RNA interference carrier used for osteoporosis and rat mesenchymal stem cells
A technology for RNA interference and osteoporosis, applied in the field of molecular biology, with good application prospects
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Embodiment 1
[0030] Example 1: Construction of an shRNA lentiviral expression vector that down-regulates the expression of peroxisome proliferator-activated receptor gamma (PPARγ)
[0031] 1. Isolation, culture, expansion and identification of rat MSC
[0032] Isolation, culture, and expansion of MSCs: adult rat femurs were washed out of the bone marrow, mixed thoroughly, centrifuged, and the precipitate was collected, and then the mononuclear cell layer was collected with Percoll separation medium with a density of 1.083, and cultured in DMEM containing 10% FCS and low sugar for 5 -7 days, replace the culture medium, and then change the medium every 4 days. After the cells were 80% confluent, they were digested with 0.25% trypsin and passaged.
[0033] Rat MSC identification: flow cytometry identification of cell surface marker antigens, mainly including CD29, CD44, CD45 (Pharmigen), in which CD29, CD44 are positive, CD45 is negative. 2. Establish an in vitro model for directional inducti...
Embodiment 2
[0047] Example 2: Preparation of Rat Bone Marrow Mesenchymal Stem Cells Down-regulating the Expression of PPARγ
[0048] 1. Virus packaging, collection:
[0049] Virus packaging was carried out according to the instructions of the lentiviral packaging kit (catalogue number K4950-00) of Invitrogen Company. The details are as follows: 293FT cells (product of Invitrogen Company, catalog number R700-07) were inoculated in a cell culture dish with a diameter of 100 mm, and when the cells reached 95% confluence, they were replaced with fresh high-glucose DMEM medium containing 10% fetal bovine serum . Use 36 μl of liposome lipo-fectamin2000 to transfect 3 μg of PPARγshRNA lentiviral expression vector plasmid and 9 μg of package mix plasmid (including VSVG, RSV-REV, pMDL g / p RRE, provided in the Invitrogen lentiviral packaging kit) into 293FT cells. Incubate in a carbon dioxide cell incubator for 24 hours, change 10 ml of complete medium, and observe the changes of 293FT cells and...
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