Micromolecule preparation for inhibiting TH1 cell polarization, and application of micromolecule preparation
A small molecule and inhibitor technology, applied in the direction of cell culture active agent, animal cells, vertebrate cells, etc., can solve problems such as infection, and achieve the effect of simple operation, efficient transfection and cell culture
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Embodiment 1
[0039] Example 1 mouse Isolation of CD4+ T lymphocytes
[0040] The method of isolating and extracting mouse spleen lymphocytes is as follows: take the spleen of a six-week-old C57BL / 6 mouse under aseptic conditions, place it in a 1640 medium, cut the spleen with ophthalmic scissors, and transfer it to a 200-mesh sterile screen , grind with a glass rod, drop 1640 medium while grinding, until all the splenocytes that are ground and dispersed flow out through the filter, collect the splenocytes and centrifuge at 450g for 10min, remove the supernatant and resuspend, add an equal volume of lymphocyte separation medium, and centrifuge at 450g for 30min , absorb the lymphocyte layer, add PBS to resuspend, centrifuge at 400g for 10min, add red blood cell lysate to stand for 5min after washing with PBS, centrifuge at 1500rpm for 10min, collect cells for counting, and use Miltenyi CD4 + T cell isolation kit, isolated from splenic lymphocytes CD4 + T lymphocytes.
Embodiment 2
[0041] Example 2 Induced differentiation of mouse Th1 cells
[0042] CD4 + After T cell sorting, counting, according to 1 × 10 6 / mL seeded in a 6-well plate, placed in 1640 complete medium containing 10% FBS, adding mixed factors to induce Th1 cell differentiation, the concentration of each factor is: IL12: 10ng / mL, IL2: 5ng / mL, anti CD28: 0.5 μg / mL, anti IL4: 1 μg / mL, anti CD3ε: 1 μg / ml, add NEAA and 55 μM β-mercaptoethanol, and culture for 72h to 96h. When the cells were cultured for 36 h, the medium was changed in half, 1 mL of the original medium was removed, and 1 mL of 1640 complete medium containing 10% FBS was mixed with 2 times the concentration of factor, NEAA and 55 μM β-mercaptoethanol.
Embodiment 3
[0043] Example 3 Preparation and transfection of miR-148a-3p small molecule preparation
[0044] 1. Dilute miR-148a-3p mimic at 1.00 OD260 with 125 μL DEPC water to make the final concentration 20 μM. The sequence of miR-148a-3p is shown in Table 1, and the above sequence was purchased from Shanghai Sangon Bioengineering (Shanghai) Co., Ltd.
[0045] Table 1 RNA and primer sequences
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[0048] 2. Transfection
[0049] CD4+T cells by 1×10 6 / mL seeded in 6-well plate, after adding 1640 complete medium and Th1 inducer. Dilute 3 μL miR-148a-3p mimic into 100 μL Jet Prime Buffer, add 6 μL Jet Prime, mix gently, and let stand at room temperature for 15 minutes to form a complex. The liposome complex was added to a six-well plate, the transfection system was 100 μL, and the final miRNA concentration was 100 nM. About 36 hours half of the liquid change.
[0050] 3. Flow cytometry analysis
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