| [Objective]Tenofovir disoproxil fumarate (TDF) is a key agent for antiretroviral therapy, but it has been proved to be an inducer of osteoporosis. In this study, the effects of TDF on the osteogenesis of Human Mesenchymal Stem Cells-bone marrow (hBMSCs) were examined, and the mRNA expression of factors involved in wnt/β-catenin canonical pathway from cells treated by TDF at different concentration were tested too.[Methods]This study includes two experiments:Part one1. To examine the osteogenetic potentiality of cultured hBMSCs in vitro. The hBMSCs cells were culture with mesenchymal stem cell osteogenic differentiation medium (MODM) for28days. The morphology was assessed by phase-contrast microscopy during the cultivation, osteogenetic potentiality was evaluated by alkaline phosphatase (ALP) staining, also determination of calcium nodules in extracellular matrix by alizarin red S.2. To evaluate the effects of TDF on proliferation and apoptosis of osteoblast, the cultured cells were divided into four groups according to the concentration of TDF:OnM TDF,50nM TDF,500nM TDF,5000nM TDF, and MTT assay was employed. The apoptosis of cells in each group were detected using Annexin V&PI staining and TUNEL assay.3. To detect the effect of TDF on the osteogensis of hBMSC. The ALP activity and calcium nodules were compared between different groups. Alkaline phosphatase staining was conducted in the3th,7th,9th and10th days of induction culture, alizarin red S staining was conducted in10th,14th,21th,28th days.4. The mRNA expression of tissue-nonspecific alkaline phosphatase (TNAP), Collagen type I (COL-1), osteoprotegerin(OPG) and receptor activator for nuclear factor-κ B Ligand(RANKL) were detected by realtime-PCR.Part two. Researches on mechanism of TDF involved in osteogenesis. The cell groups were the same as in part2. The mRNA expression of factors involved in wnt/β-catenin canonical pathway are tested by realtime-PCR, including wnt3a, Low density lipoprotein receptor related protein5/6(LRP5/6), frizzled-related protein4(FZD4) and β-catenin.[Results]Part one. First, the osteogenetic potentiality of hBMSCs was present when cultured in MODM. Cells get positive result of ALP staining and alizarin red S. Second, the proliferation ability of cultured cells was inhibited by TDF at concentration of5000nM, but not50nM TDF and500nM TDF. Third, cells treated with500nM TDF showed lower percentage of ALP positive compared with that treated with OnM TDF (17.5±4.4%vs.12.35±2.67%, P=0.005)at early stage of osteogenesis, and poor shape of calcium nodule, which means for disturbed mineralization. Cells treated by50nM TDF exhibit intermediate characteristics between500nM TDF and OnM TDF. Fourth, levels of TNAP mRNA from cells treated at50nM TDF (8.15±0.7vs.6.89±0.35, P=0.001) and500nM TDF(8.15±0.70vs.4.64±0.45, P=0.000) were lower than that in group OnM TDF. Also the levels of COL-I mRNA from cells treated at50nM TDF (9.28±0.26vs.2.81±0.17, P=0.000) and500nM TDF (9.28±0.26vs.1.98±0.15, P=0.000). Contrarily, there are higher levels of RANKL mRNA in group50nM TDF (4.98±0.20vs7.89±0.41, P=0.00) and group500nM TDF (4.98±0.20vs6.82±0.03, P=0.000) than that in group OnM TDF.Part Two. Expression of LRP5and FzD4mRNA were disarranged by TDF at concentration of50nM and500nM. And mRNA express of β-catenin in group50nM TDF and group500nM TDF were lower than that in group OnM TDF (5.01±1.69vs.2.31±0.24, P=0.001and5.01±1.69vs.3.39±0.79, P=0.021)[Conclusions]It was found in this study:1. Osteogenetic potentiality of hBMSCs and synthesis ability of osteoblast may be the targets of TDF.2. Desynchronization of factors in Wnt/β-catenin canonical pathway may contribute to the disturbed osteogenesis suffered from TDF, especially LRP5, FzD4and P-catenin. |
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