BMMSCs were obtained from 10 patients with MM (age range, 40–80 years; mean age, 63±9.7 years; 4 females and 6 males) recruited at the Tongde Hospital of Zhejiang Province (Hangzhou, China) between October 2014 and September 2015. The study was approved by the Ethics Committee of Tongde Hospital of Zhejiang Province (Hangzhou, China) and patients provided written informed consent.

The following diagnostic criteria applied: i) Symptomatic MM: Presence of M protein in serum or urine; cloned plasma cell or plasma cell tumor in the BM; relevant organ or tissue damage, including anemia, hypercalcemia, renal damage and bone lesions. ii) Asymptomatic myeloma (smoldering myeloma); serum M protein reach myeloma level (>30 g/l); ≥10% clonal plasma cells in the BM; no associated organ or tissue damage or MM-associated symptoms.

Inclusion criteria were defined as follows: i) Meet diagnostic criteria of MM; ii) onset of disease without remission or relapse; iii) aged 18–80 years; iv) Eastern Cooperative Oncology Group score <2 (19). Exclusion criteria were as follows: i) Perinatal women or mentally ill patients; ii) history of underlying liver or kidney disease; iii) other hematological malignancies.

The majority of patients with MM had M protein levels >30 g/l, IgA >25 g/l and 24-h urine light chains >1 g, some patients exhibted lower M protein levels. A previous study reported that the the number of plasmacytoid plasma cells in the BM were >10% in 95% of patients (20). Clinical manifestations of associated target organ damage, including anemia, osteolytic lesions, hypercalcemia, renal insufficiency, hyperviscosity syndrome, amyloidosis and recurrent infections, were further important diagnostic criteria in patients with MM.

TLP and thalidomide were purchased from Sigma-Aldrich (purity, >98%; Merck KGaA, Darmstadt, Germany). Low-glucose Dulbecco's modified Eagle's medium (DMEM) was purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Fetal bovine serum (FBS) was purchased from Boehringer Ingelheim GmbH (Ingelheim, Germany) and the Cell Counting kit-8 (CCK-8) was from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). Dimethyl sulfoxide (DMSO) was from Sangon Biotech Co., Ltd. (Shanghai, China), the Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) kit was supplied by Hangzhou MultiSciences (Lianke) Biotech Co., Ltd. (Hangzhou, China). Human BM mononuclear lymphocytes separating medium was obtained from Tianjin Haoyang Biological Products Technology Co., Ltd. (TBD2013LHU; Tianjin, China) and antibodies for CD34 phycoerythrin (PE; cat. no. A07776), CD45 PE (cat. no. IM3548; both Beckman Coulter, Inc., Brea, CA, USA) and CD105 PE (MHCD10504; Invitrogen; Thermo Fisher Scientific, Inc.) were. The RNA Extraction kit (antibody total RNA fast extraction kit) was from Generay Biotech Co., Ltd. (Shanghai, China), the reverse transcription (RT) PrimeScript™ RT Master mix was from Takara Biotechnology Co., Ltd. (lot no. RR036A; Dalian, China) and the quantitative polymerase chain reaction (qPCR) SuperRealPreMix color was from Tiangen Biotech Co., Ltd. (SYBR Green; lot no. FP215; Beijing, China). Polyvinylidene difluoride (PVDF) membranes were from Merck KGaA (0.45 µm) and Bio-Rad Laboratories, Inc. (0.2 µm; Hercules, CA, USA). Color pre-dyed molecular weight standard were provided by Fermentas (Thermo Fisher Scientific, Inc.), and the electrochemiluminescence (ECL) Plus luminescent kit, the SDS-PAGE loading buffer (5X), Western and IP cell lysis buffer, phenylmethylsulfonyl fluoride and the bicinchoninic acid (BCA) kit were obtained from Beyotime Institute of Biotechnology (Haimen, China). Primer sequences were as follows: IL-6, forward 5′-TAGTGAGGAACAAGCCAGAG-3′ and reverse 3′-ATGCTACATTTGCCGAAGAG-5′; IL-1β, forward 5′-TTACAGTGGCAATGAGGATG-3′ and reverse 3′-TGTAGTGGTGGTCGGAGATT-5′; stem cell factor (SCF or Kit ligand), forward 5′-ATGGCTCCAGAAATGTCTAA-3′ and reverse 3′-AATCCAATCACAGTTCCCAC-5′; and GAPDH forward, 5′-AGAAGGCTGGGGCTCATTTG-3′ and reverse, 3′-AGGGGCCATCCACAGTCTTC-5′. The following ELISA kits were used: VEGF [cat. no. EK1831; Hangzhou MultiSciences (Lianke) Biotech Co., Ltd.].

Under aseptic conditions, BM from patients with MM (3–4 ml) was collected and placed into EDTA anticoagulant tubes or directly into chromosome culture bottles. To prepare single cell suspensions of the BM, BM cells were collected and mixed with 4–5 ml F solution (Tianjin Hao Yang Biological Products Technology Co., Ltd.), centrifuged for 20 min at 4°C and 500 × g. The supernatant was discarded, then the precipitate was washed twice with F solution and suspended with whole blood and tissue diluent containing 20% FBS, which as also a part of the F soltuion kit. Separating medium (≥3 ml) was added to a 15-ml centrifuge tube, followed by the addition of an equal volume BM single cell suspension using a straw. The solution was centrifuged at 500 × g and 4°C for 30 min, and the annular milk and white lymphocytes, which formed the second layer from the top, were extracted and placed into a fresh 15-ml centrifuge tube. A total of 10 ml cleaning solution (cat. no. 2010X1118; Tianjin Hao Yang Biological Products Technology Co., Ltd.) was added and mixed with the cells. Following centrifugation at 250 × g and 4°C for 10 min, the supernatant was discarded. Cells were resuspended in 0.5 ml DMEM. Above steps were repeated 3 times. Following the final centrifugation step, cells were suspended with 0.5 ml DMEM. Resuspended cells were counted and seeded at 10⁵ cells/cm in a counting plate, containing 10% FBS and low-glucose DMEM. Cells were incubated at 37°C in 5% CO₂ with saturated humidity. Following 24 h, ~25% of cells adhered onto the walls and 75–80% of cells adhered to the walls over the second and third days. Medium was replaced following 4 days. Following 1 week, ~80% cell densities were achieved and cell passage was performed using trypsin.

Second generation BMMSCs were digested using trypsin, collected by centrifugation at 800 × g and 4°C for 5 min and the cellular morphology was observed under an optical microscope at a magnification of ×40 and ×100. CD34 PE, CD45 PE and CD105 PE antibodies were added to the cells in the experimental group, and ISO IgG1 antibodies (cat. no. IM0670U; Beckman Coulter) were added to the control group at a density of at 1 µg antibody/10⁶ for 30 min at 4°C. Cells were incubated for 30 min at 4°C, detected by flow cytometry and analysed by BD Accuri™ C6 software (version 1.0.264.21; BD Biosciences, Franklin Lakes, NJ, USA).

Second generation BMMSCs were digested with 0.25% trypsin, centrifuged at 800 × g and 4°C for 5 min, counted and seeded in 96-well plates at 5×10³ cells/well. Cells were incubated standing still for 24 h at 37°C. Equal volumes of 10% FBS in low-glucose DMEM (control group) or TPL at 6.25, 12.5, 25, 50 and 100 ng/ml in 10% FBS in low-glucose DMEM were added to the cells. Six replicates were performed in each group. Cells were cultured for 12, 24 and 48 h, and harvested by centrifugation at 800 × g and 4°C for 5 min. The supernatant was discarded and 10 µl CCK-8 in low-glucose DMEM (100 µl) was added to each well; cells in DMEM and 10% FBS were prepared as blanks. Reactions were incubated for 2 h at 37°C in the dark and optical density (OD) values was recorded at 450 nm. Half maximal inhibitory concentration (IC₅₀) was calculated by SPSS software (version 17; SPSS, Inc., Chicago, IL, USA).

Second generation BMMSCs were digested with 0.25% trypsin, centrifuged at 800 × g and 4°C for 5 min, counted and seeded in 6-well plates at 1×10⁵ cells/well. Cells were further incubated for 24 h at 37°C. Cells were incubated in 10% FBS in low-glucose DMEM with TPL at 25, 50 and 100 ng/ml or without TPL (the control group). Following 48 h, cells were collected by centrifugation at 800 × g and 4°C for 5 min. Cells were washed twice with PBS and resuspended in binding buffer [cat. no. AP101-100-BB; 500 µl; Hangzhou MultiSciences (Lianke) Biotech Co., Ltd.]. A total of 5 µl Annexin V-FITC and 10 µl PI were added prior to incubation for 5 min at 4°C in the dark. Samples were detected using flow cytometry. All the experiments were repeated 3 times. Samples were analysed by BD Accuri™ C6 software.

TRIzol (cat. no. GK3016; Shanghai Generay Biotech Co., Ltd.) was used to extract total RNA from BMMSCs in the TPL group. RT of the total RNA was performed according to the manufacturer's instructions provided by the cDNA RT kit (42°C). The total volume of the qPCR mixture was 30 µl, including RT product (1 µl), MgCl₂ (1.5 mM), 1X PCR buffer, dNTPs (0.5 mM), up- and downstream primers (0.25 µM) and Taq DNA synthetase (1 U). IL-6, IL-1β and SCF reaction parameters were as follows: 94°C for 35 sec, followed by 40 cycles of 59°C for 45 sec and 72°C for 30 sec, with a final extension at 72°C for 10 min. The internal reference GAPDH. Products were directly used in electrophoresis or stored at 4°C. All the experiments were repeated thrice. Relative gene expression data were analysed using Bio-Rad CFX Manager 3.1 (Bio-Rad Laboratories, Inc.) and the 2^−ΔΔCt method (21).

BMMSCs were treated with TPL and thalidomide (both 0, 25, 50 and 100 ng/ml) for 48 h at 37°C. Cells were harvested by centrifugation at 800 × g and 4°C for 5 min and the supernatant of the control group, TPL treatment group or thalidomide treatment group was discarded. Cells were washed twice with PBS and lysis buffer and protease inhibitor were added. The resulting solution was mixed by shaking for 1 min and then placed on ice for 10 min. The mixing and incubation on ice were repeated thrice. Following centrifugation at 1,000 × g and 4°C for 5 min, the protein concentration in the supernatant was detected using a BCA assay. Proteins (40 µg) were separated by SDS-PAGE on a 12% separation gel and 5% concentrated gel, and then transferred to the 0.45 µm PVDF membranes. Membranes were incubated with 5% skimmed milk in TBST at room temperature for 2 h. Anti-P65 (cat. no. ab7970; Abcam, Cambridge, UK) and anti-GAPDH (AP0063; Bioworld Technology, Inc., St. Louis Park, MN, USA) primary antibodies were added and incubated overnight at 4°C. Subsequently, membranes were immersed in TBST and incubated with shaking 3 times for 15 min at 25°C. Horseradish peroxidase-conjugated goat anti-mouse secondary antibodies (cat. no. bs12478; 1:5,000; Bioworld Technology, Inc.) were added and membranes were incubated with shaking for 2 h at room temperature. Membranes were immersed in TBST and incubated with shaking 3 times for 15 min at 25°C. ECL detection reagent was added according to the manufacturer's instructions. The grey value of each band was analyzed using ImageJ (version 1.4; National Institutes of Health, Bethesda, MD, USA), following exporting the image in TIFF format. All the experiments were repeated thrice.

Cells from the control, and 25, 50 and 100 ng/ml TPL groups were analysed. According to the manufacturer's instructions, cell culture supernatant was removed from −80°C and left to thaw at room temperature for 30 min. The samples of the cells and standards were added into each well in a 96-well plate, except the blank well (with only contained DMEM). Plates were sealed and incubated at 37°C for 120 min to react to the antibodies prior to washing (6 times). Except for the blank, TBST working solution (100 µl) was added, plates were sealed and incubated at 37°C for 45 min prior to washing (6 times). Then, except for the blank, enzyme binding working solution (100 µl) was added, plates were sealed and incubated at 37°C for 30 min prior to washing (6 times). Substrate color developing agent (100 µl) was added and plates were incubated at 37°C for 20 min. Termination reaction solution (100 µl) was added at 25°C for 30 min and OD values were determined at 490, 570 or 630 nm. All experiments were repeated thrice.

SPSS software was used to analyze the data. Data are expressed as mean ± standard deviation. Univariate data was analyzed by one-way analysis of variance followed by the Least Significant Difference post hoc test. P<0.05 was considered to indicate a statistically significant difference.

BMMSCs are adherent cells and in the current study they were separated from the BM following the addition of lymphocyte separating medium and screening with low-glucose DMEM. Cells exhibited a long-spindle shaped morphology and flow cytometry results indicated a CD34-negative, CD45 weak positive and CD105-positive expression (Fig. 1). All results complied with the characteristics of BMMSCs, namely a uniform long shuttle shape (22).

In order to investigate proliferation and apoptosis in TPL-treated BMMSCs, CCK-8 assays and flow cytometry were performed at varying TLP concentrations (0, 25, 50 and 100 ng/ml) over time (12, 24 and 48 h). At 12, 24 and 48 h, the OD in TLP-treated BMMSCs (all concentrations) was significantly reduced compared with the untreated control sample (F=3.085, P=0.023; F=8.896, P<0.001; F=7.873, P<0.001, respectively; Fig. 2A). These results indicated that TPL inhibited the proliferation of BMMSCs compared with the untreated control. The IC₅₀ of TPL at 48 h was 101.55±2.45 ng/ml. Apoptosis in BMMSCs significantly increased in a concentration-dependent manner at 48 h, with rates of 1.43±0.15, 3.70±0.20, 3.77±0.32 and 5.33±0.68% for 0, 25, 50 and 100 ng/ml TPL, respectively (F=49.078, P<0.001; Fig. 2B). These results suggested that TPL may induce apoptosis in BMMSCs.

To investigate the effect of TLP on cytokines, RT-qPCR was performed to detect mRNA expression levels of IL-6, IL-1β and SCF. BMMSCs were treated with TPL (0, 25, 50 and 100 ng/ml) for 48 h. Analysis revealed that IL-6, IL-1β and SCF expression were significantly decreased with TPL treatment compared with the untreated controls in what appeared to be a dose-dependent manner (F=1433.475, P<0.001; F=90.057, P<0.001; F=11117.534, P<0.001; Table I). These findings suggested that TPL may decrease cytokine mRNA expression.

To detect the effect of TPL and thalidomide on P65 protein levels, BMMSCs were treated with different TPL and thalidomide concentrations (0, 25, 50 and 100 ng/ml) for 48 h and western blots were generated. Results indicated that with increasing TPL, P65 expression decreased (F=7.489, P=0.010; Fig. 3). With increasing thalidomide, P65 expression markedly increased compared with the blank control (F=3.916, P=0.054; Fig. 3C). These results suggested that TPL downregulated P65 expression.

To detect effects of TPL on VEGF levels, BMMSCs were treated with TPL (0, 25, 50 and 100 ng/ml) for 48 h. VEGF levels significantly decreased with TPL treatment compared with the untreated controls in what appeared to be a dose-dependent manner (F=10.500, P=0.023; Table II). The lowest VEGF level (561±59 pg/ml) was measured at 100 ng/ml TPL.