Experimental animals
This study was approved by the animal ethics committee of Guangxi Medical University. Healthy 1 to 2-day newborn New Zealand rabbits (without gender limitations) were supplied by the Experimental Animal Center of Guangxi Medical University in Nanning, China (License No.: SCXK GUI 2009–0002).
Reagents and instruments
Reagents and instruments were showed as following, including PNS (freeze-drying Xue Shuan Tong injection, Wuzhou Pharmaceutical Group Co. Ltd, Wuzhou, China, No.Z20025652, 150 mg/bottle); L-DMEM culture medium and fetal bovine serum (FBS) (GE Healthcare HyClone™ Cell Culture Co., Logan, USA); 0.25 % trypsin (Shanghai Bi Yun Tian Biotechnological Co. Ltd, Shanghai, China); osteogenesis-induced liquid (Nanning Weierkai Biotechnological Co. Ltd, Nanning, China); Methylthiazoletetrazolium (MTT) assay and percoll separating medium (Sigma-Aldrich Co., St. Louis, USA); rabbit anti-human CD29 polyclonal antibody (FITC) (Beijing Biosynthesis Biotechnological Co. Ltd, Beijing, China); rat anti-human and rabbit CD45 monoclonal antibody (FITC) (AbD Serotec Co., Oxford, UK); rat anti-human and rabbit HLA-DR monoclonal antibody(FITC) (Bio-Rad Co., Hercules, USA); rat anti-human IgG1(FITC) and IgG2b(FITC) (Lianke Biotechnological Co., Hangzhou, China); total RNA-extracted kit (Corning Incorporated Co., New York, USA); reverse transcription kit (TaKaRa Co., Tokyo, Japan); real-time PCR primer (Shanghai Bioengineering Co. Ltd, Shanghai, China); DAB kit (Beijing Zhongshan Biotechnological Co. Ltd, Beijing, China); rat anti-rabbit TGF beta 1 antibody (Novus Biologicals Co., Littleton, USA); goat anti-rabbit IgG (Bio-Techne Co., Minneapolis, USA); Alkaline Phosphatase(ALP) detection kit (Nanjing Jiancheng Biotechnological Co. Ltd, Nanjing, China); alizarin red kit (Shanghai SSS Reagent Co. Ltd, Shanghai, China); CO2 calorstat (Themo Forma Co., Waltham, USA); inverted aberration microscope (Zeiss Co., Oberkochen, Germany); PCR detection system (Bio-Rad Co., Hercules, USA).
Isolation, culture and identification of rabbit bone marrow mesenchymal stem cells (BMSCs)
Following a protocol approved by the animal ethics committee of Guangxi Medical University, rabbit bone marrow were obtained from tibia and femur of new born New Zealand rabbit by flushing the marrow cavity using a 5 ml syringe filled with L-DMEM-medium (including 10 % FBS and 1 % penicillin-streptomycin) attached with a 23G needle. Then the medium with bone marrow was added into the centrifuge tube (pouring slowly along the tube wall and avoiding shaking) with isopyknic Percoll cell separation solution (1.073 g/ml). After 20-minute 2000 rpm centrifugalization, the nebulous liquid in the middle layer was carefully moved into a new centrifuge tube. And medium was added into the tube for 10-minute 1000 rpm centrifugalization. Then resuspended the cells and regulated the cell concentration to 5 × 105/ml. Afterwards, the cells were collected and cultured in a 50 ml culture flask with L-DMEM-medium supplemented with 10 % FBS and 1 % penicillin-streptomycin at 37°Cin a 5%CO2 humidified incubator. The nonadherent cells were removed after 48 h and the medium was changed every 3 days. The cell passaging was achieved when the cell confluence reached to 80–90 %. After that, the growing status of cells was observed under the inverted aberration microscope. The monoclonal antibody, such as CD29, CD45 and HLA-DR, was added in 3rd passage of cells which grew in a good condition, and the FCM was applied to detect the positive rate of each surface marker in cultured cells.
Construction of lentivirus-mediated siRNA interference cell model
On the basis of NCBI-GenBank (National Center for Biotechnology Information GenBank) information for TGF-β1 related gene sequence of New Zealand rabbit, we chose the relatively complete mRNA sequence (Oryctolagus cuniculus transforming growth factor, beta 1(TGF-β1), mRNA, NCBI Reference Sequence: XM_00272231312, 1164 bp), as the RNA interference target sequence. Nanning Weierkai Biotechnological Co. Ltd. was responsible for the synthesis of siRNA sequence, including the screening of optimal sequence (LipofectamineTM RNAi MAX transient transfection), the construction of TGF-β1 shRNA lentivirus vector (including ZsGreen green-fluorescent protein), the lentiviral packaging and titering and detection of the virus multiplicity of infection (MOI).
The verification of silencing effect via q-PCR
The total RNA was extracted from 48-hour transfected BMSCs by Trizol method. Extracted RNA(2 μg) was converted into cDNA via reverse transcriptase and was put into the real-time q-PCR: primer TGF-β1-F: 5’ AAGTCGGCACAGCGTCTATA, TGF-β1-R: 5’ TTGCTGCATTTCTGGTACAG, amplified fragment 150 bp; β-actin-75 bp, β-actin-F: 5’ ACTGGAACGGTGAAGGTGACA, β-actin-R: 5’ TCGGCCACATTGCAGAACT; the reaction condition of PCR was showed as following: 50 °C 2 min; 95 °C 2 min; 95 °C 15 s, 60 °C 32 s, reading plates, 40 circulations; 60 °C-95 °C melting curve analysis. The RQ value should be calculated and repeated three times for each sample.
The verification of silencing effect via Western blot
The total protein was extracted from 48-hour infected BMSCs by cluster-cracking reaction, and those protein samples were quantified via BCA method. 30 μg protein was transferred to PVDF membrane by SDS-PAGE electrophoresis. After Western blotting and development(compared with GAPDH), the sample was taken a picture via infrared imaging system. The gray value of plaques was analyzed and compared among groups.
Measurement of transfected BMSCs proliferation via methylthiazoletetrazolium (MTT) assay
After two passages, the cultured nontransfected BMSCs, growing in a good condition, were seeded in 50 ml culture flasks(Corning Co.). When the confluence of cells reached 40 % in the flask bottom, the pre-diluted blank virus solution or virus solution was added into the culture flask. After culturing the cells in incubator at 37 °C in 5 % CO2 for 24 h, we added normal culture medium for cell amplification. Then, the transfected cells were seeded in 96-well culture plates. Comparing with normal cells cultured in the same density, the cell growth curves of the 1st, 3rd, 5th, 7th, 9th and 11th day were drew via MTT method, so as to observe the effect of gene silencing on BMSCs proliferation.
Effects of PNS on intracellular ALP activities
With the density of 103–104/ml, BMSCs, growing in a good condition, were cultured in a 24-well plate. When the confluence of cells reached to 30 %, we divided the cells into five groups, including blank group, 100 mg group, blank virus group, RNAi group and 100 mg RNAi group. The blank virus group was transfected by blank virus diluents, and RNAi group and 100 mg RNAi group were transfected by virus diluents(silencing method) for 24 h. The medium was changed on the next day of all groups, and cells in 2nd and 5th group were also treated with PNS at 100 mg/L. After the 3rd, 5th and 7th day, the medium was eliminated, and each well was washed by 1 ml PBS once. Then, 500 μl TritonX-100 cell lysis solution was added for 40-minute ice-bath lysis. Under the instruction of ALP detection kit, the OD value of 30 μl lysis solution from each well was detected by ELISA in 520 nm wave length. Finally, the King Unit of ALP in each sample should be calculated in accordance with the formula.
Effects of PNS on the formation of calcium nodes of gene silencing BMSCs via alizarin red-staining method
With the density of 103–104/ml, BMSCs, growing in a good condition, were cultured in the 6-well plate. When cells were occupied 30 % of the bottom, those wells were divided into six groups, including blank group, positive group, 100 mg group, blank virus group, RNAi group and 100 mg RNAi group. The blank virus group was infected by blank virus diluents and the last two groups by virus diluents(silencing method) for 24 h. Next day, the osteogenesis-induced solution was put into the positive group, and PNS 100 mg/L pure culture solution into both 100 mg group and 100 mg RNAi group, while the pure culture solution into the rest three groups. Each solution above was changed every 2 days to maintain the induction. After the next 21-day culture, the alizarin red was added into each well for staining under the instruction of kit. The quantity of calcium nodes should be calculated via 8 visual fields which were chose from each sample randomly under the microscope, so as to use both mean and standard deviation to make the comparison among those groups.
Statistical analysis
Quantitative data were presented as mean + SD. Statistical significance was determined by two-tailed student’s t-test or one-way ANOVA followed by the LSD t-test for multiple comparisons. A P-value of 0.05 was considered statistically significant.