| Products/Services Used | Details | Operation |
|---|---|---|
| Gene Synthesis | The overexpression vector containing the complete coding sequence of rat Hif1a (GenBank Accession NM_024359) was constructed using pcDNA3.1 vector (Thermo Scientific, Carlsbad, CA, USA) and screened by Ampicillin (100 μg/mL, Solarbio, Beijing, China). The correct sequence was further verified by sequencing. The blank vector was also prepared as a negative control for transfection (BC). The specific small interfering RNA (siRNA) for Hif1a and the negative siRNA control (siNC) were synthesized by Genscript (Nanjing, China). Cell transfection was performed at 48 h after hypoxia treatment was started using LipofectamineTM 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. MSCs (1 × 105) were seeded in each well of 24-well plates 1 d before transfection. In each well, 1 μg of the overexpression vector or 10 pmol of siRNA were added, and the plates were incubated at 37°C for 48 h, after which MTT assay, flow cytometry, qPCR and Western blot were performed on these transfected MSCs. | Get A Quote |
Mesenchymal stem cells (MSCs) are ideal materials for cell therapy. Research has indicated that hypoxia benefits MSC survival, but little is known about the underlying mechanism. This study aims to uncover potential mechanisms involving hypoxia inducible factor 1α (HIF1A) to explain the promoted MSC survival under hypoxia. MSCs were obtained from Sprague-Dawley rats and cultured under normoxia or hypoxia condition. The overexpression vector or small interfering RNA of Hif1a gene was transfected to MSCs, after which cell viability, apoptosis and expression of HIF1A were analyzed by MTT assay, flow cytometry, qRT-PCR and Western blot. Factors in p53 pathway were detected to reveal the related mechanisms. Resul... More
