CN110777203A - Application of Kctd10 gene in treatment of liver diseases - Google Patents
Application of Kctd10 gene in treatment of liver diseases Download PDFInfo
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Abstract
The invention relates to the field of gene application and a pharmaceutical preparation for experiments, in particular to a method for establishing and identifying a specific knockout model of a hepatic stem cell regulatory gene Kctd10 in mouse liver, and application of the method in screening drugs for treating liver diseases and treating liver diseases. Kctd10 was first constructed using the FLP/Frt, Cre/Loxp recombinase system
flox/floxThe mouse model is then crossed with an Alb-cre + transgenic mouse strain to obtain a Kctd10 liver-specific gene knockout strain, and the results are confirmed. The invention provides an in-vivo animal model for the research of human liver stem cell diseases, provides an effective research approach and method for deeply knowing the occurrence of liver diseases, provides a drug screening method for specific treatment of liver diseases, and lays a foundation for the treatment of liver diseases.
Description
Technical Field
The invention relates to the field of gene application, and relates to a medicinal preparation used in vivo tests. In particular to a method for establishing and identifying a specific knockout model of a hepatic stem cell regulatory gene Kctd10 in mouse liver, which is applied to screening drugs for treating liver diseases and treating liver diseases.
Background
The function and characteristics of hepatic stem cells are closely related to the development and progression of liver disease: in the case of liver damage, hepatic stem cells proliferate and differentiate to maintain the normal function of the liver, and hepatic stem cells are actually one functional state of hepatic cells in a specific environment. The hepatic stem cell has self-replication and bidirectional differentiation potential, is expected to become a serious liver disease cell replacement therapy and a donor cell constituting a bioartificial liver, and thus becomes one of the current research hotspots. The research on the molecular development mechanism of the hepatic stem cells and the establishment of related animal models have very important significance for clinical liver disease treatment, drug research and the like. The mouse model is an ideal model for researching the formation and differentiation of the hepatic stem cells. The international peers knock out key genes in mice to cause liver stem cells to excessively proliferate and stem cell marker gene expression changes to induce the generation of liver cancer of the mice.
KCTD10(potassium channel tetramerization domain-conjugation 10) is a potassium tetramer channel protein belonging to PDIP1(polymerase delta-interacting protein 1) gene family. This gene family is highly similar in protein structure, with common BTB/POZ and K-tetra domains at the N-terminus, and a well-conserved PCNA binding site at the C-terminus. The Kctd10 gene is highly conserved evolutionarily, and encodes a highly conserved protein in humans, cattle, mice, chickens, African toads, zebrafish, and the like. At present, the Cre/loxp recombinase system is a gene-targeted recombination system used in conditional gene knockdown. The inventor surprisingly finds that by conditional gene knockout mice, two loxP sites are firstly placed on two sides of one or more important exons of a target gene to construct chimeric mice, and then the chimeric mice are hybridized with mice expressing Cre enzyme in a specific tissue, sequences between two LoxP with the same direction can be knocked out, and tissues or cells not expressing Cre enzyme cannot generate the knocking-out phenomenon, so that the purpose of knocking out the gene in the specific tissue or cells is realized, and meanwhile, the expression of the gene in other tissues or cells is not influenced. Therefore, the influence of the embryo lethal gene on the growth and development of the mouse can be avoided, so that the relevance of the gene and the physiological and pathological functions of the gene in specific tissues or cells can be better researched, and the invention is created.
The inventor firstly establishes Kctd10 Flox (Kctd 10 for short)
flox/+) A mouse model was knocked out, and homozygous Kctd10 knockout mice obtained by mating the mice with EIIa-Cre died at the embryonic stage, and it was confirmed that the protein regulates the Notch signaling pathway. In order to further research the biological function of KCTD10 in liver and prove the mechanism of KCTD10 on liver stem cell influence, the research firstly constructs Kctd10 by using FLP/Frt, Cre/Loxp recombinase system
flox/floxThe mouse model is then crossed with an Alb-cre + transgenic mouse strain to obtain a Kctd10 liver-specific knockout strain.
The invention provides an in-vivo animal model for the research of human liver stem cell diseases, provides an effective research approach and method for deeply understanding the occurrence of liver diseases, provides a drug screening method for specific treatment of liver diseases, and lays a foundation for gene therapy of liver diseases.
Disclosure of Invention
The invention aims to provide a method for constructing a liver stem cell regulatory gene liver specificity knockout mouse model.
The invention also aims to provide application of the liver stem cell regulatory gene liver-specific gene knockout mouse model.
The invention aims to provide a drug screening method for specific treatment of liver diseases.
The application of the Kctd10 gene in treating liver diseases is a non-therapeutic and non-diagnostic application.
The application of the liver stem cell regulatory gene Kctd10 liver specificity knock-out non-human mammal model in screening liver disease treating medicine is not directly related to the disease treating and diagnosing application process.
Wherein the non-human mammal is mouse, rat, guinea pig, rabbit, monkey, sheep, pig.
A method for liver stem cell regulatory gene Kctd10 liver specific gene knockdown in a non-human mammalian model, comprising the steps of:
(I) at Kctd10
flox/floxIn the mouse model of (3), two Loxp sequences flank the second exon of the Kctd10 gene;
(II) mixing Kctd10
flox/floxThe resulting mice were mated with FLP ER mice to obtain Neo gene-deleted Kctd10
flox /floxA mouse;
(III) Kctd10 obtained in step (II)
flox/+A mouse; mating with an Alb-Cre mouse, and screening Alb-Cre +; kctd10
flox/+A mouse;
(IV) Kctd10 obtained in step (II)
flox/+And (III) the obtained Alb-cre +; kctd10
flox/+Mating the mice, and screening out Alb-cre +; kctd10
floxfloxThe mouse is a liver-specific knockout homozygote mouse with the Kctd10 gene.
In step (I), the Loxp sequence is inserted into the intron on both sides of exon 2, so that the normal expression of the gene is not affected in the absence of Cre recombinase.
Liver stem cell regulatory gene Kctd10 application in liver-specific knockout of non-human mammal model.
The identification method of the liver stem cell regulatory gene Kctd10 liver specificity knocking non-human mammal model comprises the following steps:
(I) identifying the genotype of the Kctd10 liver-specific knockout non-human mammal model by PCR-agarose gel electrophoresis;
(II) determining the KCTD10 protein expression condition of a Kctd10 liver specificity knocking non-human mammal model by a protein imprinting experiment, thereby verifying the Kctd10 gene knocking-out effect;
(III) the experiment of the real-time fluorescent quantitative PCR technology verifies the KCTD10 mRNA expression condition of the Kctd10 liver-specific gene knockout non-human mammal model, thereby verifying the Kctd10 gene knockout effect.
The liver stem cell regulatory gene Kctd10 is used for specifically knocking non-human mammal model and finding the abnormal expression of liver stem cell marker gene.
The application of the hepatic stem cell regulatory gene Kctd10 in gene therapy of liver diseases.
The use of any of the foregoing, such that KCTD10 mRNA levels are reduced to around 30% of normal levels; KCTD10 positive cells were reduced to about 30% of normal levels.
The use of any one of the preceding, wherein the hepatic stem cell marker gene Bmi1, Nanog, CD44, ALDH1 mRNA level (A, B), protein level (C) is significantly reduced.
The use of any one of the preceding claims, wherein the number of positive cells of the hepatic stem cell marker gene Bmi1 is reduced to about 60% of the normal level.
Use according to any one of the preceding claims to modulate alterations in Notch signaling pathway protein levels and mRNA levels in stem cells.
The use of any one of the foregoing to modulate the level of stem cell Notch signaling pathway ligands DLL3, Jagged1, Jagged2 protein reduced to about 50% of normal levels.
The use of any one of the above, wherein the signal intensity of TACE protein, a gene downstream of the Notch signaling pathway in stem cells is reduced to about 40% of the normal level.
All of the above applications are for non-therapeutic, non-diagnostic purposes.
Accordingly, the invention provides a liver stem cell regulatory gene liver specificity knockout animal model which is used for determining biological functions of liver stem cells and livers, so that identification methods of a Kctd10 liver specificity knockout animal model and a Kctd10 liver specificity knockout animal model are provided, the application of the liver stem cell regulatory gene liver specificity knockout animal model and the identification methods of the Kctd10 liver specificity knockout animal model and the Kctd10 liver specificity knockout animal model are used for screening drugs for treating liver diseases, and a foundation is laid for gene therapy of the liver diseases.
Drawings
FIG. 1: schematic representation of mating protocol for Kctd10 liver-specific knockout mice.
FIG. 2: performing PCR agarose gel electrophoresis identification on Kctd10 liver-specific gene knockout mice genotype; alb cre +; kctd10
flox/floxRepresents KCTD10 liver-specific knockout mouse homozygote, Alb cre-; kctd10
flox/floxRepresents KCTD10 liver-specific knockout mouse wild type.
FIG. 3: and (3) specifically knocking out mouse liver Kctd10 gene, and then, expressing KCTD10 protein.
FIG. 4: after the Kctd10 gene of mouse liver is knocked out specifically, KCTD10 mRNA level is reduced to about 30% of normal level.
FIG. 5: after the mouse liver Kctd10 gene is specifically knocked out, KCTD10 positive cells are reduced to about 30% of normal level.
FIG. 6: the liver stem cell marker genes Bmi1, Nanog, CD44 and ALDH1 mRNA level (A, B) and protein level (C) in the mouse model obtained by the method are obviously reduced.
FIG. 7: the liver stem cell marker gene Bmi1 positive cells in the mouse model obtained by the method are reduced to about 60 percent of the normal level.
FIG. 8: the level of Notch signaling pathway protein and mRNA regulating stem cells in the mouse model obtained by the method are changed.
FIG. 9: the protein levels of Notch signal pathway ligands DLL3, Jagged1 and Jagged2 for regulating stem cells in the mouse model obtained by the method are reduced to about 50% of the normal level.
FIG. 10: the signal intensity of the TACE protein of the downstream gene of the Notch signal path of the regulatory stem cells in the mouse model obtained by the method is reduced to about 40 percent of the normal level.
Detailed Description
The inventor establishes a Kctd10 liver-specific gene knockout mouse model through extensive and intensive research, and has good guiding significance for identifying a method for knocking out a mouse genotype. The invention is further illustrated by the following examples.
Table 1: PCR primer set
Table 2: qPCR primer list
Example 1: establishment of Kctd10 liver-specific gene knockout mouse model
Materials and sources: KCTD10 against C57BL/6J background
loxP/+Mice, Alb-Cre and FLP ER mice were purchased from the Nanjing model animal institute. The mice were raised in the experimental animals center SPF level animal house of the university of faculty of medical science in hunan, license number: SYXK (xiang) 2014-. The temperature of the animal room is controlled at 20-25 ℃, the humidity is controlled at 40-70%, the noise is less than 60dB, the mouse is illuminated for 15-20lx 12h (12h light and shade period), and the laboratory working illumination is 150-300 lx. Mouse feed padding is subjected to high-temperature disinfection treatment, drinking water is subjected to high-temperature sterilization treatment, the padding is replaced for 2 times every 7 days, and the feed and the drinking water are supplemented every day.
The method comprises the following steps: the mating schedule of mice is shown in FIG. 1 (FIG. 1: schematic representation of the mating schedule of Kctd10 liver-specific knock-out mice), Kctd10
flox/+Selfing the mouse, and screening out the filial generation genotype Kctd10
flox/floxThe mouse of (1); mating with FLPER mice to obtain Kctd10
flox/floxA mouse; then mating with C57BL/6 mice to remove FLP enzyme, and screening Kctd10
flox/+A mouse; mating with an Alb-Cre mouse, and screening Alb-Cre +; kctd10
flox/+A mouse; the obtained Kctd10
flox/+And Alb-cre +; kctd10
flox/+Mating the mice, and screening out Alb-cre +; kctd10
flox/floxMouse, namely liver specificity Kctd10 gene knockout mouse.
Example 2: extraction of knockout mouse model genome DNA
Materials and sources: the resulting rat tail digest (500ml formulation: 1M Tris. HCl (pH8.0)25ml, 0.5M EDTA (pH8.0)100ml, NaCl 2.925g, 10% SDS 50ml, water to 500ml, room temperature storage), proteinase K (MERCK, Cat No:1245680100,100mg, 4 ℃ storage) diluted to 10% with pure water and stored at-20 ℃.
The method comprises the following steps: shearing tail of 8-12 days old mouse about 0.5-1.0cm, cutting toe number, placing toe and tail together into 1.5ml EP tube, adding 500ul tail digestive juice and 10ul proteinase K into each tube, placing into water bath kettle, adjusting temperature to 56 deg.C, and digesting overnight. After the digested tail is shaken up, phenol chloroform with the same volume (500ul) is added into each tube and mixed evenly, and the mixture is centrifuged at 12000rpm for 10min at 4 ℃; transferring 200ul of the supernatant to a sterilized and labeled 1.5ml EP tube, adding 2 times of the volume of the supernatant (namely 400ul) of absolute ethyl alcohol, turning upside down, and mixing uniformly to obtain white filaments which are DNA; centrifuging to remove anhydrous ethanol at 12000rpm for 5min at 4 deg.C; discarding the supernatant, adding 2 times of the volume of the supernatant (400 ul) of 70% ethanol, centrifuging to remove anhydrous ethanol, 12000rpm, 5min, 4 ℃; discarding the supernatant again, and air-drying for more than 30 min; 100ul of TE was added to each tube to dissolve the DNA, and the DNA was stored at 4 ℃.
Example 3: PCR identification of Kctd10 liver-specific knockout mouse model and wild type genotype (see FIG. 2. FIG. 2: PCR agarose gel electrophoresis identification of Kctd10 liver-specific knockout mouse genotype; Alb cre +; Kctd 10)
flox/floxRepresents KCTD10 liver-specific knockout mouse homozygote, Alb cre-; kctd10
flox/floxRepresents KCTD10 liver-specific knockout mouse wild type. )
Materials and sources: primer synthesis and Biotechnology engineering (Shanghai) Ltd: the primers are shown in the table 1; 2 XTagPlus Master Mix II (Dye Plus) was stored at-20 ℃ and Ultra GelRed (10000 ×) (Vazyme code: GR 501-010.5 ml L/N7E 142H7) was purchased from Vazyme Inc., DNA marker (stored at-20 ℃ C., Ming., Beijing Dyson, NMW 016), agarose powder, electrophoresis solution TBE, grade I pure water from this laboratory, 0.2ml PCR octal tubing (Axygen), 1.5ml centrifuge tubes were sterilized and used, tips (Inc.) were sterilized and used, nucleic acid dyes, microwave oven. The method comprises the following steps: preparing the system, adding PCR 8 connecting pipes, and loading. After PCR, 1% (w/v) agarose gel (1g added with 100ml of TBE solution) is prepared, after being heated for three times by microwave, the agarose gel is cooled until the agarose gel is not coagulated, nucleic acid dye (1: 10000, namely 10ul) is added, and the agarose gel is poured into a gel-making film for coagulation, and then sample loading can be carried out. The strip can be observed by ultraviolet projection at constant voltage of 120mv for 20 min.
Example 4: extracting protein of Kctd10 liver specificity gene knockout mouse and wild type
Materials and sources: RIPA strong lysate (cloudby day), PMSF protease inhibitor, ice, centrifuge method: the frozen mouse liver tissue added with (200: 1) RIPA strong lysis buffer (Biyunshi) and PMSF protease inhibitor is homogenized by a tissue homogenizer and placed on ice (prepared in the laboratory) for 30min, a centrifuge is used for centrifugation for 12000rPm for 10min at 4 ℃, and then supernatant and a new EP tube are sucked and added with 5 × loading buffer (WB-0091, 5ml, stored at 20 ℃ below zero, produced by Beijing ancient China Changchang company) (4: 1, namely tissue: loading buffer).
Example 5: protein concentration of Kctd10 liver-specific knockout mouse and wild type
Determination of materials and sources: and (3) determining the solubility of the protein before denaturation by using a BCA kit (Biyun day), a 96-well plate, pure water and an enzyme-labeling instrument. The method comprises the following steps: preparing a system according to BCA (burst cutting edge) instructions, adding a 96-pore plate, incubating in a 37 ℃ incubator for 30min, measuring the light absorption value of each sample adding hole at 562nm by using an enzyme-labeling instrument, and calculating the solubility of a sample and the sample loading volume of the next Western blotting experiment according to the measured standard curve.
Example 6: western blot experiment for determining expression conditions of KCTD10 protein and stem cell marker protein of Kctd10 liver-specific knockout mouse and wild type KCTD10 protein and related protein of Notch signal pathway
Materials and sources: 10% polyacrylamide gel premix (Xinsaimei, Cat: P40650, 600ml), protein marker, electrophoresis solution (1L formulation: Tris base 3.03g, glycine 18.77g, SDS 1g, water to constant volume to 1L), membrane transfer solution (1L formulation: Tris base 3.03g, glycine 14.4g, methanol 200ml, water to 1L), Bio-constant volume rad electrophoresis membrane transfer tank, PVDF membrane (Immobilon), methanol, ECL chemiluminescence solution (Vazyme, E411-04,4 ℃ storage), KCTD10 antibody (inventor subject group extraction, Rabbit source), GAPDH antibody (Google, GB11002, 100ul, -20 ℃ storage, Rabbit anti GAPDANTIBODY), Rabbit secondary antibody, milk powder, BSA, TBS, Tween, standing red liquid (Biyun day, P0022, Lot: 41698, 120ml, 062698, 120 ml) and shaking table storage at room temperature. The method comprises the following steps: denaturing proteins of Kctd10 liver-specific gene knockout mice and wild type mice with well-determined concentrations at 100 ℃ for 10min, preparing 10% gel according to gel preparation instructions, performing constant-pressure 150mv electrophoresis for 50min after spotting, performing membrane transfer with constant flow of 150mA for 60min, sealing with 5% (2.5g plus 50ml of TBST solution) (w/v) of skimmed milk powder for 1h after membrane transfer, washing with TBST, incubating GAPDH and KCTD10 primary antibody, and standing overnight at 4 ℃; the next day, washing the membrane with TBST on a shaking table for 10min × 3 times at a rotation speed of 90 times/min; incubating rabbit secondary antibody for 1h at room temperature on a shaking bed after washing, and rotating speed is 60 times/min; TBST was washed 10min X3 times, and ECL chemiluminescence developer was prepared and developed, the results are shown in FIG. 3 and FIG. 6 (FIG. 3: KCTD10 protein expression after mouse liver Kctd10 gene was knocked out specifically).
Example 7: extraction of Kctd10 liver-specific gene knockout mouse model and wild type RNA
Materials and sources: trizol reagent (Invitrogen Lot: 00389377, USA, 100ml, 4 ℃ storage), DEPC water (Producer, Lot: B226BA4474, 100ml, storage at room temperature), isopropanol, chloroform, 75% ethanol, ice, timer, marker pen, electric abrasive drill (Tiangen), enzyme-free 1.5ml EP tube, concentration measured by microplate reader after RNA extraction and purity measured by agarose gel. The method comprises the following steps: after removing the tissue frozen and stored in a refrigerator at-80 ℃, 500ul trizol (1mltrizol tissue weight is not more than 50mg) is added into each tube, after uniform electric grinding, 500ul trizol is added, and the mixture is kept standing for 5min at room temperature for 1ml in total. Adding 200ul chloroform, shaking for 15s, incubating at room temperature for 3min, 12000g, 15min, and centrifuging at 4 deg.C. 500ul of the supernatant was added to a fresh EP tube and an equal volume of 500ul of isopropanol was added and incubated at room temperature for 10min, 12000g, 10min,4 ℃. The supernatant was removed and 1ml of 75% ethanol, 7500g, 5min, centrifuged at 4 ℃. The supernatant was removed, dried briefly and dissolved in 100ul of DEPC water. The concentration was measured by taking 2ul and the purity was measured by running agarose (1%) gel 2 ul.
Example 8: reverse transcription Kctd10 liver-specific gene knockout mouse model and wild type RNA
Materials and sources: hiscript II Reverse Transcriptase Supermix for qPCR (Vazyme, R223-01, stored at-20 ℃), PCR apparatus (eppendorf). The method comprises the following steps: 10ul reverse transcription system method: the volume of 500ng template plus (diluted to the appropriate concentration as the case may be) was calculated and RNase free ddH2O was added to each template (on ice) for a total of 6ul, followed by 4 Xg DNA wiper Mix2ul and mixing at 42 ℃ for 2 min. Adding 5 XHiscript II ReverseTranscriptase Supermix II 2ul into each tube, blowing, beating, mixing, setting reverse transcription PCR program, and storing the product at-20 deg.C.
Example 9: real-time fluorescent quantitative PCR (polymerase chain reaction) experiment for determining Kctd10 liver-specific gene knockout mouse model and wild-type KCTD10 mRNA and stem cell marker gene mRNA expression conditions
Materials and sources: the primer 36B4 was synthesized by Biotechnology Ltd, Shanghai, KCTD10 was synthesized by Biotechnology Ltd, SyBR GreenMortermix (Vazyme, Q111-02, stored at-20 ℃), 96-well PCR lower tube (Bio-rad), qPCR apparatus (Bio-rad), and enzyme-free tip, belonging to Hannan Biotech Ltd (see Table 2 below). The method comprises the following steps: real-time fluorescent quantitative PCR 10ul system, (note: Bio-rad instrument does not require Rox correction), sample template 1: 20(1ul plus 20ul of DEPC water) diluted, spotted three biological replicates and three technical replicates, 2 NC (blank control), with results as shown in FIG. 4, FIG. 5 (FIG. 4: KCTD10 mRNA levels decreased to around 30% of normal levels after specific knock-out of mouse liver Kctd10 gene; FIG. 5: KCTD10 positive cells decreased to around 30% of normal levels after specific knock-out of mouse liver Kctd10 gene).
By measurement, as shown in fig. 6, the liver stem cell marker genes Bmi1, Nanog, CD44, ALDH1 mRNA levels (A, B), protein levels (C) were significantly reduced in the mouse model obtained by the method herein. As shown in fig. 7, the obtained mouse model reduced hepatic stem cell marker gene Bmi1 positive cells to about 60% of the normal level. As shown in fig. 8, the obtained mouse model changed the levels of Notch signaling pathway proteins and mRNA that regulate stem cells. As shown in fig. 9, the levels of the Notch signaling pathway ligands DLL3, Jagged1, Jagged2 protein that regulate stem cells in the obtained mouse model were reduced to around 50% of the normal levels. As shown in fig. 10, the signal intensity of TACE protein, a gene downstream of the Notch signaling pathway that regulates stem cells, was reduced to about 40% of the normal level in the obtained mouse model.
Claims (5)
- Application of Kctd10 gene in preparing medicine for treating liver diseases.
- 2. The application of the liver stem cell regulatory gene Kctd10 liver specificity knock-out non-human mammal model in screening liver disease treating medicine is not directly related to the disease treating and diagnosing application process.
- 3. The use according to claim 2, wherein the non-human mammal is a mouse, rat, guinea pig, rabbit, monkey, sheep, pig.
- 4. The use according to claim 2 or 3, wherein the method for preparing the liver stem cell regulatory gene Kctd10 liver-specific gene knockdown non-human mammal model comprises the following steps:(I) at Kctd10 flox/floxIn the mouse model of (3), two Loxp sequences flank the second exon of the Kctd10 gene;(II) mixing Kctd10 flox/floxThe resulting mice were mated with FLP ER mice to obtain Neo gene-deleted Kctd10 flox/floxA mouse;(III) Kctd10 obtained in step (II) flox/+A mouse; mating with an Alb-Cre mouse, and screening Alb-Cre +; kctd10 flox /+A mouse;(IV) Kctd10 obtained in step (II) flox/+And (III) the obtained Alb-cre +; kctd10 flox/+Mating the mice, and screening out Alb-cre +; kctd10 floxfloxA mouse, namely a Kctd10 gene liver-specific knockout homozygote mouse;in step (I), the Loxp sequence is inserted into the intron on both sides of exon 2, so that the normal expression of the gene is not affected in the absence of Cre recombinase.
- 5. The use of claim 2 or 3, wherein the hepatic stem cell regulatory gene Kctd10 is used in a method for liver-specific knock-out of non-human mammal model, comprising the steps of:(I) identifying the genotype of the Kctd10 liver-specific knockout non-human mammal model by PCR-agarose gel electrophoresis;(II) determining the KCTD10 protein expression condition of a Kctd10 liver specificity knocking non-human mammal model by a protein imprinting experiment, thereby verifying the Kctd10 gene knocking-out effect;(III) the experiment of the real-time fluorescent quantitative PCR technology verifies the KCTD10 mRNA expression condition of the Kctd10 liver-specific gene knockout non-human mammal model, thereby verifying the Kctd10 gene knockout effect.
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