WO2022068248A1 - 降低kat7含量或活性的物质在预防衰老和治疗肝纤维化中的应用 - Google Patents

降低kat7含量或活性的物质在预防衰老和治疗肝纤维化中的应用 Download PDF

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WO2022068248A1
WO2022068248A1 PCT/CN2021/098217 CN2021098217W WO2022068248A1 WO 2022068248 A1 WO2022068248 A1 WO 2022068248A1 CN 2021098217 W CN2021098217 W CN 2021098217W WO 2022068248 A1 WO2022068248 A1 WO 2022068248A1
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kat7
gene
substance
cells
sgkat7
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刘光慧
曲静
张维绮
汪伟
孙淑慧
李维
宋默识
王思
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中国科学院动物研究所
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Definitions

  • the present invention relates to the application of substances reducing the content or activity of KAT7 in preventing aging and treating liver fibrosis in the field of biomedicine.
  • the essence of aging is the gradual decline of the function of various organ systems in the body, the final result of which is the death of the individual.
  • the degenerative changes in organ function are driven by both genetic and environmental factors.
  • Cell senescence refers to the change process in which the cell proliferation and differentiation ability and physiological function gradually decline with the passage of time during the process of cell execution of life activities.
  • a certain number of senescent cells accumulate in aging organs and tissues, and the accumulated senescent cells lose their original normal physiological functions, thereby affecting the function of organs and tissues.
  • SASP senescence-associated secretory phenotype
  • Liver fibrosis is an aging-related disease, and liver fibrosis is more severe in older individuals than in younger individuals.
  • the specific manifestations are the regeneration of hepatocytes after repeated destruction, and the diffuse excessive deposition and abnormal distribution of extracellular matrix such as collagen, glycoproteins, and proteoglycans in the liver, which is the pathological repair response of the liver to chronic injury.
  • Liver fibrosis is histologically reversible, but can progress to cirrhosis without treatment.
  • the only treatment for cirrhosis is liver transplantation, however, due to the shortage of donors, the vast majority of patients are not well treated. Therefore, the period of liver fibrosis is a window for treatment.
  • gene therapy can be used to introduce factors such as Foxa1, Foxa2, Foxa3, Gata4, Hnfla and Hnf4a into the liver to alleviate CCL4 (carbon tetrachloride)-induced liver fibrosis.
  • the technical problem to be solved by the present invention is how to prevent aging and treat liver fibrosis.
  • the present invention provides any of the following applications:
  • the substance may be a CRISPR/Cas9 gene knockout system targeting the KAT7 gene.
  • the substance may be a recombinant vector containing the CRISPR/Cas9 gene knockout system.
  • the recombinant vector can be a sgRNA plasmid that can express a specific targeting KAT7 gene obtained by inserting a specific fragment of the KAT7 gene into lenti-CRISPRv2 using Esp3I.
  • the substance may be a KAT7 inhibitor.
  • the KAT7 inhibitor can be WM-3835.
  • the senescence may be the senescence of animal cells, tissues, organs or individuals.
  • the senescence of the cells may be senescence of mesenchymal precursor cells or mesenchymal stem cells or senescence of hepatocytes.
  • the senescence of the mesenchymal precursor cells or mesenchymal stem cells can be pathological senescence or replicative senescence.
  • the pathological senescence of the mesenchymal precursor cells or mesenchymal stem cells is senescence caused by mutations in the human adult progeria gene (WRN loss of function).
  • the senescence of the hepatocytes is the senescence of primary cells.
  • the aging of the tissue or organ may be the natural aging of the tissue or organ.
  • the aging of the individual may be natural aging.
  • the animal can be a mammal, such as a human or a mouse.
  • the present invention also provides the application of the substance that reduces the content or activity of KAT7 or the substance that inhibits the expression of KAT7 gene or knocks out the KAT7 gene in the preparation of a product for inhibiting liver fibrosis.
  • the liver fibrosis is CCL4-induced liver fibrosis.
  • the present invention also provides a product for treating and/or preventing aging, and the active ingredient of the product can be the substance that reduces the content or activity of KAT7 or the substance that inhibits the expression of KAT7 gene or knocks out KAT7 gene.
  • the present invention also provides a product for inhibiting liver fibrosis, and the active ingredient of the product can be a substance that reduces the content or activity of KAT7 or a substance that inhibits KAT7 gene expression or knocks out KAT7 gene.
  • the present invention also provides a method for delaying and/or treating and/or preventing aging, the method comprising: administering to animal cells, tissues, organs or individuals a substance that reduces the content or activity of KAT7 or inhibits KAT7 gene expression or knocks out KAT7 gene A substance to delay and/or treat and/or prevent the aging of the animal cells, tissues, organs or individuals.
  • the animal cells, tissues, organs or individuals may be normal animal cells, tissues, organs or individuals, or may be aged animal cells, tissues, organs or individuals.
  • the animal can be a mammal.
  • the mammal can be human or mouse.
  • the cells may be mesenchymal precursor cells or mesenchymal stem cells or hepatocytes.
  • the senescence of the mesenchymal precursor cells or mesenchymal stem cells can be pathological senescence or replicative senescence.
  • the pathological senescence of the mesenchymal precursor cells or mesenchymal stem cells is senescence caused by mutations in the human adult progeria gene (WRN loss of function).
  • the senescence of the hepatocytes is the senescence of primary cells.
  • the aging of the tissue or organ may be the natural aging of the tissue or organ.
  • the aging of the individual may be natural aging.
  • the product can be a medicine.
  • the mesenchymal cells can be mesenchymal precursor cells or mesenchymal stem cells.
  • Figure 1 shows that knockdown of KAT7 gene can delay the accelerated aging phenotype of mesenchymal precursor cells carrying human progeria gene mutations.
  • sg-NTC represents cells infected with sgNTC virus
  • sg-KAT7#1 and sg-KAT7#2 represent two types of cells infected with sgKAT7-human lentivirus.
  • the Bar value in b is 25mm. *** indicates that the difference reached a significant level (p ⁇ 0.001).
  • Figure 2 shows that knockdown of KAT7 can delay replicative senescence of mesenchymal precursor cells.
  • sg-NTC indicates cells infected with sgNTC virus
  • sg-KAT7 indicates cells infected with sg-KAT7#1 lentivirus.
  • the Bar values in a and b are 25mm and 100mm. *** indicates that the difference reached a significant level (p ⁇ 0.001).
  • Figure 3 shows the therapeutic effect of knockdown of KAT7 gene on delaying aging in aged mice.
  • Young means young mice
  • Aged means old mice
  • sg-NTC means old mice infected with sgNTC virus
  • sg-KAT7 means old mice infected with sgKAT7-mouse lentivirus.
  • the Bar values in f are 300mm and 200mm. * means the difference reaches a significant level (p ⁇ 0.05), ** means a difference reaches a significant level (p ⁇ 0.01).
  • Figure 4 shows that knockdown of KAT7 can delay the senescence of human primary hepatocytes.
  • sg-NTC indicates cells infected with sgNTC virus
  • sg-KAT7 indicates cells infected with sgKAT7-human lentivirus.
  • the Bar value in b is 100mm. *** means that the difference reaches a significant level (p ⁇ 0.001), ** means that the difference reaches a significant level (p ⁇ 0.01).
  • Figure 5 shows the therapeutic effect of KAT7 knockdown on delaying CCL4-induced liver fibrosis.
  • sg-NTC represents mice infected with sgNTC virus
  • sg-KAT7 represents mice infected with sgKAT7-mouse lentivirus.
  • the Bar values in a are 300mm, 50mm, 100mm and 50mm from top to bottom. * indicates that the difference reaches a significant level (p ⁇ 0.05), ** indicates that the difference reaches a significant level (p ⁇ 0.01), and *** indicates that the difference reaches a significant level (p ⁇ 0.001).
  • Figure 6 shows the effect of KAT7 inhibitor WM-3835 in delaying the senescence of mesenchymal precursor cells and hepatocytes.
  • DMSO indicates control-treated mesenchymal precursor cells or hepatocytes
  • WM-3835 indicates inhibitor-treated mesenchymal precursor cells or hepatocytes.
  • the Bar value in b is 25mm
  • the Bar value in c is 50mm. ** means that the difference reaches a significant level (p ⁇ 0.01)
  • *** means that the difference reaches a significant level (p ⁇ 0.001).
  • Human embryonic kidney cell line 293T ATCC product, Cat. No. CRL-3216.
  • 293T cell culture medium 89 parts DMEM high glucose medium (Hyclone), 10 parts fetal bovine serum (Gibco, 10100-147), 1 part penicillin/streptomycin (Invitrogen, 15070-063).
  • MPC medium Mesenchymal precursor cell culture medium: 88 parts by volume ⁇ -MEM+GlutaMAX (Invitrogen, 12571071), 10 parts by volume fetal bovine serum (Gibco, 10100-147), 1 part by volume non-essential amino acids ( Invitrogen, 11140-050), 1 volume part penicillin/streptomycin (Invitrogen, 15070-063), and recombinant human fibroblast growth factor (JPC) was added to the system at a concentration of 1 ng/ml.
  • MPC medium Mesenchymal precursor cell culture medium
  • the WS mesenchymal precursor cells in the following examples are the mesenchymal stem cells with loss of WRN function in the Chinese patent (pluripotent stem cells carrying human progeria gene mutation and its preparation method, ZL201510137846.4).
  • the biological material is obtained from the applicant, and the biological material is only used for repeating the relevant experiments of the present invention, and cannot be used for other purposes.
  • the human embryonic stem cell H9 cell line (product of WiCell, product number: WA09(H9)-DL-7) was used to prepare mesenchymal precursor cells. The specific steps are as follows:
  • step (1) (2) culturing the embryoid body obtained in step (1) until fibrous cells appear;
  • step (2) cells are collected, passaged and cultured;
  • step (3) the cells that are positive for CD73, CD90 and CD105 are sorted, which are mesenchymal precursor cells.
  • the mesenchymal precursor cells are cultured according to the following method: the culture of the mesenchymal precursor cells is cultured with the above-mentioned mesenchymal precursor cell culture medium, and the cells are passaged once every 4-5 days. Each passage was seeded into one well in a 6-well plate at a density of 1 x 105 cells.
  • the SA- ⁇ -gal staining steps for detecting cell senescence are as follows:
  • SA- ⁇ -gal senescence-associated beta-galactosidase
  • SA- ⁇ -gal is a hydrolase in lysosomes, and its activity is enhanced in senescent cells.
  • the enzyme can hydrolyze it and make cells Appears blue.
  • SA- ⁇ -gal has thus become a "gold standard" for rapid and rapid detection of cellular senescence.
  • the specific method is as follows: 1) Mesenchymal precursor cells were seeded into one well of a gelatin (sigma)-coated 6-well plate at a density of 1 ⁇ 10 5 /well, and stained on the second day.
  • step 2) After step 1), fix with fixative solution (2% (volume percentage, v/v) formaldehyde + 0.2% (volume percentage, v/v) glutaraldehyde + 97.8% (volume percentage, v/v) PBS) Cells were incubated for 4 minutes (must not exceed 5 minutes) and washed 2 times with PBS.
  • fix with fixative solution 2% (volume percentage, v/v) formaldehyde + 0.2% (volume percentage, v/v) glutaraldehyde + 97.8% (volume percentage, v/v) PBS
  • step 2) After step 2), add 2mL staining solution (40mM citric acid/sodium phosphate buffer, 5mM K4[Fe(CN)6], 5mM K3[Fe(CN)6], 150mM NaCl, 2mM MgCl2, 1mg to each well after the completion of step 2) /mL X-gal), incubate overnight in the dark in a 37°C bacterial incubator. 4) After step 3), wash twice with PBS, observe under an inverted microscope, and take pictures.
  • 2mL staining solution 40mM citric acid/sodium phosphate buffer, 5mM K4[Fe(CN)6], 5mM K3[Fe(CN)6], 150mM NaCl, 2mM MgCl2, 1mg
  • Example 1 KAT7 knockdown can delay the accelerated aging phenotype of mesenchymal precursor cells carrying human progeria gene mutation
  • the present invention finds that knocking down the KAT7 (lysine acetyltransferase 7) gene can delay the accelerated aging phenotype of the mesenchymal precursor cells carrying the human adult progeria gene mutation.
  • the experimental steps are as follows:
  • sgKAT7-huamn-F1 5′-CACCGTTCAATCTCTGTGTTTGAAG-3′ (sequence 1 in the sequence listing);
  • sgKAT7-human-R1 5'-AAACCTTCAAACACAGAGATTGAAC-3' (Sequence 2 in the Sequence Listing);
  • sgKAT7-huamn-F2 5'-CACCGACGATCTGCTCGAGTCACCC-3' (sequence 3 in the sequence listing);
  • sgKAT7-human-R2 5'-AAACGGGTGACTCGAGCAGATCGTC-3' (SEQ ID NO: 4 in the Sequence Listing).
  • the oligonucleotide sequence of the above-mentioned gRNA targeting KAT7 gene was synthesized in the company (Thermo Fisher), sgKAT7-huamn-F1 was annealed with sgKAT7-human-R1 to obtain sgKAT7-human-1, and T4 ligase (NEB) was used to connect to the FastDigest_Esp3I (NEB) digested lenti-CRISPRv2 (Addgene product, #52961) on the backbone of the vector, and the obtained recombinant vector with the correct sequence was denoted as the recombinant vector sgKAT7-human-1.
  • T4 ligase N4 ligase
  • the lenti-CRISPRv2 vector backbone contains the gene encoding Cas9 endonuclease and can express Cas9 endonuclease, and also contains the insertion site of exogenous DNA fragment for guiding Cas9 to a specific site in the genome and the encoding DNA of the gRNA backbone.
  • the recombinant vector sgKAT7-human-1 can encode sgRNA targeting KAT7 gene.
  • sgKAT7-huamn-F2 was annealed with sgKAT7-human-R2 to obtain sgKAT7-human-2, which was ligated to lenti-CRISPRv2 (Addgene product, #52961) digested with FastDigest_Esp3I (NEB) using T4 ligase (NEB). ) on the backbone of the vector, the obtained recombinant vector with the correct sequence is denoted as the recombinant vector sgKAT7-human-2.
  • the recombinant vector sgKAT7-human-2 can encode sgRNA targeting KAT7 gene.
  • sgNTC-F1 was annealed with sgNTC-R1 to obtain sgNTC, which was connected to the vector backbone obtained by cutting lenti-CRISPRv2 (Addgene product, #52961) with FastDigest_Esp3I (NEB) with T4 ligase (NEB).
  • the correct recombinant vector is the control vector.
  • sgNTC-F1 5'-CACCGACGGAGGCTAAGCGTCGCAA-3' (sequence 5 in the sequence listing);
  • sgNTC-R1 5'-AAACTTGCGACGCTTAGCCTCCGTC-3' (sequence 6 in the sequence listing).
  • the lentiviral plasmid sgKAT7-human, lentiviral packaging vector psPAX2 and pMD2G were co-transfected into 293T cells (the ratio is: 1 10cm dish 293T cell: 9 ⁇ g lentiviral plasmid sgKAT7 -human, 6 ⁇ g psPAX2 and 3 ⁇ g pMD2G) for 8 hours.
  • step 1.2.1 After completing step 1.2.1, replace with fresh 293T cell culture medium and continue to culture for 48-54 hours.
  • step 1.2.3 After completing step 1.2.2, collect the supernatant, filter with a 0.22 ⁇ m filter membrane, and collect the filtrate.
  • step 1.2.4 Take the filtrate obtained in step 1.2.3, centrifuge at 4°C and 19400rpm for 2.15 hours, discard the supernatant, and resuspend the pellet in MPC medium, which is the virus liquid containing sgKAT7-human recombinant lentivirus, referred to as sgKAT7-human virus
  • MPC medium which is the virus liquid containing sgKAT7-human recombinant lentivirus
  • the virus solution obtained by using the recombinant vector sgKAT7-human-1 was designated as sg-KAT7#1 virus solution
  • the virus solution obtained by using the recombinant vector sgKAT7-human-2 was designated as sg-KAT7#2 virus solution.
  • step 1.2 the lentiviral plasmid sgKAT7-human was replaced with the control vector, and other steps remained unchanged to obtain a control virus solution, which was recorded as sgNTC virus solution.
  • sgKAT7-human lentivirus infects mesenchymal precursor cells.
  • the fourth-generation WS mesenchymal precursor cells were used as test cells to infect sgNTC and two sgKAT7-human viruses, respectively.
  • the specific method is as follows: 2 ⁇ L of sgKAT7-human lentivirus (or sgNTC virus) and 2 ⁇ L of Polybrene are added to the culture wells (one well of a 6-well plate) inoculated with the fourth passage of WS mesenchymal precursor cells. The medium was changed the next day, and then the cells were cultured normally and passaged.
  • sgNTC sgKAT7-human lentivirus
  • sgKAT7-human lentivirus After infection with sgNTC or sgKAT7-human lentivirus, the resulting cells were serially passaged to passages 3-4. Cells were collected, KAT7 protein knockdown efficiency was detected, and cell senescence marker SA- ⁇ -gal staining was performed to detect IL6, cell proliferation molecular marker (Ki67) and senescence-related genes (p16 INK4A , p21 CIP1 , LMNB1, LAP2) mRNA levels.
  • Ki67 cell proliferation molecular marker
  • senescence-related genes p16 INK4A , p21 CIP1 , LMNB1, LAP2
  • the reagent used for the detection of Ki67 is the product of Zhongshan Jinqiao Company, the product number is ZM-0166.
  • the primers used to detect the mRNA levels of p16 INK4A , p21 CIP1 , LMNB1, and LAP2 are as follows:
  • p16 INK4A -F1 5'-ATGGAGCCTTCGGCTGACT-3' (sequence 7 in the sequence listing);
  • p16 INK4A -R1 5'-GTAACTATTCGGTGCGTTGGG-3' (sequence 8 in the sequence listing);
  • p21 CIP1 -F1 5'-CGATGGAACTTCGACTTTGTCA-3' (sequence 9 in the sequence listing);
  • p21 CIP1 -R1 5'-GCACAAGGGTACAAGACAGTG-3' (sequence 10 in the sequence listing);
  • LMNB1-F1 5'-GAAAAAGACAACTCTCGTCGCA-3' (sequence 11 in the sequence listing);
  • LMNB1-R1 5'-GTAAGCACTGATTTCCATGTCCA-3' (sequence 12 in the sequence listing);
  • LAP2-F1 5'-CCCCTCGGTCCTGACAAAAG-3' (sequence 13 in the sequence listing);
  • LAP2-R1 5'-CGCTCTTCGTCACTGGAGAA-3' (sequence 14 in the sequence listing).
  • the reagent used to detect IL6 secretion was produced by BioLegend Company, the product number is 504601.
  • the antibody used to detect KAT7 protein expression was produced by abcam company, the product number is ab70183.
  • Example 2 KAT7 knockdown can delay replicative senescence of mesenchymal precursor cells
  • the 7th generation mesenchymal precursor cells were used as test cells to infect sgNTC and sgKAT7-human virus (that is, sg-KAT7#1 lentivirus) obtained in Example 1 respectively.
  • the lentivirus preparation method and infection method were the same as the embodiment. 1.
  • the cells were collected for SA- ⁇ -gal staining, a cell senescence marker, and the mRNA levels of cell proliferation molecular markers (Ki67) and senescence-related genes (p16 INK4A , p21 CIP1 , LMNB1) were detected etc., the detection method is the same as that in Example 1.
  • the detection method of KAT7 protein expression level is the same as that of Example 1.
  • sgKAT7-mouse-F 5'-CACCGAGCCGCCGGCAATGCCGCGA-3' (sequence 15 in the sequence listing);
  • sgKAT7-mouse-R 5'-AAACTCGCGGCATTGCCGGCGGCTC-3' (SEQ ID NO: 16 in the Sequence Listing).
  • the oligonucleotide sequence of the above-mentioned gRNA targeting KAT7 gene was synthesized in the company (Thermo Fisher), sgKAT7-mouse-F was annealed with sgKAT7-mouse-R to obtain sgKAT7-mouse, and T4 ligase (NEB) was used to connect sgKAT7-mouse to FastDigest_Esp3I (NEB) was used to digest the vector backbone obtained by lenti-CRISPRv2 (Addgene product, #52961) to construct the sgKAT7-mouse lentiviral plasmid with correct sequence capable of specifically knocking down mouse KAT7.
  • the sgKAT7-mouse lentiviral plasmid can be used to knock down the mouse KAT7 gene.
  • mice Male, 20-month-old SPF mice (C57BL6), Suzhou Aiermaite Technology Co., Ltd. Mice rearing conditions: 23°C, 12 hours light/12 hours dark, free access to food and water. Experimental equipment and materials include: isoflurane gas anesthesia apparatus, insulin syringe.
  • sgKAT7 group (17 model mice): On the 1st and 60th day of the experiment, each mouse was injected with 100 ⁇ l of sgKAT7-mouse lentivirus solution through the tail vein.
  • sgNTC group (17 model mice): On the 1st and 60th day of the experiment, 100 ⁇ l of sgNTC virus solution was injected into the tail vein of each mouse.
  • Aged group (6 SPF mice (C57BL6): 28 months old, no treatment, normal feeding.
  • sgKAT7-mouse lentivirus and control virus were infected with MPCs (i.e. mesenchymal precursor cells) isolated from the bone marrow of mice (C57BL6), respectively. Proteins were extracted 7 days later, and western blotting was performed. KAT7 expression was detected, and the knockdown efficiency of sgKAT7-mouse was identified (a in Figure 3). The results showed that sgKAT7-mouse lentivirus could reduce cellular KAT7 content.
  • Antibody used Anti-human/mouse KAT7 antibody (ab70183), Abcam.
  • Anti-human actin antibody (sc-69879), Santa Cruz Biotechnology.
  • mice Six months after injection into aged mice, the health of the mice was examined using open-field and grip strength tests.
  • Open field experiment First, prepare an open black wooden box (internal field size is 81cm ⁇ 81cm, height 28cm. The bottom of the box is painted with 3 white lines (width 3mm) vertically and horizontally, thus forming 16 equal-sized squares (each The size of the grid is 20cm ⁇ 20cm). After that, each mouse is tested once, and the mouse is placed in one of the 4 corner squares facing the wall, and it is allowed to freely explore the environment for 5 minutes. The software analyzes that the mouse is in the middle of the 4 squares. Grid time and distance.
  • Grip strength test measure the grip strength of each mouse's forelimb, grab the mouse's tail, pull the mouse's tail backward when its forelimb grabs the grip tester, read the value of the grip tester, and repeat 10 times for each mouse ,take the average.
  • mice injected with sgKAT7-mouse virus performed better, and continued feeding found that the lifespan of mice injected with sgKAT7-mouse virus was prolonged.
  • sgKAT7-mouse virus and control virus (sgNTC) mice had median survival times of 143 and 114 weeks, respectively.
  • mice in each group were sacrificed, and the serum was extracted to detect the levels of inflammatory factors tumor necrosis factor TNFa and monocyte chemotactic protein MCP1 in serum. Compared with mice with control virus, the serum levels of TNFa and MCP1 in aged mice injected with sgKAT7-mouse virus were significantly reduced (e in Figure 3). The young and old groups were used as controls.
  • TNFa and MCP1 were performed according to conventional methods, and the services were provided by Beijing Zhongyuan Biological Co., Ltd.
  • liver tissue of mice was stained, using the young group and the old group. as comparison.
  • mice were perfused with normal saline until the liver became white and a part of the liver tissue was taken.
  • Iron hematoxylin (Zhongshan Jinqiao, ZLI-9610) was stained for 8 minutes, and washed with running water for 2 minutes.
  • the hepatic inflammatory cells of the aged group (Aged) mice were increased compared with the young group (Young).
  • the hepatic inflammatory cells of the mice injected with sgKAT7-mouse decreased.
  • p16 staining indicated a decrease in senescent cells (Fig. 3, f).
  • mice were harvested, and a part of the liver tissue was extracted with TRIZOL (gibco, 15596018) and sent to the company (Nuohe) to build a library for transcriptome RNA-seq sequencing.
  • Bioinformatics analysis showed that the up-regulated genes in the liver of aged mice were mainly concentrated in the inflammatory response, and the down-regulated genes were mainly concentrated in the metabolic response. It was found that knocking down KAT7 reversed about one-third of the genes that were altered in the liver tissue of aged mice, and most of these reversed genes were focused on inflammation and metabolism. In addition, the expression of some SASP-related genes was also found to be down-regulated after KAT7 knockdown (g-h in Figure 3).
  • Example 4 KAT7 knockdown can delay the senescence of human primary hepatocytes
  • Human primary hepatocytes were used as test cells (product of lonza company, product number: HUCPI), and the sgNTC and sgKAT7-human viruses of Example 1 were respectively infected.
  • the lentivirus preparation method and infection method were the same as those of Example 1. After infection with the virus, after 8 days of continuous culture, the medium used is the product of lonza company, the product number is CC-3198.
  • RNA-seq analysis showed that, consistent with the results of liver tissue of aged mice, knockdown of KAT7 showed that a large number of inflammatory genes, SASP-related genes were down-regulated, and metabolism-related genes were up-regulated. These results indicate that knocking down KAT7 can delay primary liver disease Cell senescence (c-f in Figure 4).
  • Example 5 Therapeutic effect of knockdown of KAT7 gene on delaying CCL4-induced liver fibrosis
  • mice Male, 10-week-old SPF mice (C57BL6), weighing 20 ⁇ 2 g, Beijing Huafukang Biotechnology Co., Ltd. Mice rearing conditions: 23°C, 12 hours light/12 hours dark, free access to food and water.
  • Experimental equipment and materials include: isoflurane gas anesthesia apparatus, surgical scissors, scalpel, suture, penicillin, asanascope, insulin syringe.
  • Steps 1.1, 1.2 and 1.3 are consecutive days.
  • CCL4-induced liver fibrosis surgery was performed on day 1 of the experiment.
  • sgKAT7 group (12 model mice): In the third week, 200 ⁇ l of the sgKAT7-mouse lentivirus of Example 3 was injected through the hepatic portal vein, and the outer skin was tightly sutured; penicillin was applied to the surgical site to prevent infection.
  • sgNTC group 8 model mice: In the third week, 200 ⁇ l of the sgNTC lentivirus of Example 3 was injected through the hepatic portal vein, and the outer skin was tightly sutured; penicillin was applied to the surgical site to prevent infection.
  • liver fibrosis One of the manifestations of liver fibrosis in mice is the diffuse excessive deposition and abnormal distribution of extracellular matrix such as collagen in the liver. Eight weeks after CLL4-induced hepatic fibrosis modeling, the mice were sacrificed, and a part of the hepatic lobule was taken for section staining.
  • mice were perfused with normal saline until the liver became white, and a part of the liver tissue was taken.
  • Iron hematoxylin (Zhongshan Jinqiao, ZLI-9610) was stained for 8 minutes, and washed with running water for 2 minutes.
  • the Sirius red staining of the mice in the sgNTC group was significantly deepened. Compared with the sgNTC group, the Sirius red staining was attenuated in the sgKAT7-mouse group. In addition, senescence-related staining of SA-b-gal and p16-positive cells was found to be reduced compared with the sgNTC group (Fig. 5a).
  • HA hyaluronic acid
  • PIIINP procollagen type III N-terminal peptide
  • ALP alkaline phosphatase
  • AST aspartate Transaminase
  • HA, PIIINP, ALP, ALT and AST content was carried out according to conventional methods and completed by Beijing Zhongyuan Biological Co., Ltd.
  • Example 6 The effect of KAT7 inhibitor WM-3835 in delaying WS hMPC and hepatocyte senescence
  • the fourth-generation WS mesenchymal precursor cells in Example 1 were used as test cells, and the cells were treated with 50 nM WM-3835 in DMSO. , using the solvent DMSO as a control.
  • the resulting cells were serially passaged to passages 2-3 after treatment.
  • Cells were collected, KAT7 protein knockdown efficiency was detected (b in Figure 6), and cell senescence marker SA- ⁇ -gal staining and cell proliferation molecular marker (Ki67) were detected, and it was found that the inhibitor WM-3835 can delay WS Senescence of hMPCs (Fig. 6 c-d).
  • HHEP Human primary hepatocytes
  • RNA-seq analysis showed that, consistent with KAT7 knockdown by sgKAT7-human lentivirus, a large number of inflammatory genes, SASP-related genes were down-regulated, and metabolism-related genes were up-regulated after WM-3835 treatment.
  • the experiments of the present invention confirm that knocking down the KAT7 gene has a definite effect in delaying aging and treating liver fibrosis diseases; KAT7 inhibitor WM-3835 has a definite effect in delaying the aging of mesenchymal precursor cells and hepatocytes.
  • the invention provides a new idea for developing gene therapy for delaying aging and liver fibrosis, and expands the optional range of clinical gene therapy and drug therapy.

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Abstract

提供了降低KAT7含量或活性的物质在预防衰老和治疗肝纤维化中的应用。通过敲低KAT7基因在延缓衰老和治疗肝纤维化疾病中具有明确的效果；KAT7抑制剂WM-3835在延缓肝细胞衰老中具有明确的效果。

Description

降低KAT7含量或活性的物质在预防衰老和治疗肝纤维化中的应用 技术领域
本发明涉及生物医学领域中，降低KAT7含量或活性的物质在预防衰老和治疗肝纤维化中的应用。
背景技术
衰老的本质是身体各部分器官系统的功能逐渐衰退的过程，其最终结果会导致个体死亡。导致器官功能退行性变化是由遗传和环境因素共同驱动的，伴随机体衰老，组成组织器官的最小功能单位细胞也发生着衰老。细胞衰老是指细胞在执行生命活动过程中，随着时间的推移，细胞增殖与分化能力和生理功能逐渐发生衰退的变化过程。衰老器官组织中积累着一定的衰老细胞，积累的衰老细胞失去细胞原有正常的生理功能，进而影响器官组织功能。同时衰老细胞会分泌的大量炎症因子，被称为衰老相关的分泌表型(senescence-associated secretory phenotype,SASP)，SASP的产生会恶化组织微环境。但是，利用药物或者基因治疗手段清除衰老细胞或者延缓细胞的衰老可以延缓个体衰老或衰老相关的退行性改变。
细胞衰老是生命进程中的必然过程，受到遗传和环境双重因素的影响。正向遗传筛选是确定哪些人类基因参与特定生物学过程的理想工具。近年来发展的CRISPR/Cas9基因编辑技术可以特意切割目标基因组导致相应基因功能失活。因此，可以应用于对衰老相关基因的筛选。
肝纤维化是一种衰老相关的疾病，老年个体肝纤维化较年轻个体严重。具体表现为肝细胞被反复破坏后再生，胶原，糖蛋白，蛋白多糖等细胞外基质在肝脏中弥漫性过度沉积与异常分布，是肝脏对慢性损伤的病理性修复反应。肝纤维化在组织学上是可逆的，但是不加治疗可能发展成肝硬化。肝硬化的唯一治疗方法是肝移植，但是，由于供体短缺，绝大部分病人得不到良好治疗。因此，肝纤维化时期是进行治疗的一个窗口。已有的报道表明可以利用基因治疗的方法将Foxa1,Foxa2,Foxa3,Gata4,Hnfla和Hnf4a等因子导入到肝脏中可以减轻CCL4(carbon tetrachloride)诱导的肝纤维化。
发明公开
本发明所要解决的技术问题是如何预防衰老以及治疗肝纤维化。
为解决上述技术问题，本发明提供了如下任一应用：
1、降低KAT7含量或活性的物质在制备治疗和/或预防衰老产品中的应用；
2、抑制KAT7基因表达或敲除KAT7基因的物质在制备治疗和/或预防衰老产品中的应用。
上述应用中，所述物质可为靶向KAT7基因的CRISPR/Cas9基因敲除系统。
上述应用中，所述物质可为含有所述CRISPR/Cas9基因敲除系统的重组载体。
所述重组载体可为利用Esp3I将KAT7基因的特异片段插入至lenti-CRISPRv2中得到的能表达特异靶向KAT7基因的sgRNA质粒。
上述应用中，所述物质可为KAT7抑制剂。
具体的，所述KAT7抑制剂可为WM-3835。
上述应用中，所述衰老可为动物细胞、组织、器官或个体的衰老。
所述细胞的衰老可为间充质前体细胞或间充质干细胞的衰老或肝细胞的衰老。
所述间充质前体细胞或间充质干细胞的衰老可为病理性衰老或复制性衰老。
在本发明的一个实施例中，所述间充质前体细胞或间充质干细胞的病理性衰老为人类成年早衰症基因(WRN功能丧失)突变引发的衰老。
在本发明的一个实施例中，所述肝细胞的衰老为原代细胞的衰老。
所述组织或器官的衰老可为组织或器官的自然衰老。
所述个体的衰老可为自然衰老。
所述动物可为哺乳动物，如人或小鼠。
本发明还提供了降低KAT7含量或活性的物质或抑制KAT7基因表达或敲除KAT7基因的物质在制备抑制肝纤维化产品中的应用。
在本发明的一个实施例中，所述肝纤维化为CCL4诱导的肝纤维化。
本发明还提供了治疗和/或预防衰老产品，所述产品的活性成分可为所述降低KAT7含量或活性的物质或抑制KAT7基因表达或敲除KAT7基因的物质。
本发明还提供了抑制肝纤维化产品，所述产品的活性成分可为降低KAT7含量或活性的物质或抑制KAT7基因表达或敲除KAT7基因的物质。
本发明还提供了延缓和/或治疗和/或预防衰老的方法，所述方法包括：对动物细胞、组织、器官或个体施用降低KAT7含量或活性的物质或抑制KAT7基因表达或敲除KAT7基因的物质，实现对所述动物细胞、组织、器官或个体衰老的延缓和/或治疗和/或预防。
上述方法中，所述动物细胞、组织、器官或个体可为正常的动物细胞、组织、器官或个体，也可为衰老的动物细胞、组织、器官或个体。
所述动物可为哺乳动物。所述哺乳动物可为人或小鼠。
所述细胞可为间充质前体细胞或间充质干细胞或肝细胞。
所述间充质前体细胞或间充质干细胞的衰老可为病理性衰老或复制性衰老。
在本发明的一个实施例中，所述间充质前体细胞或间充质干细胞的病理性衰老为人类成年早衰症基因(WRN功能丧失)突变引发的衰老。
在本发明的一个实施例中，所述肝细胞的衰老为原代细胞的衰老。
所述组织或器官的衰老可为组织或器官的自然衰老。
所述个体的衰老可为自然衰老。
本发明中，所述产品可为药物。所述间充质细胞可为间充质前体细胞或间充质干细胞。
附图说明
图1为敲低KAT7基因可以延缓携带人类成年早衰症基因突变的间充质前体细胞的加速衰老表型。sg-NTC表示感染sgNTC病毒的细胞，sg-KAT7#1与sg-KAT7#2表示两种感染sgKAT7-human慢病毒的细胞。b中Bar值为25mm。***表示差异达到显著水平(p＜0.001)。
图2为敲低KAT7可延缓间充质前体细胞的复制性衰老。sg-NTC表示感染sgNTC病毒的细胞，sg-KAT7表示感染sg-KAT7#1慢病毒的细胞。a与b中Bar值为25mm和100mm。***表示差异达到显著水平(p＜0.001)。
图3为敲低KAT7基因对于延缓老年小鼠衰老的治疗作用。Young表示年轻小鼠，Aged表示年老小鼠，sg-NTC表示感染sgNTC病毒的老年小鼠，sg-KAT7表示感染sgKAT7-mouse慢病毒的老年小鼠。f中Bar值为300mm和200mm。*表示差异达到显著水平(p＜0.05)，**表示差异达到显著水平(p＜0.01)。
图4为敲低KAT7可以延缓人原代肝细胞的衰老。sg-NTC表示感染sgNTC病毒的细胞，sg-KAT7表示感染sgKAT7-human慢病毒的细胞。b中Bar值为100mm。***表示差异达到显著水平(p＜0.001)，**表示差异达到显著水平(p＜0.01)。
图5为敲低KAT7基因对于延缓CCL4诱导的肝纤维化的治疗作用。sg-NTC表示感染sgNTC病毒的小鼠，sg-KAT7表示感染sgKAT7-mouse慢病毒的小鼠。a中Bar值从上至下依次为300mm,50mm,100mm和50mm。*表示差异达到显著水平(p＜0.05)，**表示差异达到显著水平(p＜0.01)，***表示差异达到显著水平(p＜0.001)。
图6为KAT7抑制剂WM-3835在延缓间充质前体细胞和肝细胞衰老方面的作用。DMSO表示对照处理间充质前体细胞或肝细胞，WM-3835表示抑制剂处理间充质前体细胞或肝细胞。b中Bar值为25mm,c中Bar值为50mm。**表示差异达到显著水平(p＜0.01)，***表示差异达到显著水平(p＜0.001)。
实施发明的最佳方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。
下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂、仪器等，如无特殊说明，均可从商业途径得到。以下实施例中的定量试验，均设置三次重复实验。所有的动物实验均获得中国科学院动物研究所动物福利委员会的批准。实验数据以平均值±标准差表示，用GraghPad Prism6统计软件分析，*P<0.05,**P<0.01,***P<0.001。
人胚胎肾细胞293T系：ATCC产品，货号CRL-3216。
293T细胞培养基：89体积份DMEM高糖培养基(Hyclone)，10体积份胎牛血清(Gibco,10100-147)，1体积份青霉素/链霉素(Invitrogen，15070-063)。
间充质前体细胞培养基(即MPC培养基)：88体积份α-MEM+GlutaMAX(Invitrogen，12571071),10体积份胎牛血清(Gibco,10100-147),1体积份非必需氨基酸(Invitrogen，11140-050)，1体积份青霉素/链霉素(Invitrogen，15070-063),加入重组人成纤维细胞生长因子(JPC)并使其在体系中的浓度为1ng/ml。
下述实施例中的WS间充质前体细胞为中国专利(携带人类成年早衰症基因突变的多能干细胞及制备方法，ZL201510137846.4)中的WRN功能丧失的间充质干细胞，公众可从申请人处获得该生物材料，该生物材料只为重复本发明的相关实验所用，不可作为其它用途使用。
用人胚胎干细胞H9细胞系(WiCell公司产品，货号：WA09(H9)-DL-7)制备获得间充质前体细胞，具体步骤如下：
(1)将人胚胎干细胞H9细胞进行拟胚体分化，得到拟胚体；
(2)将步骤(1)获得的拟胚体进行培养，至纤维状细胞出现；
(3)完成步骤(2)后，收集细胞，传代并培养；
(4)完成步骤(3)后，分选CD73、CD90和CD105均为阳性的细胞，即为间充质前体细胞。
间充质前体细胞按照如下方法培养：间充质前体细胞的培养用上述的间充质前体细胞培养基进行培养，4-5天传一次代。每次传代以1×10 5细胞密度接种到6孔板中的一个孔中。
下述实施例中，检测细胞衰老的SA-β-gal染色步骤如下：
SA-β-gal(senescence-associated beta-galactosidase)是溶酶体中的一种水解酶，在衰老细胞中活性增强，当添加底物X-Gal时，该酶可将其酶解并使细胞显现蓝色。SA-β-gal也因此成为一种快捷迅速检测细胞衰老的“金标准”。具体方法如下：1)以1×10 5/孔的密度将间充质前体细胞接种到明胶(sigma)包被的6孔板的一个孔中，第2天进行染色。2)步骤1)完成后用固定液(2％(体积百分比，v/v)甲醛+0.2％(体积百分比，v/v)戊二醛+97.8％(体积百分比，v/v)PBS)固定细胞4分钟(一定不要超过5分钟)，用PBS洗2遍。3)步骤2)完成后每孔加入2mL染色液(40mM柠檬酸/磷酸钠缓冲液、5mM K4[Fe(CN)6]、5mM K3[Fe(CN)6]、150mM NaCl、2mM MgCl2、1mg/mL X-gal)，在37℃细菌培养箱避光孵育过夜。4)步骤3)完成后用PBS洗2遍后，倒置显微镜下观察，拍照。
实施例1、敲低KAT7基因可以延缓携带人类成年早衰症基因突变的间充质前体细胞的加速衰老表型
本发明发现，敲低KAT7(lysine acetyltransferase 7)基因可以延缓携带人类成年早衰症基因突变的间充质前体细胞的加速衰老表型，实验步骤如下：
1、制备敲低人KAT7基因的重组慢病毒
1.1从GeCKOv2.0(1000000048)质粒库中获得敲低KAT7基因的sgRNA序列，如下：
sgKAT7-huamn-F1：5′-CACCGTTCAATCTCTGTGTTTGAAG-3′(序列表中序列1)；
sgKAT7-human-R1：5′-AAACCTTCAAACACAGAGATTGAAC-3′(序列表中序列2)；
sgKAT7-huamn-F2：5′-CACCGACGATCTGCTCGAGTCACCC-3′(序列表中序列3)；
sgKAT7-human-R2：5′-AAACGGGTGACTCGAGCAGATCGTC-3′(序列表中序列4)。
在公司(Thermo Fisher)合成上述靶定KAT7基因的gRNA的寡核苷酸序列，sgKAT7-huamn-F1与sgKAT7-human-R1退火得到sgKAT7-human-1，用T4连接酶(NEB)连接到用FastDigest_Esp3I(NEB)酶切lenti-CRISPRv2(Addgene产品，#52961)得到的载体骨架上，将得到的序列正确的重组载体记为重组载体sgKAT7-human-1。lenti-CRISPRv2载体骨架中含有Cas9内切酶编码基因并能表达Cas9内切酶，还含有用于引导Cas9到基因组特定位点的外源DNA片段插入位点以及gRNA骨架的编码DNA。重组载体sgKAT7-human-1能编码导向KAT7基因的sgRNA。
按照上述方法，sgKAT7-huamn-F2与sgKAT7-human-R2退火得到sgKAT7-human-2，用T4连接酶(NEB)连接到用FastDigest_Esp3I(NEB)酶切过后的lenti-CRISPRv2(Addgene产品，#52961)载体骨架上，将得到的序列正确的重组载体记为重组载体sgKAT7-human-2。重组载体sgKAT7-human-2能编码导向KAT7基因的sgRNA。
按照上述方法，sgNTC-F1与sgNTC-R1退火得到sgNTC，用T4连接酶(NEB)连接到用FastDigest_Esp3I(NEB)酶切lenti-CRISPRv2(Addgene产品，#52961)得到的载体骨架上，得到的序列正确的重组载体即为对照载体。
sgNTC-F1：5′-CACCGACGGAGGCTAAGCGTCGCAA-3′(序列表中序列5)；
sgNTC-R1：5′-AAACTTGCGACGCTTAGCCTCCGTC-3′(序列表中序列6)。
1.2、将慢病毒质粒sgKAT7-human(即重组载体sgKAT7-human-1或重组载体sgKAT7-human-2)、慢病毒包装载体psPAX2(Addgene产品，#12260)和pMD2G(Addgene产品，#12259)共转染293T细胞,得到sgKAT7-human病毒，步骤如下：
1.2.1采用Lipo3000转染试剂盒(ThermoFisher)，将慢病毒质粒sgKAT7-human、慢病毒包装载体psPAX2和pMD2G共转染293T细胞(配比为：1 个10cm dish 293T细胞：9μg慢病毒质粒sgKAT7-human、6μg psPAX2和3μg pMD2G)，培养8小时。
1.2.2完成步骤1.2.1后，更换为新鲜的293T细胞培养基，继续培养48-54小时。
1.2.3完成步骤1.2.2后，收集上清，采用0.22μm滤膜过滤，收集滤液。
1.2.4取步骤1.2.3得到的滤液，4℃、19400rpm离心2.15小时，弃上清，用MPC培养基重悬沉淀，即为含有sgKAT7-human重组慢病毒的病毒液，简称sgKAT7-human病毒液，将利用重组载体sgKAT7-human-1得到的病毒液记为sg-KAT7#1病毒液，将利用重组载体sgKAT7-human-2得到的病毒液记为sg-KAT7#2病毒液。
按照上述步骤1.2的方法，将慢病毒质粒sgKAT7-human替换为对照载体，其他步骤均不变，得到对照病毒液，记为sgNTC病毒液。
2、sgKAT7-human慢病毒感染间充质前体细胞。
以第4代的WS间充质前体细胞为供试细胞，分别感染sgNTC和两种sgKAT7-human病毒。具体方法为：2μL sgKAT7-human慢病毒(或sgNTC病毒)和2μL Polybrene加到接种有第4代的WS间充质前体细胞的培养孔(6孔板的一个孔)中。次日换液，之后正常培养，传代。
感染sgNTC或sgKAT7-human慢病毒后，将所得细胞连续传代至3-4代。收集细胞，进行KAT7蛋白敲低效率检测，并进行细胞衰老标志物SA-β-gal染色，检测IL6、细胞增殖分子标志物(Ki67)以及衰老相关基因(p16 INK4A、p21 CIP1、LMNB1、LAP2)的mRNA水平。
检测Ki67所用试剂为中杉金桥公司产品，货号是ZM-0166。
检测p16 INK4A、p21 CIP1、LMNB1、LAP2的mRNA水平的引物如下：
p16 INK4A-F1：5′-ATGGAGCCTTCGGCTGACT-3′(序列表中序列7)；
p16 INK4A-R1：5′-GTAACTATTCGGTGCGTTGGG-3′(序列表中序列8)；
p21 CIP1-F1：5′-CGATGGAACTTCGACTTTGTCA-3′(序列表中序列9)；
p21 CIP1-R1：5′-GCACAAGGGTACAAGACAGTG-3′(序列表中序列10)；
LMNB1-F1：5′-GAAAAAGACAACTCTCGTCGCA-3′(序列表中序列11)；
LMNB1-R1：5′-GTAAGCACTGATTTCCATGTCCA-3′(序列表中序列12)；
LAP2-F1：5′-CCCCTCGGTCCTGACAAAAG-3′(序列表中序列13)；
LAP2-R1：5′-CGCTCTTCGTCACTGGAGAA-3′(序列表中序列14)。
检测IL6分泌所用试剂为BioLegend公司生产，货号是504601。
检测KAT7蛋白表达所用抗体为abcam公司生产，货号是ab70183。
衰老相关SASP等检测等衰老生物学研究方法检测的结果(图1中a-e)表明，与对照组sgNTC慢病毒感染细胞相比，sgKAT7-human慢病毒感染细胞KAT7蛋白表达降低(图1中a)，Ki67阳性细胞数显著增加，两个sgKAT7-human慢病毒感染细胞Ki67阳性细胞数分别为sgNTC慢病毒感染细胞的2.9倍和3.1倍 (图1中b)；sgKAT7-human慢病毒感染细胞SA-β-gal染色阳性细胞数显著下降，两个sgKAT7-human慢病毒感染细胞SA-β-gal阳性细胞数分别为sgNTC慢病毒感染细胞的0.3倍和0.3倍(图1中c)；sgKAT7-human慢病毒感染细胞中p16 INK4A、p21 CIP1的表达量均显著下降，LMNB1、LAP2的表达量均显著增加(图1中d)；sgKAT7-human慢病毒感染细胞中IL6分泌显著下降，两个sgKAT7-human慢病毒感染细胞IL6分泌分别为sgNTC慢病毒感染细胞的0.1倍和0.3倍(图1中e)。
说明sgKAT7-human慢病毒能够明显延缓WS间充质前体细胞的病理性衰老进程。
实施例2、敲低KAT7可延缓间充质前体细胞的复制性衰老
以第7代的间充质前体细胞作为供试细胞，分别感染实施例1所得sgNTC和sgKAT7-human病毒(即sg-KAT7#1慢病毒),慢病毒制备方法和感染方法均同实施例1。感染病毒后，经过3-4次传代，收集细胞进行细胞衰老标志物SA-β-gal染色，并检测细胞增殖分子标志物(Ki67),衰老相关基因(p16 INK4A、p21 CIP1、LMNB1)mRNA水平等，检测方法均同实施例1。KAT7蛋白表达水平检测方法同实施例1。
结果显示，sgKAT7-human慢病毒感染细胞KAT7蛋白表达降低，经过3-4次传代后感染sgNTC的间充质前体细胞生长完全阻滞，而感染sgKAT7-human病毒的间充质前体细胞则可快速生长(感染sgKAT7-human病毒的Ki67阳性细胞数为sgNTC慢病毒感染细胞的5.2倍，图2中a)，并且细胞衰老标志物SA-β-gal染色、细胞增殖能力检测以及p16 INK4A、p21 CIP1等衰老分子标志物RT-qPCR检测结果表明，与sgNTC慢病毒相比，sgKAT7-human慢病毒够明显延缓间充质前体细胞的复制性衰老(图2中a-c)：sgKAT7-human慢病毒感染细胞SA-β-gal阳性细胞数为sgNTC慢病毒感染细胞的0.4倍(图2中b)；与sgNTC感染细胞相比，sgKAT7-human慢病毒感染细胞中p16 INK4A、p21 CIP1的表达量均显著下降，LMNB1的表达量显著增加(图2中c)。
实施例3、敲低KAT7基因对于延缓老年小鼠衰老的治疗作用
1、制备敲低小鼠KAT7基因的慢病毒
1.1、重组载体的制备
sgKAT7-mouse-F：5′-CACCGAGCCGCCGGCAATGCCGCGA-3′(序列表中序列15)；
sgKAT7-mouse-R：5′-AAACTCGCGGCATTGCCGGCGGCTC-3′(序列表中序列16)。
在公司(Thermo Fisher)合成上述靶定KAT7基因的gRNA的寡核苷酸序列，sgKAT7-mouse-F与sgKAT7-mouse-R退火得到sgKAT7-mouse，用T4连接酶(NEB) 连接sgKAT7-mouse到用FastDigest_Esp3I(NEB)酶切lenti-CRISPRv2(Addgene产品，#52961)得到的载体骨架上，构建成序列正确的能够特异敲低小鼠KAT7的sgKAT7-mouse慢病毒质粒。sgKAT7-mouse慢病毒质粒能用于敲低小鼠KAT7基因。
1.2、将慢病毒质粒sgKAT7-mouse、慢病毒包装载体psPAX2和pMD2G共转染293T细胞,按照实施例1中步骤1.2获得sgKAT7-mouse病毒，而后获得对照病毒(sgNTC)，两种病毒用1X PBS重悬，得到的两种病毒液中病毒量相等。
2、建立正常自然衰老小鼠模型
雄性，20个月龄的SPF级小鼠(C57BL6)，苏州爱尔麦特科技有限公司。小鼠饲养条件：23℃，12小时光照/12小时黑暗，自由摄食和饮水。实验器械和材料包括：异氟烷气体麻醉仪，胰岛素注射器。
2.1、分组处理
sgKAT7组(17只模型鼠)：试验第1天和60天，每只小鼠尾静脉分别注射100μl sgKAT7-mouse慢病毒液。
sgNTC组(17只模型鼠)：试验第1天和60天，每只小鼠尾静脉分别注射100μl sgNTC病毒液。
年轻组(Young)(6只SPF级小鼠(C57BL6))：2月龄，不做处理，正常饲养。
老年组(Aged)(6只SPF级小鼠(C57BL6))：28月龄，不做处理，正常饲养。
3、效果评价
试验第6个月后，对治疗的效果进行评价。具体方法如下：
3.1、Western blot检测sgKAT7-mouse敲低效率
在进行小鼠体内实验前，将所得sgKAT7-mouse慢病毒与对照病毒(sgNTC)分别感染从小鼠(C57BL6)骨髓分离的MPC(即间充质前体细胞)，7天后提取蛋白，进行western blot检测KAT7表达，鉴定sgKAT7-mouse敲低效率(图3中a)，结果显示sgKAT7-mouse慢病毒可以降低细胞KAT7含量。
所用抗体：抗人/鼠KAT7抗体(ab70183)，Abcam。
抗人actin抗体(sc-69879)，Santa Cruz Biotechnology。
3.2、行为学检测衰老相关的退行性改变
在注射到老年小鼠体内6个月后，利用旷场实验和握力实验检测小鼠健康状况。
旷场实验：首先，准备开口的黑色木盒(内部场地大小为81cm×81cm，高28cm。盒底漆成纵横各3条白线(宽3mm)，从而形成16个等大的方格(每格大小20cm×20cm)。之后每只小鼠接受一次测试，将小鼠面朝墙放入4个拐角方格之一，并让其自由探索环境5min，通过软件分析出小鼠处于中间4个方格的时间和距离。
握力实验：检测每一只小鼠前肢握力，抓住小鼠尾部，当其前肢抓住抓力测定仪后往后拉动小鼠尾巴，读取抓力测定仪数值，每只小鼠重复10次，取平均值。
结果(图3中b-d)发现，注射sgKAT7-mouse病毒的老年小鼠表现更好,并且持续喂养发现注射sgKAT7-mouse病毒小鼠寿命延长，在143周龄时，注射sgKAT7-mouse病毒与对照病毒(sgNTC)小鼠的中位生存时间分别为143周和114周。
3.3、小鼠血液炎症因子检测
在病毒注射到老年小鼠体内8个月后，每组处死6只小鼠，提取血清，检测炎性因子肿瘤坏死因子TNFa和单核细胞趋化蛋白MCP1在血清中的含量，结果表明与注射对照病毒的小鼠相比，注射sgKAT7-mouse病毒的老年小鼠血清中TNFa和MCP1含量显著降低(图3中e)，利用年轻组和老年组作为对照。
TNFa和MCP1检测均按常规方法进行，由北京中原生物有限公司提供服务。
3.4、小鼠肝脏组织染色
老年小鼠状态改善的一个重要的指标是肝脏组织炎性沁润细胞减少，衰老细胞比例减少，在病毒注射到老年小鼠体内8个月后，小鼠肝脏组织染色，利用年轻组和老年组作为对照。
(1)小鼠用生理盐水灌流至肝脏偏白后取肝脏组织一部分。
(2)用4％多聚甲醛(PFA)固定1周。
(3)用30％蔗糖溶液处理2周，脱水。
(4)石蜡包埋，切片。切片厚度为5mm。
(5)对切片进行脱蜡，复水。
(6)铁苏木素(中杉金桥，ZLI-9610)染色8分钟，流水冲洗2分钟。
(7)0.5％伊红(中杉金桥，ZLI-9613)染色2分钟。
(8)脱水，封片，镜下观察。
未注射病毒时，与年轻组(Young)相比，老年组(Aged)小鼠肝脏炎性细胞增加。与注射sgNTC病毒小鼠组相比，注射sgKAT7-mouse组小鼠肝脏炎性细胞减少。此外p16染色表明衰老细胞减少(图3中f)。
3.5老年小鼠肝脏组织RNA-seq分析
8个月后小鼠取材，取一部分肝脏组织用TRIZOL(gibco,15596018)提RNA后送公司(诺禾)建库，进行转录组RNA-seq测序。生物信息学分析表明年老小鼠肝脏中上调基因主要集中在炎性反应上，下调基因主要集中在代谢反应上。发现敲低KAT7能逆转大约1/3的在老年小鼠肝脏组织中变化的基因，并且这些逆转的基因大部分都是集中在炎症和代谢方面。此外，还发现一些SASP相关基因的表达在敲低KAT7之后也会表达下调(图3中g-h)。
实施例4、敲低KAT7可以延缓人原代肝细胞的衰老
以人原代肝细胞(hHEP)作为供试细胞(lonza公司产品，货号是HUCPI)， 分别感染实施例1的sgNTC和sgKAT7-human病毒,慢病毒制备方法和感染方法同实施例1。感染病毒后，经过8天持续培养，所用培养基为lonza公司产品，货号是CC-3198。
培养结束后，细胞衰老标志物SA-β-gal染色、细胞培养上清中IL6等检测结果表明，与sgNTC慢病毒相比，sgKAT7-human慢病毒够明显延缓肝细胞的衰老(图4中a-b)。此外，通过RNA-seq分析表明和老年小鼠肝脏组织结果一致，敲低KAT7后大量炎性基因，SASP相关基因表达下调，代谢相关的基因表达上调，这些结果表明敲低KAT7可以延缓原代肝细胞的衰老(图4中c-f)。
实施例5、敲低KAT7基因对于延缓CCL4诱导的肝纤维化的治疗作用
1建立CCL4诱导肝纤维化小鼠模型
雄性，10周龄的SPF级小鼠(C57BL6)，体重20±2g，北京华阜康生物科技股份有限公司。小鼠饲养条件：23℃，12小时光照/12小时黑暗，自由摄食和饮水。实验器械和材料包括：异氟烷气体麻醉仪，手术剪刀，手术刀，缝合线，青霉素，体式镜，胰岛素注射器。
步骤1.1、步骤1.2和步骤1.3是连续记天数。
1.1、建立CCL4诱导肝纤维化小鼠模型
实验的第1天进行CCL4诱导肝纤维化手术。手术方法如下：首先将小鼠麻醉，用脱毛器对小鼠腹部进行脱毛；按照1ml/kg体重腹腔注射体积百分比为10％的CCL4油剂溶液(CCL4:橄榄油＝1：9)，每周两次。连续注射8周。进行CCL4诱导肝纤维化手术后的小鼠命名为模型鼠。对照组动物腹腔注射同等剂量的橄榄油溶剂。
未进行手术的小鼠命名为正常鼠。
1.2、分组处理
sgKAT7组(12只模型鼠)：在第三周时通过肝门静脉注射200μl实施例3的sgKAT7-mouse慢病毒，严密缝合外层皮肤；在手术部位涂抹青霉素，防止感染。
sgNTC组(8只模型鼠)：在第三周时通过肝门静脉注射200μl实施例3的sgNTC慢病毒，严密缝合外层皮肤；在手术部位涂抹青霉素，防止感染。
正常组(12只正常鼠，Control)：无处理。
1.3、效果评价
试验第8周，对治疗CCL4诱导肝纤维化的效果进行评价。具体方法如下：
1.3.1、小鼠肝脏的天狼星红染色
小鼠肝脏发生纤维化的表现之一就是胶原等胞外基质在肝脏中弥漫性过度沉积与异常分布。CLL4诱导肝纤维化造模后第8周，处死小鼠，取肝小叶一部分进行切片染色。
1)小鼠用生理盐水灌流至肝脏偏白后取肝脏组织一部分。
(2)用4％多聚甲醛(PFA)固定1周。
(3)用30％蔗糖溶液处理2周，脱水。
(4)石蜡包埋，切片。切片厚度为5mm。
(5)对切片进行脱蜡，复水。
(6)铁苏木素(中杉金桥，ZLI-9610)染色8分钟，流水冲洗2分钟。
(7)用天狼星红染色液染色(中杉金桥，DC0041-2)染色1小时。
(8)脱水，封片，镜下观察。
与正常组相比，sgNTC组小鼠天狼星红染色明显加深。与sgNTC组相比，sgKAT7-mouse组天狼星红染色减弱。此外，发现与sgNTC组相比，衰老相关的染色SA-b-gal和p16阳性细胞减少(图5中a)。
1.3.2小鼠血液肝功能检测
处死小鼠后，眼球取血，检测表征肝功能的一些因子透明质酸(HA)，III型前胶原N端肽(PIIINP)，碱性磷酸酶(ALP)，(谷丙转氨酶)ALT和谷草转氨酶(AST)含量。结果发现相比于正常组，表征肝细胞功能的因子下调，sgKAT7-mouse病毒感染组表达上调(图5中b)。
检测HA，PIIINP，ALP，ALT和AST含量检测均按常规方法进行，由北京中原生物有限公司完成。
说明，敲低KAT7基因对于延缓CCL4诱导的肝纤维化的具有治疗作用。
实施例6、KAT7抑制剂WM-3835在延缓WS hMPC和肝细胞衰老方面的作用
以实施例1中第4代的WS间充质前体细胞为供试细胞，用50nM的WM-3835的DMSO溶液处理细胞，WM-3835(结构式如图6中a)委托药明康德公司合成，使用溶剂DMSO作为对照。
经过处理后将所得细胞连续传代至2-3代。收集细胞，进行KAT7蛋白敲低效率检测(图6中b)，并进行细胞衰老标志物SA-β-gal染色和细胞增殖分子标志物(Ki67)的检测，发现抑制剂WM-3835可以延缓WS hMPC的衰老(图6中c-d)。
以人原代肝细胞(hHEP)作为供试细胞(lonza公司产品，货号是HUCPI)，用100nM的WM-3835的DMSO溶液处理细胞，并利用DMSO作为对照。
处理后经过8天培养，RT-qPCR检测P15，IL6等衰老分子标志物，检测结果表明，与DMSO组相比，WM-3835够明显延缓肝细胞的衰老。此外，通过RNA-seq分析表明和利用sgKAT7-human慢病毒敲低KAT7结果一致，WM-3835处理后大量炎性基因，SASP相关基因表达下调，代谢相关的基因表达上调，这些结果说明WM-3835可以延缓原代肝细胞的衰老(图6中e-i，其中Early表示早代结果(处理4天)，Late表示晚代结果(处理9天))。
工业应用
本发明的实验证实敲低KAT7基因在延缓衰老和治疗肝纤维化疾病中具有明 确的效果；KAT7抑制剂WM-3835在延缓间充质前体细胞和肝细胞衰老中具有明确的效果。该发明为开发用于延缓衰老和肝纤维化疾病的基因治疗提供了新的思路，且扩大了临床基因治疗和药物治疗的可选择范围。

Claims (18)

  1. 降低KAT7含量或活性的物质在制备治疗和/或预防衰老产品中的应用。
  2. 根据权利要求1所述的应用，其特征在于：所述物质为靶向KAT7基因的CRISPR/Cas9基因敲除系统。
  3. 根据权利要求2所述的应用，其特征在于：所述物质为含有所述
    CRISPR/Cas9基因敲除系统的重组载体。
  4. 根据权利要求1所述的应用，其特征在于：所述物质为KAT7抑制剂。
  5. 根据权利要求4所述的应用，其特征在于：所述KAT7抑制剂为WM-3835。
  6. 根据权利要求1-5中任一所述应用，其特征在于：所述衰老为动物细胞、组织、器官或个体的衰老。
  7. 抑制KAT7基因表达或敲除KAT7基因的物质在制备治疗和/或预防衰老产品中的应用。
  8. 根据权利要求7所述的应用，其特征在于：所述物质为靶向KAT7基因的CRISPR/Cas9基因敲除系统。
  9. 根据权利要求8所述的应用，其特征在于：所述物质为含有所述CRISPR/Cas9基因敲除系统的重组载体。
  10. 根据权利要求7所述的应用，其特征在于：所述物质为KAT7抑制剂。
  11. 根据权利要求10所述的应用，其特征在于：所述KAT7抑制剂为WM-3835。
  12. 根据权利要求7-11中任一所述应用，其特征在于：所述衰老为动物细胞、组织、器官或个体的衰老。
  13. 降低KAT7含量或活性的物质或抑制KAT7基因表达或敲除KAT7基因的物质在制备抑制肝纤维化产品中的应用。
  14. 治疗和/或预防衰老产品，其活性成分为降低KAT7含量或活性的物质或抑制KAT7基因表达或敲除KAT7基因的物质。
  15. 抑制肝纤维化产品，其活性成分为降低KAT7含量或活性的物质或抑制KAT7基因表达或敲除KAT7基因的物质。
  16. 延缓和/或治疗和/或预防衰老的方法，包括：对动物细胞、组织、器官或个体施用降低KAT7含量或活性的物质或抑制KAT7基因表达或敲除KAT7基因的物质，实现对所述动物细胞、组织、器官或个体衰老的延缓和/或治疗和/或预防。
  17. 根据权利要求16所述的方法，其特征在于：所述动物为哺乳动物。
  18. 根据权利要求16所述的方法，其特征在于：所述哺乳动物为人或小鼠。
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