The Biological Effect Of Adenoassociated Virus-mediated OP-1 And HVEGF Encoding Genes Transfer On The Goat Degenerative Intervertebral Discs

BackgroundUpper and lower end of the plate, the intermediate nucleus and the surrounding fibrous ring constitute a basic unit of the intervertebral disc. When different forms of energy load are assumed, the stress will be transmitted and distributed uniformly in all directions. Creep, stress relaxation and hysteresis phenomena are special features of disc with viscoelastic properties.The fibrous ring of the intervertebral disc is arranged in a concentric circle as a whole, and is composed of 25 radiating hub like fibers, and the cross-linked structure is composed of a network structure. This structure is suitable for counter flexion or rotation under pressure anti tension is strong; type Ⅱ collagen fibers were random arrangement, space filled with proteoglycan and water, against compression force stronger. The normal human intervertebral disc annulus collagen is made up of 60% collagen type Ⅱ collagen and 40%, and other types of collagen content are less, which makes the disc elastic perfect. In the gravity load of the spine, the core of the nucleus pulposus is dispersed in all directions, and the special structure of the fiber ring can protect the integrity of the disc structure.Water, protein, polysaccharide, collagen and so on are important components of the intervertebral disc, which constitute the framework of the extracellular matrix. The intervertebral disc cells common notochord cells, cartilage cells, fibroblasts and chondrocytes With the growth of the age, number of notochord cells decreased, fiber cells and cartilage cells gradually increased, the elderly in the nucleus pulposus cells of the notochord disappeared. These cells are filled in the extracellular matrix. PG (proteoglycan) has the capacity of absorbing water, maintaining a certain volume, and maintaining the volume of the cell matrix. PG is a negative ion carrier, and its quantity and volume density changes can control the change of osmotic pressure between the disc and the surrounding tissue, and thus affect the physiological metabolism of the extracellular matrix. Along with the growth of the age, synthetic metabolism of cells such as notochord cells reduces gradually, extracellular matrix metabolism in balance to maintain and catabolism increases, and cell number gradually reduced.Back pain is one of the common diseases affecting people’s quality of life, and lumbar disc degeneration is the main cause of disease. In order to study the pathogenesis and treatment of the disease, we must find a reliable, reproducible, easy to operate, economic, and human understanding of the high degree of lumbar intervertebral disc degeneration animal model.Surgical treatment and conservative treatment are two major measures to treat lumbar disc herniation. Surgery is a traumatic operation, the integrity and stability of the spine is affected, and conservative treatment is usually short-term effect is better and the long-term effect is not good. Biological treatment is to reverse the disc degeneration, restore its biological function, so as to achieve the purpose of treatment.By mediating gene transfer into intervertebral disc cells, the effect of disc matrix metabolism can be regulated. Promoting the synthesis and inhibition of the disc cells, and inhibiting the decomposition of the intervertebral disc cells are the conditions for the selection of gene therapy. Gene is divided into two kinds of structural genes and regulatory genes,and the gene therapy of intervertebral disc is generally used to mediate gene. It was later found that the hTGF beta 1 gene mediated by adenovirus can lead to a long term effective expression of the rabbit intervertebral disc, and the effective expression time was over 3 months. Adenovirus was treated by TGF and Nishida 1 in 1999, and it was found that the gene can be expressed in high transfection efficiency and high level of gene expression.The related virus is smaller than that of the adenovirus, and can be controlled by the surrounding genes, it can infect many kinds of cells in the stage of infection and the non dividing stage. In recent years, the related viral vectors are attracting more and more attention of scholars. Adenovirus mediated Lac2Z gene can be successfully transfected into rabbit intervertebral disc cells, although its transfection efficiency is lower than that of adenovirus vector, but its vivo and vitro test were successful, which was confirmed by Lattermann et al.ObjectiveWe establish a breeding animal model of intervertebral disc degeneration slow trough the method of fiber ring 16G needle puncture. We observe the expression of adenovirus vector for the expression of the safety and effective expression of the intervertebral disc in the intervertebral disc of goat degeneration. We observe the effect of the expression of the-1 gene in vivo on the expression of the degenerated intervertebral disc after the transfection of the recombinant cells into the sheep. We observe the effect of endothelial growth factor hVEGF on the expression of human vascular endothelial growth factor gene in vivo and to promote the repair of degenerated intervertebral disc. We also observe the effect of OP-1 gene combined with hVEGF gene on the expression of gene in vivo, and the better effect on the degeneration of intervertebral disc.Methods1. Animal grouping:specific pathogen free goat were divided into 8 groups. Group H contained 4 animals as a spare anima. Group A contained 8 goats which were treated with AAV-OP-1, and group B contained 8 goats which were treated with AAV-VEGF. Group C contained 8 goats which were treated with AAV-OP-1 and AAV-VEGF, and group D contained 8 goats which were treated with phosphate buffer saline. Group E contained 4 goats which were treated with AAV-EGFP, and group F was a Simple acupuncture group which contained 8 animals. Group G contained the remaining 8 animals which were treated by operation but no injury of intervertebral disc. Intervertebral discs of group G and the group H were intact.2. Production of fiber ring acupuncture animal model. Group G and group H were excluded, and the remaining 76 animals were treated into animal model of degeneration. Under general anesthesia, we take the side position, extraperitoneal approach to expose intervertebral disc. The 3 intervertebral discs were randomly selected, with a 16G needle puncture fibre ring, and the puncture point was located in the central part of the central part of the fiber ring, and the upper end plate of the inferior vertebral body was controlled by the central part of the fiber ring. Marking the transverse process of the corresponding intervertebral disc with black.3. Gene transfection of intervertebral disc(groupA,B,C,D and E). The time was fourth weeks (28 days) after modeling. We used the same operation approach. We injected 50ul AAV-OP-1 (1×1012vg/L) with micro syringe of 27G needle in the group A, and We injected 50ul AAV-VEGF (1×1012vg/L) in the group B. We injected 50ul AAV-OP-1 and AAV-VEGF (1×1012vg/L) in the group C, and we injected 50ul PBS in the group D. We injected 50ul AAV-EGFP (1×1012vg/L) in the group E.4 goats were executed in the group A,B,C and D respectively 6 weeks(42 days),8weeks(56days), 12 weeks(84days),and 16weeks(102days) after modeling. While we executed 2 goats in the group F and G, then we gained intervertebral disc tissue. We executed 4 goats of group E and gained intervertebral disc tissue.4. Imaging observation. We shoot DR plain film of lumbar vertebra at the time of 0 week, 4weeks (28d)、6 weeks (42d)、8 weeks(56d)、12 weeks(84d)、16 weeks(102d), and we use PACS system to analyze the data.5. Histological observation.. We used the methods of HE staining, Safranine 0 staining, Masson staining and immunohistochemical staining. The development and changes of intervertebral disc degeneration was observed by qualitative and quantitative analysis.6. Biochemical detection. We used immunofluorescence and Western blotting to detect the detect expression of gene qualitatively and quantitatively.7. Statistical analysis. Results are expressed as X±SD. To determine the significance of difference among various groups, the statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey’s test using SPSS17.0. Comparison between group differences has statistical significant (p<0.05) in all tests and comparison between group differences has remarkable statistical significant (p<0.01).Results1.Image analysis results, The intervertebral disc height of the animals in the OP-1 group was higher than that in the blank control group, and the percent DHI and blank control group were significantly different (P<0.05). The optimal height of the disc height was in the double gene combined group, and the percent DHI and blank control group were significantly different (P<0.05). The disc height of the animals in the VEGF group and the F group were decreased, and it was similar with control group, The data analysis of DHI and control group was not statistically significant (P> 0.05).2. immunohistochemical staining of collagen type Ⅱ collagen:The type Ⅱ collagen content in the nucleus pulposus of OP-1 group was higher than control group and group at the time of 6 weeks,8 weeks,12 weeks,16 weeks, especially at the time of 16 weeks. The average optical density of collagen type Ⅱ collagen immunohistochemical staining was significantly different from that of the control group (P<0.05). There was no significant difference between VEGF group and D and F group. The collagen content of VEGF group and F group was not significantly different, and the average optical density of group and blank control group were statistically significant (P> 0.05). The type Ⅱ collagen content in the nucleus pulposus of the double gene combined group was higher than that in the blank control group and group F. There were significant differences in the average optical density of collagen type Ⅱ and the blank control group (P> 0.01). OP-1 and VEGF gene can significantly improve the synthesis and metabolism of type Ⅱ collagen in animal.3.immunohistochemical staining of collagen type I collagen:The content of type I collagen in OP-1 group was higher than that in control group and F group at the timed of 6 weeks,8weeks,12 weeks and 16 weeks, especially at the time of 16 weeks. The average optical density of collagen type I collagen was significantly different from the control group (P<0.05). OP-1 can promote collagen synthesis and metabolism in animals. There was no significant difference in groups VEGF, D and F in all the time.T he collagen type I collagen in the group VEGF and group F were all decreased, and the data were similar to the blank control group. The average optical density values of type I collagen and the blank control group were statistically significant (P> 0.05). The type Ⅱ collagen content in the nucleus pulposus of the double gene combined group was higher than that in the blank control group and group F. There were significant differences in the average optical density of collagen type I and the blank control group (P> 0.01). OP-1 and VEGF gene can significantly improve the synthesis and metabolism of type I collagen in animal.4. Determination of protein synthesis rate by 35S. The content of protein in the nucleus pulposus of OP-1 group in the nucleus pulposus of OP-1 group was higher than control group and group at the time of 6 weeks,8 weeks,12 weeks,16 weeks, especially at the time of 16 weeks.there were significant difference beteen group OP-1 and control group. There was no significant difference in groups VEGF, D and F in all the time. The content of protein in the group VEGF and group F were all decreased, and the data were similar to the blank control group. The average optical density values of the content of protein and the blank control group were statistically significant (P> 0.05). The protein polysaccharide content in the nucleus pulposus of the double gene combined group was higher than that in the blank control group and group F. There were significant differences in the average optical density of protein and the blank control group (P> 0.01). OP-1 and VEGF gene can significantly improve the synthesis and metabolism of protein in animal body.5. Detection of type Ⅱ collagen by Western Blot. The type Ⅱ collagen content in the nucleus pulposus of OP-1 group was higher than control group and group at the time of 6 weeks,8 weeks,12 weeks,16 weeks, especially at the time of 16 weeks. The density of collagen type Ⅱ collagen was significantly different from that of the control group (P<0.05). OP-1 can promote collagen synthesis and metabolism in vivo. The average optical density of collagen type Ⅱ collagen immunohistochemical staining was significantly different from that of the control group (P<0.05). There was no significant difference between VEGF group and D and F group. The collagen content of VEGF group and F group was not significantly different, and the average optical density of group and blank control group were statistically significant (P> 0.05). The type Ⅱ collagen content in the nucleus pulposus of the double gene combined group was higher than that in the blank control group and group F. There were significant differences in the average optical density of collagen type Ⅱ and the blank control group (P> 0.01). OP-1 and VEGF gene can significantly improve the synthesis and metabolism of type Ⅱ collagen in animal.6. Immunofluorescence:In the group E, the expression of EGFP in the intervertebral disc of the model was still valid after 16 weeks.Conclusions1.12 weeks after transfection of AAV-OP-1 and AAV-hVEGF, the expression of the gene was sustainable.2. AAV-OP-1 transfection of degenerated intervertebral disc can delay the degeneration of the intervertebral disc.3. AAV-OP-1 and AAV-hVEGF have synergistic effect, and the double gene can delay the degeneration of intervertebral disc more effectively.