Application of reagent for detecting SNF5 gene expression in preparation of atherosclerosis diagnosis product
阅读说明：本技术 检测snf5基因表达的试剂在制备动脉粥样硬化诊断产品中的应用 (Application of reagent for detecting SNF5 gene expression in preparation of atherosclerosis diagnosis product ) 是由 姜怡邓 张慧萍 谢琳 马胜超 熊建团 张宏红 盛思琪 马飞 揭育祯 于 2021-04-22 设计创作，主要内容包括：本发明属于医学技术领域,具体涉及检测SNF5基因表达的试剂在制备动脉粥样硬化诊断产品中的应用。本发明通过检测SNF5在Hcy促进动脉粥样硬化炎症过程中的表达水平,在体外合成SNF5过表达慢病毒和SNF5小RNA干扰片段,研究其对动脉粥样硬化过程中IL-1β的影响,阐明了在Hcy促进动脉粥样硬化炎症过程中SNF5的作用。本发明涉及的SNF5在Hcy促进动脉粥样硬化炎症过程中具有重要作用,为以后从分子水平诊断动脉粥样硬化提供了理论依据,具有重大的理论意义和潜在的实用价值。该方法简单易行,容易推广。(The invention belongs to the technical field of medicine, and particularly relates to application of a reagent for detecting SNF5 gene expression in preparation of an atherosclerosis diagnosis product. The invention discloses a method for detecting the expression level of SNF5 in the process of promoting atherosclerosis by Hcy, synthesizing SNF5 overexpression lentivirus and SNF5 small RNA interference fragment in vitro, researching the influence of the SNF5 overexpression lentivirus on IL-1 beta in the process of promoting atherosclerosis, and clarifying the effect of SNF5 in the process of promoting atherosclerosis by Hcy. The SNF5 has an important role in promoting the inflammatory process of atherosclerosis by Hcy, provides a theoretical basis for diagnosing atherosclerosis from a molecular level in the future, and has important theoretical significance and potential practical value. The method is simple and easy to implement and easy to popularize.)
1. Application of a reagent for detecting SNF5 gene expression in preparing atherosclerosis diagnosis products.
2. The use of a reagent for detecting the expression of SNF5 according to claim 1 in the preparation of a product for diagnosing atherosclerosis, wherein the reagent comprises: the reagent for detecting the expression level of the SNF5 gene by qRT-PCR, western blot and immunofluorescence method.
3. The application of the reagent for detecting the expression of SNF5 in the preparation of the atherosclerosis diagnosis product according to claim 2, wherein the reagent for detecting the expression level of SNF5 gene by qRT-PCR at least comprises a pair of primers for specifically amplifying SNF5 gene.
4. The method for evaluating the efficacy of anti-atherosclerosis based on inflammatory response using SNF5 gene according to claim 1, comprising the steps of:
s1, treating human THP-1 mononuclear-derived macrophage by Hcy;
s2, adding the tested medicine into a culture system of human THP-1 mononuclear-derived macrophages treated by Hcy, and culturing for 48 h;
s3, detecting the following items as the evaluation indexes of the drug effect of the tested drug: mRNA and protein expression levels of SNF 5.
5. An anti-atherosclerosis drug based on inflammatory reaction prepared by using SNF5 gene of claim 1, wherein the drug comprises an inhibitor of SNF5 gene, and the inflammatory reaction proinflammatory cytokine is IL-1 beta.
6. The drug of claim 5, wherein the inhibitor is one of an RNA interference vector of SNF5 gene, a small RNA interference fragment, an antibody of SNF5 and other inhibitors capable of inhibiting expression of SNF 5.
Technical Field
The invention relates to the technical field of medicine, in particular to application of a reagent for detecting SNF5 gene expression in preparation of an atherosclerosis diagnosis product.
Background
Atherosclerosis (atherosclerosis) is a chronic systemic disease which is caused by factors such as injury or biochemical stimulation acting on the wall of an artery and relates to multiple cells, multiple factors and multiple links, mainly involves the large and middle arteries, and the affected vessel wall has lipid accumulation, extracellular matrix protein deposition and artery intimal calcification, so that the elasticity of the artery is reduced, the lumen is narrowed, arterial thrombosis can be formed and even broken, and the health of human beings is seriously damaged. In summary, the researchers' views about the pathogenesis of atherosclerosis include the theory of inflammatory response, the theory of smooth muscle clonal proliferation, the theory of vascular injury response, the theory of thrombus mosaicism, and other theories. There is a great deal of evidence to support the important role of inflammatory responses in the pathogenesis of atherosclerosis, however, the overall coordination of the specific links of inflammatory responses and their interaction mechanisms remain unexplained.
Atherosclerosis, one of the leading causes of death and serious disease in industrialized societies, is becoming a global health problem. Homocysteine (Hcy) is an independent risk factor for atherosclerosis, and it is involved in inflammatory responses through a synergistic effect of multiple mechanisms. The pathogenesis of atherosclerosis is related to inflammatory molecules, including the cytokines interleukin-1 beta (IL-1 beta), the cytokine interleukin 6(IL-6), and tumor necrosis factor-alpha (TNF-alpha). Of these, IL-1 β plays a key role in a wide range of inflammatory diseases, including atherosclerosis and its complications. However, the mechanism that causes elevated IL-1 β levels during the development and progression of atherosclerosis caused by Hcy is still unclear.
Switch/non-fermentable sucrose (SWI/SNF) is an evolutionarily conserved multi-subunit chromatin remodeling complex that regulates epigenetic structure and cellular properties. Recently, the role of the SWI/SNF complex in cancer has become more pronounced and has been identified as the chromatin-regulating complex that is most frequently mutated in cancer. Genomic sequencing of various cancer cells in humans has shown frequent mutations in SWI/SNF factors, particularly SNF5, SWI5 being one of the subunits of the SWI/SNF complex, playing a key role in regulating gene expression by controlling chromatin dynamics. As a potent, true tumor suppressor gene, numerous studies have shown mutations or deletions in SNF5 expression in a variety of cancers, including rhabdomyomas, prostate cancer, clear cell renal cell carcinoma, and colon cancer. In addition, studies have shown that SNF5 can regulate numerous genes associated with cancer. For example, SNF5 can regulate the cell cycle through RB-mediated repression of E2F target genes (including cyclin a and E2F 113). These findings collectively emphasize the cancer suppressing effect of SNF5 and illustrate the function of SWI/SNF in maintaining oncogene expression programs in human tumors. However, the role of SNF5 in the mechanism of atherosclerosis has not yet been elucidated.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide the application of the reagent for detecting the expression of the SNF5 gene in preparing an atherosclerosis diagnosis product.
Further, the reagent comprises: the reagent for detecting the expression level of the SNF5 gene by qRT-PCR, western blot and immunofluorescence method.
Furthermore, the reagent for detecting the expression level of the SNF5 gene by qRT-PCR at least comprises a pair of primers for specifically amplifying the SNF5 gene.
Another object of the present invention is to provide a method for evaluating an anti-atherosclerotic drug effect based on an inflammatory response using the SNF5 gene, comprising the steps of:
s1, treating human THP-1 mononuclear-derived macrophage by Hcy;
s2, adding the tested medicine into a culture system of human THP-1 mononuclear-derived macrophages treated by Hcy, and culturing for 48 h;
s3, detecting the following items as the evaluation indexes of the drug effect of the tested drug: and detecting the expression level of mRNA and protein of SNF 5.
The invention also aims to provide an anti-atherosclerosis drug based on inflammatory reaction, which is prepared by using the SNF5 gene, wherein the drug comprises an inhibitor of the SNF5 gene, and the inflammatory reaction proinflammatory cytokine is IL-1 beta.
Further, the inhibitor is one of an RNA interference vector of the SNF5 gene, a small RNA interference fragment, an antibody of SNF5 and other inhibitors capable of inhibiting the expression of SNF 5.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention discovers a new function of the SNF5 gene, namely that the SNF5 gene is highly expressed in atherosclerotic vascular plaques and can be used as a marker for atherosclerosis diagnosis.
2. Based on the function of the SNF5 gene in promoting atherosclerosis, the gene provides a target for developing and treating atherosclerosis.
3. The inhibitor of the SNF5 gene can be used for preparing medicaments for preventing and treating atherosclerosis.
Drawings
FIG. 1 shows the detection of ApoE by ELISA, respectively-/-The content of IL-1 beta in the serum of mice of different diet treatment groups,**P<0.01。
FIG. 2 shows that ELISA and qRT-PCR are used to detect IL-1 beta content (A) and mRNA expression (B) in human THP-1 monocyte-derived macrophage under Hcy action respectively,**P<0.01。
FIG. 3 ApoE visualization of qRT-PCR (A), western blot (B) and immunofluorescence staining (C)-/-Expression of SNF5 in blood vessels of mice in different diet-treated groups,**P<0.01。
FIG. 4 shows that qRT-PCR and western blot are used for respectively detecting human THP-1 single-nuclear source giantmRNA (A) and protein expression (B) of SNF5 in phagocytic cells,**P<0.01。
FIG. 5 shows that SNF5 overexpression lentivirus (LV-SNF5) and small RNA interference fragment (si-SNF5-1/2/3) are transfected in human THP-1 mononuclear-derived macrophage, respectively, qRT-PCR and western blot are used for detecting mRNA and protein expression of SNF5 respectively,*P<0.05，**P<0.01。
FIG. 6 shows IL-1. beta. content (A), mRNA expression (B) and protein expression (C) in human THP-1 monocyte-derived macrophages after transfection of an over-expressed SNF5 lentivirus (LV-SNF5),**P<0.01。
FIG. 7 shows IL-1. beta. content (A), mRNA expression (B) and protein expression (C) in human THP-1 monocyte-derived macrophages after transfection of SNF5 small RNA interference fragment (si-SNF5-1/2),*P<0.05，**P<0.01。
Detailed Description
The invention is described in detail below with reference to the figures and the specific embodiments, but the invention should not be construed as being limited thereto. The technical means used in the following examples are conventional means well known to those skilled in the art, and materials, reagents and the like used in the following examples can be commercially available unless otherwise specified.
Gene ID of the mouse SNF5 Gene involved in the following protocol: 6598 Gene ID of human SNF5 Gene: 20587.
the specific technical scheme of the invention is as follows:
(1) establishment of ApoE-/-Mouse atherosclerosis model: by random number method to convert ApoE-/-Mice were randomly assigned to the model control group (ApoE)-/-Mice + plain feed, a-control group, n ═ 10) and hyperhomocysteinemia group (ApoE)-/-Mice + homomethionine feed, HHcy group, n 10); a normal control group (C57BL/6J mice + general feed, N-control group, N-10) was also included.
(2) Blood is taken from eyeball, serum is separated, and ApoE is detected by ELISA-/-Serum IL-1. beta. content in mice of different diet-treated groups.
(3) Establishing a human THP-1 mononuclear-derived macrophage cell line, and respectively detecting the content of IL-1 beta and mRNA expression under the action of Hcy by ELISA and qRT-PCR.
(4) qRT-PCR, western blot and immunofluorescence were used to detect expression of SNF5 in mouse blood vessels and human THP-1 monocyte-derived macrophages, respectively.
(5) The over-expression of SNF5 lentivirus (LV-SNF5) and small RNA interference fragment (si-SNF5-1/2/3) were transfected in human THP-1 monocyte-derived macrophages, and the effect on IL-1 beta content and expression was examined. Wherein, the SNF5 lentivirus (LV-SNF5) is designed by Shanghai Jikai Gene GmbH, the vector is GV492, the target Gene Gene ID: 6598.
the sequence of the small RNA interference fragment (si-SNF5-1/2/3) is as follows:
si-SNF5-1:5’-GCUCCGAGGUGGGAAACUATT-3’，SEQ ID NO：5；
si-SNF5-2:5’-GCAACGAUGAGAAGUACAATT-3’，SEQ ID NO：6；
si-SNF5-3:5’-CCACCAUCGCAUACAGCAUTT-3’，SEQ ID NO：7。
the present invention further demonstrates the role of SNF5 in the atherosclerotic inflammatory process by the following experiments.
1 test subject
1.1 Experimental animals
Male ApoE-/-Mice were purchased from experimental animal technology ltd, viton, beijing.
1.2 cell lines
THP-1 cell line (human acute monocytic leukemia cells, ATCC)
2 instruments and equipment and experimental reagent
2.1 Primary reagents
Sterile cell culture flasks, pipettes, filters (Corning, usa); fetal bovine serum, RPMI 1640 medium (Gibco, usa); phorbol ester (Promega, usa); homocysteine (Sigma, usa); blood/cell/tissue gene RNA extraction reagent (Beijing Tiangen Biochemical technology Co., Ltd., China); reverse transcription kit (Takara, Japan); qRT-PCR mix (QIAGEN, Germany); SNF5 antibody (Abcam, usa); hypersensitive ECL luminescence solution, protein extraction and BCA kit (Kaiyi, China); PBS powder, mouse β -actin antibody, goat HRP-labeled secondary antibody (sequoia china, china); endogenous catalase blocking working solution, goat serum working solution and an anti-fluorescence quencher (China ); fluorescent secondary antibodies (Protein Tech, usa); ELISA kit (cloud clone, affluence); the SNF5 and GAPDH primers were synthesized by Biotechnology engineering (Shanghai) Ltd.
2.2 Main Instrument
Thermostated cell culture chambers (Thermo Fisher Scientific, usa); clean bench (Antai technologies, Inc., Suzhou, China); laser confocal fluorescence microscopy (Olympus, japan); model 5415D micro bench centrifuge (Eppendorf, Germany); precision electronic balances (Sartorius, germany); ice maker (AF10 SCOTSMAN, usa); fluorescent quantitative PCR instrument (Funglyn, shanghai); general PCR instruments (Bio-Rad, USA; Transmission Electron microscope (JEOL), micropipettor (Eppendorf, Germany), water purification instruments (Heal Force), full-automatic enzyme labeling instruments (Bio-TEK), constant temperature freezing microtomes (Leica), centrifuges (Thermo Fisher, Eppendorf, Dalong), vortex mixing instruments (Dalong), chemiluminescence gel imaging instruments, voltage-stabilized power supplies, electrophoresis instruments, electro-rotating instruments (BIO-RAD), constant temperature metal baths (Calud), negative pressure attractors (Rocker), and the like.
3 method
3.1 preparation of frozen section of mouse aortic root
Dropping proper amount of OCT embedding medium onto the tissue of aorta root, regulating tissue angle for subsequent operation, freezing in a freezing table, fixing on a support frame, regulating angle, removing excessive embedding medium, exposing tissue, slicing, observing under a mirror to ensure the cut plane to reach the position near the aorta root, slicing continuously in thickness of about 4 microns, sticking anti-dropping glass slide, and storing in a box at-80 deg.C.
3.2 immunofluorescence staining
1) Fixation of 20in RT with 4% Paraformaldehyde (cells) or 10% acetone (tissue)
2) PBS 5min X3 RT
3)0.5％Triton-X100 5min RT
4) Goat serum is sealed for 2h, and residual liquid RT is completely absorbed by filter paper
5) Add primary anti- (overnight) 4 deg.C
6) Rewarming for 30min RT
7) PBS 5min X3 RT
8) Adding secondary antibody, and keeping away from light at 1h 37 deg.C
9) PBS 5min X3 RT light avoidance
10) DAPI staining 5min RT dark
11) PBS 5min X3 RT light avoidance
12) Sealing the anti-fluorescence quencher and taking a picture under a confocal microscope for RT light protection
The whole operation process prevents the dry sheet.
3.3 fluorescent quantitative PCR (qRT-PCR) for detecting expression of SNF5
3.3.1 extraction of RNA
Extracting RNA according to the kit specification, taking out the frozen tissues and cells, placing the frozen tissues and cells on ice, adding 1ml of lysis solution RZ, homogenizing by using a homogenizer, and transferring the lysis sample into a 1.5ml RNase-free centrifuge tube; standing and cracking the sample for about 5min in a super clean bench (sterilized by ultraviolet irradiation for 20-30 min), and properly prolonging the time according to the cracking condition of the sample to completely separate the nucleic acid-protein compound; centrifuging at 4 deg.C and 12,000 rpm for 5min to remove protein, fat, etc., and transferring the supernatant into a new 1.5ml RNase-free centrifuge tube; adding 200 mul/sample of chloroform, oscillating for 15sec by a vortex oscillator, standing for 3min at room temperature, centrifuging at 12 ℃ and 000rpm for 10min at high speed at 4 ℃, wherein RNA is contained in a colorless aqueous phase, carefully sucking 400 mul of the colorless aqueous phase, and transferring to a new 1.5ml RNase-free centrifuge tube; slowly adding 200 μ l of anhydrous ethanol, slightly inverting the centrifuge tube to mix the colorless water phase and the anhydrous ethanol, transferring into an adsorption column, centrifuging at 12,000 rpm for 30sec (4 ℃), and pouring off the waste liquid; adding 500 μ l RD deproteinized solution into adsorption column, centrifuging at 4 deg.C and 12,000 rpm for 30sec, and pouring off waste liquid; adding 700 μ l RW rinse solution into adsorption column, standing at room temperature for 2min, centrifuging at 4 deg.C 12,000 rpm for 30sec, and discarding waste liquid for 2 times; centrifuging again for 2min (12 at 4 ℃ and 000rpm), taking the adsorption column out of the collection pipe, standing at normal temperature for 5-10 min, and removing the remaining rinsing liquid; preparing a new centrifugal tube without RNase with the volume of 1.5ml, marking, placing an air-dried adsorption column into the centrifugal tube, vertically suspending the centrifugal tube in the center of an adsorption membrane, dropwise adding 70 mu l of RNase-free water, standing the centrifugal tube at room temperature for 2min, and centrifuging the centrifugal tube at the speed of 12 ℃ and 000rpm for 2min to obtain RNA, wherein the RNA is stored at the temperature of-80 ℃ to prevent RNA degradation.
3.3.2 reverse transcription
This step was done in an ultra clean bench illuminated by uv light, using RNase-free tips during the operation, placing 200 μ l RNase-free centrifuge tubes on ice, and then adding the following ingredients in table 1 in sequence:
TABLE 1 reverse transcription System
After the above systems are mixed evenly, the mixture is instantaneously centrifuged to carry out RT reaction, and the reaction procedure is as follows: keeping the temperature at 37 ℃ for 15min, 85 ℃ for 5s and 4 ℃. The product after reaction is preserved at-20 ℃ in a short time and at-80 ℃ for a long time, and repeated freeze thawing is avoided.
3.3.3 qRT-PCR detection of expression of SNF5
The following systems in Table 2 were added to 200. mu.l RNase-free centrifuge tubes, respectively, in that order:
TABLE 2 qRT-PCR System
The sequence of the upstream primer for detecting the human SNF5 is shown as SEQ ID NO: 1, the sequence of the downstream primer is shown as SEQ ID NO: 2, the sequence of the upstream primer for detecting the mouse SNF5 is shown as SEQ IDNO: 3, the sequence of the downstream primer is shown as SEQ ID NO: 4, respectively. After all samples are added, vortex oscillation and uniform mixing are carried out, the samples are placed into a fluorescent quantitative PCR instrument for reaction after instantaneous centrifugation, and the reaction procedure is as follows: the reaction was run for 45 cycles at 95 ℃ for 2min, 95 ℃ for 5s, 60 ℃ for 10s, and GAPDH was used as an internal control. According to As a result, the SNF5 primer was synthesized by Shanghai's engineering design.
3.4 enzyme-linked immunosorbent assay (ELISA)
Preparing a standard substance, preparing a reagent, and treating a sample to be detected; sealing plates after sample adding, and incubating for 1h at 37 ℃; plate throwing, adding detection solution A, and incubating at 37 ℃ in an incubator for 1 h; washing with washing solution for 3 times; adding detection solution B, and incubating at 37 deg.C for 30 min; washing with washing solution for 5 times; adding TMB substrate, incubating at 37 deg.C for 10 min; adding stop solution, and detecting by an enzyme-linked immunosorbent assay (450 nm).
3.5 viral infection
24 hours before infection, the cells are re-inoculated into a new culture bottle according to a certain concentration, and the confluence degree of the cells can reach about 70 percent on the next day; sucking off the original culture medium in the culture bottle, and adding PBS for washing twice; adding 2ml of serum-free culture medium again, adding 15 μ l of each of negative control (Lv-GFP) and over-expression SNF5 lentivirus (Lv-SNF5) (virus infection conditions are determined by experiment in the early stage), shaking the flask gently to mix well, placing at constant temperature of 37 deg.C and 5% CO2After culturing for 6 hours in the cell incubator, changing the liquid; the infection efficiency was observed under an inverted fluorescence microscope after 48 hours. After puromycin screening, after infection efficiency is observed under a mirror to meet requirements, cells are collected for subsequent experimental operation.
3.6 transfection of Small RNA-infected fragments
Inoculating the cells into a new culture bottle again according to a certain concentration 24 hours before infection, and ensuring that the confluence degree of the cells can reach about 70% in the next day;
adding 200pmol siRNA into 50 mul serum-free 1640 culture medium according to the proportion, and softly and uniformly mixing;
③ mixing the Lipofectamin reagent evenly, diluting 1 mul Lipofectamin reagent with 50 mul serum-free 1640 culture medium, mixing evenly gently, and standing for 5min at room temperature;
mixing the diluted siRNA and the Lipofectamin reagent uniformly, and standing at room temperature for 20min to form an siRNA/Lipofectamin mixture;
fifthly, adding the mixed siRNA/Lipofectamin mixture into a culture bottle containing cells and a culture medium, and shaking the cell culture bottle back and forth gently;
sixthly, the cells are in CO2After incubation at 37 ℃ for 48h in the incubator, Hcy intervention was performed and cells were collected for subsequent experiments.
3.7 Western Blot
(1) The experimental principle is established on the basis of molecular biology, biochemistry and immunogenetics, a cell or biological tissue sample treated by gel electrophoresis is stained by a specific antibody, and a protein stained on polyacrylamide gel is transferred onto a nitrocellulose membrane and then incubated with a first antibody. Bound antibodies were then detected with horseradish peroxidase-conjugated goat secondary IgG. At room temperature for 2h or 4 ℃ overnight.
(2) Experimental procedure
Experimental preparation: cleaning thin and thick glass plates used for glue preparation with purified water, placing the glass plates into an oven at 60 ℃ for baking until no water or water stain exists on the surfaces of the glass plates, assembling a thin plate and a thick plate by using a glue preparation frame, and preparing gel after confirming that the glue preparation plate is clamped without errors (preventing glue leakage caused by no sealing of the glue preparation plate).
Preparation of SDS-PAGE gel: SDS-PAGE gels of optimal concentration were selected based on the molecular weight of the known target gene, as shown in Table 3.
TABLE 3 SDS-PAGE gel preparation table (1.5 mm/block)
Preparing gel according to the formula proportion, respectively adding the corresponding amount of liquid, fully and uniformly mixing by using a Pasteur pipette, and fully and uniformly mixing after adding the corresponding volume of liquid. Slowly adding the uniformly mixed separation glue liquid to the indication position of the glue preparation plate, then adding a proper amount of distilled water at a constant speed, so as to discharge redundant bubbles, flattening the separation glue, standing the glue preparation frame when a visible obvious boundary line between the separation glue and the distilled water is completely solidified, allowing the distilled water to flow out, and sucking the residual water by using filter paper. Then, uniformly mixing the concentrated glue liquid in a corresponding proportion, slowly adding the mixed liquid into the separation glue, immediately and quickly inserting a clean comb into the concentrated glue, and paying attention to the fact that no air bubbles exist while inserting the comb.
③ protein sample loading and electrophoresis
a. Two prepared gels are assembled with an electrophoresis tank frame and put into an electrophoresis tank (note that the electrode of the electrophoresis tank frame and the electrode of the electrophoresis tank need to correspond (black corresponds to black, and red corresponds to red) but not to be reversed (positive alignment, negative alignment to negative), a sufficient amount of electrophoresis liquid is added into the electrophoresis tank, and 3 mul of protein Marker and 10 mul of corresponding group of protein samples are sequentially added into the hole.
b. Electrophoresis: after the 80V voltage allowed the protein sample to run through approximately the demarcation point of the concentrating and separating gels, the voltage was raised to 120V and then electrophoresed until the Marker bromophenol blue fraction ran to the green line at the bottom of the gel plate, and the electrophoresis was immediately stopped.
Fourthly, rotating the membrane
a. Preparing a film transferring article: PVDF membrane (0.22 μm or 0.45 μm) of appropriate molecular size was selected, and thick filter paper, absolute ethanol, milk blocking solution, etc. were prepared.
b. Cutting a PVDF membrane with a proper size (marking is done so as to identify genes with different molecular weights), soaking in absolute ethyl alcohol for 5min in advance, placing in an electrotransfer solution before use, soaking for 3min, and soaking the prepared filter paper in the electrotransfer solution in advance until no dry or wet interlayer exists in the filter paper for later use.
c. Slowly taking out the electrophoresis tank, detaching the glass plate, slightly prying the glass plate by using a gel cutting plate, removing redundant gel according to Marker prompt, putting the needed gel into electric transfer liquid prepared in advance, sequentially from the positive pole, namely a sponge gasket → a thick filter paper layer → a PVDF film → a sample glue → a thick filter paper layer → a sponge gasket (note: the film and the glue are rolled flat by using a roller, no air bubble can be arranged in the middle of each layer), fastening the film transfer clamping plate, putting the electrophoresis tank into an electrophoresis tank, fully adding the electric transfer liquid, putting the electrophoresis tank into a 4 ℃ refrigerator, and carrying out 0.30A constant current electric transfer for 120 min.
Sealing: after two hours, the electrotransformation is finished, the PVDF membrane is taken out, and the membrane is sealed for about 4 hours in 5 percent pre-prepared skimmed milk powder.
Sixthly, incubation of primary antibody: PBST washing PVDF membrane, 10 min/times x 3. Primary antibody was prepared using PBST, and the dilution was poured into antibody incubators (the dilution must completely cover the PVDF membrane) on a horizontal shaker at 4 ℃ overnight, according to the instructions for dilution.
Seventhly, incubation of secondary antibodies: the overnight incubated primary antibody was recovered and the PBST washed with PVDF membrane for 10 min/times X3 (in order to wash away the unbound primary antibody completely). The secondary antibody is prepared according to the dilution ratio recommended by the antibody specification (the dilution ratio is usually 1:5000), and the corresponding secondary antibody of the mouse source/rabbit source is added into the antibody incubation box after being prepared and incubated for 2 hours at room temperature.
Exposure and gel image analysis: opening an imager for preheating half an hour in advance, operating according to a program and opening Image Lab software; the antibody was recovered according to the mass of the secondary antibody (generally three times), and PBST was washed 10 min/times.3. Preparing ECL luminous liquid according to the proportion of A to B being 1 to 1, fully and uniformly mixing, keeping out of the sun for standby, flatly placing the washed PVDF membrane on an exposure plate (one surface of the binding protein faces upwards), uniformly dripping the prepared luminous liquid on the PVDF membrane by using a pipettor, and immediately exposing (note that the whole exposure process needs to be carried out in the absence of the sun).
Ninthly, after exposure is finished, analyzing the exposure result by using a Bio-Rad 5.0 gel imager, exporting the gray value obtained by analysis by using an Excel table, counting, namely inputting the ratio of the gray value corresponding to the target protein of each lane to the gray value corresponding to the reference protein in the lane into Prism 5.0 software to draw a statistical chart and analyzing.
4. Statistical treatment
All data types are quantitative data, and the statistical results are in terms of mean ± standard deviationThe mean of two samples was tested using Student's t, the mean of multiple samples was tested using One-way ANOVA, the mean-Newman-Keuls was used for pairwise comparisons between groups, and the test level was found to be 0.05.
5 results
5.1 content of IL-1. beta. in serum of mice of different diet-treated groups
ELISA detection of ApoE-/-The serum IL-1. beta. content of the mice in the different diet-treated groups was shown in FIG. 1: compared with A-control group mice, the content of IL-1 beta in the serum of HHcy group mice is obviously increased (P)<0.01), the difference was significant.
5.2 Effect of Hcy on IL-1 β expression in macrophages
To further confirm the effect of Hcy on IL-1 β expression, after THP-1 monocyte-derived macrophages were treated with Hcy for 48h, ELISA and qRT-PCR were used to detect IL-1 β content and expression in macrophages. The results are shown in FIG. 2: compared with the control group, the IL-1 beta content and mRNA expression level of the Hcy group are obviously increased (P <0.01), and the difference is significant.
5.3 ApoE-/-Expression of SNF5 in atherosclerotic plaques in mice
Separately detecting ApoE by qRT-PCR and western blot-/-The mRNA and protein expression levels of SNF5 in mouse blood vessels are shown in fig. 3: the expression of SNF5 was significantly increased in HHcy group mice as compared with A-control group mice (P)<0.01), the difference was significant. Subsequently, SNF5 and MOMA-2 immunofluorescent staining is carried out on the aortic root section of the mouse, and the result shows that the expression of SNF5 in the macrophage of the aortic plaque of the mouse is obviously increased.
5.4 Hcy promotes expression of SNF5 in THP-1 monocyte-derived macrophages
After THP-1 mononuclear-derived macrophages are treated by Hcy, the expression of SNF5 is respectively detected by qRT-PCR and western blot, and the result is shown in figure 4: the mRNA and protein levels of SNF5 were significantly increased in macrophages after Hcy treatment (P <0.01), with the difference being significant.
5.5 validation of the efficiency of the SNF5 overexpression vector and the Small interfering fragment
To reveal the role of SNF5 in Hcy-induced atherosclerosis, we transfected cells with SNF5 overexpressing lentivirus and small interfering fragments, observed the infection efficiency of lentivirus with a fluorescence inverted microscope, and then verified the overexpression/interference efficiency with qRT-PCR and western blot, respectively, as shown in FIG. 5: the mean level of SNF5 mRNA and protein expression in macrophages was significantly increased/decreased (P <0.01) after overexpression/interference of SNF5, indicating that the vector and small interfering fragments could be used in subsequent experiments.
5.6 Effect of over-expressing SNF5 on IL-1 β expression
The content of IL-1. beta. in the supernatant of the macrophage culture medium was measured by ELISA, and the results are shown in FIG. 6: the content of IL-1 beta in the culture medium supernatant is obviously increased after over-expressing SNF5 (P < 0.01); meanwhile, qRT-PCR and western blot are adopted to respectively detect the expression level of IL-1 beta in macrophages after over-expressing SNF5, and the result indicates that: the mRNA and protein levels of IL-1 beta in macrophages were significantly increased after SNF5 overexpression (P <0.01), with significant differences.
5.7 interference with the Effect of SNF5 on IL-1 β expression
The content of IL-1. beta. in the supernatant of the macrophage culture medium was measured by ELISA, and the results are shown in FIG. 7: the content of IL-1 beta in the culture medium supernatant is obviously reduced after the SNF5 interference (P is less than 0.01); meanwhile, qRT-PCR and western blot are adopted to respectively detect the expression level of IL-1 beta in macrophages after over-expressing SNF5, and the result indicates that: the levels of IL-1 β mRNA and protein in macrophages were significantly reduced after SNF5 interference (P <0.01), with the difference being significant.
Conclusion 6
Hcy promotes the level of IL-1 beta in macrophages by up-regulating the expression of SNF5, thereby promoting the formation of atherosclerosis.
The invention feeds ApoE by high methionine feed-/-Mouse duplicating hyperhomocysteinemia atherosclerosis model, observing inflammation factor ILThe level of-1 beta and the observation of the function of Hcy in promoting the SNF5 in the process of atherosclerosis inflammation provide a new theoretical basis for clinically preventing and treating the atherosclerosis inflammation in the future, and have very important significance.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Sequence listing
<110> Ningxia medical university
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<120> application of reagent for detecting SNF5 gene expression in preparation of atherosclerosis diagnosis product
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