Unless otherwise specified, all chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies to IgG, GAPDH, ALP, Runx2, Osterix, CD44, CD34, U0126 (an inhibitor of phospho-ERK1/2) ERK1/2, JNK, p90rsk,phospho-ERK1/2, phospho-JNK and phospho-p90rsk1(Ser380) were purchased from the Abcam Corporation, USA.
Isolation and culture of rBMSC
To obtain the rBMSC, the rabbit was used. The femur from a neonatal New Zealand white rabbit was isolated, and the ends of the femur were opened. The bone marrow was flushed from the femur with low glucose Dulbecco’s modified Eagle’s medium (DMEM) using a 1mL syringe. Cells were harvested into a culture dish, suspended using a Pasteur pipette, seeded into a flask containing DMEM and 15 % fetal bovine serum, and cultured in an incubator with 5 % CO2 at 37 °C. The medium was replaced every 2days. When cells grew to a confluence of approximately 85 %, they were passaged with 0.25 % trypsin and 0.1 % EDTA (1:2). Cell growth was monitored using an inverted phase contrast microscope (Nikon Co.). The animal experimental protocols were approved by the Chongqing medical university experimental animal management committee.
RNA Extraction and RT-PCR
To detected the GHSR expression status in the rBMSC, the RT-PCR was used. Total RNA was isolated from o cells using the RNeasy kit (Qiagen, Hilden, Germany). All RNA samples were treated with RNase-free DNase I to remove genomic DNA contamination. The RNA content of samples was too low to be accurately quantified by spectrometry, and thus, 6.5-μL RNA aliquots were converted to cDNA by reverse transcription, then amplified (TaKaRa, Inc., Dalian, China). The ghrelin receptor PCR primers were: sense, 5’-TCTTCCTTCCTGTCTTCTGTC-3’;antisense, 5’-AGTCTGAACACTGCCACC-3’and the PCR condition was 95 °C 5 min, 95 °C 30 s 57 °C 30 s 72 °C 30 s 30 cycles, 72 °C 10 min.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bromide (MTT) assay
To determine the cell growth percentage, the MTT assay was operated. Cells were grown in 96-well plates (1 × 103 cells/well) supplemented with MGF. Control cells were switched from RPMI-1640 to DMEM containing 0.1 % dimethyl sulfoxide (DMSO). At 1 to 6 days following ghrelin treatment (400, 500, 600, 700 and 800ng/ml ghrelin), 20μL of MTT was added to each well to a final concentration of 0.5 %. After a 4h incubation at 37 °C in the dark, 150μL DMSO was added to each well for 10 min to dissolve the formazan crystals. The absorbance was measured using a microplate reader (EXL800, Cole-Parmer, Vernon Hills, IL, USA) at 490nm. All experiments were repeated three times. The viability of the MGF treated cells was expressed as percentage of population growth plus the standard error of the mean (SEM) relative to that of untransfected control cells. Cell growth was calculated as follows:
$$ \%\ growth = \left( mean\ \exp erimental\ absorbance\hbox{--} mean\ control\ absorbance\right)\ / mean\ control\ absorbance \times 100 $$
Immunofluorescence
To detected the CD44 and CD34 expressin status, the immunofluorescence was used. The rBMSC were fixed in 3.7 % paraformaldehyde for 30 min at room temperature, permeabilized with 0.5 % Triton X-100 in PBS for 15 min, and blocked with 1 % BSA in phosphate-buffered saline (PBS) with 10 % goat serum overnight at 4 °C. The samples were then stained with primary antibodies diluted in PBS. The primary antibody binding was detected with an Alexa Fluor 488 goat anti-rabbit IgG (H + L) secondary antibody. Images were captured with a Nikon A1 confocal microscope. Experiments were performed in triplicate.
Western blotting
The detected the protein in MAPK pathway and osteogenic, the western blot was used. The protein homogenates from rBMSC were separated using electrophoresis on 8–12 % sodium dodecyl sulphate/polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked for 30 min at room temperature in PBS buffer containing 5 % fat-free milk and 0.1 % Tween 20. Membranes were then incubated with primary antibody for at least 1 h at room temperature or overnight at 4 °C. The membranes were subsequently washed three times with PBS containing 0.1 % Tween 20, incubated with peroxidase-conjugated secondary antibodies, and developed using ECL reagents (Pierce, Rockford, IL, USA).
Osteogenic differentiation
To detect the rBMSC differentiation to osteogenic, this experiments was operated. The rBMSC were plated at a density of 5000 cells/cm2 and exposed to standard differentiation-inducing media for 21 days. The medium was changed twice per week. Osteogenic differentiation was achieved following standard in vitro protocols. Endothelial differentiation was stimulated by culturing the cells in endothelial growth medium-2 (EGM-2) [31].
Statistical analysis
Statistically significant differences between gene expression levels were determined using one-way analysis of variance (ANOVA) followed by a Newman–Keuls test with GraphPad Prism version five software (GraphPad Software, La Jolla, CA, USA, www.graphpad.com/company/). Replicates were included in the statistical model. Differences were considered statistically significant at the 95 % confidence level (P < 0.05). Data are presented as mean ± S.D.