MiRNAs composition for treating sepsis and application thereof
A composition and polymer technology, applied in antiviral agents, medical preparations containing active ingredients, pharmaceutical formulas, etc., can solve the problems of unsatisfactory prognosis, no treatment method, complicated pathogenesis, etc., and achieve good endothelial cells , Inhibit inflammatory response, protect endothelial cells
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Embodiment 1
[0058] Example 1: Screening and identification of miRNA
[0059] 1. LPS induces cells to produce functional exosomes
[0060] To avoid the influence of exogenous exosomes, the exosomes in the serum were removed by ultracentrifugation. The special tube for ultracentrifugation was pre-sterilized by soaking in 75% ethanol for 12 h, and the lid was placed on the ultra-clean workbench to evaporate and dry before use. The fetal bovine serum was placed in a sterile ultracentrifuge tube, balanced, centrifuged at 4°C, 120,000 × g, and centrifuged for 12 h. The white precipitate attached to the bottom was the exosomes in the serum. Carefully keep the supernatant in the ultra-clean workbench and filter it with a 0.22 μm filter to avoid the introduction of bacterial contamination. In subsequent experiments, all cell culture processes requiring exosome extraction were prepared using exosome-free serum to prepare complete medium.
[0061] When the melanoma cells B16-F10 were revived and ...
Embodiment 2
[0083] Example 2 Preparation of Nanoparticles Containing miRNAs Composition
[0084] The formulations of miRNAs compositions are set as five groups CI, CII, CIII, CIV and CV, and the formulations of each group are as follows: 27 parts of HA-PEI, 1 part of miRNAs composition, and 11 parts of solvent;
[0085] CI, CII, CIII, CIV and CV differ in the proportions of the 7 miRNAs:
[0086] CI: The ratio of 7 miRNAs in iExo was analyzed according to the sequencing results of iExo, miR-146a-5p: miR-466i-5p: miR-206-3p: miR-690: miR-6239: miR-615-5p : miR-7651-5p is 6:2:2:4:1:1:1 ( figure 2 A), Nanoparticles containing miRNAs in the same proportions as above are referred to as component I (CI).
[0087] CII: The ratio is the same as the CI, and the dose is doubled when used as the CII group.
[0088] CIII: According to the regulatory network of miRNAs ( figure 2 B), In the study of endothelial-related functions, four miRNAs regulating the CAM pathway: miR-466i-5p, miR-206-3p, mi...
Embodiment 3
[0100] Example 3 Nanoparticles containing miRNAs composition can inhibit LPS-induced inflammatory response 1. CCK8 experiment
[0101] 1) Preparation of cell samples
[0102] Human umbilical vein endothelial cells (HUVEC cells) were cultured in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (BI, Israel) and 100 U / mL penicillin-streptomycin mixture (Meilun, China), and 5% CO at 37°C 2 grown in an incubator;
[0103] The above cultured HUVEC cells were transfected with CI-CV nanoparticles (Example 2) and iExo (prepared in Example 1), CCK8 detection was performed after 24 hours of stimulation. details as follows:
[0104] Control group: (1) Ctrl group: the above-mentioned human umbilical vein endothelial cells (HUVEC cells) were cultured normally without any treatment (no inflammatory serum stimulation);
[0105] (2) C-NP group: Ctrl group was added with nanoparticles empty (no inflammatory serum stimulation);
[0106] No loading of nanoparticles: the method of ...
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