Protocol approval and animal treatments
The present study was approved by the Research and Animal Ethics Association of Zhongshan Hospital affiliated to Fudan University (NO: GDR452). A total of 40 one-year-old female Sprague-Dawley rats (14-~160 g) were purchased from the Animal Laboratory of the Academy of Medical Sciences (Beijing, China) and used in strict accordance with the guidelines for the Care and Use of Laboratory Animals. The rats were kept in separate cages with free access to food and water, and in a twelve/twelve-hour light/dark cycle (temperature, 24 °C; humidity, 50%). Rats were randomly divided into 4 groups of 10. The sham group received only a skin incision, which was then sutured to close. The rats in the other 3 groups received surgical development of LDD, in which the sacrospinal muscles, spinous processes, supraspinous ligaments, interspinous ligaments and posterolateral halves of the bilateral zygapophysial joints of the lumbar spine were removed. Afterwards, the rats in LDD group received an orthotopic injection of 150 µl normal saline; rats in the LDD + AAV-scr group received an orthotopic injection of 1011 AAV-scr in 150 µl volume; rats in the LDD + AAV-IGF1/VEGF group received an orthotopic injection of 1011 AAV-IGF1/VEGF in 150 µl volume. Rats were kept for 8 weeks before analysis.
Cell Culture And Aav Preparation
A human disc cell line, nucleus pulposus SV40 (HNPSV) has been described before [31]. The HNPSV cells were cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco; Life Technologies, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS, Sigma-Aldrich, St Louis, MO, USA), penicillin (100 µg/ml) and streptomycin (250 ng/ml) at 37 °C, in a 5% CO2 atmosphere. AAV serotype 2 vectors were generated by transfection of human embryonic kidney 293 cells. Rat IGF1 was prepared by PCR using cDNA from primary rat hepatic cells. Respective scramble sequence was used as control for rat IGF1. The sequence for shVEGF is 5’-TGTGAATGCAGACCAAAGA-3’, and for its scramble is 5’-GGTATCTACTAGATGTACT-3’ [32]. Transfection was performed with Lipofectamine 3000 reagent (Invitrogen, CA, Carlsbad, USA), according to the instructions of the manufacturer. The prepared virus was stored at -80 °C. Titration of viral vectors was determined using a dot-blot assay.
Sampling And Analysis By Immunohistochemistry And Elisa
After euthanized by an intraperitoneal overdose of pentobarbital sodium, the lumbar spines of the rats, including the L4 to L6 discs, were removed. The vertebral pulp and annulus fibrosus were isolated for immunohistochemical and ELISA analysis. For immunohistochemistry, the metaphysis of the vertebral pulp and annulus fibrosus specimens were fixed in a 4% paraformaldehyde for 2 hours, then paraffin-embedded followed by being cut into 5-µm-thick sections. The immunohistochemistry followed an ABC method, while DBA was used to develop IGF1 signals and fast red was used to develop VEGF signals. Primary antibodies are mouse anti-IGF1 (ab176523; Abcam, Cambridge, MA, USA; dilution: 1:300) and rabbit anti-VEGF (ab52917; Abcam; dilution: 1:800). Secondary antibodies are HRP-conjugated anti-mouse and anti-rabbit (Jackson ImmunoResearch Labs, West Grove, PA, USA). ELISA was performed using IGF1 kit (MG100; R&D System, Los Angeles, CA, USA), VEGF kit (RRV00; R&D System), proteoglycan kit (EKC39620; Biomatik, Wilmington, DE, USA) and collagen II kit (LS-F23885; LSBio, Seattle, WA, USA).
Apoptosis Assay
Cells were labeled with annexin V-FITC and propidium iodide (PI), using an apoptosis detecting kit (KeyGEN Biotech, Nanjing, China), and analyzed by flow cytometry using CellQuest software (Becton-Dickinson Biosciences, San Jose, CA, USA).
Rna Isolation, Quantitative Polymerase Chain Reaction (rt-qpcr)
RNA extraction and cDNA synthesis were routine performed. RT-qPCR primers were: Rat GAPDH forward: 5’-TGATTCTACCCACGGCAAGTT-3’, Rat GAPDH reverse: 5’-TGATGGGTTTCCCATTGATGA-3’; Rat VEGF forward: 5’-CAAGCCAAGGCGGTGAGCCA-3’, Rat VEGF reverse: 5’-TCTGCCGGAGTCTCGCCCTC-3’; Rat IGF1 forward: 5'-TCTTGAAGGTGAAGATGCACACCA-3', Rat IGF1 reverse: 5'-CCTGAGGTTCTTCACAG-3'. Values were normalized against GAPDH, which proved to be stable across the samples, and then compared to experimental controls.
Statistical Analysis And Bioinformatics
All statistical analyses were carried out using the SPSS 20.0 statistical software package. Data were investigated using one-way ANOVA with a Bonferroni correction, followed by Fisher's exact test to compare 2 sub-groups. All values are shown as mean ± standard deviation (SD) and are considered significant if p < 0.05, not significant (NS) if p > 0.05. For bioinformatics analysis, transcriptome RNA-sequencing (RNA-seq) data of human disc specimens from normal and LDD patients were downloaded from the GEO data portal (https://www.ncbi.nlm.nih.gov/geo/). RNA-seq data of specimens in published database (GSE124272) were used for analysis by the R software Linear Models for Microarray and RNA-Seq Data (Limma) package. Pairwise differential expression was quantified using Cuffdiff. Cufflinks was used to determine FPKM levels for each gene from the STAR alignment and was used as input for Cuffdiff. Next, we performed differential gene analysis of all transcriptional data, setting a log2 |fold change| > 1 and a false discovery rate (FDR) < 0.05 as the cutoff values.