| ObjectiveTo establish a rabbit model of degenerative intervertebral disc induced by puncturing the annulus with needle and aspirating some nucleus pulposus from the intervertebral discs. And to evaluate the effect of bone mesenchymal stem cell transferred by lentivirus-sox9 constructure in treatment of rabbit degenerative intervertebral disc.Methods1. The animal model of degenerative intervertebral disc of lumbar spine was established by puncturing the annulus with needle and aspirating some nucleus pulposus from the intervertebral discs.New Zealand white rabbits without spinal deformity or degenerative intervertebral disc were adopted. The lumbar intervertebral discs of L3/4,L4/5,L5/6 were exposed and punctured to aspirate nucleus pulposus by 21Gsyrige needle. The postoperative changes of surgical discs were detected by X-ray and MRI. 12 weeks after operation, the discs were sliced to detect the condition of degeneration. The expression of typeⅡcollagen and proteoglycan was measured by immunohistochemistry and spectrophotometry respectively.2. The rabbit bone mesenchymal stem cells were isolated and cultured in vitroNew Zealand white rabbits, two months age and body weight of 1.5～2.5 kg, were adopted for this study. Bone mesenchymal stem cells were collected by aspirating from the iliac crest, isolated into single cell by percoll method, washed by PBS fluid two times, and then inoculated in 40 ml culture bottle with a density of 2×105/cm2. Subculture of cells were cultured with a proportion of 1:3. Cellular morphology and grows of state were detected by inverted microscope. The MSCs of third generation were made into single cell suspension and identified by immunofluorescence.3. Lentvirus-sox9 constructure transferred bone mesenchymal stem cellsMSCs of third generation were adopted and transferred by lentivirus-sox9 constructure when their fusion achieved up to 70%-80% with a density of 5×105/cm2. Then the mixture fluid was mixed slightly and cultured over night in incubator containing a proportion of 5% carbon dioxide at 37 centigrade. After 48 hours, the expression of GFP, the transfection rate and the grows state of cells were detected by fluorescent microscope. The expression of Sox9 protein was measured by western-blot examination.4. To evaluate the effect of bone mesenchymal stem cells transferred by lentivirus-Sox9 constructure in treatment of rabbit degenerative intervertebral discAfter the animal model had been built, MSCs transferred by Sox9 gene were transplanted into the degenerative disc of 12 rabbits, MSCs were transplanted into other 12 rabbits, 12 rabbits were not treated as control group. The effect of repair the degenerative discs was evaluated by X-ray, MRI, pathological, immunohistochemical and biochemical examination.Results1. The degeneration of lumbar intervertebral disc of rabbits occurred 4 weeks after surgery of animal model. The height of the intervertebral disc and the signal of nucleus pulposus decreased increasingly 12 weeks later in study group, and the expression of typeⅡcollagen and proteoglycan was less than control group (P<0.05).2. Bone mesenchymal stem cells were isolated and cultured successfully, and its advantages including rapid prolification, better repetitiveness and so on. It is an ideal seed cell for tissue engineering.3. The cellular morphology of bone mesenchymal stem cells transferred by lentivirus-Sox9 constructure was polygon, larger cell body, growing well and GFP-positive. The transfection rate is over 90%. The apoptosis rate and the prolification of cells were not disturbed by transferred procedure according to flow cytometry and MTT examination. The expression of Sox9 protein in cells transferred by Sox9 was higher than cells without transfection and cells transferred by lentivirus according to western-blot analysis.4. There were serious decrease of the intervertebral disc height and the signal of intervertebral disc at T2 image in control group, moderate decrease in MSCs group, no decrease in study group of MSCs transferred by sox9 gene. The expression of typeⅡcollagen and proteoglycan in study group of MSCs transferred by sox9 gene was higher than MSCs group and control group(P<0.05).Conclusion 1. The rabbit model of degenerative intervertebral disc can be built by puncturing the annulus with needle and aspirating some nucleus pulposus from the intervertebral disc.2. MSCs transferred by lentivirus-sox9 constructure may be an alternative method in treatment of rabbit degenerative intervertebral disc and its therapeutic effects are better than that of MSCs group. |
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