Use of interleukin-22 in the treatment of fatty liver disease
a technology of interleukin-22 and fatty liver, which is applied in the field of medical use of interleukin22 (il22), can solve the problems of fatty liver, cirrhosis and hepatocellular carcinoma, hepatocyte metabolic dysfunction,
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example 1
Murine IL-22 Gene Cloning
[0083]Cloning of human IL-22 gene: Human peripheral blood monocytes were stimulated with anti-human CD3 mAb and cultured for 24 h. Total RNA was extracted by ultracentrifugation, and cDNA was synthesized with the dT primers. Human IL-22 gene was amplified by PCR with the sense primer (5′-GCA GAA TCT TCA GAA CAG GTT C-3′) and anti-sense primer (5′-GGC ATC TAA TTG TTA TTT CTA G-3′). The amplified DNA is cloned into E. coli expression vector.
[0084]Cloning of mouse IL-22 gene: C57BL / 6 female mice were injected with LPS (5 mg / kg, sc). The spleen was obtained after 20 hours. Total RNA was extracted and cDNA was synthesized with the dT primers. Mouse IL-22 gene was amplified by PCR with the sense primer (5′-CTC TCA CTT ATC AAC TGT TGA C-3′) and anti-sense primer (5′-GAT GAT GGA CGT TAG CTT CTC AC-3′). The amplified cDNA was cloned into E. coli expression vector pET21(+)
[0085]Both human IL-22 and murine IL-22 were verified by DNA sequencing, as shown in FIG. 1 and F...
example 2
22 and Mouse IL-22 Gene Expression
[0086]E. coli strain BL21(+) was used to express the recombinant protein. The E.coli cells were homogenized under high pressure. IL-22 inclusion bodies were obtained by centrifugation and washed with buffers (Tris-HCl 50 mM, NaCl 100 mM, EDTA 1 mM, DTT 1 mM, and sodium deoxycholate 0.5%) completely. Inclusion bodies were solubilized in 8M urea, 50 mM Mes, 10 mM EDTA, and 0.1 mM DTT, pH 6.5. Inclusion bodies was refolded 4 times for 20 hours in 100 mM Tris-HCl, 2 mM EDTA, 0.5 M L-arginine, 1 mM reduced glutathion, and 0.1 mM oxidized glutathion, pH 8. The mixture was then concentrated and purified using a Superdex75 (Amersham) column chromatography. The protein was eluted with 20 mM Tris-HCl, 50 mM NaCl, pH 7. The purity of IL-22 was determined by SDS-PAGE (>95%) as shown in FIG. 3 and FIG. 4. IL-22 protein aliquot was stored at −80 ° C.
example 3
nt IL-22 Decreases Levels of Serum Transaminase in Obese ob / ob Mice
[0087]The recombinant murine IL-22 obtained in example 2 was injected to obese ob / ob mice (8-12 weeks, 35-50 g) at a dose of 300 μg / kg / d for 14 days. Same amount of vehicle solution (0.1% BSA, PBS) was injected to the mice in control groups. The animals were sacrificed at day 15 and the serum was collected. Levels of serum ALT and AST were determined. The results are shown in FIG. 5.
[0088]The results demonstrate that IL-22 can significantly decrease the levels of serum AST and ALT in addition to the decreased levels of serum triglyceride.
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