphs are observations of aortic calcification in mice; A is an image of whole aorta stained with Alizarin red; B is an image of aorta section stained with Alizarin red and VON Kossa; C is an image of aortic calcium content detection; non-CKD group means mice received sham surgery; CKD group means chronic kidney disease mouse model group (n=3);

[0025] Figure 4 The results of Western blot detection of ALPL, Runx2, BMP2 and DJ-1 protein expression in primary mouse vascular smooth muscle cells; A is the Western blot detection result; BE are statistical graphs of ALPL, Runx2, BMP2 and DJ-1 protein expression, respectively; GAPDH is the internal reference protein; AAV-NC group represents mouse primary vascular smooth muscle cells infected with the control empty adeno-associated virus AAV-NC; AAV-DJ-1 group represents mouse primary vascular smooth muscle cells infected with DJ-1 overexpressing adeno-associated virus AAV-DJ-1 (n=6). DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] Example 1 Adeno-associated virus packaging of mouse DJ-1 gene

[0032] PCR primers were designed based on the CDS sequence of the mouse DJ-1 gene (NM_020569.3). The kozak protection sequence GCCACC was added before the CDS sequence ATG, and the Flag tag was fused to the C-terminus. At the same time, AsisI / MluI restriction sites were introduced. The CDS sequence of the mouse DJ-1 gene was constructed into the adeno-associated virus (AAV) overexpression vector pAV-SM22A-P2A-GFP (purchased from Shandong Weizhen Biotechnology Co., Ltd.), and the correct recombinant plasmid was confirmed by sequencing. After that, the correct recombinant plasmid was transformed into Escherichia coli DH5α competent cells, the recombinant plasmid was extracted, and the expression of the DJ-1 gene was verified by Western blot, and the positive clones were identified (the results are shown in Figure 1 ), after sequencing verification, the correct recombinant plasmid was extracted and sent to a biological company for virus packaging and purification. The purified adeno-associated virus was named AAV-DJ-1, and the virus titer of the virus liquid was 1.22×10 14vectorgenome(VG) / mL.

[0033] At the same time, empty plasmid was transfected to construct empty adeno-associated virus AAV-GFP as a control.

[0034] DJ-1 gene (NM_020569.3) CDS region sequence (SEQ ID NO.1):

[0035] .

[0036] The specific construction steps of adeno-associated virus AAV-DJ-1 include:

[0037] 1. Enzyme Digestion

[0038] Add the samples as shown in Table 1, mix well, and place at 37°C for 1 hour for enzyme digestion. After the reaction is completed, use 1% agarose gel electrophoresis to detect the size of the target enzyme digestion band, and use a gel recovery kit to recover the target gene fragment.

[0039] Table 1 Enzyme digestion system of plasmid containing mouse DJ-1 gene

[0040] Reaction liquid composition volume DNA fragments (0.1 μg / μL) 10μL 10×Buffer 3μL AsisI 1μL MqI 1μL <![CDATA[ddH2O]]> 15μL Total 30μL

[0041] The adeno-associated virus vector pAV-SM22A-P2A-GFP plasmid was digested with the enzyme digestion system shown in Table 2, and then the vector was recovered by gel.

[0042] Table 2 Enzyme digestion system of adeno-associated virus vector

[0043] Reaction liquid composition volume Carrier (0.5 μg / μL) 4μL 10×Buffer 5μL AsisI 1.5μL MqI 1.5μL <![CDATA[ddH2O]]> 38μL Total 50μL

[0044] 2. Connect

[0045] The recovered target gene fragment was connected to the pAV-SM22A-P2A-GFP vector that had been similarly double-digested. The connection system is shown in Table 3:

[0046] Table 3 Connection system

[0047] Element volume Target gene fragment 4μL Vector fragment 4μL 10×T4Buffer 1μL T4 DNA ligase (10U / μL) 1μL Total 10μL

[0048] Add samples as shown in Table 3, mix thoroughly, and centrifuge microcentrifuge. Connect at 22°C for 1 h.

[0049] 3. Conversion

[0050] The ligation products were transformed into E. coli DH5α competent cells, and coated on LB plates with corresponding resistance for screening. The specific steps of transformation are as follows:

[0051] Take out the DH5a competent cells prepared in advance from -80°C and place them in an ice bath.

[0052] After the DH5a competent cells melted, take 5 μL of the ligation product into 20 μL of DH5a competent cells, mix thoroughly, and let stand in an ice bath for 15 min.

[0053] Place the centrifuge tube in a 42°C water bath for 40 seconds (do not shake the centrifuge tube during this period), then quickly move it to an ice bath and let it stand for 2 minutes.

[0054] Add 200 μL of sterile LB medium (without antibiotics) to the centrifuge tube, mix well, and place in a shaker at 37°C, 220 rpm, and shake for 1 hour. The purpose is to express the relevant resistance marker gene on the plasmid and resuscitate the bacteria.

[0055] Spread onto solid culture medium plates with corresponding resistance and culture in a 37°C incubator overnight.

[0056] 4. Sequencing

[0057] After picking a single colony for culture, the plasmid was extracted and the positive clone was identified by restriction digestion. After sequencing verification, the correct recombinant plasmid was extracted and verified for use in downstream experiments.

[0058] 5. Adeno-associated virus packaging

[0059] 5.1 Cell recovery

[0060] 1) Add 10 mL of fresh DMEM medium to a 10 cm culture dish and preheat to 37°C in an incubator.

[0061] 2) Take out the HEK293T cell cryovial from liquid nitrogen and quickly put it into a 37-38°C water bath to thaw it.

[0062] 3) After the solution in the HEK293T cell cryovial melts, centrifuge at 200 g for 5 min, discard the liquid, aspirate 1 mL of preheated culture medium, gently blow up the cell pellet, and transfer it to a 10 cm culture dish.

[0063] 4) Shake well and place in an incubator for cultivation.

[0064] 5) Culture overnight and replace with fresh culture medium (to remove the toxic effect of DMSO in the freezing medium on the cells).

[0065] 5.2 Cell passaging (taking 10 cm culture dish as an example)

[0066] 1) UV sterilize the biosafety cabinet for half an hour.

[0067] 2) During sterilization, preheat DMEM medium (containing 10% FBS and 1% penicillin-streptomycin) and PBS in a 37°C water bath; keep 0.25% trypsin at room temperature and do not heat it in a water bath.

[0068] 3) Take HEK293T cells with a confluence close to 100% and good activity, discard the culture medium in the culture dish, add 5 mL 1× PBS, shake a few times, and discard the PBS.

[0069] 4) Add 1 mL of 0.25% trypsin and digest for about 1 minute in a biosafety cabinet. When the room temperature is low, the cells can be placed in an incubator for digestion. The digestion time should not be too long, otherwise it will affect the efficiency and activity of cell reattachment.

[0070] 5) Add 5 mL of pre-warmed culture medium to terminate digestion.

[0071] 6) Use a pipette to evenly mix the cells and subculture them at a ratio of 1:3. Take 2 mL of the culture medium and transfer it to a new 10 cm culture dish, then add 8 mL of pre-warmed DMEM medium.

[0072] Note: When there are many subculture plates, add the preheated DMEM medium to the 10 cm culture dish first, and then add the medium containing cells to avoid uneven distribution of cells. Gently mix the medium in the culture dish before placing it in the incubator to evenly disperse the cells in the medium.

[0073] 5.3AAV-DJ-1 virus packaging (taking 10 cm culture dish as an example)

[0074] Day 1: HEK293T cells with a confluence of more than 90% were plated at a ratio of 1:3 (approximately 2.5×10 6 The culture medium was Hydone high-glucose DMEM medium (containing 10% FBS).

[0075] The next day: 1 h before transfection, change to serum-free medium.

[0076] Use Merck's transfection kit to prepare the transfection reagent according to the following ratio:

[0077] Mix 1: 500 μL of DMEM medium (without FBS) and 60 μL of 1 μg / μL TR;

[0078] Mix 2: 500 μL of DMEM medium (without FBS), 5 μg of LTR-plasmid, 6 μg of RC-plasmid and 10 μg of AAV-heiper.

[0079] Mix Mix 1 and Mix 2, keep at room temperature for 20 minutes, and then add dropwise to a 10 cm culture dish. (At this point, the cell confluence is preferably 80%-90%, too few cells will affect the transfection efficiency).

[0080] Day 3: 24 hours after recombinant plasmid transfection, replace with new serum-free medium.

[0081] Day 5: 72 hours after transfection, the virus-producing cells and the culture medium were collected into a 50 mL centrifuge tube, centrifuged, and the culture supernatant and cell pellet were harvested separately. The virus in the culture supernatant was precipitated with PEG8000; the cell pellet was lysed to collect the virus; and the AAV-DJ-1 virus solution obtained from the cell pellet and supernatant was combined.

[0082] 5.4 AAV virus purification and concentration

[0083] 5.4.1 Iodixanol density gradient centrifugation

[0084] ①Prepare different concentrations of iodixanol;

[0085] ②Take a supercentrifuge tube and slowly add different concentrations of iodixanol layer by layer using an electric pipette;

[0086] ③ Add the treated virus solution to the top layer;

[0087] ④After balancing, overspeed and centrifuge at 18℃ and 48000rpm for 2.5h.

[0088] 5.4.2 Concentration

[0089] ①After centrifugation, puncture the bottom of the ultrafiltration tube with a needle and collect the adeno-associated virus layer into a 15mL tube;

[0090] ②Inject the collected virus solution into the concentration column, add PBS + 0.001% PF68 to the top, and mix well;

[0091] ③4000rpm, 10℃, centrifuge for 1h;

[0092] ④ Repeatedly blow and aspirate the remaining liquid in the ultrafiltration tube into the virus storage tube, and finally add the virus storage solution, and mark the name and date;

[0093] ⑤ Vortex the collected viruses to mix well and then centrifuge them, and aspirate 10 μL of the virus solution for titer detection.

[0094] 5.5 Virus titer detection method

[0095] Real-time quantitative PCR is a simple, high-throughput method for determining the number of adeno-associated virus particles in purified virus samples. The Ct value of each template is linearly related to the logarithm of the starting copy number of the template. A standard curve can be made using a standard with a known starting copy number, and finally the unknown template can be quantitatively analyzed using the standard curve.

[0096] 5.5.1 Removal of free DNA molecules

[0097] First, dilute the virus 10 times to ensure that the free DNA in the sample is fully degraded: take 5 μL of virus dilution solution to 45 μL PBS buffer and mix thoroughly. Prepare Mixture according to the following system: 4 μL ultrapure water (DNase & RNase Free), 1 μL DNAasel, 1 μL 10× DNAasel Buffer and 4 μL of virus dilution solution, then incubate at 37°C for 30 minutes and heat at 95°C for 5 minutes to inactivate the DNase.

[0098] 5.5.2 Removal of viral protein coat

[0099] Add 1 μL of proteinase K (5 μg / μL) to the above-mentioned enzyme-inactivated Mixture system, and then incubate at 37°C for 30 min; then add 30 μL of ultrapure water to dilute to 40 μL (the virus stock solution is diluted 100 times), heat at 95°C for 5 min to inactivate proteinase K, then centrifuge at 12000 rpm for 2 min, and take the supernatant for qPCR detection.

[0100] 5.5.3qPCR

[0101] Take 5 μL of the supernatant obtained in 5.5.2 and perform a 10-fold gradient dilution, that is, the virus stock solution is diluted 1000 times. Take 2 μL of the sample to be tested and the standard respectively as templates for qPCR detection.

[0102] qPCR reaction system: 2×SYBR Green mix 10μL, F and R primer mixture 0.8μL, ultrapure water 7.2μL and test sample or standard 2μL.

[0103] qPCR reaction program: 95°C for 3 min; 95°C for 5 sec, 60°C for 15 sec, 72°C for 15 sec, and 40 cycles.

[0104] Example 2 Effect of DJ-1 gene-expressing adeno-associated virus on vascular calcification

[0105] 12-week-old male C57BL / 6 mice were divided into two groups, with 6 mice in each group, and the adeno-associated virus AAV-DJ-1 prepared in Example 1 and the empty adeno-associated virus AAV-NC as a control were injected into the tail vein respectively. The mice in the two groups and the treatment conditions were as follows:

[0106] Experimental group (AAV-DJ-1): 12-week-old C57BL / 6 mice were injected with adeno-associated virus AAV-DJ-1 at a dose of 5×10 11 vg / piece;

[0107] Control group: 12-week-old C57BL / 6 mice, injected with adeno-associated virus AAV-NC, dose 5×10 11 vg / piece;

[0108] On the 14th day after the adeno-associated virus injection, the aorta of the mice in the experimental group and the control group were taken to extract proteins and detect the expression of DJ-1. Figure 1 As shown. First, primary vascular smooth muscle cells were extracted from wild-type (WT) mice and transfected with control adenovirus (AAV-NC) and DJ-1 overexpressing adenovirus (AAV-DJ-1). Then phosphorus (Pi) was added to induce cell calcification. After 6 days, Alizarin red staining was performed to detect the cell calcification level, as shown in Figure 2. Figure 2As shown, after overexpression of DJ-1, the degree of cell calcification was significantly reduced. Furthermore, the two groups of mice were fasted for one day, the surgical instruments and operating table were disinfected, the mice were caught, and 1% sodium pentobarbital was injected intraperitoneally into the mice. After the mice were anesthetized, they were placed in a prone position, fixed, and the hair on the left side of their backs was shaved, and the area around the surgical incision was disinfected with iodine. The skin on the back of the mouse was cut open to expose the left kidney, and the upper and lower poles of the left kidney were ligated and removed. The incision was sutured, and after the mouse recovered for a week, the right kidney was ligated and completely removed in the same way to achieve 5 / 6 nephrectomy in the mouse. The mice in the sham control group underwent the same surgery, but the kidney tissue was not removed. After the mice recovered for a week, the mice in the CKD group were fed a high-phosphorus diet (1.5%) for 8 weeks, while the mice in the sham control group (non-CKD) were fed a standard laboratory diet. After the model was completed, the mouse aorta was taken. The calcification of the mouse aorta was observed as shown in the figure. Figure 3 As shown. Figure 3 It can be seen that after overexpression of mouse smooth muscle DJ-1, the overall calcification of mouse blood vessels was significantly reduced. After AAV-DJ-1 was used to overexpress mouse primary vascular smooth muscle cells DJ-1, cell proteins were extracted, and 40 μg of protein samples were taken for Western blot verification. The expression of DJ-1 protein and ALPL, Runx2, and BMP2 proteins were detected respectively. The results are shown in Figure 2. Figure 4 As shown. The results showed that overexpression of DJ-1 protein reduced the expression of ALPL, Runx2, and BMP2 proteins, thereby alleviating the calcification of vascular smooth muscle cells. It can be seen that the injection of adeno-associated virus AAV-DJ-1 into the tail vein of mice can significantly alleviate aortic calcification.

[0109] The above results confirm the key role of DJ-1 gene in inhibiting the expression of ALPL, Runx2 and BMP2 genes. By injecting DJ-1 gene adeno-associated virus into the tail vein of C57BL / 6 mice to overexpress DJ-1 gene, the degree of aortic calcification in mice can be reduced.

[0110] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of a reagent for increasing the expression of DJ-1 gene in the preparation of an anti-vascular calcification drug, characterized in that: The nucleotide sequence of the DJ-1 gene is shown in SEQ ID NO.1; The reagent is an adeno-associated virus that overexpresses the DJ-1 gene.

2. The use according to claim 1, characterized in that: The method for constructing an adeno-associated virus that overexpresses the DJ-1 gene comprises the steps of constructing the DJ-1 gene into an adeno-associated virus overexpression vector to obtain a recombinant plasmid, and then packaging and purifying the recombinant plasmid with an adeno-associated virus.

3. The use according to claim 2, characterized in that: The adeno-associated virus overexpression vector is a pAV-SM22A-P2A-GFP vector.

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