ID Number		2855
Study Type		In Vitro
Model		This study investigated the ability of millimeter-wave (MMW) to promote the differentiation of bone marrow stromal cells (BMSCs) into cells with a neural phenotype; the cells were induced by ²-mercaptoethanol (BME) in combination with MMW or BME alone.
Details		AUTHORS' ABSTRACT: This study investigated the ability of millimeter-wave (MMW) to promote the differentiation of bone marrow stromal cells (BMSCs) into cells with a neural phenotype. The BMSCs were primarily cultured. At passage 3, the cells were induced by ²-mercaptoethanol (BME) in combination with MMW or BME alone. The expressions of nucleostemin (NS) and neuron-specific enolase (NSE) were detected by immunofluorescent staining and Western blotting respectively to identify the differentiation. The untreated BMSCs predominately expressed NS. After induced by BME and MMW, the BMSCs exhibited a dramatic decrease in NS expression and increase in NSE expression. The differentiation rate of the cells treated with BME and MMW in combination was significantly higher than that of the cells treated with BME alone (P<0.05). It was concluded that MMW exposure enhanced the inducing effect of BME on the differentiation of BMSCs into cells with a neural phenotype. PARTIAL PAPER: Millimeter-wave (MMW) irradiation, as a novel physical therapy, has been increasingly attracted the attention of researchers in biological medicine. Low-power MMW irradiation can induce a variety of changes in the cell proliferation and body metabolism. Advances have been made in using MMW to promote the cell apoptosis[1, 2] and kill the tumor cells[3, 4]. However, few studies on MMW inducing cell differentiation are available. The present study investigated the ability of MMW to promote the differentiation of bone marrow stromal cells (BMSCs) into cells with a neural phenotype, in an attempt to provide experimental basis for the future clinical application. 1 MATERIALS AND METHODS 1.1 Main Reagents and Animals Paraformaldehyde (4%) was bought from Fluka Chemical Co., USA. Rabbit anti-nucleostemin (NS) antibody and rabbit anti-neuron-specific enolase (NSE) were purchased from Santa Cruz Co., USA. FITC-labeled goat anti-rabbit IgG was procured from Beijing Zhongshan Golden Bridge Biological Technology Co. Ltd., China. ²-actin was provided by Sigma Co., USA. ECL reagents were from Perbio Co., UK. DMEM and fetal bovine serum (FBS) were products from Gibco Yeqing TONG, E-mail: tongyq01@yahoo.cn #Corresponding author Co., USA. Other reagents were of analytic grade and were made in China. Rats aged 46 weeks were provided by the Center of Experimental Animals of Tongji Medical College, HUST, Wuhan, China. 1.2 Isolation and Culture of BMSCs Primary BMSCs were isolated and cultured according to the technique established by Reynolds et al[5]. Briefly, the femurs of 6- to 8-week-old rats were taken and washed 3 times with Hanks solution containing antibiotics and heparin. Then, the distal end and the proximal epiphysis of the femurs were removed, and the pulp chamber was flushed with Hanks solution using a sterile syringe. The washed cells were harvested and transferred to the centrifuge tube, which was followed by centrifugation at 1 500 r/min for 5 min and removal of the supernatants. Afterwards, the cells were rinsed with Hanks solution 2 times and re-suspended with 10% new-born calf serum. The cells were plated into a 25 mL-culture bottle at a density of 1×l05/mL and cultured in an incubator at 37°C in an atmosphere of 5%CO2 [6, 7]. The culture media were replaced 24 h later and the unattached cells removed. Medium replacement was performed every 3 days. For passage, the cells were digested with 0.25% trypsin-EDTA, plated into the culture bottle at a density of l×105/mL and then incubated with 10% new-born calf serum. 1.3 Cell Inducement At passage 3, BMSCs in the logarithmic phase of growth were divided into 3 groups in terms of the different inducement protocols. In the control group, BMSCs were cultured in DMEM supplemented with 10% new-born calf serum. In the mercaptoethanol (BME)-induced group, the cells were administered BME alone. In the MMW group, the cells were treated with BME and MMW in combination. After pre-treated with 1-mmol/L BME for 24 h, the cells were treated with 5-mmol/L BME for another 6 h. Thereafter, the cells were subjected to MMW irradiation three times at an interval of 2 h. Each session lasted 10 min. 1.4 MMW Irradiation Cell suspension (3 mL) with the cell density of l×105/mL was seeded into a 24-well plate and placed under a 623-A MMW radiator (Beijing Vacuum Electronics Research Institute, China). The culture plate and the radiator were both placed in a thermotank at 37°C. After standing, the cells received MMW irradiation. The frequency of MMW was set at 36.11 GHz and the power at 10 mW/cm2. The cells were incubated for 24 h after irradiation. The relevant parameters were measured. Each experiment was conducted in triplicate. 1.5 Immunocytochemistry 1.5.1 Preparation of Cell-seeded Coverslips In the control group, the culture media were discarded when the cells of third generation reached the logarithmic phase. The cells were washed with 0.01 mol/L PBS 2 or 3 times and fixed in 4% paraformaldehyde for 30 min at 4°C. In the MMW group, all the procedures for the preparation of cell-seeded coverslips were similar to those of the control group, except that the cells were cultured for 24 h before the culture media were removed. 1.5.2 Immunofluorescent Staining BMSCs were immunofluorescently detected for the expression of NS. In brief, the cells on the coverslips were treated with Triton X-100 for 15 min and then H2O2 methanol (300 mg/L) for 20 min, which was followed by washing with PBS (0.01 mol/L) 2 or 3 times and blockade with 4% bovine serum albumin for 30 min at 37°C. Afterwards, the cells were co-cultured with anti-NS antibody (dilution at 1:300) at 4°C overnight and then with FITC-labeled goat anti-rabbit IgG (dilution at 1:300) for 2 h at room temperature Consequently, the cell-seeded coverslips were observed and photographed under an immunofluorescence microscope. 1.5.3 Immunocytochemistry The cells were measured immunocytochemistrically for the expression of NSE. Some steps were the same as those described in 1.5.2 except that the anti-NSE antibody (dilution at 1:300) was used instead of the anti-NS antibody and biotinylated goat anti-rabbit IgG was used as the second antibody. In the end, the cells were incubated with HRP-streptoavidin for 1 h at room temperature, stained with DAB and observed under a light microscope. 1.6 Western Blotting 1.6.1 Extraction of Cell Proteins The culture media were removed and the cells were washed with cold PBS 2 times. Then, the culture plate was placed on ice. Cell lysate (200 ¼L) was added at 4°C. The mixture was left on ice for 10 to 30 min with shaking. The supernatant was transferred to the centrifuge tube and shaken by ultrasonic waves. The centrifugation was performed at 20 000 r/min for 10 min at 4°C. The supernatant was stored at 20°C for later use. 1.6.2 Immunoblotting Total proteins were separated by 10% SDS-PAGE and electrophoretically transferred to a nitrocellulose membrane. Non-specific binding of antibody was blocked with 5% non-fat dry milk for 45 min at 4°C. Immunoblotting was carried out with rabbit anti-NS antibody (1:800), followed by HRP-labeled goat anti-rabbit IgG (1:3000), and the blots were developed by the enhanced chemiluminescence method. ²-actin served as internal control. 1.7 Statistical Analysis Ten visual fields were randomly selected on the cell coverslides and observed. The cell positive for NSE were counted on each of the visual field. Student-Newman- Keuls test was employed for the statistical analysis of the differences among the groups by employing SPSS 12.0 software package. The data were expressed as ±s. A P<0.01 was considered to be statistically significant. 2 RESULTS 2.1 NS Expression NS, as a marker protein of stem cells, usually expresses in the nucleoplasm or nucleolus of stem cells prior to the differentiation. When the stem cells began to differentiate, NS expression was substantially decreased and sometimes undetectable. Therefore, NS was regarded as non- or weak expression in the post-differentiation cells. Our findings showed that most of the non-induced BMSCs were positive for NS in their nuclei (fig. 1A). After treatment with BME, NS expression was significantly reduced and a great number of BMSCs were negative for NS (fig. 1B). When the cells were treated with BME and MMW irradiation in combination, the number of NS-positive BMSCs was much less than that of the differentiating cells induced by BME alone (fig. 1C). {End of partial paper]
Findings		Effects
Status		Completed With Publication
Principal Investigator		Huazhong Univ of Science and Technology, Wuhan, Ch
Funding Agency		?????
Country		CHINA
References		Tong , Y et al. J. Huazhong Univ. Sci. Technolog. Med. Sci., (2009) 29:409-412
Comments