| The incidence of degenerative disc disease is fairly high, and it becomes a fact which needs no longer to be quarrelled about. By the newest study of physiology, biochemistry and pathology, it is proved that the maintenance of water of intervertebral disc primarily depends on proteoglycan content in the matrix, and Type II collagen plays a key role in enduring pressure. Some evidence indicates that the combination of extrinsic, intrinsic and genetic factors all play a role to cause the disc degeneration, with a progressive decrease in proteoglycan content leading to dehydration of the nucleus pulposus, and Type I collagen begins to appear in the matrix, the ratio between Type I and Type II increases. All these cause the swelling pressure, disc height and the disc load bearing ability decrease. The disc progressively herniate and cause radiculopathy and myelopathy.The limited available technology for the management of intervertebral disc disorders generally is highly invasive, usually requiring surgery in which all or portions of the affected intervertebral disc or discs are removed. Examples of this type of surgery include nucleotomy and discectomy, procedures that compromise the structural integrity of the disc. Noninvasive treatments are aimed a relieving the symptoms associated with disc degeneration rather than at treating the disc itself.Is there a way to stop or reverse the disc degeneration, and the way need not to compromise the structural integrity of the disc, whereas the patients' agony can be decreased to the minimum degree?Transforming growth factor β1 (TGF-β1) can stimulate nucleus pulposus tissue in culture synthesize proteoglycan in vitro, and it can also modulate the content of Type II. On the basis of these results, we developed some researches.The TGF- β1 gene fragment were obtained from plasmid pcDNA3. 1(+)-TGF β1 by PCR amplification. The recombinant adenovirus vector containing TGF- β1 gene was (Ad/CMV-hTGF β1constructed with the way of homologous recombination. Rabbit nucleus pulposus cells were cultured in monolayer, the morphology of the cells were observed, and the enchanced green fluorescent protein (EGFP) gene was transduced intocells, and the expression of the exogenous marker gene was detected, The result: The cells cultured in vitro are similar with the nucleus pulposus tissue in vivo, and the cells became feeble after subculture. With the cells culturing extended, the viability of the cells declined. After the EGFP gene was transduced into cells, the green light could be observed the next day, and the cells could give out light three month until they dead. With the success of rabbit nucleus pulosus cells' culture, the model of cells culture had been established. The successful expression of EGFP gene demonstrated the exogenous gene can be expressed in nucleus pulposus cells.On the basis of the above result, the human degenerative nucleus pulposus cells were cultured in monolayer and treated with Ad/CMV-TGFβ1.Immunohistochemical staining for human transforming growth factor β1 and Type II collagen were performed to determine TGF- β1 production and the content of Type II collagen. Cells treated with Ad/CMV-hTGF β1 exhibited extensive and intense positive immunostaining for transforming growth factor and Type II collagen, while the control groups exhibited weak positive immunostaining for TGF-β1 and Type II collagen . It can be demonstrated that human degenerative nucleus pulposus cells can not only express exogenous TGF- β1 gene, but also enhance the synthesis of Type II collagen.In vivo study, Ad/CMV-TGF β1 ,saline and Ad/CMV-LacZ were injected into lumbar discs of the rabbits. Immunohistochemical staining for TGF- β1 was performed at 10 days and 20 days . Bioassays were performed to determine TGF- β1 and hyaluronic acid at 10 days, 20 days and 45 days. The nucleus pulposus tissue injected with Ad/CMV-TGF β1 ( including 1 Odays and 20 days ) exhibited extensive and intensive positive immunostaining for TGF- β1. The nucleus pulposus tissue injected...
|
PDF Full Text Request