A drug target for implant osseointegration in diabetic patients
A technology for diabetes and implants, which is applied in the field of biomedicine, can solve problems such as the unclear mechanism of insulin action, and achieve the effect of promoting osseointegration of implants
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Embodiment 1
[0051] Example 1 Insulin Promotes Wound Healing Experiment
[0052] 1. Culture of THP-1 cells
[0053] Cells were cultured in complete medium (containing RPMI1640: FBS: streptomycin double antibody = 90:10:1, without B-mercaptoethanol), and the culture conditions were 37°C, 5% CO 2 The daily subculture operation is to draw the culture medium to a new culture bottle when the cells grow well under normal circumstances, and add a new medium; centrifuge the next day, centrifuge at 1000rpm for 3min, remove the old medium and wash with 1× PBS solution for 1 Then, centrifuge at 1000rpm for 3min, add new medium to continue passage.
[0054] 2. PMA induces THP-1 cell differentiation
[0055] Culture mononuclear cell line THP-1 cells, pass to the third generation, and use 5×10 6 Inoculate THP-1 cells in 12-well plate and culture in RPMI1640 medium containing 10% fetal bovine serum, 1% glutamine, 1% penicillin and streptomycin, using 60 μg / L myristyl phorbol acetate After (PMA) treat...
Embodiment 2
[0071] Example 2 Screening for genes related to osseointegration
[0072] 1. Cell culture
[0073] 1) The culture steps of THP-1 cells are the same as in Example 1;
[0074] 2) The step of PMA-induced THP-1 cell differentiation is the same as in Example 1;
[0075]3) The steps of treating macrophages with insulin are the same as in Example 1, and the concentration of insulin used is 200 ng / ml.
[0076] 2. Extraction of RNA
[0077] 1) Collection of cells
[0078] Remove the cell culture medium, wash once quickly with PBS buffer, remove the PBS buffer, and the adherent cells can be collected with a cell scraper. The collected cells were transferred to 1.5mL RNase-free EP tubes, and 3 tubes were taken for each group of cells as parallel samples.
[0079] 2) Add TRIzol Lysis Solution to the EP tube, so that the cells are completely dissolved in TRIzol and fully lysed;
[0080] 3) Transfer the lysate to a 1.5 mL RNase-free sterile centrifuge tube, add 200 μl chloroform, shak...
Embodiment 3
[0097] The overexpression of embodiment 3ZNF774 gene
[0098] 1. Cell culture and differentiation steps are the same as in Example 1
[0099] 2. Transfection
[0100] 1) Treatment of cells before transfection
[0101] The day before transfection, seed 3-5×10 on a 6-well culture plate 5 cells / well, cultured in antibiotic-free medium for one day, and replaced with serum-free medium before transfection.
[0102] 2) Construction of gene overexpression vector
[0103] Synthesize specific PCR amplification primers according to the ZNF774 sequence in GeneBank, and the primer sequences are as follows:
[0104] Forward primer: 5'-CCGGGTACCGCCACCATGT GGCTGGGGACTTC-3' (SEQ ID NO.1)
[0105] Reverse primer: 5'-CGGCTCGAGAATCAAATGGGTGTTTTGATGGC-3' (SEQ ID NO.2)
[0106] Two restriction enzyme sites, KpnI and XhoI, were added to the 5' end primer and the 3' end primer, respectively. The cDNA extracted and reverse-transcribed from a Parkinson's patient was used as an amplification temp...
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