ABSTRACT
BACKGROUND: Recent studies have demonstrated that energy metabolism-related genes play important roles in intervertebral disc (IVD) cell adaptation to negative environmental factors, such as hypoxia and insufficiency of nutrient. But the effects of these genes in pressure-induced intervertebral disc degeneration remain uncertain.OBJECTTVE: To investigate continuous pressure-induced expression of energy metabolism genes: hypoxia-inducible factor 1α(HIF-lo), glucose transporter-1 (GLUT-1) and vascular endothelial growth factor (VEGF) in rabbit annulus fibrosus (AF).DESIGN: A randomized controlled experiment.SETTING: The Central Laboratory and the Laboratory of Department of Orthopaedics, Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: The experiment was carried out in the Central Laboratory and the Laboratory of Department of Orthopaedics, Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology from May to October 2005. Twenty-five Japanese white rabbits(about 4 months old, weighting 2.5-3.0 kg, provided by the Experimental Animal Center of Tongji Medical College, Huazhong University of Science and Technology) were selected to establish the animal model.METHODS: A controllable pressure-induced rabbit intervertebral disc degeneration model was adopted to impose various pressured on rabbit IVDs in vivo. The survived animals whose IVDs were compressed successfully were divided randomly into 4 groups. The IVDs were treated with no pressure as control (control group), with 15 kg axial load for 24 hours (24hours group), 72 hours (72 hours group), and 24 hours with 48 hours free for self-reparation (reparation group). Reverse transcriptase-polymerase chain reaction was used to detect the expression of HIF-1α and GLUT-1. Western Blot and immunohistochemical test were carried out for the content and distribution of VEGF.MAIN OUTCOME MEASURES: Expression of HIF-1α and GLUT-1, content and distribution of VEGF.RESULTS: Twenty of 25 rabbits entered result analysis. Two rabbits were missed because of vertebral fracture, while death of 3 rabbits within 1-3 days postoperatively caused another loss. ①HIF-1α. A very low expression was detected in the control group, while the expression in the 24 hours group was raised over 20 times than that in the control group (t=25.022, P＜0.01). The expression in the 72 hours group and reparation group decreased as compared with the 24hours group. ②GLUT-1 expressed weakly in the control group. The expression in the 24 hours group rose a lot as compared with the control group (t=18.314, P＜0.01) and the expression in the 72 hours group rose slightly than that in the 24 hours group (t =2.819, P＜0.05). The expression in the reparation group is close to that in the 24 hours group. ③Littie VEGF content was detected in the control group, while the content rose significantly in the other 3 groups. Immunohistochemical staining showed more VEGF positive stained cells in outer AF than in inner AF.CONCLUSION: Continuous pressure can strongly up-regulate the expression of energy metabolism gene: HIF-1α,GLUT-1 and VEGF in vivo. These genes play important roles in AF adaptation and reparation in over load-caused damage.
ABSTRACT
The protective effect of niacinamide on interleukin-1beta (IL-1beta)-induced annulus fibrosus (AF) type II collagen degeneration in vitro and the mechanism were investigated. Chiba's intervertebral disc (IVD) culture models in rabbits were established and 48 IVDs from 12 adult Japanese white rabbits were randomly divided into 4 groups: normal control group, niacinamide-treated group, type II collagen degneration group (IL-1beta) and treatment group (niacinamide+IL-1beta). After culture for one week, AFs were collected for inducible nitric oxide synthase (iNOS), cysteine containing aspartate specific protease-3 (Caspase-3) and type II collagen immunohistochemical examination, and type II collagen reverse transcription polymerase chain reaction (RT-PCR). The results showed that rate of iNOS positive staining AF cells in the 4 groups was 17.6%, 10.9%, 73.9% and 19.3% respectively. The positive rate in treatment group was significantly lower than in the type II collagen degeneration group (P<0.01). Rate of Caspase-3 positive staining AF cells in the 4 groups was 3.4%, 4.2%, 17.6% and 10.3% respectively. The positive rate in treatment group was lower than in the type II collagen degeneration group (P<0.01). Type II collagen staining demonstrated that lamellar structure and continuity of collagen in treatment group was better reversed than in the degeneration group. RT-PCR revealed that the expression of type II collagen in treatment group was significantly stronger than that in type II collagen degeneration group (P<0.01). It was concluded that niacinamide could effectively inhibit IL-1beta stimulated increase of iNOS and Caspase-3 in AF, and alleviate IL-1beta-caused destruction and synthesis inhibition of type II collagen. Niacinamide is of potential for clinical treatment of IVD degeneration.
ABSTRACT
The protective effect of niacinamide on interleukin-1β (IL-1β)-induced annulus fibrosus (AF) type Ⅱ collagen degeneration in vitro and the mechanism were investigated. Chiba's intervertebrai disc (IVD) culture models in rabbits were established and 48 IVDs from 12 adult Japanese white rabbits were randomly divided into 4 groups: normal control group, niacinamide-treated group, type Ⅱ collagen degneration group (IL-1β) and treatment group (niacinamide+IL-1β). After culture for one week, AFs were collected for inducible nitric oxide synthase (iNOS), cysteine containing aspartate specific protease-3 (Caspase-3) and type Ⅱ collagen immunohistochemical examination, and type Ⅱ collagen reverse transcription polymerase chain reaction (RT-PCR). The results showed that rate of iNOS positive staining AF cells in the 4 groups was 17.6%, 10.9%, 73.9% and 19.3% respectively. The positive rate in treatment group was significantly lower than in the type Ⅱ collagen degeneration group (P＜0.01). Rate of Caspase-3 positive staining AF cells in the 4 groups was 3.4%, 4.2%, 17.6% and 10.3% respectively. The positive rate in treatment group was lower than in the type Ⅱ collagen degeneration group (P＜0.01). Type Ⅱ collagen staining demonstrated that lamellar structure and continuity of collagen in treatment group was better reversed than in the degeneration group. RT-PCR revealed that the expression of type Ⅱ collagen in treatment group was significantly stronger than that in type Ⅱ collagen degeneration group (P＜0.01). It was concluded that niacinamide could effectively inhibit IL-1β stimulated increase of iNOS and Caspase-3 in AF, and alleviate IL-1β-caused destruction and synthesis inhibition of type Ⅱ collagen. Niacinamide is of potential for clinical treatment of IVD degeneration.
ABSTRACT
The regulatory effects of niacinamide (Nia) on intervertebral disc (IVD) aggrecan in vitro was investigated. Chiba's 10 ng/mL interleukin-1 (IL-1)-induced rabbit IVD degeneration model in vitro was established. 0.5, 0.25 and 0.05 mg/mL Nia was added to normal and degenerated IVDs for intervention. On the first and second week after intervention, safranin O-fast green staining intensity and glycosaminoglycan (GS) content were measured. The expression of aggrecan core protein was detected by RT-PCR. The results showed: (1) After treatment with 0.5 mg/mL Nia for one week, the GS content in nucleus pulposus (NP) was increased by 44.8 % as compared with control group (P＜0.01); The GS content in IL-1 induction groups was increased with the increase of Nia concentrations: After treatment with 0.5 mg/mL for one week, the GS content in NP was increased by 68.3 % as compared with control group (P＜0.01). After two weeks, GS content in NP and fibrous rings was still higher than in control group at the same period (P＜0.01)and untreated group (P＜0.01). (2) Safranin O-fast green staining revealed that with the increase of Nia concentrations, staining density in NP and fibrous rings was increased and histological structure damage to IVDs by IL-1β was alleviated. (3) RT-PCR showed that the expression of core protein gene in IL-1β-induced degenerated IVDS was increased with the increase of Nia concentrations.It was concluded that under conditions in vitro, Nia could up-regulate the expression of aggrecan in IVDs and protect IVDs from IL-1β-induced degeneration at least partially, which offers a potential choice for IVD degeneration clinical therapy.
ABSTRACT
To study the effect on regulation of cell cycle of osteosarcoma cell line MG63 tranceduced with exogenous p16ink4a and hRb1 genes, pIRES-p16ink4a-hRb1, pIRES-p16ink4a and pIRES-hRb1 plasmids were constructed by gene recombination technology. The recombinant plasmid was transferred into osteosarcoma cell line MG63 by metafectene, and the resistant clones were selected by G418 selective medium. mRNA and protein expression of osteosarcoma cell line were assayed by RT-PCR and Western Blot respectively. Cell cycle and apoptosis were analyzed by subG1 flow cytometric. Cell proliferation was tested by MTT. In the genome of these transfected target cells, the expression of p16ink4a and hRb1 mRNA and protein were detected respectively in vitro. It was demonstrated with subG1 flow cytometric analysis and MTT method that p16ink4a and hRb1 genes cooperation more significantly inhibited cell growth and induced a more marked G1 arrest and apoptosis than p16ink4a/hRb1 alone (P＜0.01). Coexpression of exogenous p16ink4a with hRb1 broke the regulatory feedback loop of p16ink4a-cyclinD1/CDK-hRb1 and played a more significant role in inhibiting cell growth as well as inducing cell apoptosis than p16ink4a or hRb1 did alone in vitro.
ABSTRACT
To investigate therapeutic efficiency of Ad/CMV- hTGF-beta1 gene for rabbit intervertebral disc degeneration model. 60 Japanese white rabbits were selected to form the 1.5-L6 Anterior-Lateral-Anulus-Fibrosus-Incision-Induced model in order to simulate human intervertebral disc degeneration. 36 rabbits, whose corresponding intervertebral discs were injected with 20 microl (10 x 10(6) pfu) of Ad/CMV- hTGF-beta1 gene, constituted the therapy group, 12 were injected with 20 microl (10 x 10(6) pfu)of Ad/CMV-LacZ gene as comparison group, while 12 were only injected with equivalent capacity of saline for empty comparison group, 3 weeks after injection, examples were taken for investigation of HE staining, MRI, Western Blotting and immunohistochemical research TGF-beta1. Wide distribution of TGF-beta1 was detected by immunohistochemical research in the degenerated annulus fibrosus after injection. Western Blotting research showed significant increase of TGF-beta1 content in intervertebral discs treated with TGF-beta1 gene than comparison groups. MRI signal transformed from low to comparatively high and that intervertebral disc pathological degree improved. Ad/CMV- hTGF-beta1 gene transfection is a potential method to increase TGF-beta1 content and reverse intervertebral disc degeneration.
Subject(s)
Animals , Rabbits , Adenoviridae , Genetics , Gene Transfer Techniques , Genetic Therapy , Genetic Vectors , Intervertebral Disc , Pathology , Lumbar Vertebrae , Spinal Diseases , Drug Therapy , Transfection , Transforming Growth Factor beta , Genetics , Transforming Growth Factor beta1ABSTRACT
To investigate therapeutic efficiency of Ad/CMV- hTGF-beta1 gene for rabbit intervertebral disc degeneration model. 60 Japanese white rabbits were selected to form the 1.5-L6 Anterior-Lateral-Anulus-Fibrosus-Incision-Induced model in order to simulate human intervertebral disc degeneration. 36 rabbits, whose corresponding intervertebral discs were injected with 20 microl (10 x 10(6) pfu) of Ad/CMV- hTGF-beta1 gene, constituted the therapy group, 12 were injected with 20 microl (10 x 10(6) pfu)of Ad/CMV-LacZ gene as comparison group, while 12 were only injected with equivalent capacity of saline for empty comparison group, 3 weeks after injection, examples were taken for investigation of HE staining, MRI, Western Blotting and immunohistochemical research TGF-beta1. Wide distribution of TGF-beta1 was detected by immunohistochemical research in the degenerated annulus fibrosus after injection. Western Blotting research showed significant increase of TGF-beta1 content in intervertebral discs treated with TGF-beta1 gene than comparison groups. MRI signal transformed from low to comparatively high and that intervertebral disc pathological degree improved. Ad/CMV- hTGF-beta1 gene transfection is a potential method to increase TGF-beta1 content and reverse intervertebral disc degeneration.