The Effect Of PPAR δ Agonist And SiRNA On The Differentiation Of Rats Bone Marrow Stromal Stem Cells And Primary Osteoblast

Objective To investigate the effect of peroxisome proliferators-activated receptor δ(PPAR δ) agonist GW501516 or low expression of PPAR δ on the adipogenic and osteogenic differentiation of rats bone marrow stromal stem cells(BMSCs), as well as on the differentiation and mineralization of primary osteoblast. In order to clarify the role of PPAR δ in the regulation of bone mass and osteoporosis, thus providing a new target for the treatment of osteoporosis.Methods ① Bone marrow tissue were obtain from the femoral of 3-week-old male SD rats under sterile conditions. Rat BMSCs were cμltured using whole marrow adhesion method after filtered, and then Then purified cells use their different adherent speed. The biomarkers(CD29, CD90, CD11 b, CD45) on the cell membrane were identified using flow cytometry.② Rat BMSCs of P3 generation were randomly divided into control group, PPAR δ agonist group and si RNA interference group. The expression of osteoblast differentiation markers(alkaline phosphatase, osteocalcin and) and adipocyte differentiation markers(AP2, fat vitronectin) were detected using Real Time PCR after osteogenic and adipogenic induced culture. Mineralized nodules and lipid droplets were detected using Alizarin Red and Oil Red O staining.③ Primary osteoblasts were cultured inα-MEM culture medium containing ascorbic acid. They were randomly divided into control group, PPAR δ agonist group and si RNA group. The expression of osteoblast differentiation markers(alkaline phosphatase, osteocalcin and) were detected using Real Time PCR. Mineralized nodules were detected using Alizarin Red staining.Results ①BMSCs had fusiform shape, and arranged in spiral or waterfall shape. BMSCs had high expression of CD29 and CD90, and had low expression of CD11 b and CD45 when using flow cytometry detect cell surface markers. ②Compared with control group, BMSCs in PPAR δ agonist group have more expression of osteoblast differentiation markers and less expression of adipocyte differentiation marker. There are more mineralized nodules and fewer lipid droplets in the PPAR δ agonist group. Compared with control group, BMSCs in si RNA group have less expression of osteoblast differentiation markers and more expression of adipocyte differentiation marker. There are fewer mineralized nodules and more lipid droplets in the si RNA group.③ Primary osteoblasts had triangular or polygonal shape, and arranged in cobblestone shape. Compared with control group, Primary osteoblasts in PPAR δ agonist group have more expression of osteoblast differentiation markers. There are more mineralized nodules in the PPAR δ agonist group. Compared with control group, Primary osteoblasts in si RNA group have less expression of osteoblast differentiation markers. There are fewer mineralized nodules in the si RNA group.Conclusion ①BMSCs of rat can amplify and differentiate in vitro. BMSCs cell surface have high expression of CD29 and CD90.And have low expression of CD11 b and CD45. ②PPAR δ agonist can promote osteogenic differentiation of BMSCs, and inhibit their differentiation into adipocytes direction. ③ PPAR δ agonists can promote differentiation and mineralization of osteoblast.