Correction of post-traumatic thoracolumbar kyphosis using pedicle subtraction osteotomy.

The aim of this study was to retrospectively analyze and evaluate the effect of treatment employing pedicle subtraction osteotomy for chronic, posttraumatic thoracolumbar kyphosis. This study included 19 patients, 11 males and 8 females, with chronic, posttraumatic thoracolumbar kyphosis. Pre-operative kyphosis ranged from 31° to 63°. The history of trauma ranged from 8 to 63 months. All patients were treated with pedicle subtraction osteotomy. A mean 40.2° improvement in sagittal alignment was achieved with a mean correction rate of 85.8 %. Perioperative complications were encountered in two patients, one with cerebrospinal fluid leakage followed by encephalitic infection and one with a wound infection. Both were treated conservatively with antibiotics and local wound care. There were no other severe complications. The average follow-up period was 15 months (range 6-41 months). At the last follow-up, clinical symptoms and neurological function were significantly improved. Of 14 patients presenting with intractable back pain, VAS scores improved from a preoperative mean of 6.7 (range 5.0-8.0) to an average 2.0 (range 0-3.0) at final follow-up. No significant loss of correction was observed (loss of 1.7°), and solid fusion was achieved in all 19 patients. A single-stage posterior pedicle subtraction osteotomy is a safe and effective procedure for correction of posttraumatic thoracolumbar kyphosis. Using this technique, it is possible to safely obtain no greater than 55° of correction at a single level.

Monitoring Stub1-Mediated Pexophagy.

Mammalian cells can turn over peroxisomes through Stub1-mediated pexophagy. The pathway potentially permits cellular control of the quantity and quality of peroxisomes. During this process, heat shock protein 70 and the ubiquitin E3 ligase, Stub1, translocate onto peroxisomes to be turned over to initiate pexophagy. The Stub1 ligase activity allows the accumulation of ubiquitin and other autophagy-related modules on targeted peroxisomes. Elevating reactive oxygen species (ROS) levels within the peroxisomal lumen can activate Stub1-mediated pexophagy. One can, therefore, use dye-assisted ROS generation to trigger and monitor this pathway. This article outlines the procedures for using two classes of dyes, fluorescent proteins and synthetic fluorophores, to initiate pexophagy within mammalian cell cultures. These dye-assisted ROS generation-based protocols can not only be used to target all the peroxisomes within a cell population globally but can also permit the manipulation of individual peroxisomes within single cells. We also describe how Stub1-mediated pexophagy can be followed using live-cell microscopy.

[Construction and biological assessment of tissue engineering intervertebral disc].

OBJECTIVE: To investigate the value of bone marrow-mesenchymal stem cells (BM-MSCs) transformed by nucleus pulposus (NPs) for construction of tissue engineering disc. METHODS: BM-MSCs and fetal NPs were cultured in vitro, planted on polylactic acid-polyglycolic acid copolymer (PLGA), and observed with inverted microscope and scanning electronic microscope. PLGA scaffolds with adherent BM-MSCs and NPs, as well as BM-MSCs and NPs suspension were implanted into intervertebral discs of New Zealand white rabbits, respectively. Intervertebral signal intensity was evaluated by Thompson grading 12 weeks later. Proteoglycan and type IIcollagen were determined by spectrophotometric method and immunohistochemistry, respectively. RESULTS: Spindle or multi-angular BM-MSCs turned into fibro-like phenotype coculture of BM-MSCs and NPs, which grew well with normal morphology when they attached on PLGA scaffolds. There was statistical difference in intervertebral signal intensity, and the expression of proteoglycan and type IIcollagen between PLGA scaffolds group and control group (P < 0.05), the content of proteoglycan was (3.93 ± 0.31) mg/100 mg in the PLGA scaffolds group whereas (3.52 ± 0.26) mg/100 mg in the control group. CONCLUSIONS: BM-MSCs can be induced into NPs by cocultivation, and PLGA scaffolds can provide good growing conditions, and maintain high mechanical properties and spacial structure which meet the requirement of tissue engineering disc to prevent degeneration.

Clinical Comparison of Selective versus Nonselective Decompression for Symptomatic Tandem Stenosis of the Cervical and Thoracic Spine: A Retrospective Cohort Study.

OBJECTIVE: The aim of the present paper was to reveal the clinical differences between selective and nonselective decompression for symptomatic tandem stenosis of the cervical and thoracic spine (TSCTS). METHODS: A total of 34 patients were eligible and included in the study. Among them, 8 patients underwent selective cervical decompression (CD), 15 patients underwent selective thoracic decompression (TD), and 11 patients underwent combined CD and TD (CTD) surgery. Age, sex, operative time, intraoperative blood loss, postoperative hospital stay, inpatient expenditure, preoperative upper Japanese Orthopaedic Association (JOA) rate, canal occupation rate, high-intensity T2-weighted image (T2WI) of the spinal cord, and preoperative and postoperative JOA scores were compared among the three groups. RESULTS: The CD group had shorter operative time (138.8 ± 36.1 vs 229.7 ± 95.8 vs 328.6 ± 94.8, min, P < 0.001), less intraoperative blood loss (141.3 ± 116.7 vs 496.7 ± 361.8 vs 654.6 ± 320.5, mL, P = 0.004), and shorter postoperative hospital stay (4.6 ± 1.6 vs 9.0 ± 3.5 vs 10.3 ± 6.6, days, P = 0.008), as well as lower preoperative upper JOA rate (34.1 ± 5.6 vs 53.9 ± 8.4 vs 48.2 ± 15.2, %, P = 0.001) than the TD and CTD groups. The CTD group had higher inpatient expenditure than the CD and TD groups (87,850 ± 18,379 vs 55,100 ± 12,890 vs 55,772 ± 15,715, CNY, P < 0.001). The cervical canal occupation rates were similar among different groups (P > 0.05); however, the TD group showed a higher thoracic canal occupation rate than the CD group (58.3 ± 14.7 vs 43.3 ± 12.3, %, P = 0.035). All positive levels in high-intensity T2WI of the spinal cord were decompressed. The preoperative JOA scores as well as the postoperative JOA scores at 6 months and at last follow-up were comparable among the three groups (P > 0.05). Similarly, the JOA recovery rate showed no significant difference among the groups (P > 0.05). CONCLUSION: Selective CD or TD alone demonstrated similar clinical effectiveness to nonselective and combined CTD for TSCTS. Individualized surgical decision should be made after meticulous assessments of clinical and radiological manifestations, general patient condition, and socioeconomic factors.

The correlation study on homocysteine, blood lipids and blood glucose levels in patients with cerebral infarction.

PURPOSE: To explore the correlation of changes in homocysteine (HCY) l, blood lipids and blood glucose levels in patients with cerebral infarction. METHODS: 120 patients with cerebral infarction admitted to our hospital from February 2018 to February 2020 were selected as the experimental group, and 120 healthy volunteers in the same period were selected as the control group. The blood pressure and the homocysteine, blood lipids, blood glucose levels were compared; the experimental group was subdivided into single cerebral infarction group, combined diabetes group and combined hypertension group, and their blood lipid levels were compared with the control group; Spearman method was used to analyze the relationship between HCY, blood lipid, blood sugar levels and cerebral infarction. RESULTS: ① The diastolic and systolic blood pressure levels of the experimental group were higher, whereas the control group were lower (P<0.05). ② The levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C) and TC/[high density lipoprotein cholesterol (HDL-C)] of the experimental group were higher, but the level of HDL-C was lower than that of the control group (all P<0.05). ③ The fasting blood glucose (FBG) and glycosylated hemoglobin (GHb) levels of the experimental group were higher than those of the control group (all P<0.05). ④ The HCY level in the experimental group was higher than that in the control group (P<0.05). ⑤ The levels of TC, TG, LDL-C and TC/HDL-C in single cerebral infarction group, combined diabetes group and combined hypertension group were higher, and HDL-C level was lower than that in control group (all P<0.05). ⑥ Spearman analysis revealed that HCY was positively correlated with TC, TG, LDL-C and FBG (all P<0.05). CONCLUSION: The level of HCY is positively correlated with the levels of TC, TG, LDL-C and FBG in patients with cerebral infarction.

Profiling carbohydrate-receptor interaction with recombinant innate immunity receptor-Fc fusion proteins.

The recognition of bacteria, viruses, fungi, and other microbes is controlled by host immune cells, which are equipped with many innate immunity receptors, such as Toll-like receptors, C-type lectin receptors, and immunoglobulin-like receptors. Our studies indicate that the immune modulating properties of many herbal drugs, for instance, the medicinal fungus Reishi (Ganoderma lucidum) and Cordyceps sinensis, could be attributed to their polysaccharide components. These polysaccharides specifically interact with and activate surface receptors involved in innate immunity. However, due to the complexity of polysaccharides and their various sources from medicinal fungi, quantitative analysis of medicinal polysaccharide extracts with regard to their functions represents a major challenge. To profile carbohydrate-immune receptor interactions, the extracellular domains of 17 receptors were cloned as Fc-fusion proteins, such that their interactions with immobilized polysaccharides could be probed in an enzyme-linked immunosorbent assay. The results show that several innate immune receptors, including Dectin-1, DC-SIGN, Langerin, Kupffer cell receptor, macrophage mannose receptor, TLR2, and TLR4, interact with the polysaccharide extracts from G. lucidum (GLPS). This analysis revealed distinct polysaccharide profiles from different sources of medicinal fungi, and the innate immune receptor-based enzyme-linked immunosorbent assay described here can serve as a high-throughput profiling method for the characterization and quality control of medicinal polysaccharides. It also provides a means to dissect the molecular mechanism of medicinal polysaccharide-induced immunomodulation events.

[Diagnosis and treatment of disc herniation at cervical vertebra 7-thoracic vertebra 1 (C(7)-T(1))].

OBJECTIVE: To study the outcome of laminoforaminotomy with posterolateral discectomy for patients with lateral disc herniation at C(7)-T(1). METHODS: From August 2000 to August 2008, 12 patients with lateral disc herniation at C(7)-T(1) underwent posterolateral discectomy were analyzed retrospectively. Neurologic function were evaluated with the Motor Scoring System. Preoperative motor were compared with postoperative one. The unique clinical manifestation, imageology features and intraoperative findings were analyzed. RESULTS: All these twelve patients were lateral type. All the patients showed hand intrinsic muscles atrophy and hand weakness. Nine patients had no paraesthesia. The average follow-up period was 26 months. Postoperative scores were significantly higher than preoperative ones. CONCLUSIONS: Disc herniation at C(7)-T(1) is predominantly lateral type and present C(8) nerve motor deficit (hand intrinsic muscles atrophy and hand weakness) and only minority has paraesthesia in C(8) nerve dermatome. Posterolateral cervical discectomy technique is safe and effective for patients with lateral disc herniation at C(7)-T(1).

Hsc70/Stub1 promotes the removal of individual oxidatively stressed peroxisomes.

Peroxisomes perform beta-oxidation of branched and very-long chain fatty acids, which leads to the formation of reactive oxygen species (ROS) within the peroxisomal lumen. Peroxisomes are therefore prone to ROS-mediated damages. Here, using light to specifically and acutely induce ROS formation within the peroxisomal lumen, we find that cells individually remove ROS-stressed peroxisomes through ubiquitin-dependent pexophagy. Heat shock protein 70 s mediates the translocation of the ubiquitin E3 ligase Stub1 (STIP1 Homology and U-Box Containing Protein 1) onto oxidatively-stressed peroxisomes to promote their selective ubiquitination and autophagic degradation. Artificially targeting Stub1 to healthy peroxisomes is sufficient to trigger pexophagy, suggesting a key role Stub1 plays in regulating peroxisome quality. We further determine that Stub1 mutants found in Ataxia patients are defective in pexophagy induction. Dysfunctional peroxisomal quality control may therefore contribute to the development of Ataxia.

Expression of HSPA8 in Nucleus Pulposus of Lumbar Intervertebral Disc and Its Effect on Degree of Degeneration.

INTRODUCTION: This study aimed to investigate the expression of a 70-kDa heat shock protein [heat shock 70-kDa protein 8 (HSPA8)/heat shock protein 70 (Hsc70)] in human degenerative lumbar intervertebral discs and its relationship with the degree of degeneration of human intervertebral discs. METHODS: A total of 72 cases of lumbar intervertebral disc nucleus pulposus tissues were collected. Among these, 18 cases of nucleus pulposus tissue were assigned to the control group, while 54 cases of nucleus pulposus tissues were assigned to the experimental group. According to the preoperative MRI, cases in the experimental group were further divided into three groups: protrusion group (n = 18), extrusion group (n = 18), and sequestration group (n = 18). Western blot was performed to determine the relative expression of HSPA8 in the nucleus pulposus in each group. Hematoxylin and eosin staining was performed to determine the number of nucleus pulposus cells, morphological differences, and cell densities of the degenerated intervertebral discs and normal intervertebral discs. Immunohistochemistry was performed to determine the expression of HSPA8 in nucleus pulposus tissues in each group. RESULTS: Hematoxylin and eosin staining results: There were significant differences in cell morphology and number between the control group and the experimental group. Furthermore, there were significant differences in cell density (F = 936.80, P < 0.01). Immunohistochemistry results: HSPA8 was expressed in lumbar intervertebral disc nucleus pulposus tissues, and its expression of gradually decreased with the severity of the disease, and the differences were significant (F = 2110.43, P < 0.01). Western blot results: The expression of HSPA8 in human degenerative nucleus pulposus tissues gradually decreased, and the differences were significant (F = 1841.72, P < 0.01). CONCLUSION: HSPA8 is stably expressed in human intervertebral disc nucleus pulposus tissues, and its expression is associated with the degree of intervertebral disc degeneration.

Dual Therapeutic Effects of Interferon-α Gene Therapy in a Rat Hepatocellular Carcinoma Model With Liver Cirrhosis.

Human hepatocellular carcinoma (HCC) often arises from a background of liver cirrhosis. Therefore, in order to develop therapeutic strategies for HCC, an animal model bearing multifocal liver tumors accompanied by liver cirrhosis is a preferred experimental setting. In this study, we developed a rapid and reproducible method for generating such a model in rats by weekly administration of diethylnitrosamine (DEN) at doses based on body weight (BW). By adjusting the duration of administration of DEN, the animals could be induced to develop HCC alone, or HCC and liver cirrhosis simultaneously. The latter model was used for evaluating the therapeutic effects of adenoviral delivery of interferon-α (IFN-α). Our results demonstrated that targeting of IFN-α expression to the liver significantly reduced liver tumor volume and ameliorated liver cirrhosis. Mechanistic studies revealed that IFN-α gene therapy induced immunomodulatory, antiproliferative, and proapoptotic activities that were effective in the control of tumor growth, and reduced the expressions of transforming growth factor-ß (TGF-ß) and tissue inhibitor of metalloproteinase-1 (TIMP-1), leading to amelioration of liver cirrhosis. These results suggest that IFN-α gene therapy is a promising strategy to treat HCC patients who have concomitant liver cirrhosis.

Dual therapeutic effects of interferon-alpha gene therapy in a rat hepatocellular carcinoma model with liver cirrhosis.

Human hepatocellular carcinoma (HCC) often arises from a background of liver cirrhosis. Therefore, in order to develop therapeutic strategies for HCC, an animal model bearing multifocal liver tumors accompanied by liver cirrhosis is a preferred experimental setting. In this study, we developed a rapid and reproducible method for generating such a model in rats by weekly administration of diethylnitrosamine (DEN) at doses based on body weight (BW). By adjusting the duration of administration of DEN, the animals could be induced to develop HCC alone, or HCC and liver cirrhosis simultaneously. The latter model was used for evaluating the therapeutic effects of adenoviral delivery of interferon-alpha (IFN-alpha). Our results demonstrated that targeting of IFN-alpha expression to the liver significantly reduced liver tumor volume and ameliorated liver cirrhosis. Mechanistic studies revealed that IFN-alpha gene therapy induced immunomodulatory, antiproliferative, and proapoptotic activities that were effective in the control of tumor growth, and reduced the expressions of transforming growth factor-beta (TGF-beta) and tissue inhibitor of metalloproteinase-1 (TIMP-1), leading to amelioration of liver cirrhosis. These results suggest that IFN-alpha gene therapy is a promising strategy to treat HCC patients who have concomitant liver cirrhosis.

Immune therapy with cultured microglia grafting into the injured spinal cord promoting the recovery of rat's hind limb motor function.

OBJECTIVE: To study the effect of activated microglia grafting on rats' hind limb motor function recovery after spinal cord injury. METHODS: Microglia were separated from primary culture and subcultured for 3 generations. Lipopolysaccharide was added to the culture medium with the terminal concentration of 10 microl/L for microglia activation 3 days before transplantation. Totally 80 adult Wistar rats were divided into transplantation group and control group, with 40 rats in each group. Spinal cord injury model of rats was set by hitting onto the spinal cord using a modified Allen impactor. With a 5 microl micro-syringe, the activated microglia suspension was injected into the injured area 7 days after the first operation. Basso, Beattie and Bresnahan (BBB) scoring for hind limb motor function was taken on the 1st, 7th, 14th, 21st, and 28th day after microglia transplantation, and 8 rats were sacrificed at each time point mentioned above, respectively. Frozen sections of the spinal cord were made for haematoxylin-eosin (HE) and Naoumenko-Feigin stainings. SPSS 11.0 software was used for statistical analysis. RESULTS: BBB scores for hind limb motor function on the 14th, 21st, and 28th day were significantly higher compared with the control group. Most liquefaction necrosis areas disappeared and only a few multicystic cavities surrounded by aggregated microglia remained in the transplantation group. Naoumenko-Feigin staining for microglia showed that the transplantation group had significantly more positive cells (P < 0.05). CONCLUSIONS: Grafting of activated microglia into the injured spinal cord can significantly promote the hind limb motor function recovery in rats with spinal cord injury and reduce the size of liquefaction necrosis area. The extent of lower limb motor function improvement has a positive correlation with the number of aggregated microglia.

[Outcomes of posterior C(1-2) fusion for atlantoaxial instability or dislocation using pedicle screws of atlas and axis].

OBJECTIVE: To retrospectively analyze and evaluate the results of treatment for atlantoaxial instability or dislocation employing pedicle screws of atlas and axis. METHODS: Thirty-one patients (23 male and 8 female) with atlantoaxial instability or dislocation were stabilized using pedicle screws of atlas and axis between May 2005 to January 2008. The patients ranged in age from 17 to 67 years (mean 43.5 years). Patients consisted of chronic odontoid fracture in 17, Os odontoideum in 8, fresh odontoid fracture in 4, transverse ligament rupture in 1, rheumatoid arthritis in 1. Clinical features included neck pain in 31; restricted neck movement in 28, varying degrees of spastic quadriparesis in 19. All patients underwent posterior C(1) to C(2) pedicle screw fixation. Operative time, intraoperative blood loss, complications were recorded, neurological and radiographic studies were carried. RESULTS: Mean follow-up time was 13 months. Operative time averaged 2.5 h. Mean intraoperative blood loss was 300 ml. A patient had postoperative wound infection and was treated conservatively with antibiotics and local wound care. A patient developed pulmonary artery embolism and got well with anticoagulation. Satisfactory stability was achieved in all cases with no vascular and C(2) neuralgia. Average JOA score in 19 cases increased at final follow-up (P < 0.01). Solid fusion was achieved in 29 cases, fusion rate was 93.6%. CONCLUSIONS: Stabilization of atlantoaxial complex via pedicle screws of atlas and axis has advantages of intraoperative restoration, easier placement of screw, solid fixation. It is a safe and effective treatment modality for posterior C(1-2) fusion.

Identification of pathways significantly associated with spondyloarthropathy/ankylosing spondylitis using the sub­pathway method.

The aim of the present study was to extract potential sub­pathway biomarkers for spondyloarthropathy (SpA)/ankylosing spondylitis (AS) using a sub­pathway strategy. SpA/AS­relevant data, reference pathways and long non­coding (lnc)RNA­micro (mi)RNA­mRNA interactions were downloaded. The seed pathways based on Kyoto Encyclopedia of Genes and Genomes pathways and the mRNAs in the co­expressed lncRNA­mRNA interactions were extracted. Sub­pathways regulated by lncRNA were selected after establishing condition­specific lncRNA competitively regulated pathways (LCRP) network. Significant sub­pathways were further identified using the attract method. These significant sub­pathways were evaluated in the other independent published AS microarray data (E­GEOD­25101) using in silico validation. In addition, to uncover SpA/AS­relevant lncRNAs, the degree analysis for all nodes in the LCRP network was conducted. A total of 35 lncRNAs, 131 mRNAs and 145 co­expressed interactions were identified. When entering these 131 mRNAs into the reference pathways, 82 seed pathways were extracted, which were transformed into undirected graphs, and the 35 lncRNAs were mapped to the pathway graphs to further establish the condition­specific LCRP network. Based on degree analysis, four hub lncRNAs were selected, including C14orf169, LINC00242, LINC00116 and LINC00482. It was identified that 35 lncRNAs competitively regulating sub­pathways were involved in 56 complete pathways. Among these, the top three sub­pathways were path: 04010_1, which was a subregion of the mitogen­activated protein kinase (MAPK) signaling pathway; path: 04062­1, an important subregion in the chemokine signaling pathway; and path: 04066_2, was a part of HIF­1 signaling pathway. Furthermore, it was validated consistently in the separate microarray data set E­GEOD­25101. Cancer­associated pathways and hub node C14orf169 were identified in validation. Sub­pathways, including the MAPK signaling pathway and chemokine signaling pathway, and hub lncRNA (C14orf169) may serve important roles in SpA/AS.

Diffusion-weighted 7.0T Magnetic Resonance Imaging in Assessment of Intervertebral Disc Degeneration in Rats.

BACKGROUND: Intervertebral disc degeneration (IDD) is a major cause of disc protrusion, likely to be associated with decrease of water content. This research aimed to evaluate IDD by diffusion-weighted imaging (DWI) with a 7.0 Tesla (T) magnetic resonance imaging (MRI) machine. METHODS: A total of 24 healthy Sprague-Dawley rats were randomly selected and divided into four groups (A, B, C, and D), each consisting of 3 male and 3 female rats (28, 42, 56, and 70 days old, respectively). All the rats were imaged with a 7.0T MRI, producing T2WI, T1WI, and functional DWI sequences. Data were collected and apparent diffusion coefficient (ADC) charts were constructed. Nucleus pulposus and annulus fibrosus regions were identified, several regions of interest were chosen, and their ADC values were obtained. After imaging, rats were sacrificed and their intervertebral discs (L1-L6) were dissected, yielding a total of 144 discs. Protein was extracted for the purpose of Western blotting. Comparison among multiple samples used one-way analysis of variance and least significant difference methods. RESULTS: 7.0T MRI revealed evident decrease in signal intensity within intervertebral discs of Sprague-Dawley rats with age. Intervertebral disc ADC values significantly decreased from Group A (0.00154 ± 0.00008) to Group D (0.00107 ± 0.00007; P < 0.01); nucleus pulposus ADC values significantly decreased from Group A (0.00164 ± 0.00005) to Group D (0.00140 ± 0.00007; P < 0.01) and annulus fibrosus ADC values significantly decreased from Group A (0.00129 ± 0.00014) to Group D (0.00082 ± 0.00012; P < 0.01). Meanwhile, it also revealed evident decrease from high spinal level to low spinal level: nucleus pulposus ADC values in Group A significantly decreased from L1/L2 (0.00163 ± 0.00006) to L6/S1 (0.00139 ± 0.00004; P < 0.01). While annulus fibrosus ADC values did not differ significantly between levels in Group A (P > 0.05). Western blotting showed that aggrecan content of intervertebral discs decreased from Group A (1.88 ± 0.16) to Group D (0.17 ± 0.04) with age (P < 0.01); Type II collagen content of intervertebral discs decreased from Group A (2.22 ± 0.04) to Group D (0.20 ± 0.01) with age (P < 0.01). No significant differences in aggrecan and Type II collagen content of L1-L6 intervertebral discs in Group A were noted (P > 0.05). Mean ADC values of different intervertebral regions were positively correlated with aggrecan and Type II collagen content (aggrecan: r = 0.631, P < 0.01; Type II collagen: r = 0.680, P < 0.01). CONCLUSION: 7.0T MRI-DWI could be applied to effectively diagnose and research early IDD in tiny variations.

Staining of Lipid Droplets with Monodansylpentane.

Monodansylpentane (MDH) is a fluorophore that displays selective labeling of lipid droplets (LDs). The dye preferentially segregates into the neutral lipid cores of LDs and emits blue fluorescence, compatible with the simultaneous use of green and red fluorescent reporters in multi-color live-cell imaging. MDH can be used for visualizing LDs not only in cell cultures, but also in fixed tissues such as the fat body and ovaries from Drosophila. MDH is therefore a versatile marker for LDs in fluorescence microscopy.

Lentivirus-mediated TGF-ß3, CTGF and TIMP1 gene transduction as a gene therapy for intervertebral disc degeneration in an in vivo rabbit model.

The present study examined the effects of transforming growth factor (TGF)-ß3, connective tissue growth factor (CTGF) and tissue inhibitor of metalloproteinase 1 (TIMP1) gene transduction, using a lentiviral vector, on rabbit intervertebral disc degeneration in vivo, with the intention of investigating their potential use in gene therapy. A model of lumbar intervertebral disc degeneration was created by needle puncture into the annulus fibrosus of 15 New Zealand white rabbits. Empty lentivirus or recombinant lentiviral plasmid lenti-TGFß3-P2A-CTGF-T2A-TIMP1 was injected into degenerative lumbar intervertebral discs (representing the control and experimental groups, respectively), whilst untreated degenerative lumbar intervertebral discs served as the puncture group. After 16 and 20 weeks, magnetic resonance imaging (MRI) was conducted and the changes in intensity on micrographs of degenerative intervertebral discs were measured. The mRNA levels of aggrecan and type II collagen in nucleus pulposus tissue were determined by reverse transcription-polymerase chain reaction, and protein expression levels of type II collagen and aggrecan were determined by western blot analysis. MRI results indicated that intervertebral disc degeneration was ameliorated in the experimental group when compared with the control and the puncture group. Furthermore, the expression levels of type II collagen and aggrecan in the puncture and control groups were significantly lower than in the experimental group (P<0.05). In conclusion, lenti-TGFß3-P2A-CTGF-T2A-TIMP1 co-transduction can promote synthesis of aggrecan and type II collagen in degenerative intervertebral discs, thereby delaying intervertebral disc degeneration. These results indicate the potential of gene therapy in treatment of intervertebral disc degeneration.

Combined expression of CTGF and tissue inhibitor of metalloprotease-1 promotes synthesis of proteoglycan and collagen type II in rhesus monkey lumbar intervertebral disc cells in vitro.

BACKGROUND: Low back pain has emerged as a widespread disease often caused by intervertebral disc degeneration. This study aimed to establish an in vitro cell culture model of rhesus monkey lumbar intervertebral discs and to investigate the effect of combined connective tissue growth factor (CTGF) and tissue inhibitor of metalloprotease-1 (TIMP-1) expression mediated by adeno-associated virus (AAV) on collagen type II and proteoglycan levels. The purpose of these investigations was to explore potential methods for relieving the degeneration of lumbar intervertebral disc cells. METHODS: Rhesus monkey lumbar intervertebral disc nucleus pulposus cells (NPCs) were isolated by enzyme digestion, cultured, and transduced with rAAV2-CTGF-IRES-TIMP-1, rAAV2-CTGF, or rAAV2-TIMP-1 at a multiplicity of infection (MOI) of 10(6). The expression of collagen type II and proteoglycan was measured using RT-PCR and Western blotting. The synthetic rate of proteoglycan was measured using (35)S incorporation. RESULTS: Rhesus monkey lumbar intervertebral disc NPCs were transduced with rAAV2-CTGF-IRES-TIMP-1, rAAV2-CTGF, and rAAV2-TIMP-1 and the transduced genes were expressed and detected. Compared to the control, CTGF promoted the synthesis of collagen type II and proteoglycan. TIMP-1 showed an enhancing effect on the expression of proteoglycan but no effect on collagen type II. Expression of both genes in rhesus monkey lumbar intervertebral disc NPCs significantly enhances the synthesis of proteoglycan and collagen type II. CONCLUSIONS: Single gene transduction of CTGF or TIMP-1 can enhanced synthesis of proteoglycan. CTGF expression can also enhance collagen type II protein synthesis. Combined transduction of both CTGF and TIMP1 can significantly promote the expression of proteoglycan and collagen type II to levels greater than transduction of a single gene alone. Our study provides a good basis for multi-gene therapy to treat lumbar intervertebral disc degeneration.