Effects of leptin on differentiation of bone marrow mesenchymal stem cells into osteoblasts

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Abstract

BACKGROUND:

Leptin and its receptor express in many central and peripheral sites, have various biological functions, and greatly affect bone metabolism. However, the mechanism of leptin effects on bone reconstruction is still unclear.

OBJECTIVE:

To observe the effects of leptin on differentiation of bone marrow mesenchymal stem cells (BMSCs) into osteoblasts.

DESIGN, TIME AND SETTING:

The cytological, in vitro, study was performed at the Laboratory of the Department of Biochemistry and Molecular Biology, Shanxi Medical University from March to September 2008.

MATERIALS:

Iliac bone marrow from healthy volunteers was obtained from the Second Hospital, Shanxi Medical University. Leptin (Sigma, USA) was used in this study.

METHODS:

Normal human BMSCs were isolated and cultured by the whole bone marrow method. Cells of the third passage at 1×108/L were incubated on coverslip in a 6-well plate, and randomly divided into 3 groups: osteogenic induction group, which was added with primary medium and osteogenic inducer, supplemented with 10−8 mol/L dexamethasone, 10 mmol/L β-phosphoglycerol and 50 mg/L vitamin C; leptin osteogenic induction group was added with 50 nmol/l leptin in addition to osteogenic inducer. Cells in the blank control group were only treated with LG-DMEM containing 10% fetal bovine serum.

MAIN OUTCOME MEASURES:

The following parameters were measured: surface marker expression of BMSCs; results of calcified nodules staining; activity of alkaline phosphatase; contents of collagen-I and osteocalcin; expression of osteoprotegerin and RANKL protein.

RESULTS:

Positive rate of CD44 and CD71 were respectively 95.21% and 72.31% in BMSCs at passage 3, but positive rate of CD34 was only 0.39%. Von Kossa staining showed brown extracellular matrix calcium salt deposition at 21 days following osteogenic induction. Compared with the blank control group, activity of alkaline phosphatase; contents of collagen-I and osteocalcin were significantly increased in the osteogenic induction and leptin osteogenic induction groups at 14 days (P < 0.05). The increase of each index in the leptin osteogenic induction group was significantly higher than in the osteogenic induction group (P < 0.05). Compared with the osteogenic induction group, osteoprotegerin expression was significantly upregulated in the leptin osteogenic induction group (P < 0.05), but RANKL protein expression was significantly dowregulated (P < 0.05).

CONCLUSION:

Leptin could improve osteogenic differentiation of human BMSCs, and regulate bone metabolism through osteoprotegerin/RANK way

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