Objective
To investigate the effects of human insulin-like growth factor 1 (hIGF-1) gene transfected by recombinant adenovirus vector (Ad-hIGF-1) on the apoptosis of rabbit nucleus pulposus cells induced by tumor necrosis factor α (TNF-α).
Methods
The intervertebral disc nucleus pulposus were harvested from 8 healthy adult domestic rabbits (male or female, weighing 2.0-2.5 kg). The nucleus pulposus cells were isolated with collagenase II digestion and the passage 2 cells were cultured to logarithm growing period, and then they were divided into 3 groups according to culture condition: DMEM/F12 medium containing 10% PBS, DMEM/F12 medium containing 10% PBS and 100 ng/mL TNF-α, and DMEM/ F12 medium containing 10% PBS, 100 ng/ mL TNF-α, and Ad-hIGF-1 (multiplicity of infection of 50) were used in control group, TNF-α group, and Ad-hIGF-1 group, respectively. The results of transfection by adenovirus vector carrying hIGF-1 gene were observed by fluorescent microscopy; the expression of hIGF-1 protein was detected by Western blot, hIGF-1 mRNA expression by RT-PCR, and the cell apoptosis rate by TUNEL and flow cytometry.
Results
Green fluorescence was observed by fluorescent microscopy in Ad-hIGF-1 group, indicating that successful cell transfection. The expressions of hIGF-1 protein and mRNA were detected in Ad-hIGF-1 group by Western blot and RT-PCR, while the control group and TNF-α group had no expression. The cell apoptosis rates of TNF-α group, Ad-hIGF-1 group, and control group were 34.24% ± 4.60%, 6.59% ± 1.03%, and 0.40% ± 0.15%, respectively. The early apoptosis rates of TNF-α group, Ad-hIGF-1 group, and control group were 22.16% ± 2.69%, 5.03% ± 0.96%, and 0.49% ± 0.05%, respectively; the late cell apoptosis rates were 13.96% ± 4.86%, 10.68% ± 3.42%, and 0.29% ± 0.06%, respectively. Compared with TNF-α group, the cell apoptosis rates of Ad-hIGF-1 group and control group were significantly reduced (P lt; 0.05); the cell apoptosis rate of Ad-hIGF-1 group was significantly higher than that of control group (P lt; 0.05).
Conclusion
Ad-hIGF-1 could inhibit the apoptosis of nucleus pulposus cells induced by TNF-α.
Objective To explorer the survival time of autogeneic BMSCs labeled by superparamagnetic iron oxide (SPIO) in rabbit intervertebral discs and the rule of migration so as to prove bases of gene therapy preventing intervertebral disc degeneration. Methods Twelve rabbits were used in this experiment, aged 8-10 weeks, weighing 1.5-2.0 kg and neglecting their gender. BMSCs were separated from rabbits bone marrow by density gradient centrifugation and cultivated, and the 3rd generation of BMSCs were harvested and labeled with SPIO, which was mixed with poly-l-lysine. The label ing efficiency was evaluated by Prussian blue staining and transmission electron microscope. Trypanblau stain and MTT were performed to calculate the cell’ s activity. Rabbits were randomly divided into experimental group (n=8) and control group (n=4), the labeled BMSCs and non-labeled BMSCs (5 × 105/mL) were injected into their own intervertebral discs (L1,2, L2,3, L3,4 and L4,5), respectively. At 2, 4, 6 and 8 weeks, the discs were treated with Perl’s fluid to observe cell survival and distribution. Results The label ing efficiency of BMSCs with SPIO was 95.65% ± 1.06%, the cell activity was 98.28% ± 0.85%. There was no statistically significant difference in cell prol iferation within 7 days between non-labeled and labeled cells (P gt; 0.05). After 8 weeks of operation, the injected cells was al ive. ConclusionLabeled BMSCs with SPIO is feasible in vitro and in vivo, and the cells can survive more than 8 weeks in rabbit discs.
Objective
To summarize the research progress of microRNA (miRNA) and its non-viral vector in intervertebral disc degeneration (IDD) and to investigate the potential of non-viral vector delivery of miRNA in clinical application.
Methods
The related literature about the role of miRNA in IDD and its non-viral delivery system was reviewed and analyzed.
Results
MiRNA can regulate the related gene expression level and further participate in the pathophysiologic process in degenerated intervertebral disc, miRNA delivered by various non-viral vectors has obtained an ideal effect in some diseases.
Conclusion
MiRNA plays a great role in the cellular and molecular mechanisms of IDD, as a safe and effective strategy for gene therapy, non-viral vector provides new possibilities for IDD treated with miRNA.
Objective To introduce the latest advances of research on repair of the degenerative intervertebral disc with gene transduction.Methods The recentlypublished articles about the treatment of degenerative disc with gene transduction were reviewed, especially the articles published during the recent 5 years about the application of this therapy to regulating the synthesisand degradation of the extracellular matrix of the degenerative intervertebral disc.Results The shape and function of the normal intervertebral disc were reported to be closely related to the synthesis and degradation of the extracellular matrix of the intervertebral disc. The extracellular matrix of the intervertebral disc was a target for the gene transduction to repair the degenerative intervertebral disc. There was a great development of the treatment with gene transduction, especially in vector choice, target gene transduction, and transgene regulation and safety. Conclusion The advances of the research have indicated that repair of the degenerative intervertebral disc with gene transduction is a keyto curing the disease of the degenerative intervertebral disc.
Objective To study the adenovirus-mediated human bone morphogenetic protein-2 gene （Ad-hBMP-2）transferred to the intervertebral disc cells of the New Zealand rabbit in vitro. Methods The cells of New Zealand white rabbitswere isolated from their lumbar discs. The cells were grown in the monolayer and treated with an adenovirus encoding the LacZ gene (Ad-LacZ) and Ad-hBMP-2 (50,100, 150 MOI,multiplicity of infection) in the Dulbecco’s Modified Eagle Medium and the Ham’s F-12 Medium in vitro. Three days after the Ad-hBMP-2 treatment,the expression of hBMP-2 in the cells that had been infected by different dosesof MOI was determined by immunofluorescence and the Western blot analysis, and the expression was determined in the cells with the Ad-LacZ treatment in a dose of 150 MOI. Six days after the Ad-hBMP-2 treatment, mRNA was extracted for the reverse transcription polymerase chain reaction (RT-PCR) and the difference was detected between the control group and the culture group that was treated withAd-hBMP-2 in doses of 50, 100 and 150 MOI so that the expressions of aggrecan and collagen ⅡmRNA could be observed. Results The expression of hBMP-2 in the cells was gradually increased after the transfection in an increasing dose, which was observed by immunofluorescence and the Western blot analysis. At 6 days the aggrecan and collagen type Ⅱ mRNA expressions were up-regulated by Ad-hBMP-2 after the transfection at an increasing viral concentration in the dosedependent manner. Conclusion The results show that Ad-hBMP-2 can transfect the rabbit intervertebral disc cells in vitro with a high efficiency rate and the expression of hBMP-2 after theinfection is dose-dependent in the manner. AdhBMP-2 after transfection can up-regulate the expression of aggrecan and collagen Ⅱ mRNA at an increasing viral concentration.
Objective To investigate the effect of local injection of curcumin-loaded mesoporous silica nanoparticles (Cur@MSN) on the repair and treatment of degenerative intervertebral disc tissue in rats, and provide a new strategy for the treatment of intervertebral disc degeneration. Methods Mesoporous silica nanoparticles (MSN) and Cur@MSN were prepared according to the method reported in the literature. Rat nucleus pulposus cells were co-cultured with curcumin and Cur@MSN, respectively, and the growth status and activity of cells in normal environment and inflammatory environment (adding lipopolysaccharide) were observed respectively. Twelve 8-week-old SD rats were randomly divided into 4 groups, including normal group, degeneration group, curcumin group, and Cur@MSN group, with 3 rats in each group. Acupuncture degeneration model was established in coccygeal intervertebral discs (Co7-8, Co8-9) of rats, and corresponding intervention were injected. Imaging, gross pathology, and histological examination were performed after 4 weeks, respectively, to observe the tissue structure and pathological changes of intervertebral discs. Results Under scanning electron microscope, Cur@MSN was spherical in shape, with groove-like pores on its surface. Particle size analysis showed that the particle size of MSN was concentrated in 120-160 nm, and that of Cur@MSN was distributed in 130-170 nm. Zeta potential analysis showed that the average Zeta potential of MSN, curcumin, and Cur@MSN was ?12.5, ?22.5 and ?13.5 mV, respectively. The entrapment efficiency of Cur@MSN was 20.4%, and the drug loading rate was 0.2%. Curcumin released by Cur@MSN in 12 h accounted for about 60% of the total drug dose, and curcumin released in 28 h accounted for about 70%. In cell experiment, there was no significant difference in cell proliferation absorbance among the groups in normal environment (P>0.05), but the cell proliferation absorbance in the Cur@MSN group on the 3rd and 5th day in inflammatory environment was significantly higher than that in the control group and the curcumin group (P<0.01). The percentage of disc height index and the Pfirrmann grade of the Cur@MSN group were better than those of the degeneration group and the curcumin group (P<0.01). The histological score of the Cur@MSN group was lower than that of the degeneration group and the curcumin group (P<0.01). Conclusions Cur@MSN can delay the degeneration process of rat coccygeal intervertebral disc, and has certain repair and treatment effects on its degenerated intervertebral disc. Among them, curcumin can delay the degeneration of intervertebral disc by inhibiting inflammation, and the loading of MSN is helpful for curcumin to exert its biological effects.
The material properties and volume proportion of the fibers as well as the cross-sectional area proportion of nucleus pulposus vary greatly in different studies. The effect of these factors on the mechanical behavior of intervertebral discs (IVDs) are uncertain. The IVDs finite element models with different parameters were created to investigate the pressure, height, rotation, stress, and strain of the IVDs under loads: pure compression, rotation after compression or axial moment after compression. The results showed that the material properties of fibers had great impact on the mechanical behavior of IVDs, especially on the rotation angle. When the fiber volume ratio was small, its changes had a significant impact on the rotation angle of the IVDs. The area proportions of nucleus pulposus had relatively little effect on the mechanical behavior of IVDs. The IVDs rotation should be observed when validating the model. By adjusting the elastic modulus or volume ratio of fibers within a reasonable range, a model that could simulate the mechanical behavior of normal IVDs could be obtained. It was reasonable to make the area proportion of nucleus pulposus within 25%–50% for the IVDs finite element model. This study provides guidance and reference for finite element modeling of the IVDs and the investigation of the IVDs degeneration mechanism.
ObjectiveTo comprehensively analyze the relationship between microRNAs and intervertebral disc degeneration at home and abroad.
MethodsThe literature about the relationship between microRNAs and intervertebral disc degeneration was reviewed and analyzed.
ResultsMicroRNA can lead to intervertebral disc degeneration by regulating the gene expression, thus influencing the cell's apoptosis and proliferation, increasing of the production of inflammatory mediator and protease, which play important roles in intervertebral disc degeneration.
ConclusionMicroRNA is a research focus in the field of intervertebral disc degeneration. Further research of the relationship between microRNAs and intervertebral disc degeneration will help to identify the pathogenesis of intervertebral disc degeneration and furnish the new ideal for the diagnosis and treatment of intervertebral disc degeneration.
Objective To introduce the research of nucleus pulposus cells for treating intervertebral disc degeneration. Methods The original articles in recent years about nucleus pulposus cells for treating intervertebral disc degeneration were extensively reviewed, and retrospective and comprehensive analysis was performed. Results Nucleus pulposus cells are not only simply a remnant of embryonic notochordal cells, but have also an important influence on the well-being of the whole disc. The biological treatment strategies aim to regenerate the disc by either trying to improve the micro-enviroment within the disc or to increase the popoulation of the nucleus pulposus, which includes transplanting mesenchymal stem cellsto differentiate into nucleus-l ike cells in the degenerated intervertebral disc. Conclusion Nucleus pulposus cells or ucleus pulposus l ike cells based cell transplantation methods prove to be a promising and real istic approach for the intervertebral disc regeneration.
Objective To review the research progress of the seed cells, scaffolds, growth factors, and the prospects for clinical application of the intervertebral disc regeneration. Methods The recent literature concerning the regeneration strategies and tissue engineering for treatment of degenerative intervertebral disc disease was extensively reviewed and summarized. Results Seed cells based on mesenchymal stem cells (MSCs) and multiple-designed biomimetic scaffolds are the hot topic in the field of intervertebral disc regeneration. It needs to be further investigated how to effectively combine the interactions of seed cells, scaffolds, and growth factors and to play their regulation function. Conclusion The biological regeneration of intervertebral disc would have a very broad prospects for clinical application in future.
