WO2021169812A1 - Pax4的抑制剂在制备抑制纤维化的药物中的应用 - Google Patents

Pax4的抑制剂在制备抑制纤维化的药物中的应用 Download PDF

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WO2021169812A1
WO2021169812A1 PCT/CN2021/076352 CN2021076352W WO2021169812A1 WO 2021169812 A1 WO2021169812 A1 WO 2021169812A1 CN 2021076352 W CN2021076352 W CN 2021076352W WO 2021169812 A1 WO2021169812 A1 WO 2021169812A1
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pax4
fibrosis
cardiac
inhibiting
protein
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French (fr)
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肖晗
张幼怡
李明喆
胡国民
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北京大学第三医院（北京大学第三临床医学院）
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    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the present invention relates to the field of genes, in particular to the application of PAX4 inhibitors in the preparation of drugs for inhibiting fibrosis.
  • Cardiac fibrosis is an important part of most cardiac conditions.
  • the manifestation of cardiac fibrosis is the excessive deposition of extracellular matrix in the heart tissue, which leads to the destruction of the physiological heart tissue structure, and ultimately leads to heart failure, which seriously threatens human health and life.
  • Transdifferentiation of fibroblasts into myofibroblasts is the key to the initiation and maintenance of cardiac fibrosis.
  • Myofibroblasts have important contraction and secretion functions, and are characterized by the expression of ⁇ -smooth actin ( ⁇ SMA), fibronectin and type I collagen (Collagen I, Col I) .
  • Angiotensin II is a polypeptide preparation recognized in current research that regulates vasoconstriction, affects cardiac function, and can induce cardiac fibrosis.
  • This preparation can usually be used to treat mice or fibroblasts to simulate the state of cardiac fibrosis.
  • researchers from various countries have carried out a lot of research around the mechanism of cardiac fibrosis, but its specific molecular mechanism is still not very clear.
  • Cardiac fibrosis is still an important target for clinical treatment of heart disease. Since the treatment of cardiac fibrosis can delay the occurrence and development of heart failure, it is an important issue to be solved to fill the gap in the research mechanism of cardiac fibrosis.
  • the transcription factor PAX4 is a member of subfamily IV of the Paired box (PAX) family.
  • PAX4 gene In the human genome, the PAX4 gene is located in the long arm of chromosome 7, zone 2, zone 1 subband, and consists of 12 exons and 11 introns. PAX4 is also known as KPD, MODY9.
  • the PAX4 protein is located in the cell nucleus and can form a transcription factor complex that binds to the 5'end specific sequence of the target gene to regulate the expression of the downstream target gene.
  • Current research believes that members of the PAX family perform important functions in many stages of embryonic development and organ formation, and also function in all aspects of the body after adulthood.
  • Members of the PAX family from insects, amphibians, birds, and mammals, have a fairly conservative sequence of evolution.
  • the full-length PAX4 contains 349 amino acids. Its protein structure includes a 128-amino acid bipartite paired domain (PD) and a homeodomain (HD). Its C-terminus has not only a common PAX family The transcription activation domain also has a unique negative regulatory domain.
  • PD 128-amino acid bipartite paired domain
  • HD homeodomain
  • Current research believes that members of the PAX family are important regulators of tissue development and cell differentiation. The research on PAX4 mainly focuses on the research related to pancreatic islets, cancer and retina. Current research shows that PAX4 participates in the differentiation of pancreatic islet beta cells and delta cells during embryonic development, and participates in insulin secretion under normal conditions. Loss of PAX4 can induce type 1 and type 2 diabetes.
  • PAX4 is a potent tumor suppressor for human insulinoma and melanoma. PAX4 can promote the migration and invasion of human epithelial cancer. Studies have reported that PAX4 is expressed at high levels in the photoreceptors of the rat retina, suggesting that PAX4 may play a role in the retina. However, the research on the function of PAX4 in the heart is still blank.
  • the purpose of the present invention is to provide a new drug for inhibiting cell fibrosis.
  • the following technical solutions are proposed:.
  • One of the objectives of the present invention is to provide the application of PAX4 inhibitors in the preparation of drugs for inhibiting fibrosis.
  • a drug for inhibiting fibrosis which includes an inhibitor of PAX4.
  • it also relates to a method for inhibiting fibrosis, which is characterized by including the step of inhibiting PAX4 gene expression.
  • the inventors of the present application used biochemistry, molecular biology, and cytology research methods to find that PAX4 expression levels are highly expressed in pathological models such as mice, mouse fibroblast fibrosis models, and myocardial infarction models.
  • Subsequent use of small interfering RNA technology means respectively transfecting fibroblasts with small interfering RNA or injecting into the tail vein of mice, using small interfering RNA knockdown to reduce the level of PAX4 protein in cells or heart tissues, so as to clarify the function of PAX4 on mouse heart.
  • bioinformatics methods are used to analyze and predict the target genes involved in cardiac fibrosis that can be bound and regulated by PAX4.
  • PAX4 was knocked down by small interfering RNA to clarify its regulatory effect on downstream genes, and the mechanism of action was clarified. This discovery was made by the inventor of the present application for the first time and was unexpected.
  • the PAX4 inhibitor includes siRNA and PAX4 gene knockout reagent.
  • the PAX4 gene knockout reagent is siRNA (small interfering RNA).
  • the fibrosis refers to cardiac fibrosis, pancreatic fibrosis or lung fibrosis.
  • the present invention also relates to the application of PAX4 gene or its expression product in promoting the proliferation of cardiac fibroblasts in vitro.
  • the present invention also relates to the use of PAX4 gene knockout reagents to inhibit the proliferation of cardiac fibroblasts in vitro.
  • the present invention also relates to the inhibition or blocking of the expression of the fibrosis-promoting factor TGF ⁇ and the promotion of fibrosis by PAX4 gene knockout reagents in vitro in cardiac fibroblasts.
  • Inhibitors IL1R2 and CXCL10 expression in the application are preferably applied to cell fibrosis in humans or animals.
  • the present invention also relates to PAX4 gene knockout reagents in promoting cell fibrosis by inhibiting fibrosis promoting factor TGF ⁇ and promoting fibrosis inhibiting factors IL1R2 and TGIF2.
  • the experimental results of this application confirm that after PAX4 is knocked down in cardiac fibroblasts, TGF ⁇ protein levels decrease, and IL1R2 and TGIF2 protein levels increase, which suggests that PAX4 increases fibrosis-promoting factor TGF ⁇ and inhibits fibrosis-inhibiting factors IL1R2 and TGIF2 So as to play a multi-dimensional role in promoting fibrosis.
  • the present invention provides for the first time the new application of PAX4 as a new and important target for the treatment of cardiac fibrosis.
  • the present invention uses biochemistry, molecular biology, and cytology research methods to find that PAX4 expression levels are highly expressed in pathological models such as mice, mouse fibroblast fibrosis models, and myocardial infarction models.
  • Subsequent use of small interfering RNA technology means respectively transfecting fibroblasts with small interfering RNA or injecting into the tail vein of mice, using small interfering RNA knockdown to reduce the level of PAX4 protein in cells or heart tissues, so as to clarify the function of PAX4 on mouse heart. The role of.
  • PAX4 is a potentially new and important target for the treatment of cardiac fibrosis and the prevention of heart failure in the heart.
  • Figure 1 The analysis of immunohistochemical experiments found that PAX4 expression level increased under the pathological stimulus environment of fibroblasts.
  • Figure 1A Immunohistochemistry uses PAX4 antibody to label PAX4 protein level and its location.
  • Figure 1B IOD analysis results of immunohistochemistry using PAX4 antibody to label PAX4 quantitatively.
  • Figure 2 Western blotting verifies that the expression level of transcription factor PAX4 is elevated in a mouse fibrosis model.
  • Figure 2A Western blotting uses PAX4 antibody to compare PAX4 protein levels in cardiac tissues in the cardiac fibrosis model and its control group, with GAPDH as an internal control.
  • Figure 2B Quantitative and statistical analysis results of protein content detected by PAX4 Western blotting.
  • Figure 3 Western blot method to detect the protein levels of cardiac fibrosis markers fibronectin, ⁇ SMA and Col I in a mouse fibrosis model.
  • Figure 3A Western blotting uses fibronectin, ⁇ SMA and Col I antibodies to compare fibronectin, ⁇ SMA and Col I protein levels in cardiac tissues in the cardiac fibrosis model and its control group, with GAPDH as an internal control.
  • Figure 3B Quantitative and statistical analysis results of protein content detected by fibronectin, ⁇ SMA, and Col I Western blotting.
  • Figure 4 Western blot method to detect the protein levels of PAX4 in the heart tissues of the infarct zone, border zone and remote zone of the mouse myocardial infarction model at different periods.
  • Figure 5 Immunofluorescence experiment detects the changes in the fluorescence intensity of the transcription factor PAX4 and the myofibroblast markers fibronectin, ⁇ SMA, and Col I after stimulation with 1 ⁇ M angiotensin II on cardiac fibroblasts for three days.
  • Figure 5A Transcription factor PAX4, and myofibroblast marker fibronectin, ⁇ SMA, Col I fluorescence intensity, the circled fluorescence is the nucleus, and the remaining fluorescence is the target gene for detection.
  • Figure 5B Relative quantitative and statistical analysis results of immunofluorescence.
  • Figure 6 Western blotting verified that the expression level of transcription factor PAX4 increased under the stimulation of angiotensin II in fibroblasts.
  • Figure 6A Western blot experiment to detect the protein level of PAX4 after angiotensin stimulation.
  • Figure 6B Quantitative and statistical analysis results of PAX4 protein content detected by Western blotting.
  • Figure 7 Western blotting verified that the levels of myofibroblast markers fibronectin, ⁇ SMA, and Col I protein increased under the stimulation of angiotensin II in fibroblasts.
  • Figure 7A Western blot experiment to detect the protein levels of fibronectin, ⁇ SMA, and Col I after angiotensin stimulation.
  • Figure 7B Quantitative and statistical analysis results of fibronectin, ⁇ SMA, and Col I protein content detected by Western blotting.
  • Figure 8 Western blotting was used to detect PAX4 protein levels in the heart tissue of PAX4 knockdown mice after intravenous injection of small interfering RNA in the mouse tail vein to verify the knockdown efficiency of experimental small interfering RNA knockdown PAX4.
  • Figure 8A Western blot experiment to detect the protein level of PAX4 after injection of small interfering RNA into the tail vein of the mouse.
  • Figure 8B Quantitative and statistical analysis results of PAX4 protein content detected by Western blotting.
  • Figure 9 PAX4 small interfering RNA was injected through the tail vein of the mouse to construct a fibrosis model by injecting AngII into the embedded micro-osmotic pump. The results of Sirius red staining showed that knocking down PAX4 inhibited the production of fibrosis.
  • Figure 9A Results of tissue sections stained with Sirius Red.
  • Figure 9B Quantitative and statistical analysis results of the area of cardiac fibrosis.
  • FIG. 10 PAX4 small interfering RNA was injected through the tail vein of the mouse and AngII was injected into the mouse embedded micro-osmotic pump to construct a fibrosis model, and the fluorescence intensity of Col I was detected by immunofluorescence experiment.
  • the upper image shows the fluorescence intensity of Col I
  • the lower image shows the co-stained image of Col I and the nuclear dye Hoechst.
  • Figure 11 PAX4 small interfering RNA was injected through the tail vein of the mouse to construct a fibrosis model by injecting AngII into the buried micro-osmotic pump. Echocardiography revealed that knockdown of PAX4 had a protective effect on the mouse heart function.
  • Figure 11A Left ventricular posterior wall thickness
  • Figure 11B EF value
  • Figure 11C FS value
  • Figure 11D E/E'.
  • Figure 12 Western blotting was used to detect the protein level of PAX4 in cardiac fibroblasts after small interfering RNA was transfected to verify the knockdown efficiency of PAX4 knockdown.
  • Figure 13 Immunofluorescence staining method to detect the protein expression levels of myofibroblast markers fibronectin (Figure 13A), ⁇ SMA ( Figure 13B), and Col I ( Figure 13C) after PAX4 knockdown in fibroblasts transfected by small interfering RNA , To verify the inhibitory effect of knocking down PAX4 on fibrosis.
  • Figure 14 Western blotting experiments to detect the protein expression levels of myofibroblast markers fibronectin, ⁇ SMA, and Col I after adenovirus infected fibroblasts overexpressing PAX4, verifying the promoting effect of PAX4 on fibrosis.
  • Figure 15 Western blot experiment to detect the influence of the possible downstream genes TGF ⁇ , IL1R2, and TGIF2 protein expression levels after PAX4 knockdown in fibroblasts transfected with small interfering RNA.
  • Example 1 Animal pathological model experiment, constructing mouse heart fibrosis model, extracting heart tissue, using immunohistochemistry and western blotting experiment methods to detect the location and content of PAX4 protein expression, as well as myofibroblast markers fibronectin and ⁇ SMA, the content of extracellular matrix Col I.
  • angiotensin II-induced mouse cardiac fibrosis model 10-week-old male C57BL/6 mice were randomly divided into two groups, an operation group and a sham operation group. The mice received angiotensin (3mg ⁇ kg-1 ⁇ day-1)
  • the micro-osmotic pump buried pump (Alzet MODEL 1007D, DURECT, Cupertino, CA) was used to construct the fibrosis model for 7 days.
  • Preparation of the micro-osmotic pump 1 day before the operation, inject angiotensin II (dissolved in sterile PBS buffer) into the micro-osmotic pump with a 1 mL syringe, soak the micro-osmotic pump in sterile PBS buffer, and equilibrate at 37 degrees Celsius overnight.
  • the mice were anesthetized with 2% to 3% isoflurane, a 0.7cm-long transverse incision was made in the back neck of the mouse, and the subcutaneous tissue was bluntly separated with forceps to reduce the micro-osmotic pressure.
  • the pump is buried, the wound is sutured, and neomycin ointment is applied to prevent infection.
  • the operation group was continuously infused with angiotensin II at a concentration of 3 mg/kg/d for 7 days.
  • Antigen retrieval place the slices in a citrate (PH 6.0) antigen retrieval solution and heat it in a pressure cooker to fully expand the antigen epitope (after the pressure cooker continues to vent gas, count for 2 minutes). After the heat repair is completed, place it at room temperature. When the section temperature drops to room temperature, wash with PBS 3 times for 5 minutes each time.
  • Serum blocking place the slices in a wet box, and block with 10% goat serum for 30 minutes at room temperature.
  • Primary antibody incubation After discarding the serum, add the prepared primary antibody to the slices, and incubate in a refrigerator at 4 degrees Celsius overnight (or incubate in an incubator at 37 degrees Celsius for 2 hours).
  • Extraction of total protein from myocardial tissue Take the myocardial tissue stored in liquid nitrogen, put it in a mortar and grind it with liquid nitrogen, take two thirds (the other third is used to extract RNA) and add it to the tissue lysate (20mmol /L Tris-HCl pH 7.4, 150mmol/L NaCl, 2.5mmol/L EDTA, 50mmol/L NaF, 0.1mmol/L Na 4 P 2 O 7 , 1mmol/L Na 3 VO 4 , 1% Triton X-100, 10% Glycerol, 0.1% SDS, 1% deoxycholic acid, 1mmol/L PMSF, 1g/mL aprotinin.) After mixing, let it stand on ice for 15 minutes, and add 800 microliters of lysis solution for every 50 mg of myocardial tissue.
  • TBST wash the membrane after washing three times and change the corresponding
  • the second antibody of the species at room temperature for 1 hour, wash the membrane with TBST and develop it, put the membrane in a developer (Millipore Corporation), and then dry it and place it in a luminescence detection machine for exposure.
  • Band intensity was quantified using NIH ImageJ software.
  • angiotensin II micro-osmotic pump buried pump method select 10-week male C57BL/6 mice to construct a mouse cardiac fibrosis model, detect the expression level of PAX4 in cardiac tissue in the cardiac fibrosis model, and detect fibrosis markers at the same time To correspond to the degree of fibrosis in the heart tissue.
  • Example 2 Detection of PAX4 protein levels in the infarct area and remote area at different time points of cardiac tissue surgery in a mouse myocardial infarction model.
  • mice at 10 weeks Male C57BL/6 mice at 10 weeks were used for the establishment of myocardial infarction model.
  • the mice were randomly divided into myocardial infarction group and sham operation control group.
  • the myocardial infarction group used left coronary artery stenosis to induce the occurrence of myocardial infarction.
  • the control group underwent sham operation. The operation was performed under gas anesthesia, and the mice were gas anesthetized by inhaling 2% isoflurane.
  • the infarct area, border area and remote area of the mouse heart tissue were collected at 1, 4, and 7 days after the operation.
  • Example 3 Using cardiac fibroblasts and treating the cells with 1 ⁇ M angiotensin for three days, immunofluorescence and western blot experiments were used to detect the protein levels of PAX4, fibrosis markers fibronectin, ⁇ SMA and Col I.
  • Isolation and culture of adult mouse cardiac fibroblasts Male C57/BL6 mice about 8 weeks old were severed and put to death. They were quickly immersed in 75% alcohol for about half a minute, and the heart was immediately opened in a clean bench to take out the heart. Wash it twice in PBS buffer at 4 degrees Celsius, cut off the blood vessels at the atrium and the bottom of the heart, then cut the ventricle into small pieces, and wash it with PBS once to wash away part of the residual blood. Add 0.1% type II collagenase (330U, Worthington, Columbia, NJ, USA/Sigma, St. Louis, MO, USA) prepared with PBS balanced salt solution for digestion. The entire digestion process is carried out under constant temperature stirring at 36-37 degrees Celsius.
  • the supernatant digestion solution is taken and added to the same amount of DMEM culture medium containing 10% FBS, and mixed evenly. Repeat this process about 7 to 8 times until the tissue block is completely digested. Centrifuge the collected tubes at 1000 rpm for 5 minutes at room temperature, discard the supernatant, resuspend the cells in DMEM medium containing 10% FBS, and combine the cardiomyocyte suspension obtained each time.
  • the solution was seeded in a petri dish with a diameter of 100 mm, and placed in an incubator at 37 degrees Celsius and 5% CO 2 for 2 hours to make the fibroblasts basically adhere to the wall. Aspirate and discard the culture fluid in the petri dish, and add new DMEM culture fluid containing 10% FBS to continue the culture. After 3 days, the cells were full, passaged and proceeded to follow-up experiments.
  • the extraction method of cardiac fibroblast protein the cells are first digested from the base gel with trypsin, centrifuged, the supernatant is taken, washed three times with cold PBS, and then cell lysate (20mmol/L Tris-HCl PH7.4, 150mmol /L NaCl, 2.5mmol/L EDTA, 50mmol/L NaF, 0.1mmol/L Na4P2O7, 1mmol/L Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% SDS, 1% deoxycholic acid, 1mmol/L PMSF ,and 1mg/ml aprotinin) lyse the cells, sonicate the cells and centrifuge at 12000g for 15 minutes at 4 degrees Celsius. Collect the supernatant. After taking 5 microliters for protein quantification, add 5X gel loading buffer to the remaining supernatant at 100 degrees Celsius for 5 minutes to ensure protein denaturation.
  • Immunofluorescence staining experiment the cells were fixed with 4% paraformaldehyde at 37 degrees Celsius for 15 minutes, then washed with warm PBS for 3 times, and then 0.2% Triton X-100 was used to rupture the membrane for 20-30 minutes. After washing with warm PBS for 3 times, blocking solution (5% BSA) was added to block for 30 minutes. After that, the primary antibody ⁇ SMA (ab32575, abcam, Cambridge, MA, USA), fibronectin (ab2413, abcam, Cambridge, MA, USA), PAX4 (ab101721, abcam, Cambridge, MA, USA) was used to incubate overnight at 4 degrees Celsius.
  • the experimental results show that angiotensin II stimulates cardiac fibroblasts for three days can promote fibrosis markers fibronectin, ⁇ SMA and Col I
  • the statistical analysis of the quantitative results showed that the fibrosis markers increased significantly compared with the protein level of the control group after angiotensin II stimulation.
  • Example 4 Using mouse tail vein injection of small interfering RNA to knock down PAX4 levels in mice, and then construct a fibrosis model, using echocardiography, comparison of heart-body ratio, Sirius red (PSR) staining, western blotting, immunofluorescence The experimental method detects the effect of PAX4 on cardiac function, as well as the effect on the protein levels of myofibroblast markers fibronectin and ⁇ SMA, and extracellular matrix Col I.
  • PSR Sirius red
  • Mouse tail vein injection of small interfering RNA wild-type C57BL/6 adult mice (11 weeks old, male, weighing about 27g) are selected, and the method of tail vein injection is given daily with 10 nmol of PAX4 knockdown to reduce the interference RNA sequence (chemically modified Sequences, including knockdown sequences and meaningless control sequences) (Ribobio Co., Ltd (Guangzhou, China)), each injection volume is 0.12mL (dissolved in saline), and the injection is continued for 3 days. On the fourth day, angiotensin II was infused using the above-mentioned buried pump method.
  • PAX4 knockdown chemically modified Sequences, including knockdown sequences and meaningless control sequences
  • mice were injected with control and knock-down interfering RNA sequences into the tail vein every other day until angiotensin II was continuously infused for 7 days. Measure the ultrasound index, body weight, and heart weight, and collect myocardial tissue for follow-up testing.
  • Echocardiography Place the mouse in an anesthesia box and give isoflurane (2.5% isoflurane, 0.8L/min) for anesthesia. After anesthesia, remove the mouse from the anesthesia box and quickly place it in a supine position on heating Put on the nasal mask connected to the anesthetic on the board, fix the limbs of the mouse with adhesive strips, and adjust the concentration of isoflurane to 1% to maintain anesthesia. Use depilatory cream (Nail, Canada) to depilate the breasts. Vevo 2100 ultrasound system (Fujifilm Visual sonics, Canada) was used for mouse echocardiography.
  • Heart weighing After the blood collection of the mouse, the thorax was cut quickly, and the heart was irrigated with ice-cold PBS (0.8% NaCl, 0.02% KCl, 0.02% KH2PO4, 0.4% Na2HPO4) with an indwelling needle, and then the whole heart was taken out and placed on ice. Cut off the blood vessels, fat and other tissues on the heart in the PBS, absorb the water with filter paper, weigh the whole heart weight (HW), after weighing, cut off the auricles on the heart, leaving the entire ventricle.
  • PBS ice-cold PBS
  • HW whole heart weight
  • Tissue sectioning and staining the cross-section of the heart papillary muscle level is fixed in 4% paraformaldehyde solution (W/V%, prepared with PBS) for 6 to 8 hours, then the paraformaldehyde is discarded, and 20% sucrose solution is added ( W/V%, prepared with PBS) dehydrated. Then put it into 70% (3 hours), 80% (3 hours) ethanol solution for gradient dehydration, and finally put it in 90% ethanol plus n-butanol solution (volume ratio 1:1) overnight.
  • W/V% paraformaldehyde solution
  • knocking down PAX4 can relieve the increase of extracellular matrix, inhibit the occurrence of fibrosis, and improve the function of the heart.
  • Example 5 Intervening PAX4 protein levels at the level of cardiac fibroblasts, using immunofluorescence experiments or western blot experiments to detect the effect on the protein levels of myofibroblast markers fibronectin, ⁇ SMA and Col I.
  • Cardiac fibroblasts transfected with small interfering RNA The P2 generation of cardiac fibroblasts was passaged from P1 to a 6-well plate the night before the experiment was transfected with small interfering RNA, and cultured overnight at 37 degrees Celsius and 5% carbon dioxide to ensure Fibroblasts spread morphologically and have not yet produced extracellular matrix that affects transfection efficiency.
  • fibroblasts in the 6-well plate were gently washed with warm PBS at 37 degrees Celsius, and the washing was repeated three times to ensure that the culture medium was thoroughly washed off, and then 500 ⁇ l OPTI-MEM (Opti-MEM I Reduced Serum Medium, 31985070, Life), then add 80nmol/L PAX4 small interfering RNA (sc-152040, Santa Cruz Biotech, CA, USA)) or control small interfering RNA (sc-37007, Santa Cruz Biotech, CA, USA) and 3 ⁇ l HiPerFect transfection Reagents (301705, QIAGEN, Beijing, China). 6 hours after transfection, DMEM medium containing 10% fetal calf serum was added to culture. The protein detection experiment used cell samples transfected with small interfering RNA for three days.
  • Adenovirus cardiac fibroblast infection PAX4 The P2 generation of cardiac fibroblasts was passaged from P1 to a 6-well plate the night before the experiment plus virus infection, and cultured overnight at 37 degrees Celsius and 5% carbon dioxide to ensure the spread of fibroblasts. , And has not produced extracellular matrix that affects transfection efficiency.
  • PAX4 When the cells grow at a density of 60%, use 2MOI PAX4 or control adenovirus to infect the cells under serum-free conditions, change the medium after 6 hours, change to DMEM containing 10% fetal bovine serum and culture for three days before proceeding with subsequent experiments.
  • PAX4 can promote the occurrence of cardiac fibrosis, and knocking down PAX4 can reduce the content of fibronectin, ⁇ SMA and Col I.
  • Example 6 In cardiac fibroblasts, the protein level of PAX4 in the cell was knocked down, so as to detect the expression level of its downstream regulatory genes by means of western blot experiments.

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Abstract

PAX4的抑制剂在制备抑制纤维化的药物中的应用。通过小干扰RNA干扰转录因子PAX4的表达可以阻断其对下游基因的抑制作用，使得其下游多个纤维化抑制因子发挥功能；小干扰RNA降低的PAX4的表达同时也降低纤维化促进因子的表达水平，抑制其促纤维化的作用，从而抑制心脏纤维化的发生。因此，PAX4在心脏中是一个潜在的全新的治疗心脏纤维化从而预防心衰的重要靶点。

Description

PAX4的抑制剂在制备抑制纤维化的药物中的应用
相关申请的交叉引用
本申请要求于2020年02月25日提交的中国发明专利申请CN202010115236.5的优先权，在此通过引用整体并入本文。
技术领域
本发明涉及基因领域，尤其涉及PAX4的抑制剂在制备抑制纤维化的药物中的应用。
背景技术
心脏纤维化是大多数心脏病理状况的重要组成部分。心脏纤维化的表现是心脏组织中细胞外基质的过度沉积，导致生理性心脏组织结构的破坏，最终导致心力衰竭，严重威胁人类的健康和生命。成纤维细胞向肌成纤维细胞转分化是心脏纤维化反应的启动和维持关键。肌成纤维细胞具有重要的收缩和分泌功能，并以表达α-平滑肌动蛋白(α-smooth actin,αSMA)、纤连蛋白(fibronectin)以及I型胶原蛋白(Collagen I，Col I)为其特点。血管紧张素II是一种现今研究中公认的调节血管收缩、影响心脏功能，能够诱发心脏纤维化的多肽制剂，通常可以使用该制剂处理小鼠或者成纤维细胞，模拟心脏纤维化的状态。尽管迄今为止，各国研究者围绕着心脏纤维化的机制开展了大量的研究，但是其具体的分子机制仍不十分清楚。心脏纤维化仍是当今临床治疗心脏疾病的重要靶点。由于治疗心脏纤维化能够延缓心力衰竭的发生、发展，因此去填补心脏纤维化研究机制中尚存的空白就是一个尚待解决的重要问题。
转录因子PAX4是Paired box(PAX)家族第IV亚族的成员。在人类基因组中，PAX4基因位于7号染色体长臂3区2带1亚带，由12个外显子、11个内含子组成。PAX4也被称为KPD，MODY9。PAX4蛋白位于细胞核内，可形成转录因子复合体与目标基因的5’端特异性序列结合调控下游靶基因的表达。目前的研究认为PAX家族的成员在胚胎发育以及器官形成的诸多阶段行使重要功能，并且也在成年后机体的方方面面发挥功能。PAX家族的成员，从昆虫、两栖动物、鸟类、哺乳动物中， 其序列的演化过程是相当保守的。全长的PAX4包含349个氨基酸，它的蛋白结构包括一个128个氨基酸的bipartite paired结构域(PD)，和一个同源结构域(homeodomain，HD)，其C-端不仅有一个PAX家族常见的转录激活域，还有一个独特的负调节结构域。目前的研究认为，PAX家族的成员是组织发育以及细胞分化的重要调控因子。而对于PAX4的研究，主要集中在胰岛、癌症以及视网膜相关的研究中。目前的研究表明，PAX4参与胚胎发育时期胰岛beta细胞以及delta细胞的分化，参与正常条件下的胰岛素分泌。缺失PAX4会诱发一型、二型糖尿病。在肿瘤相关的研究者，PAX4是人的胰岛素瘤和黑色素瘤的强效肿瘤抑制因子。PAX4能够促进人上皮癌的迁移和侵袭。有研究报道PAX4在大鼠视网膜光感受器中高水平表达，提示PAX4可能在视网膜中发挥作用。然而，关于PAX4在心脏中的功能研究尚属空白。
发明内容
本发明的目的在于提供一种新的抑制细胞纤维化的药物。为了实现本发明的目的，拟采用如下技术方案：。
本发明的目的之一是提供PAX4的抑制剂在制备抑制纤维化的药物中的应用。
在本发明的另一方面，还涉及抑制纤维化的药物，其包括PAX4的抑制剂。
在本发明的另一方面，还涉及抑制纤维化的方法，其特征在于包括抑制PAX4基因表达的步骤。
本申请的发明人利用生物化学、分子生物学、细胞学研究手段，研究发现PAX4表达水平在小鼠以及小鼠成纤维细胞纤维化模型、心肌梗死模型等病理模型中高表达。后续使用小干扰RNA的技术手段分别通过小干扰RNA转染成纤维细胞或者鼠尾静脉注射的方式，利用小干扰RNA敲减降低细胞或心脏组织中PAX4蛋白水平，从而明确PAX4对小鼠心脏功能的作用。此后利用生物信息学手段分析预测能够被PAX4结合并调控的参与心脏纤维化的靶基因。此后，再通过小干扰RNA敲减PAX4的方法明确其对下游基因的调控作用，明确了作用机制。这一发现是本申请的发明人首次发现的，并且是出乎意料的。
本发明的一个优选实施方式中，所述的PAX4的抑制剂包括siRNA、PAX4基因敲除试剂。
本发明的一个优选实施方式中，所述的PAX4基因敲除试剂为siRNA即小干扰RNA。
本发明的一个优选实施方式中，所述的纤维化是指心脏纤维化、胰脏纤维化或肺脏纤维化。
虽然本发明的发现优选应用于人体或动物体的细胞纤维化，但是，本发明还涉及PAX4基因或其表达产物在促进体外心脏成纤维细胞增殖中的应用。
虽然本发明的发现优选应用于人体或动物体的细胞纤维化，但是，本发明还涉及PAX4基因敲除试剂在抑制体外心脏成纤维细胞增殖中的应用。
虽然本发明的发现优选应用于人体或动物体的细胞纤维化，但是，本发明还涉及PAX4基因敲除试剂在体外心脏成纤维细胞中抑制或阻断纤维化促进因子TGF β的表达以及促进纤维化抑制因子IL1R2和CXCL10的表达中的应用。
虽然本发明的发现优选应用于人体或动物体的细胞纤维化，但是，本发明还涉及PAX4基因敲除试剂通过抑制纤维化促进因子TGF β以及促进纤维化抑制因子IL1R2和TGIF2在促进细胞纤维化中的应用。本申请的实验结果证实，心脏成纤维细胞被敲减PAX4后，TGFβ蛋白水平降低，IL1R2以及TGIF2蛋白水平升高，这提示PAX4是通过增加纤维化促进因子TGFβ、抑制纤维化抑制因子IL1R2以及TGIF2从而起到多维度促进纤维化的作用。
本发明首次提供了PAX4作为新的重要的治疗心脏纤维化的靶点的新应用。特别是，本发明利用生物化学、分子生物学、细胞学研究手段，研究发现PAX4表达水平在小鼠以及小鼠成纤维细胞纤维化模型、心肌梗死模型等病理模型中高表达。后续使用小干扰RNA的技术手段分别通过小干扰RNA转染成纤维细胞或者鼠尾静脉注射的方式，利用小干扰RNA敲减降低细胞或心脏组织中PAX4蛋白水平，从而明确PAX4对小鼠心脏功能的作用。此后利用生物信息学手段分析预测能够被PAX4结合并调控的参与心脏纤维化的靶基因。此后，再通过小干扰RNA敲减PAX4的方法明确其对下游基因的调控作用，明确了作用机制。因此PAX4在心脏中是一个潜在的全新的治疗心脏纤维化从而预防心衰的重要靶点。
附图说明
图1：免疫组化实验分析发现PAX4在成纤维细胞的病理刺激环境下，表达水平升高。图1A：免疫组化利用PAX4抗体标记PAX4蛋白水平及其定位。图1B：免疫组化利用PAX4抗体标记PAX4定量的IOD分析结果。
图2：蛋白质印迹法验证转录因子PAX4在小鼠纤维化模型中表达水平升高。图2A：蛋白质印迹法利用PAX4抗体比较心脏纤维化模型及其对照组中心脏组织中PAX4蛋白水平，GAPDH作为内参。图2B：PAX4蛋白印迹法检测的蛋白含量的定量及统计分析结果。
图3：蛋白质印迹法检测小鼠纤维化模型中心脏纤维化标志物fibronectin、αSMA以及Col I的蛋白水平。图3A：蛋白质印迹法利用fibronectin、αSMA以及Col I抗体比较心脏纤维化模型及其对照组中心脏组织中fibronectin、αSMA以及Col I蛋白水平，GAPDH作为内参。图3B：fibronectin、αSMA以及Col I蛋白印迹法检测的蛋白含量的定量及统计分析结果。
图4：蛋白质印记法检测小鼠心梗模型梗死区、边界区和远隔区不同时期心脏组织中PAX4的蛋白水平。
图5：免疫荧光实验检测心脏成纤维细胞给予1μM血管紧张素II刺激三天后，转录因子PAX4、以及肌成纤维细胞标志物fibronectin、αSMA、Col I荧光强度的变化。图5A：转录因子PAX4、以及肌成纤维细胞标志物fibronectin、αSMA、Col I荧光强度，圈出的荧光为细胞核，其余荧光为检测的目的基因。图5B：免疫荧光的相对定量及统计分析结果。
图6：蛋白质印迹法验证转录因子PAX4在成纤维细胞的血管紧张素II刺激下，表达水平升高。图6A：蛋白印迹实验检测PAX4在血管紧张素刺激后的蛋白水平。图6B：蛋白印迹法检测的PAX4蛋白含量的定量及统计分析结果。
图7：蛋白质印迹法验证在成纤维细胞的血管紧张素II刺激下，肌成纤维细胞标志物fibronectin、αSMA、Col I蛋白水平升高。图7A：蛋白印迹实验检测fibronectin、αSMA、Col I在血管紧张素刺激后的蛋白水平。图7B：蛋白印迹法检测的fibronectin、αSMA、Col I蛋白含量的定量及统计分析结果。
图8：蛋白质印迹法检测鼠尾静脉注射小干扰RNA转染敲减小鼠PAX4后心脏组织中PAX4的蛋白水平，验证实验小干扰RNA敲减PAX4的敲减效率。图8A：蛋白印迹实验检测PAX4在鼠尾静脉注射小干扰RNA后的蛋白水平。图8B：蛋白印迹法 检测的PAX4蛋白含量的定量及统计分析结果。
图9：通过鼠尾静脉注射PAX4小干扰RNA后给小鼠埋微渗透泵注射AngII构建纤维化模型，天狼星红染色结果发现敲减PAX4抑制纤维化的产生。图9A：天狼星红染色的组织切片结果。图9B：心脏纤维化面积的定量和统计分析结果。
图10：通过鼠尾静脉注射PAX4小干扰RNA后给小鼠埋微渗透泵注射AngII构建纤维化模型，利用免疫荧光实验检测Col I的荧光强度。上图表示Col I的荧光强度，下图是Col I与细胞核染料Hoechst共染的图。
图11：通过鼠尾静脉注射PAX4小干扰RNA后给小鼠埋微渗透泵注射AngII构建纤维化模型，超声心动检测发现敲减PAX4对小鼠心功能有保护作用。图11A：左室后壁厚度，图11B：EF值，图11C：FS值，图11D：E/E’。
图12：蛋白质印迹法检测小干扰RNA转染心脏成纤维细胞后中PAX4的蛋白水平，验证敲减PAX4的敲减效率。
图13：免疫荧光染色法检测小干扰RNA转染敲减成纤维细胞中的PAX4后肌成纤维细胞标志物fibronectin(图13A)、αSMA(图13B)、Col I(图13C)的蛋白表达水平，验证敲减PAX4对于纤维化的抑制作用。
图14：蛋白质印迹实验检测腺病毒侵染成纤维细胞过表达PAX4后肌成纤维细胞标志物fibronectin、αSMA、Col I的蛋白表达水平，验证PAX4对于纤维化的促进作用。
图15：蛋白质印迹实验检测小干扰RNA转染敲减成纤维细胞中的PAX4后可能的下游基因TGFβ、IL1R2以及TGIF2蛋白表达水平的影响。
具体实施方式
下面结合具体实施例对本发明做进一步的详细说明，所述是对本发明的解释而不是限定，本发明中所用的方法及其相关的试剂，可以有其他可选择和替代方案，能够达到相同技术结果即可。
下列实施例中未注明具体条件的实验方法，按照所属领域的常规操作进行，或按照制造厂商所建议的条件进行。
实施例1、动物病理模型实验，构建小鼠心脏纤维化模型，取材心脏组织，利用免疫组化以及蛋白质印记实验的方法，检测PAX4蛋白表达的位置和含量，以及肌成 纤维细胞标志物fibronectin和αSMA，细胞外基质Col I的含量。
血管紧张素II诱导小鼠心脏纤维化模型的制备：10周周龄雄性C57BL/6小鼠随机分为两组，手术组和假手术组，小鼠使用血管紧张素(3mg·kg-1·day-1)微渗透泵埋泵(Alzet MODEL 1007D,DURECT,Cupertino,CA)7天的方式构建纤维化模型。微渗透压泵的准备：手术前1天，将血管紧张素II(无菌PBS缓冲液溶解)用1mL注射器注入微渗泵，将微渗透压泵浸泡于无菌PBS缓冲液中，37摄氏度平衡过夜。手术时，用2％～3％的异氟烷麻醉小鼠，在小鼠后颈部剪开一长约0.7cm的横切口，用镊子伸入皮下，钝性分离皮下组织，将微渗透压泵埋入，缝合伤口，涂上新霉素软膏防止感染。手术组持续输注血管紧张素II，浓度为3mg/kg/d，持续7天。
PAX4免疫组织化学的实验方法：样品：多聚甲醛固定石蜡包埋后切片。(1)脱蜡：将组织切片用二甲苯处理15min 3次，100％乙醇5min 2次，95％乙醇5min 2次，80％乙醇5min 1次。最后用蒸馏水洗2min。(2)去除内源性过氧化氢酶：将切片置于3％过氧化氢溶液(用100％甲醇配置)中10-15min，以去除内源性过氧化氢酶。随后用PBS清洗3次，每次5min。(3)抗原热修复：将切片置于柠檬酸盐(PH 6.0)抗原修复液中，高压锅中热修复，将抗原表位充分展开(待高压锅连续冒气之后，计时2分钟)。热修复完毕后放置于常温，待切片温度降至常温时用PBS清洗3次，每次5分钟。(4)血清封闭：将切片置于湿盒内，用10％山羊血清室温封闭30min。(5)一抗孵育：弃去血清后在切片上加入配好的一抗，在4摄氏度冰箱中过夜(或在37摄氏度温箱中孵育2小时)。(6)二抗孵育：于4摄氏度冰箱中取出湿盒，凉至常温后，使用PBS清洗3次，每次5min。随后加入辣根过氧化物酶标记的二抗(中杉金桥公司，兔二步法)，在室温孵育30min。(7)DAB显色：用PBS清洗3次，利用DAB显色液进行显色。DAB工作液配制方法：1mLDAB稀释液+50uLDAB显色液。(8)复染核：先在蒸馏水中浸泡2min,随后放入苏木精溶液中30s，自来水冲洗3次。在70％盐酸酒精分化5-6s，水洗30s。在1％氨水中返蓝60s，蒸馏水中洗去浮色。(可于镜下观察核是否已蓝染)。(9)脱水、透明、使用中性树脂封片剂封片。95％乙醇2min两次，无水乙醇2min两次，最后在二甲苯替代物中5min两次。(10)待片子凉干后扫描，利用软件进行阳性面积统计。
心肌组织总蛋白的提取：取保存于液氮中的心肌组织，放入研钵中用液氮研磨，取三分之二(另外三分之一用来提取RNA)加入组织裂解液中(20mmol/L Tris-HCl pH  7.4，150mmol/L NaCl,2.5mmol/L EDTA,50mmol/L NaF，0.1mmol/L Na 4P 2O 7，1mmol/L Na 3VO 4,1％Triton X-100,10％Glycerol,0.1％SDS,1％deoxycholic acid,1mmol/L PMSF，1g/mL aprotinin.)混匀后冰上静置15分钟，大约每50毫克心肌组织加入800微升裂解液。收集匀浆液，超声破碎(45％，5s on,5s off for 4cycles)后于4摄氏度12000rpm离心15分钟，将上清一部分移入新EP管中，蛋白定量后-80摄氏度冻存，一部分加入四分之一体积的5Xloading buffer，混匀，100摄氏度煮5分钟，冻存，留作后续用western blot检测相关蛋白。
蛋白质印迹实验：使用10％SDS-PAGE胶电泳后硝酸纤维素膜转膜，5％脱脂牛奶室温封闭1小时，一抗4摄氏度冷室过夜孵育，一抗货号分别为：Fibronectin(ab2413,abcam,Cambridge,MA,USA),αSMA(ab32575,abcam,Cambridge,MA,USA),Col I(203002,MD Biosciences),PAX4(ab101721,abcam,Cambridge,MA,USA),TGFβ(10804-MM33,Sino biological,Beijing,China)，IL1R2(sc-376247,Santa Cruz Biotech,CA,USA),TGIF2(ab190152,abcam,Cambridge,MA,USA),GAPDH(2118S,CST).TBST洗膜洗三遍后换对应的种属二抗，室温1小时，TBST再洗膜后显影，将膜至于显影液中(Millipore Corporation)，然后控干放入发光检测机器中曝光。条带强度使用NIH ImageJ software软件定量。
使用血管紧张素II微渗透泵埋泵的方式，选用10周雄性C57BL/6小鼠构建小鼠心脏纤维化模型，检测心脏纤维化模型中心脏组织中PAX4的表达水平，同时检测纤维化标志物的水平来对应心脏组织中的纤维化程度。
首先取用埋血管紧张素II微渗透泵构建的心脏纤维化模型组织和假手术组的心脏组织，使用PAX4抗体进行免疫组化的实验，实验结果如图1A所示，在心脏发生纤维化的组织中，PAX4的蛋白的水平无论是心肌细胞还是心脏成纤维细胞中都有所上升，统计结果如图1B所示，定量结果统计分析表明,与健康心脏组织中PAX4的蛋白水平相比较，纤维化心脏组织中的PAX4蛋白水平显著地升高。
此后利用心脏纤维化模型组织和假手术组的心脏组织总蛋白进行蛋白质免疫印迹实验，检测PAX4的蛋白水平实验结果如图2所示，实验得到了与免疫组化实验一致的结果，转录因子PAX4的蛋白水平在纤维化的心脏组织中明显增多(图2A)。定量结果统计分析(图2B)表明和健康的心脏组织相比较，纤维化心脏组织中的PAX4蛋白水平显著地升高。
同时也利用蛋白质免疫印迹实验检测了心脏组织中纤维化标志物fibronectin、αSMA和Col I的蛋白水平。实验结果如图3A所示，实验表明，纤维化的心脏组织中纤维化标志物fibronectin、αSMA和Col I的蛋白水平较正常心脏组织中含量，均明显增多。定量结果统计分析(图3B)表明纤维化心脏组织中fibronectin、αSMA和Col I的蛋白水平显著地升高。但PAX4的增多与纤维化标志物fibronectin、αSMA和Col I的增多是否有因果关系，仍需后续实验结果实验验证。
实施例2、检测小鼠心梗模型心脏组织手术不同时间点梗死区、远隔区中PAX4蛋白水平。
10周雄性C57BL/6小鼠用来进行心梗模型的建立手术。小鼠随机的分组，分为心梗组和假手术对照组。心梗组利用左冠状动脉狭窄术从而诱发心梗的发生。对照组进行假手术。该手术在气体麻醉下进行，小鼠通过吸入2％的异氟烷进行气体麻醉。实验组分别在手术后1天、4天、7天时取小鼠心脏组织的梗死区、边界区和远隔区收集心脏组织。
通过蛋白质免疫印迹法检测小鼠心梗模型手术后1天、4天、7天梗死区、边界区和远隔区心脏组织内源的转录因子PAX4的蛋白水平。图4左侧展示了心脏梗死的梗死区、边界区、远隔区的具体位置。实验结果表明，与假手术组相比较，心梗手术的各个区域的PAX4蛋白水平均不同程度的升高。结合该心梗模型结果与实施例1纤维化病理条件的结果提示，在心脏发生病理变化时，心脏组织中PAX4蛋白水平均会升高，这个升高的具体功能在下面后续所示的试验中验证。
实施例3、利用心脏成纤维细胞，使用1μM血管紧张素处理细胞三天后，利用免疫荧光和蛋白质印记实验检测PAX4、纤维化标志物fibronectin、αSMA和Col I的蛋白水平。
成年小鼠心脏成纤维细胞的分离和培养：将约8周龄的雄性C57/BL6小鼠断颈处死，迅速浸泡于75％的酒精约半分钟，立即于超净工作台中开胸取出心脏，置入4摄氏度的PBS缓冲液中清洗两次，剪掉心房和心底部的血管，然后将心室剪碎成小块，用PBS洗一遍，以洗去部分残血。加入PBS平衡盐溶液配制的0.1％II型胶原酶(330U,Worthington,Columbia,NJ,USA/Sigma,St.Louis,MO,USA)进行消化。整个消化过程在36～37摄氏度恒温搅拌条件下进行，每消化8分钟后取上清消化液，加入到等量的含10％FBS的DMEM培养液中，混合均匀。重复该过程约7～8次直到组织块消化完 全，将收集的几管细胞室温1000rpm离心5分钟，弃上清，用含10％FBS的DMEM培养液重悬细胞，合并每次所得心肌细胞悬液，接种于直径100mm的培养皿中，在37摄氏度、5％CO 2的培养箱内放置2小时使成纤维细胞基本贴壁。吸弃培养皿中的培养液，加入新的含10％FBS的DMEM培养液继续培养。3天后细胞长满，传代并进行后续实验。
心脏成纤维细胞蛋白质的提取方法：细胞先用胰酶从基底胶上消化下来，离心后取上清，使用冷PBS清洗三遍，后用细胞裂解液(20mmol/L Tris-HCl PH7.4,150mmol/L NaCl,2.5mmol/L EDTA,50mmol/L NaF,0.1mmol/L Na4P2O7,1mmol/L Na3VO4,1％Triton X-100,10％glycerol,0.1％SDS,1％deoxycholic acid,1mmol/L PMSF,and 1mg/ml aprotinin)裂解细胞，超声破碎后在4摄氏度条件下，12000g离心15分钟。收上清。取5微升进行蛋白定量后，其余上清加入5X凝胶上样缓冲液，100摄氏度5分钟确保蛋白变性。
免疫荧光染色实验：细胞在37摄氏度条件下，用37摄氏度温热4％多聚甲醛固定15分钟后，使用温热PBS清洗3次，再用0.2％Triton X-100破膜20-30分钟。温热PBS清洗3次后加入封闭液(5％BSA)封闭30分钟。此后使用一抗αSMA(ab32575,abcam,Cambridge,MA,USA),fibronectin(ab2413,abcam,Cambridge,MA,USA),PAX4(ab101721,abcam,Cambridge,MA,USA)在4摄氏度条件下过夜孵育。回收储存一抗后，PBS清洗3次，然后室温孵育二抗Alexa Fluor 488 1小时。室温条件下使用Hoechst(Invitrogen,Carlsbad,CA,USA)染核8分钟。使用高内涵筛选成像系统Cellomics ArrayScan VTI HCS Reader(Thermo Fisher Scientific,Rockford,IL,USA)的Morphology Explorer BioApplication模块统计和分析荧光强度。
细胞水平，我们提取小鼠原代心脏成纤维细胞，在12孔板中培养至P2代，使用1μM浓度血管紧张素II刺激细胞三天后收样。首先固定样本，利用免疫荧光检测其内源PAX4以及纤维化标志物fibronectin、αSMA和Col I蛋白水平。图5A中圈出的荧光为细胞核位置，其余荧光分别表示特定抗体识别的特异目的蛋白(PAX4、fibronectin、Col I和αSMA)的定位和含量。实验结果可以看到转录因子PAX4主要表达于细胞核中。实验结果提示在血管紧张素II刺激下，PAX4、fibronectin、αSMA和Col I荧光强度均有不同程度的增强。图5B中定量结果显示在血管紧张素II刺激三天后，转录因子PAX4以及心脏纤维化发生时常见的标志物fibronectin、αSMA和Col I含量显著 增长。
此后利用同处理条件下培养的心脏成纤维细胞总蛋白进行蛋白质印迹实验，首先检测PAX4的蛋白水平。实验结果如图6所示，实验得到了与免疫荧光实验接近的结果，转录因子PAX4蛋白水平在血管紧张素II刺激后升高(图6A)，定量的结果统计分析表明其蛋白水平升高的程度具有显著性。同时也检测了肌成纤维细胞标志物fibronectin、αSMA和Col I的蛋白水平(图7)，实验结果表明血管紧张素II刺激心脏成纤维细胞三天能够促进纤维化标志物fibronectin、αSMA和Col I的蛋白水平(图7A)，定量结果统计分析表明纤维化标志物在血管紧张素II刺激后，较对照组蛋白水平显著的升高。
实施例4、利用鼠尾静脉注射小干扰RNA的方式敲减小鼠体内PAX4水平，之后构建纤维化模型，利用超声心动、比较心体比、天狼星红(PSR)染色、蛋白质印迹实验、免疫荧光实验的方法检测PAX4对于心功能的作用，以及对于肌成纤维细胞标志物fibronectin和αSMA，细胞外基质Col I的蛋白水平的影响。
鼠尾静脉注射小干扰RNA：选用野生型C57BL/6成年小鼠(11周龄，雄性，体重约27g)，尾静脉注射的方法每日给予10nmol的PAX4敲减小干扰RNA序列(化学修饰的序列，包括敲减序列和无意义的对照序列)(Ribobio Co.,Ltd(Guangzhou,China))，每次注射体积0.12mL(生理盐水溶解)，持续注射3天。第四天用上述埋泵方法埋泵开始输注血管紧张素II。从第五天开始每隔1天给小鼠尾静脉注射对照和敲减小干扰RNA序列，直到血管紧张素II持续输注7天。测其超声指标、体重、心脏重量，收取心肌组织进行后续检测。
超声心动：将小鼠置于麻醉箱中给予异氟烷(2.5％异氟烷，0.8L/min)进行麻醉，麻醉完毕将小鼠从麻醉箱中取出，迅速将其以仰卧位放置于加热板上并将连接麻醉药的鼻罩戴好，胶条固定小鼠四肢，将异氟烷浓度调至1％进行维持麻醉。胸部用脱毛膏(Nail,Canada)进行脱毛。采用Vevo 2100超声仪(Fujifilm Visual sonics，Canada)进行小鼠超声心动图检测。
心脏称重：小鼠采血完毕，迅速剪开其胸廓，用留置针以冰的PBS(0.8％NaCl,0.02％KCl,0.02％KH2PO4,0.4％Na2HPO4)灌洗心脏，之后取出完整心脏，在冰的PBS中剪掉心脏上的血管、脂肪等组织，用滤纸吸干水分，称量全心脏重量(heart weight,HW)，称重后，剪掉心脏上的心耳，剩下整个心室。用手术刀片留取心脏中部乳头肌 横切部分，置于4％多聚甲醛(W/V％，以PBS配制)中固定以做组织切片，其余心肌组织放入冻存管于液氮中冻存。
组织切片及染色：心脏乳头肌水平的横断面于4％多聚甲醛溶液(W/V％，以PBS配制)中固定6～8小时后，弃去多聚甲醛，加入20％的蔗糖溶液(W/V％，以PBS配制)中脱水。然后再依次放入70％(3小时)，80％(3小时)乙醇溶液中梯度脱水，最后置于90％乙醇加正丁醇溶液(体积比1：1)中过夜。次日依次置入95％乙醇加正丁醇溶液(45分钟2次)、正丁醇(30分钟)、丁醇(20分钟)，用滤纸吸干表面液体，用石蜡包埋组织块。之后用切片机做心脏切片，石蜡切片厚度5μm，于乳头肌水平横切，进行天狼星红染色，以检测胶原沉积情况。首先进行脱蜡，二甲苯10分钟3次，100％乙醇3分钟2次，95％乙醇3分钟2次，80％乙醇3分钟1次，70％乙醇3分钟1次。最后用蒸馏水洗3次。之后进行天狼星红染色，先把水蘸干，放入天狼星红溶液中染色1分钟，蒸馏水中洗去浮色(3次)，用95％乙醇快速洗1次，再放入100％乙醇中1分钟2次(注意不要让黄染的颜色洗掉)，最后用80％二甲苯进行透明处理(10分钟2次)，中性树脂覆盖切片表面，加盖玻片封存。最后组织切片观察及定量分析。采用NanoZoomer-SQ(Hamamatsu,Japan)图像分析系统分析定量胶原纤维面积(天狼星红染色)。观察天狼星红染色切片，每个标本测量胶原纤维化面积(红染部分)，测量整个心脏横断面面积，用纤维化面积除以心脏总面积即纤维化的百分数。
在进行了超声心动等指标检测后，称取体重，收取心脏组织称量重量，取部分心脏组织进行冰冻切片和石蜡切片的组织包埋准备后续的免疫组化实验，收心脏组织的蛋白样品进行蛋白质印迹实验。
首先检测PAX4的蛋白敲减效率(图8)，同时检测血管紧张素II刺激对于PAX4蛋白水平的影响。实验结果表明，PAX4的蛋白敲减效率良好，注射无义序列的小鼠在给予血管紧张素II后，其心肌PAX4蛋白表达量增加，与之前实施例1中的实验结果一致。定量结果统计分析表明血管紧张素II显著促进PAX4蛋白水平，敲减后蛋白水平为敲减前的三分之一(图8B)。
其次，天狼星红染色的结果如图9所示，注射无义序列的小鼠给予血管紧张素II后，代表心脏纤维化的胶原面积增多。注射PAX4小干扰RNA的小鼠与注射无义序列的小鼠心脏纤维化面积没有明显变化。注射PAX4小干扰RNA的小鼠再给予血管紧张素II，与注射PAX4小干扰RNA不给予血管紧张素II的小鼠比较，心脏纤维化 面积增多；与注射无义小干扰RNA后给予血管紧张素II的小鼠比较，心脏纤维化面积减少(图9A)。定量结果统计小鼠的心脏纤维化面积分析表明敲减PAX4能够显著抑制血管紧张素II所引起的心脏纤维化(图9B)。
使用心脏组织冰冻切片进行了Col I的免疫荧光实验(图10)，实验结果与天狼猩红染色的结果一致，注射无义序列的小鼠给予血管紧张素II后心脏组织中Col I红色荧光强度增高，注射PAX4小干扰RNA后给予血管紧张素II小鼠的心脏组织Col I荧光强度明显减弱。
超声心动图的结果如图11所示，敲减PAX4能够显著减少血管紧张素II诱导的左室后壁厚度(LVPWD；d)的增厚(图11A)，能够显著改善血管紧张素II造成的EF的下降(图11B)，有改善血管紧张素造成的FS下降的趋势(图11C),能够显著改善血管紧张素II造成的E/E’的升高(图11D)。以上超声心动结果提示敲减PAX4对于小鼠心脏的收缩功能、舒张功能均起到保护作用。
综合以上实验结果，提示敲减PAX4能够起到缓解细胞外基质增多，抑制纤维化发生，改善心脏功能的作用。
实施例5、在心脏成纤维细胞水平干预PAX4蛋白水平，使用免疫荧光实验或者蛋白质印迹实验检测对于对于肌成纤维细胞标志物fibronectin、αSMA和Col I的蛋白水平的影响。
心脏成纤维细胞转染小干扰RNA：心脏成纤维细胞P2代在实验转染小干扰RNA的前一日晚上由P1代传代到6孔板中，37摄氏度，5％二氧化碳环境下过夜培养，确保成纤维细胞形态铺展，并且尚未产生影响转染效率的细胞外基质。转染当日37摄氏度温热PBS温和清洗6孔板中的成纤维细胞，重复清洗三遍，确保彻底洗去培养液,之后每孔加入500μl OPTI-MEM(Opti-MEM I Reduced Serum Medium,31985070,Life)，再分别加入80nmol/L PAX4小干扰RNA(sc-152040,Santa Cruz Biotech,CA,USA))或者对照小干扰RNA(sc-37007,Santa Cruz Biotech,CA,USA)和3μl HiPerFect转染试剂(301705,QIAGEN,Beijing,China)。转染6小时后加含10％胎牛血清的DMEM培养液培养。检测蛋白的实验使用转染小干扰RNA三天时的细胞样品。
腺病毒心脏成纤维感染PAX4：心脏成纤维细胞P2代在实验加病毒感染前一日晚上由P1代传代到6孔板中，37摄氏度，5％二氧化碳环境下过夜培养，确保成纤维细胞形态铺展，并且尚未产生影响转染效率的细胞外基质。细胞生长60％密度时，使用 2MOI PAX4或对照腺病毒在无血清条件下感染细胞，6小时后换液，换含10％胎牛血清DMEM培养三天后进行后续实验。
为了进一步确定PAX4在心脏成纤维细胞中的作用，首先设计实验通过在心脏成纤维细胞中转染PAX4小干扰RNA的方式敲减PAX4，蛋白质印迹实验证实心脏成纤维细胞中敲减PAX4有效(图12)。此后使用免疫荧光的实验检测敲减PAX4后肌成纤维细胞标志物fibronectin、αSMA和Col I的荧光强度。实验结果表明敲减PAX4后，肌成纤维细胞标志物fibronectin(图13A)、αSMA(图13B)和Col I(图13C)的荧光减弱。
在成纤维细胞中过表达PAX4三天后进行蛋白质印迹实验，实验结果如图14所示，过表达PAX4后，肌成纤维细胞标志物fibronectin、αSMA和Col I蛋白水平均有不同程度的升高。
以上实验均提示，PAX4能够促进心脏纤维化的发生，敲减PAX4能够降低纤维化标志物fibronectin、αSMA和Col I的含量。
实施例6、在心脏成纤维细胞中，敲减细胞内PAX4的蛋白水平，从而利用蛋白质印记实验的手段检测其下游调控基因的表达水平。
由于动物实验和细胞实验均提示敲减PAX4能够对心脏功能起到保护作用。然而其中的作用机制尚不清楚。因此我们利用生物信息学手段分析找到PAX4可能调控的三个影响心脏纤维化的作用靶点TGFβ(促纤维化)、IL1R2以及TGIF2(纤维化抑制因子)。TRANSFAC预测PAX4能够结合在TGFβ、IL1R2以及TGIF2的启动子上，因此我们利用蛋白质印迹实验的方法检测了敲减PAX4时这三个基因的蛋白水平。
如图15所示，心脏成纤维细胞敲减PAX4后，TGFβ蛋白水平降低，IL1R2以及TGIF2蛋白水平升高，这提示PAX4是通过促进纤维化促进因子TGFβ，抑制纤维化抑制因子IL1R2以及TGIF2从而起到多维度促进纤维化的作用。
以上具体实施方式仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神及原则之内所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。以上所述是本发明的优选实施例，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明所述原理的前提下，还可以作出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (8)

  1. PAX4的抑制剂在制备抑制纤维化的药物中的应用。
  2. 根据权利要求1所述的应用，所述的PAX4的抑制剂包括siRNA、PAX4基因敲除试剂。
  3. 根据权利要求2所述的应用，所述的PAX4基因敲除试剂为siRNA。
  4. 根据权利要求1～3任意一项所述的应用，所述的纤维化是指心脏纤维化、胰脏纤维化或肺脏纤维化。
  5. PAX4基因或其表达产物在促进体外心脏成纤维细胞增殖中的应用。
  6. PAX4基因敲除试剂在抑制体外心脏成纤维细胞增殖中的应用。
  7. PAX4基因敲除试剂在体外心脏成纤维细胞中抑制或阻断纤维化促进因子TGFβ的表达以及促进纤维化抑制因子IL1R2和TGIF2的表达中的应用。
  8. PAX4基因敲除试剂通过抑制纤维化促进因子TGFβ以及促进纤维化抑制因子IL1R2和TGIF2在促进细胞纤维化中的应用。
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