Inhibition of miR-17-5p promotes mesenchymal stem cells to repair spinal cord injury.

OBJECTIVE: The aim of this study was to explore the role of microRNA-17-5p (miR-17-5p) in the pathogenesis of spinal cord injury repair by mesenchymal stem cells (MSCs), and to investigate the possible underlying mechanism. MATERIALS AND METHODS: MiR-17-5p mimics and negative controls were transfected into MSCs. Dual-luciferase reporter gene assay was used to verify the functional binding between miR-17-5p and its target mRNA. After overexpression or knockdown of miR-17-5p, the expression level of target genes in MSC cells was analyzed by Real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot. The proliferation ability of cells was detected by cell counting kit-8 (CCK-8) assay. The effect of miR-17-5p and VEGF-A on angiogenesis was assessed by HUVEC assay. T8 spinal cord injury model was constructed in nude mice. All mice were divided into the negative control group, the SCI group, the miR-17-5p-NC group, the miR-17-5p-inhibitor group, and the miR-17-5p-inhibitor + sh-VEGF-A group. After injection of different treated MSCs at the lesion site, the proportion of intact tissue as well as reduced lumen volume was measured at 28 d. Meanwhile, the motor function of hind limbs was scored based on the Basso Beattie Bresnahan (BBB) standard scale at 7 d, 14 d, 21 d, and 28 d after transplantation, respectively. RESULTS: A binding site of miR-17-5p was found on the mRNA of VEGF-A. The protein expression of VEGF-A was strikingly altered after overexpression or knockdown of miR-17-5p. Knocking down miR-17-5p expression significantly increased the protein level of VEGF-A and GDNF. Meanwhile, miR-17-5p down-regulation significantly enhanced the viability and the angiogenic ability of MSCs. However, simultaneous knockdown of miR-17-5p and VEGF-A showed the opposite results. After spinal cord injury, the proportion of intact spinal cord tissues in mice was significantly reduced, whereas reduced lumen volume was remarkably increased. After injection of MSCs alone, the proportion of intact tissues was significantly increased. After knocking down miR-17-5p, the proportion was further increased. However, no significant effect was found on the amount of intact tissues after knocking out VEGF-A. Moreover, the reduction in cavity volume appeared to present an opposite trend comparable to the proportion of intact tissues. The BBB scores were significantly decreased in the mice model, while remarkably increased after MSC transplantation. Furthermore, the BBB score was the highest in the miR-17-5p knockout group, while VEGF-A knockout had little effect on it. In addition, no significant difference was found in the mRNA expression GFP in the spinal cord of mice in different groups after MSCs treatment. CONCLUSIONS: Inhibition of miR-17-5p up-regulates the expression of VEGF-A and GDNF in MSCs, and promotes the repair of spinal cord injury by MSCs.

Selecting key genes associated with osteosarcoma based on a differential expression network.

Despite recent advances in osteosarcoma diagnosis and therapy, much remains unclear about the molecular mechanisms involved in the disorder, and the discovery of novel drug-targeted genes is essential. We explored the potential molecular mechanisms and target genes involved in the development and progression of osteosarcoma. First, we identified the differentially expressed genes in osteosarcoma patients and matching normal controls. We then constructed a differential expression network based on differential and non-differential interactions. Pathway-enrichment analysis was performed based on the nodes contained in the main differential expression network. Centrality analysis was used to select hub genes that may play vital roles in the progression of human osteosarcoma. Our research revealed a total of 176 differentially expressed genes including 82 upregulated and 94 downregulated genes. A differential expression network was constructed that included 992 gene pairs (1043 nodes). Pathway-enrichment analysis indicated that the nodes in the differential expression network were mainly enriched in several pathways such as those involved in cancer, cell cycle, ubiquitin-mediated proteolysis, DNA replication, ribosomes, T-cell receptor signaling, spliceosomes, neurotrophin signaling, oxidative phosphorylation, and tight junctions. Six hub genes (APP, UBC, CAND1, RPA, YWHAG, and NEDD8) were discovered; of these, two genes (UBC and RPA) were also found to be disease genes. Our study predicted that UBC and RPA had potential as target genes for the diagnosis and treatment of osteosarcoma.

Combination gene therapy targeting on interleukin-1ß and RANKL for wear debris-induced aseptic loosening.

This study investigated the efficacy of a combination gene therapy to repress interleukin-1 (IL-1) and receptor activator of nuclear factor NF-kappa B ligand (RANKL) for the treatment of particulate debris-induced aseptic loosening, and tried to explore the molecular mechanism of the exogenous gene modifications on osteoclastogenesis. RAW cells activated by titanium particles were transduced with DFG-IL-1Ra (retroviral vector encoding IL-1 receptor antagonist) and AAV-OPG (adeno-associated viral vectors-osteoprotegerin) individually or in combination for 4 weeks. Pro-inflammatory cytokines in culture media were determined by enzyme-linked immunosorbent assay, and gene expressions of RANK, IL-1ß, c-Fos, TRAF6, JNK1 and CPK were examined using real-time PCR. An established knee-implant-failure mouse model was employed to evaluate the efficacy of the in vivo double-gene therapy. The surgical implantation of a titanium alloy pin into the proximal tibia was followed by monthly challenge with titanium debris. Peri-implant gene transfers of IL-1Ra and OPG (respectively or in combination) were given 3 weeks after surgery. The combination of OPG and IL-1Ra gene transfer exhibited strong synergetic effects in blockage of inflammation and osteoclastogenesis at 8 weeks after gene modification. The combination therapy reversed peri-implant bone resorption and restored implant stability when compared with either single gene transduction. Real-time PCR data indicated that the action of IL-1Ra gene therapy may be mediated via the JNK1 pathway, while the reduction of osteoclastogenesis by OPG gene modification may be regulated by c-Fos expression. In addition, both gene modifications resulted in significant diminishment of TRAF6 expression.

Urodynamic study of bladder function for patients with lumbar spinal stenosis treated by surgical decompression.

Lumbar spinal stenosis usually leads to different degrees of nerve damage, presenting with back and leg pain, and/or neurogenic bladder symptoms. To determine whether lumbar decompression improved urological function, bladder dysfunction was evaluated in this retrospective study of 26 patients with lumbar spinal stenosis who had undergone lumbar decompression surgery. Urodynamic study procedures were performed pre-operatively and 6 months post-operatively. The Japanese Orthopaedic Association score rating system and Oswestry Disability Index were employed for clinical evaluation. Following surgery, post-voiding residual urine, maximum cystometric capacity and maximum flow rate improved significantly. There was no statistically significant improvement in voided volume, bladder compliance, maximum detrusor pressure or upper urinary tract damage. Urodynamic study was important in the diagnosis of neurogenic bladder dysfunction, prevention of renal deterioration and assessment of post-operative effects after surgical decompression for patients with lumbar spinal stenosis.

Cell-based osteoprotegerin therapy for debris-induced aseptic prosthetic loosening on a murine model.

Exogenous osteoprotegerin (OPG) gene modification appears a therapeutic strategy for osteolytic aseptic loosening. The feasibility and efficacy of a cell-based OPG gene delivery approach were investigated using a murine model of knee prosthesis failure. A titanium pin was implanted into mouse proximal tibia to mimic a weight-bearing knee arthroplasty, followed by titanium particles challenge to induce periprosthetic osteolysis. Mouse fibroblast-like synoviocytes were transduced in vitro with either AAV-OPG or AAV-LacZ before transfused into the osteolytic prosthetic joint 3 weeks post surgery. Successful transgene expression at the local site was confirmed 4 weeks later after killing. Biomechanical pullout test indicated a significant restoration of implant stability after the cell-based OPG gene therapy. Histology revealed that inflammatory pseudo-membranes existed ubiquitously at bone-implant interface in control groups, whereas only observed sporadically in OPG gene-modified groups. Tartrate-resistant acid phosphatase+osteoclasts and tumor necrosis factor α, interleukin-1ß, CD68+ expressing cells were significantly reduced in periprosthetic tissues of OPG gene-modified mice. No transgene dissemination or tumorigenesis was detected in remote organs and tissues. Data suggest that cell-based ex vivo OPG gene therapy was comparable in efficacy with in vivo local gene transfer technique to deliver functional therapeutic OPG activities, effectively halted the debris-induced osteolysis and regained the implant stability in this model.

Liver involvement in epidemic haemorrhagic fever: in situ hybridization, immunohistochemical and pathological studies.

In the present study, liver biopsy specimens from 37 patients with epidemic haemorrhagic fever (EHF) were investigated by using light and electron microscopy, immunohistochemistry for the EHF virus envelope protein G2 and in situ hybridization (ISH) for EHF viral RNA. Immunostaining and in situ hybridization were both positive, and a few of the aetiological agents, the EHF virus (EHFV) particles, were found individually within the dilated Golgi cisternae and vesicles, and the dilated rough endoplasmic reticulum of the hepatocyte. The pathological alterations of the liver cells infected with EHFV were: hepatocellular degeneration, cytoplasmic vacuolation, and spotty necrosis; moreover, zonal necrosis was present and adjacent to the narrowed and occluded sinusoids. In degenerated and spotty necrotic cells, the positive signals from of ISH for EHFV RNA and those from immunostaining for the virus protein were both stronger than those in the zonal liver cell necrosis area. The results showed that hepatocellular degeneration and spotty necrosis might be directly related to the virus and its duplication inside the liver cells, while the zonal necrosis was ischaemic and thought to be caused by microcirculation dysfunction.

Ultrastructural observation on macrophage-lymphocyte interactions in semen from Tripterygium wilfordii Hook.f. takers.

Sixty-nine specimens from Tripterygium Wilfordii Hook.f. (TWH) users were investigated by electron microscopy. No macrophages were demonstrated in the 21 specimens collected prior to the administration of TWH. However, it was found in 23 out of the 48 semen specimens obtained following the TWH administration. The macrophages were functionally active as shown by the presence of a large number of cytoplasmic processes and pseudopodia on the surface, and primary and secondary lysosomes in the cytoplasm. The macrophages phagocytized sperm debris and degenerated or dead spermatids with formation of specific phagosomes. Around those macrophages, lymphocytes were commonly noted. The cytoplasmic processes of the two cell types could come into contact or even fuse with each other, leading to tight junction-like structure; in some of the contacts, the plasma membranes were found dissolved so as to form direct cytoplasmic linkage.