MiRNA of phellinus linteus and application thereof
A technology of P. clefthoof, mir-cm2, which is applied in the fields of biotechnology and medicine, can solve the problems of exacerbation of acne inflammation, aggravation of hair follicle blockage, etc., and achieve the effect of reducing expression
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Embodiment 1
[0053] Example 1 Screening and identification of miRNA
[0054] 1. Preparation of Samples
[0055] Extraction of RNA samples can be derived from lysates of X. pyogenes or exosomes from X. pyogenes. For the lysate: high-pressure shearing of Xylomicron in PBS, and high-speed centrifugation to obtain a supernatant, the supernatant is the lysate of Xylem. For exosomes: The exosomes of Xylomicron are obtained by the differential centrifugation method after homogenizing Xylem. First centrifuge at 3000 x g for 30 min to remove dead cells, collect the supernatant and centrifuge at 10000 x g for 60 min to remove cell debris. The resulting supernatant was further centrifuged at 150,000 × g for 90 minutes, and the exosomes (the pellet fraction) were suspended in PBS buffer.
[0056] 2. RNA extraction and quality inspection
[0057] Total RNA from exosome samples was extracted by Trizol (Thermo Fisher, USA). The integrity of RNA was detected by agarose gel electrophoresis, and the el...
Embodiment 2
[0070] Example 2 miR-CM2~CM5 reduce the inflammatory damage of HaCaT cells induced by LPS
[0071] 1. CCK8 experiment
[0072] 1) Preparation of cell samples
[0073] Human immortalized epidermal cells (HaCaT 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 at 37 °C, 5% CO 2 grown in an incubator;
[0074] The cultured HaCaT cells were transfected with miR-CM2 mimics, miR-CM3 mimics, miR-CM4 mimics, and miR-CM5 mimics by Lipo2000 transfection reagent (Thermo Fisher, USA), respectively, and pre-protected for 24 h. LPS with a final concentration of 1 μg / mL was added to each well (96-well plate), and CCK8 detection was performed after stimulation for 6 h (experimental group).
[0075] Control group (Ctrl group): The above-mentioned human immortalized epidermal cells (HaCaT cells) were cultured normally without any treatment (no pre-protection, no LPS stimulation); mo...
Embodiment 3
[0125]Example 3 miR-CM2, miR-CM3, miR-CM4 or miR-CM5 skin care lotions reduce epidermal thickness in UV-irradiated mouse skin inflammation
[0126] 1) Preparation of miR-CM2, miR-CM3, miR-CM4 or miR-CM5 skin care lotion
[0127] The formula of miR-CM2 skin care lotion: by weight percentage, disodium EDTA 0.03%, glycerin 4%, xanthan gum 0.1%, p-hydroxyacetophenone 0.2%, Montanov L-emulsifier 1%, ARLACEL170 emulsifier 1% , Glyceryl Stearate 0.3%, Cetearyl Alcohol 1%, Caprylic / Capric Triglyceride 4%, Dimethicone 1%, Methyl Propylene Glycol 0.35%, Polyethyleneimine-1500 1 %, sodium hyaluronate 1%, miR-CM2 0.75%, and the balance is deionized water.
[0128] The composition of the miR-CM3 skin care lotion is the same as the formula of the above-mentioned miR-CM2 skin care lotion, only miR-CM2 is replaced by miR-CM3;
[0129] The composition of the miR-CM4 skin care lotion is the same as the formula of the above-mentioned miR-CM2 skin care lotion, only replace miR-CM2 with miR-CM4;...
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