Healthy adult rabbits were purchased from the Experimental Animal Feeding and Management Center of Bengbu Medical College, China.
Main materials and instruments
Cells: rabbit NPMSCs were isolated and cultured in our laboratory.
Instruments: ultra-clean working table (HVAC Purification Equipment, Bengbu, China), cell culture box (Thermo Fisher Scientific, Waltham, MA, USA), micro-centrifuge (Allsheng Instruments, Hangzhou, China), PCR instrument (Lattice Scientific Instrument, Hangzhou, China), and flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).
Reagents: DMEM (C11995500CP; Gibco, Billings, MT, USA), RPMI 1640 (C11875500BT; Gibco) fetal bovine serum (#04-002-1a; BioIND, Beit-Haemek, Israel), antibiotic–antimycotic (#15240-112; Life Technologies, Carlsbad, CA, USA); PBS, pH 7.4 (#10010-023; Gibco); Trypsin–EDTA (0.05%) (#25300-054; Life Technologies), bovine serum albumin (#15561012; Life Technologies), Lipofectamine® 2000 transfection reagent (#11668-019; Life Technologies), Opti-mem® I reduced serum medium (#31985-062; Life Technologies); western blot (WB) and IP cell lysis solution, predye protein marker (Fermentas, Vilnius, Lithuania); and polyvinylidene membrane (Millipore, Burlington, MA, USA).
Antibodies: CD90 antibody (ab225; Abcam, Cambridge, UK), CD105 antibody (ab221675; Abcam), CD34 antibody (bs-0646R; Bioss, Woburn, MA, USA), CD45 antibody (bs-0522R; Bioss), FITC-labeled sheep anti-mouse IgG (bs-0296g-fitc; Bioss), FITC-labeled sheep anti-rabbit IgG (bs-0295g-fitc; Bioss), GDF5 antibody (ab93855; Abcam), and anti-actin antibody (SAB4200248; Sigma-Aldrich, Burlington, MA, USA).
Kits: Ultrapure RNA extraction kit (CW0581S; Kangwei Century Biotechnology, Beijing, China), SuperRT cDNA synthesis kit (CW0741S; Kangwei Century Biotechnology), UltraSYBR Mixture (High ROX, CW2602M; Kangwei Century Biotechnology), and BCA protein quantitative kit (Beyotime Biotechnology, Shanghai, China).
Cell isolation culture and identification
Acquisition of NPMSCs: briefly, the rabbit NP tissues were harvested, digested by collagenase, centrifuged, resuspended, and the NPCs were obtained. Then the suspension of NPCs was centrifuged. Subsequently, the supernatant fluid was removed, and the complete medium of mesenchymal stem cells was added. Finally, the suspension was filtered to obtain NPMSCs. When the cell fusion rate reached 80–90%, they were digested by trypsin and re-inoculated for the subculture of the NPMSCs. The morphological and biological characteristics of the cells were observed under an inverted microscope.
The surface immunophenotype of the NPMSCs in rabbits was identified by flow cytometry (FCM). The third generation of NPMSCs cells was digested with trypsin and then centrifuged. Subsequently, the precipitated cells were collected, washed with PBS, and diluted to the cell suspension. Anti-rabbit CD90, CD105, CD34, and CD45 antibodies were added, respectively. The suspension was incubated at 25 °C, washed with PBS, incubated with FITC-labeled sheep anti-mouse/sheep anti-rabbit IgG secondary antibody (in the absence of light), and washed with PBS. After the mixing procedure, the mixture was detected by FCM.
Quantitative real-time PCR (qRT-PCR): samples were collected to extract the RNA, and the mRNA level of the target gene was detected by qRT-PCR. RNA was extracted from the NPMSCs, and then the cDNA was synthesized. Subsequently, qRT-PCR was performed, using the GAPDH gene as an internal reference, and the experiment was performed in triplicate for each of the genes per sample. After adding each component to the PCR tube, we carefully sealed the plate membrane, mixed it evenly, and centrifuged the solution to generate a pellet at the bottom of the tube. PCR amplification conditions were based on the instructions of the qRT-PCR kit. According to the instructions of the instrument, the PCR experiment was performed three times. The data were collected and analyzed.
Cell transfection and observation
NPMSCs were divided into three groups: a transfection group (lentiviral vector carrying the GDF5 gene used to transfect NPMSCs); a control virus group (NPMSCs transfected with an ordinary lentiviral vector); and a normal group (the NPMSCs alone).
Transfection methods: the lentivirus plasmids were diluted to 25 L of Opti-MEM® I reduced serum medium, and 0.5 L of Lipofectamine 2000 was diluted to 25 L of Opti-MEM® I reduced serum medium. The solutions were gently mixed, followed by a period of keeping them static, before adding the mixtures to the three groups for culturing.
The gene transfection rate was identified by FCM. All cell groups were digested with trypsin and centrifuged. The precipitated cells were collected followed by washing with PBS and diluting the cell suspension. Anti-rabbit GDF5 was added and incubated for 30 min. The cells were incubated with FITC-labeled sheep anti-rabbit IgG secondary antibody and washed with PBS. Following mixing, the positivity rate of GDF5 in each group was determined by FCM.
Cell counting: Kit-8 (CCK8; Invitrogen, Waltham, MA, USA) was used to measure cell proliferation on days 1, 4, and 7, respectively. The absorbance of the solutions was measured at 450 nm using a microplate reader (BioTek, Winooski, VT, USA).
qRT-PCR was used to detect the mRNA levels of keratin 8, 18, 19 (Krt8, Krt18, and Krt19) in all groups. Cell samples were collected, followed by RNA extraction, and finally, cDNA synthesis was performed. The procedures were identical to those described previously.
The protein levels of KRT8, KRT18, and KRT19 in all groups were detected by WB. The steps include the extraction of the total protein of cells, quantification of protein, protein electrophoresis, film transferring, sealing and incubation, PVDF membrane chemiluminescence, followed by developing and fixing. Finally, the image analysis and statistics were performed: the developing strip was analyzed, and the optical density was scanned by Image J v1.8.0. The relative expression was calculated as the ratio between the optical density of the target band and the corresponding value of the β-actin.
All the experiments were performed in triplicate for each of the genes per sample. The mean values obtained were compared by analysis of variance (ANOVA). Data were presented as mean ± standard deviation. One-way ANOVA was used for comparisons between the groups, and independent sample t-tests were used for pairwise comparisons. Differences were statistically significant at P < 0.05.
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