| BackgroundLower back pain is is one of the most common orthopaedic diseases with trouble human today, its reason is intervertebral disc degeneration, strain of lumbar muscles, etc. About 4/5 of the patients go to hospital because of lower back pain as the main symptoms, the main reason of which is intervertebral disc degeneration. Now scholars and clinicians mainly research the treatment of intervertebral disc degeneration, but the basic research on the mechanism of intervertebral disc degeneration is rarely researched, relatively.Sox9 gene occupies the important position in the Sox gene family, it mainly regulates the growth and development of the people and vertebrate, as well as playing a decisive role in the cartilage repairment and gender determination. In 1994, Foster, etc. found a human Sox9 gene in the chromosome 17q24 using the technology of fluorescence in situ hybridization and so on. Wagner, etc successfully separated and cloned the human Sox9 gene by amplificating rapidly cDNA terminal binding to human embryonic brain cDNA.With the development of molecular biology related technology and the deepening of the research on mechanism of intervertebral disc degeneration, scholars found that the Sox9 gene participates in the whole changing process of upgrowth maturity and degeneration of intervertebral disc, and from more and more the relevant tests evidence. Now, it is very clear that the Sox9 gene promotes the formation of type II collagen, proteoglycan and cell proliferation, and scholars also have a lot of understanding to relevant regulatory mechanismGruber etc. collected 37 cases(aged from 15 to 76) human intervertebral disc annulus to conduct the Sox9 gene immunohistochemical staining, founding that Sox9 gene stains homogeneously in young people's annulus fibrosus cells and nucleus pulposus cells and with the increasement of age and intervertebral disc degeneration, Sox9 gene in the annulus fibrosus cells dyeing gradually reduces or even disappears.It is verified by scholars that, pro-inflammatory cytokine highly expressed in related diseases such as arthritis can significantly inhibit biological function of the Sox9 gene, thus weakened the regeneration and repairment of abnormal cartilage, and further research is remained that the biological function of leading to difficultly repair itself is also suppressed in the process of intervertebral disc degeneration and whether there exists the Sox9 gene point mutation and regulation of defects and why the Sox9 gene expression reduces in the process of intervertebral disc degeneration.So building safe, reliable and effective Sox9 gene recombinant adenovirus vectors of relevant research is quite necessary, and previous scholars use more AdEasy system for the construction of adenovirus vector, which exists defects in the preservation and producing efficiency. AdMax system has advantage of easy operation, higher efficiency of virus recombination, lower rate of mutation, higher efficiency of productivity and so on. It can provide stable and reliable adenovirus vector for further study of the role and related mechanism in the Intervertebral disc,s degeneration using adenovirus vectors related to the Sox9 gene by AdEasy system.Using SOX9 adenovirus vector infection of intervertebral disc nucleus pulposus cells, and observing the behavior of the cells, to acquaintance the biological effect of Sox9 gene on intervertebral disc. ObjectiveUsing AdMax system to build adenovirus vector related to Sox9 gene, and carry on the appraisal, provide reliable adenovirus vector for relative research of Sox9 gene. Infecting human intervertebral disc nucleus pulposus cells, to acquaintance the biological effect of Sox9 gene on intervertebral disc. Method1. The establishment method of adenovirus vector. First,using PCR to amplificate Sox9 gene, then restructed it into linear pAV- MCMV- GFP- 3 flag after screening, transfered it into DH5 a cells,and we could get the transformant. Indentifing the transformant by PCR technology. After that, ransfected the transformant into HEK293 cells by AdMax adenovirus packaging system, In which the Sox9 gene adenovirus vectors can be packaged, matured and amplified.2. the detection method of Adenovirus vector:Using Western Blot method to test the Sox9 gene recombinant adenovirus, applyed established adenovirus vector virus DNA for PCR identification and gene sequencing, and measured the titer of the virus.3. Preparation of degenerative intervertebral disc nucleus pulposus cells, through infecting intervertebral disc nucleus pulposus cells, to realize the relationship of the gene and intervertebral disc nucleus pulposus cells. ResultWith fragment of 1565 bp gained, which size is consistent with the Sox9 gene, It means that Sox9 gene had been restructed in the adenovirus vectors successfully. With PCR detection methods, we got virus DNA of 1565 bp fragments, it declared that Sox9 gene fragments had been recombinant into adenovirus. In Western Blot testing, there were positive bands from 72 to 95 ×103,which had the same size with Sox9-GFP-Flag protein(56 KDa +28 KDa +2 KDa = 86 KDa), and the, so the expression of Sox9 protein has been detected successfully. It has improved the concent of type II collagen ConclusionWe have Successfully identified correctly. It has improved the concent of type II collagen... |
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