Construction method and application of liver fibrosis disease model
A liver fibrosis and construction method technology, applied in the biological field, can solve the problems of high cost, long modeling time, and long time consumption, and achieve the effects of non-toxic, harmless, environment-friendly, low mortality, and stable models
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Embodiment 1
[0036]Embodiment 1, the construction of the liver fibrosis model of the present invention
[0037] Using mice as target animals, proceed as follows:
[0038] 1. Use the Cre-loxP knockout technology to knock out the 3rd-8th exon sequence of the mouse NF-YA gene at one time to obtain NF-YA Loxp / Loxp Conditional homozygous mice. Transgenic Alb-Cre mice and NF-YA Loxp / Loxp When the conditional homozygous mice were mated, half of the offspring carried both Alb-Cre and NF-YA conditional knockout genes. This animal then interacts with NF-YA Loxp / Loxp Conditional homozygous mice were mated to obtain liver parenchymal cell NF-YA gene conditional knockout homozygous mice (NF-YA Loxp / Loxp / Alb-Cre)( figure 1 A), that is, as the liver fibrosis model of the present invention.
[0039] The knockout strategy in the above examples is to knock out the NF-YA gene using Cre-loxP knockout technology. However, in other embodiments, there are many technical means to achieve gene knockout, su...
experiment example 1
[0042] Experimental example 1. Gene identification of liver NF-YA gene conditional knockout mice
[0043] 1. Experimental method
[0044] (1) Mouse genomic DNA extraction: the tail tip of the liver NF-YA gene conditional knockout mouse obtained in Example 1 of the present invention was clipped to extract genomic DNA.
[0045] (2) PCR amplification: PCR reaction system includes 2 μL 2×Master Mix; 2 μL genomic DNA; 1 μL each of primer 1 (NF-YAF1 or F2 or Alb F) and primer 2 (NF-YA R or Alb R); 6 μL ddH2O . PCR reaction program: 94°C pre-denaturation for 2min, 94°C denaturation for 3min, 55°C annealing for 30s, 72°C extension for 30s, after 25-40 cycles, extend at 72°C for 20min, and finally store at 4°C. Primer sequences: NF-YA F1, 5'-GTAAGTCAGGCTCCAGGG-3'; NF-YA F2, 5'AGGCAAGGCAGATTTAGGAAGGTC-3'; NF-YA R, 5'-GGGTTGTCAGGATGTTCGCAG-3'; Alb F, 5'-ATTTGCCTGCATTACCGGT-3 '; Alb R, 5'-ATCAACGTTTTCTTTTCGG-3'.
[0046] (3) Gel electrophoresis: 10 μl of the PCR product was taken in t...
experiment example 2
[0050] Experimental example 2. Western blot detection of NF-YA protein expression in liver parenchymal cells of NF-YA gene conditional knockout mice
[0051] 1. Experimental method
[0052] (1) Preparation of liver tissue protein samples. Isolation Example 2 Liver NF-YA Gene Conditional Knockout Homozygous Mice (NF-YAL Loxp / Loxp / Alb-Cre) liver tissue, while separating NF-YA wt / wt Wild-type genetic mice, NF-YA Loxp / Loxp Conditional homozygous mice, NF-YA Loxp / wt / Alb-Cre liver tissue was used as a control. Protein lysate RIPA was added to the liver tissue, which was ultrasonically broken and lysed on ice for 20 min, and then centrifuged at 4°C and 12000 g for 10 min. The supernatant was aspirated, and the protein concentration in the supernatant was determined by BCA method.
[0053] (2) Separation of proteins by polyacrylamide gel electrophoresis (SDS-PAGE). Take 20 μL of protein samples, run 80V constant voltage electrophoresis for about 15 minutes, and adjust the volt...
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