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Transplantation of CTGF and TIMP1 Gene-modified Nucleus Pulposus Cells Reverses Rabbit Intervertebral Disc Degeneration

Author: LiuYong
Tutor: LiJianMin
School: Shandong University
Course: Surgery
Keywords: nucleus pulposus cells transplantation gene-modified disc degeneration
CLC: R681.53
Type: PhD thesis
Year: 2011
Downloads: 25
Quote: 0
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Abstract

[Objective] Disc degeneration is the main cause of low back pain. Given the fact that the disc herniation is casused by the disc dengeneration, finding an effective method to retard disc gegeneration is considered a meaningful attempt. Based on rabbit disc degeneration model which was built by using minimally invasive CT-guided puncture, adeno-associated virus (adeno-associated virus, AAV)-mediated connective tissue growth factor (connective tissue growth factor, CTGF) and matrix metalloproteinase tissue inhibitor 1 (tissue inhibitor of metalloproteinases 1, TIMP1) were selected to co-transfect rabbit intervertebral disc cells in vitro, then the gene-modified cells were transplanted into rabbit degenetative lumbar intervertebral discs. Disc height, disc signals, collagen typeⅡand proteoglycan were observed at different times. [Methods]63 New Zealand White rabbits were used for experiment, of which 60 rabbits were applied for minimally invasive CT-guided puncture in rabbit disc degeneration model:Select L2-3, L3-4, L4-5 disc level, under CT scan to puncture, LI-2, L5-6 intervertebral disc does not puncture as a control group. The left rear was the needle point, we measure puncture angle and depth. After disinfection and the CT location, the percutaneous puncture with 18G needle from right rear puncture lumbar intervertebral disc. When puncture needle coming close to the annul us then we used CT scan and adjust the angle of puncture. After penetrating into the nucleus cavity, we used CT scan again to determine the tip located in the central nucleus. After 2 weeks MRI scan was used to check the status of disc degeneration. Another three rabbits were used to disc cell culture. Mixed the rAAV2-CTGF-TIMPl 106 per cell and the amount of viral particles in serum-free cell culture medium HAMF12/DMEM, the mixture will be washed by adding PBS buffer to the cell nucleus of the second generation of transfected cells. Using the minimally invasive method, select the L2-3, L3-4, L4-5 disc,32G needle to penetrate the nucleus, the 22u 1 of cell suspension were injected slowly, then using the micro-injection in degeneration of the intervertebral disc as the cell transplantation group;using the degenerative intervertebral disc into phosphate buffered saline (PBS) as a degeneration control group.normal disc as a control group.Observing the changes of intervertebral height, measuring and comparing % DHI in the 6,10,14 weeks respectively. Using MRI to check the change of disc signal. Using HE staining, Safranine 0 staining and Masson staining to observe the changes of transgenic tissue before and after the gene-modified cells transplantation. Immunohistochemical staining was used to observe the collagen typeⅡexpression of nucleus pulposus cells. Western-blot was used to observe the CTGF and TIMP1 expression in the intervertebral disc.35S integration method was used to observe proteoglycan synthesis rate of the rabbit nucleus pulposus. RT-PCR was used to observe typeⅡcollagen and proteoglycan mRNA expression. Analysis of variance and multiple pairwise comparison of the mean undergo with Statistical software SPSS15.0. P<0.05 was considered statistically significant. [Results] CT guided percutaneous puncture was characterized by safety and less trauma, and so on. MRI confirmed that 2 weeks after the the puncture, disc degeneration began, which testify the method for the establishment of rabbit intervertebral disc degeneration model to be new and effective.Adeno-associated virus (AAV) can efficiently transfected into rabbit nucleus pulposus cells and realized rapid-mediated gene expression, with a safe, efficient and so on. X-ray examination showed % DHIs of blank control group were remarkably decreased compared with cell transplantation group respectly after 6,10,14 weeks (p<0.05), which showed that the disc height of cell transplantation group could be maintained. MRI confirmed that the longer the observation time, the more progressive disc degeneration in the degeneration control group,6 weeks,10 weeks,14 weeks after that, based on modified Thompson classification method, there was statistical difference between the transgenic cell transplantation group and the degeneration control group (P<0.05). HE staining showed that after transplantation of genetically modified cells, within the cell nucleus there were more divisions than the uniform. Safranine 0 staining showed that after transplantation proteoglycan was significantly increased. Masson staining showed after transplantation of genetically modified cells, intervertebral disc degeneration slowed down obviously. Immunohistochemical staining results showed after the rabbit nucleus pulposus cells of transgenic that type II collagen synthesis and secretion increased compared to the previous capacity, and there was significant statistical difference between the cell transplantation group and the degeneration control group (P<0.05). After transgenic cell transplantation, Western-Blot test of cytokine expression in the intervertebral disc showed after 6 weeks,10 weeks and 14 weeks:CTGF expression was positive, and TIMP1 expression was positive in 6 weeks and 10 weeks. In 14 weeks, TIMP1’s positive expression was significantly decreased, which suggested that the expression of CTGF in vivo disc was more durable.In 6weeks,10 weeks and 14 weeks after transgenic cells transplantation, RT-PCR detection of proteoglycan and type II collagen mRNA expression showed that there was tatistical differensce between cell transplantation group and degeneration control group (P<0.05), there was statistical difference between the normal control group and the degeneration control group (P<0.05), there was no statistical difference between cell transplantation group and normal control group (P> 0.05). In 6 weeks,10 weeks and 14 weeks after transgenic cells transplantation,35S integration assay was applied to detect the rate of proteoglycan synthesis. There was statistical difference between Cell transplantation group and degeneration control group (P<0.05), there was no statistical difference between cell transplantation group and blank control group (P> 0.05).[Conclusion] The minimally invasive percutaneous CT-guided puncture disc can successfully build a rabbit disc degeneration model. Adeno-associated virus-mediated CTGF and TIMP1 in the rabbit nucleus pulposus cells can be successfully expressed. CTGF gene and TIMP1 gene co-transferration in vitro and in vivo transplantation again, can contribute to the maintenance of disc height, the recovery of normal disc MRI signal. Immunohistochemistry, RT-PCR and integrated method 35S were found to transfer double-gene cell transplantation can promote the biosynthesis of proteoglycan and typeⅡcollagen in the intervertebral disc, suggesting that can delay or reverse the disc degeneration. This study provides a new way for gene therapy combined cell therapy to reverse disc degeneration. [Objective] Disc degeneration is the main cause of low back pain. Given the fact that the disc herniation is casused by the disc dengeneration,finding an effective method to retard disc gegeneration is considered a meaningful attempt. Based on rabbit disc degeneration model which was built by using minimally invasive CT-guided puncture, adeno-associated virus (adeno-associated virus, AAV)-mediated connective tissue growth factor (connective tissue growth factor, CTGF) and matrix metalloproteinase tissue inhibitor 1 (tissue inhibitor of metalloproteinases 1, TIMP1) were selected to co-transfect rabbit intervertebral disc cells in vitro, then the gene-modified cells were transplanted into rabbit degenetative lumbar intervertebral discs. Disc height, disc signals, collagen type II and proteoglycan were observed at different times.[Methods]63 New Zealand White rabbits were used for experiment, of which 60 rabbits were applied for minimally invasive CT-guided puncture in rabbit disc degeneration model:Select L2-3, L3-4, L4-5 disc level, under CT scan to puncture, L1-2, L5-6 intervertebral disc does not puncture as a control group. The left rear was the needle point, we measure puncture angle and depth. After disinfection and the CT location, the percutaneous puncture with 18G needle from right rear puncture lumbar intervertebral disc. When puncture needle coming close to the annulus then we used CT scan and adjust the angle of puncture. After penetrating into the nucleus cavity,we used CT scan again to determine the tip located in the central nucleus. After 2 weeks MRI scan was used to check the status of disc degeneration. Another three rabbits were used to disc cell culture.Mixed the rAAV2-CTGF-TIMP1 106 per cell and the amount of viral particles in serum-free cell culture medium HAMF12/DMEM, the mixture will be washed by adding PBS buffer to the cell nucleus of the second generation of transfected cells.Using the minimally invasive method, select the L2-3, L3-4, L4-5 disc,32G needle to penetrate the nucleus, the 22μl of cell suspension were injected slowly, then using the micro-injection in degeneration of the intervertebral disc as the cell transplantation group;using the degenerative intervertebral disc into phosphate buffered saline (PBS) as a degeneration control group.normal disc as a control group.Observing the changes of intervertebral height, measuring and comparing % DHI in the 6,10,14 weeks respectively. Using MRI to check the change of disc signal. Using HE staining, Safranine O staining and Masson staining to observe the changes of transgenic tissue before and after the gene-modified cells transplantation. Immunohistochemical staining was used to observe the collagen type II expression of nucleus pulposus cells. Western-blot was used to observe the CTGF and TIMP1 expression in the intervertebral disc.35S integration method was used to observe proteoglycan synthesis rate of the rabbit nucleus pulposus. RT-PCR was used to observe typeⅡcollagen and proteoglycan mRNA expression. Analysis of variance and multiple pairwise comparison of the mean undergo with Statistical software SPSS15.0. P<0.05 was considered statistically significant.[Results] CT guided percutaneous puncture was characterized by safety and less trauma, and so on. MRI confirmed that 2 weeks after the the puncture,disc degeneration began, which testify the method for the establishment of rabbit intervertebral disc degeneration model to be new and effective. Adeno-associated virus (AAV) can efficiently transfected into rabbit nucleus pulposus cells and realized rapid-mediated gene expression, with a safe, efficient and so on.X-ray examination showed % DHIs of blank control group were remarkably decreased compared with cell transplantation group respectly after 6,10,14 weeks(p<0.05), which showed that the disc height of cell transplantation group could be maintained.MRI confirmed that the longer the observation time, the more progressive disc degeneration in the degeneration control group,6 weeks,10 weeks,14 weeks after that, based on modified Thompson classification method, there was statistical difference between the transgenic cell transplantation group and the degeneration control group (P <0.05). HE staining showed that after transplantation of genetically modified cells, within the cell nucleus there were more divisions than the uniform. Safranine O staining showed that after transplantation proteoglycan was significantly increased. Masson staining showed after transplantation of genetically modified cells, intervertebral disc degeneration slowed down obviously.lmmunohistochemical staining results showed after the rabbit nucleus pulposus cells of transgenic that type II collagen synthesis and secretion increased compared to the previous capacity, and there was significant statistical difference between the cell transplantation group and the degeneration control group (P<0.05).After transgenic cell transplantation, Western-Blot test of cytokine expression in the intervertebral disc showed after 6 weeks,10 weeks and 14 weeks:CTGF expression was positive, and TIMP1 expression was positive in 6 weeks and 10 weeks. In 14 weeks,TIMP1’s positive expression was significantly decreased, which suggested that the expression of CTGF in vivo disc was more durable.In 6weeks,10 weeks and 14 weeks after transgenic cells transplantation, RT-PCR detection of proteoglycan and type II collagen mRNA expression showed that there was tatistical differensce between cell transplantation group and degeneration control group (P<0.05), there was statistical difference between the normal control group and the degeneration control group (P<0.05), there was no statistical difference between cell transplantation group and normal control group (P> 0.05). In 6 weeks,10 weeks and 14 weeks after transgenic cells transplantation,35S integration assay was applied to detect the rate of proteoglycan synthesis. There was statistical difference between Cell transplantation group and degeneration control group (P<0.05), there was no statistical difference between cell transplantation group and blank control group (P> 0.05).[Conclusion] The minimally invasive percutaneous CT-guided puncture disc can successfully build a rabbit disc degeneration model. Adeno-associated virus-mediated CTGF and TIMP1 in the rabbit nucleus pulposus cells can be successfully expressed. CTGF gene and TIMP1 gene co-transferration in vitro and in vivo transplantation again, can contribute to the maintenance of disc height, the recovery of normal disc MRI signal.Immunohistochemistry, RT-PCR and integrated method 35S were found to transfer double-gene cell transplantation can promote the biosynthesis of proteoglycan and type II collagen in the intervertebral disc, suggesting that can delay or reverse the disc degeneration. This study provides a new way for gene therapy combined cell therapy to reverse disc degeneration.

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CLC: > Medicine, health > Surgery > Orthopaedic Surgery ( movement system diseases,orthopedic surgery ) > Bone diseases > Spine and back disorders > Spinal joint disease
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