| PartⅠ. Establishment of the rat model of lumbar intervertebral discdegenerationObjective:To establish a rat model of lumbar intervertebral disc degeneration by puncturing theannulus fibrosus using a27-gauge (27G) needle to simulate the human intervertebral discdegeneration, and provide a large number of moderately degenerative nucleus pulposuscells for study of next part.Methods:Using27G puncture needles to injury annulus fibrosus of L45and L56discs in60Sprague Dawley rats under C-arm fluoroscopy guiding. At pre-operation and2weeks,4weeks,8weeks after surgery, respectively, modified Thompson classification by MRI, HEstaining and Masson staining were performed to determine the degenerative degree ofpunctured discs, and RT-PCR was performed to test the mRNA expression of Aggrecan,type Ⅱ collagen, Sox-9and type I collagen in nucleus pulposus.Results:At2weeks after surgery, signal intensity from T2-weighted MR images of mostpunctured discs changed non-significantly, only few disc exhibited slight decreased signalintensity. At4weeks after surgery, T2-weighted MR images of nucleus pulposus showedmild to moderate decrease of signal intensity, and the degenerative degree of most punctured discs were classified to grade2to3in modified Thompson classification. At8weeks after surgery, T2-weighted MR images of nucleus pulposus demonstrated severedecrease of signal intensity, and ‘black disc’ changes can be found in many discs. The MRIresults showed a gradually rising trend of modified Thompson grading along with the timeextending after puncture procedure (p<0.05).Histological HE staining and Masson staining showed cracks among layers ofcollagen fibers of annulus fibrous, even the rupture of collagen fibers along with the timeextending after puncture procedure. The amount of notochord cells and chondroid cellswithin the nucleus pulposus reduced gradually, while the boundary between nucleuspulposus and annulus fibrosus was not clear gradually.RT-PCR analysis showed that the mRNA expression of Aggrecan, type Ⅱ collagenand Sox-9decreased gradually except for the type I collagen which showed a graduallyincreasing trend.Conclusions:The procedure of posterior lumbar intervertebral disc punctured by a27G needleunder C-arm fluoroscopic guidance is of high success rate with less invasive, less bleedingand strong controllability. MRI, histology and RT-PCR results revealed that the rat modelof disc degeneration showed a slow pathological process which was similar with humanlumbar disc degeneration. The rat model of lumbar disc degeneration can provide a largenumber of degenerative nucleus pulposus cells for following study in vitro. PartⅡ IGF-1regulates expression of Aggrecan and type Ⅱcollagen inrat degenerative nucleus pulposus cells Objective:To identify the effect of IGF-1on the gene transcription and protein expression ofAggrecan and type Ⅱ collagen, and the proliferation of rat degenerative nucleus pulposuscells.Methods:Nucleus pulposus cells from mile to moderate degenerative discs (modifiedThompson grade2to3) were cultured under monolayer culture conditions. Cells shapewas observed and cell viability was determined by Trypan blue staining.Passage2nucleus pulposus cells were given IGF-1at different concentrations (0,20,50,100,200ng/ml). After3days, the proliferation of nucleus pulposus cells was assessedby MTT assay.Passage2nucleus pulposus cells were cultured in alginate beads under3-dimensionalconditions, and were given IGF-1at different concentrations (0,20,50,100,200ng/ml) for12hours. Then, the mRNA expression of Aggrecan and type Ⅱ collagen in nucleuspulposus cells were assessed by RT-PCR.Degenerative nucleus pulposus cells cultured in alginate beads were given IGF-1(100ng/ml) for24hours. Then, the protein expression of Aggrecan and type Ⅱ collagen innucleus pulposus cells were assessed by western-blot.Results:It took a longer time for degenerative nucleus pulposus cells to achieve adherencethan other type cells. The nucleus pulposus cells were spindle or polygonal. The viabilityof primary cells was91%to96%, and reduced slightly after cells were passaged.The OD570nm value increased gradually with concentration of IGF-1increasing.IGF-1at100ng/ml can increase cells proliferation capacity by approximately66%(p<0.05). However, there was no significant difference of OD570nm value between100ng/ml group and200ng/ml group (p>0.05).The mRNA expression of Aggrecan and type Ⅱ collagen in rat degenerative nucleus pulposus cells increased gradually with concentration of IGF-1increasing（p<0.05）.However, there was no significant difference between100ng/ml group and200ng/ml group(p>0.05).IGF-1at100ng/ml can significantly increase the protein level of Aggrecan and type Ⅱcollagen in rat degenerative nucleus pulposus cells（p<0.01）.Conclusions:IGF-1can promote the proliferation of rat degenerative nucleus pulposus cells, andincrease the mRNA and protein expression of Aggrecan and type Ⅱ collagen in ratdegenerative nucleus pulposus cells. As a growth factor, IGF-1may have considerablepromise in the biological treatment of disc degeneration. PartⅢ IGF-1up-regulates expression of Aggrecan and type Ⅱcollagenin degenerative nucleus pulposus cells via PI3K/Akt signaling pathwayObjective:To investigate the possible signaling pathway through which IGF-1increased theexpression of Aggrecan and type Ⅱ collagen in rat degenerative nucleus pulposus cells.Methods:Passage2nucleus pulposus cells in3-dimensional culture were switched toserum-free media for12h and then divided into4groups: control group; IGF-1group(100ng/ml); LY294002group (50μM, PI3K/Akt pathway inhibitor); IGF-1+LY294002group, according to the processing methods. We performed a western-blot using antibodiesagainst phospho PI3K or phospho Akt to detect activation of PI3K/Akt pathway by IGF-1,and inhibition of PI3K/Akt signaling pathway by LY294002.Passage2nucleus pulposus cells in3-dimensional culture were switched to serum-free media for12h and then divided into4groups: control group; IGF-1group;LY294002group and IGF-1+LY294002group. Then western-blot was used to detect theeffect of activation or inhibition of PI3K/Akt signaling pathway on expression of Aggrecanand type Ⅱ collagen in degenerative nucleus pulposus cells.Results:Compared with control group, the IGF-1can activate the PI3K/Akt signaling pathwayas evidenced by the phosphorylation of PI3K and Akt (p<0.01). However, when comparedwith IGF-1group, the protein expression of p-PI3K and p-Akt decreased significantly inIGF-1+LY294002group (p<0.01).Western-blot results showed that IGF-1can up-regulate protein expression ofAggrecan and type Ⅱ collagen in degenerative nucleus pulposus cells when compared withcontrol group (p<0.01). However, when compared with IGF-1group, the proteinexpression of Aggrecan and type Ⅱ collagen decreased significantly in IGF-1+LY294002group (p<0.01).Conclusions:IGF-1can activate the PI3K/Akt signaling pathway, and up-regulate expression ofAggrecan and type Ⅱ collagen in rat degenerative nucleus pulposus cells. However,LY294002can significantly inhibit these effects of IGF-1. So, we concluded that IGF-1can up-regulate expression of Aggrecan and type Ⅱ collagen via PI3K/Akt signalingpathway. |
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