Experimental Study Of CXCR4 Gene Transfected Bone Marrow Mesenchymal Stem Cells In The Treatment Of Rabbit Intervertebral Disc Degeneration

Part I:Culture and identification of rabbit bone marrow mesenchymal stem cellsObjective:To isolate, culture and amplify the bone marrow mesenchymal stem cells (MSCs) from New Zealand white rabbits, and to identify the cultured cells and to supply MSCs for the subsequent experiments.Methods:After general anesthesia, sterilization bone marrow was taken from the medullary cavity of femur and tibia of 2 weeks old New Zealand white rabbits. Adherent cultured and passage was done to purify MSCs. MTT assay method was used to draw cell growth curve. Cell morphology and flow cytometry analysis of surface markers, and osteogenic, adipogenic, chondrogenic differentiation of MSCs were done to identify cultured MSCs.Results:After passaging to third generations, MSCs was gradually purified. Mature MSCs under the microscope were seen as short fusiform shape, clear contour and with rounded nucleus. Cells growth rapidly after first passage. Flow cytometry results showed MSCs with good homogeneity. More than 97% cells expressed stem cell specific antigen CD29, CD90, did not express hematopoietic cell surface markers CD45 and CD34. MSCs differentiated into osteoblasts, adipocytes and chondrocytes-like cells after being induced, which demonstrated that MSCs has the ability of multiply differentiation potential.Conclusion:After adherence and passage culturing, high purity MSCs can be obtained. The cultured MSCs have characteristics of stem cells, which has the ability of multipotential differentiation and can be used as seed cells for regenerative medicine.Part Ⅱ CXCR4 gene were transfered to rabbit bone marrow mesenchymal stem cells by lentiviral vectorObjective:To investigate the feasibility of CXCR4 gene transfer to rabbit MSCs by lentiviral vector and improve the migration ability of CXCR4 over-expressed MSCs to SDF-1 in vitro.Methods:Recombinant rabbit CXCR4-pIRES2-EGFP and packaging plasmids were used to infect 293T cells. Packaged, collected virus suspension, concentrated, detected viral titer and infected MSCs. The viability of transducted MSCs was detected by the trypan blue staining. CXCR4 mRNA and protein content of MSCs was detected by QPCR and Western blot, respectively. The migration of CXCR4-MSCs to SDF-1 was detected by the transwell chemotaxis assay.Results:Both CXCR4 mRNA and protein expression of transducted MSCs were significantly increased than that of non-transducted MSCs. Trypan blue staining showed that viability of transducted MSCs did not change significantly before and after transduction. Transwell chemotaxis assay showed that the migration of CXCR4-MSCs to SDF-1 was significantly improved than that of non-transducted MSCs (P<0.05). CXCR4 blocker AMD3100 inhibited the migration of CXCR4-MSCs to SDF-1.Conclusion:CXCR4 could be transducted to rabbit MSCs effectively with lentiviral vector, which didn’t affect the cell’s ability obviously. Transducted MSCs can high expression of CXCR4 mRNA and protein and significantly improve the migrating ability of CXCR4-MSCs to SDF-1.Part Ⅲ Efficacy of CXCR4 over-expressed MSCs in the regeneration of normal intervertebral discObjective:To explore the role of over-expression of CXCR4 on MSCs therapy of intervertebral disc degeneration.Methods:CXCR gene was transducted to MSCs by recombinant virus vector. CXCR4-MSCs were injected to intervertebral disc after annulus fibrosus being punctured to create degeneration model.15 New Zealand white rabbits were divided into three groups:CXCR4-MSCs groups, MSCs groups and PBS groups depending on the substance of injection. Disc height Index, histology and mRNA of Aggrecan and type Ⅱ collagen were obtained at 4 and 8 weeks after transplantation.Results:The protein over-expression of CXCR4 in MSCs were detected by Western Blot.4 weeks after transplantation, the disc height of three groups decreased significantly than pre-operation. But there were no significant difference among the three groups. Disc height index in CXCR4-MSCs groups didn’t decrease obviously at 8 weeks after transplantation. But there were significantly decrease in MSCs groups and PBS groups. There were significant difference among three groups. Disc degeneration in histology was significantly slowed down in CXCR4-MSCs groups and MSCs groups at 4 and 8 weeks after transplantation rather than PBS groups. RT-PCR results showed that, Aggrecan and type Ⅱ collagen mRNA were both expressed at 8 weeks after transplantation. Aggrecan mRNA expression of CXCR4-MSCs groups was more than that of MSCs groups and PBS groups, respectively. There was significant difference. There were no significant difference among three groups of type Ⅱ collagen mRNA expression.Conclusion:CXCR4 gene could be transferred to MSCs. CXCR4-MSCs transplantation could significantly slow down intervertebral disc degeneration.Part Ⅳ Development and evaluation of rabbit intervertebral disc degeneration modelObjective:To develop a simple, effective and reproducible model of intervertebral disc degeneration model of New Zealand white rabbits.Methods:18 healthy adult New Zealand white rabbits, excluding spinal deformity, weighing from 4.4+ 0.27kg, were selected for experiment. After general anesthesia using 1% sodium pentobarbital, L3/4, L4/5, L5/6 disc were exposed by retroperitoneal approach. Disc was punctured from anterolateral side by a 21 gauge needle and stayed for 5 seconds, repeating 3 times per disc. Using self-made limiter to control the depth of puncture in 5mm. L2/3 and L6/7 were exposed and didn’t puncture as control. Six rabbits were selected randomly for X ray and MRI to measure disc height and T2 signal intensity at 2,4,8 weeks post-operation, respectively. Then the rabbits were sacrificed by giving overdose anesthesia. Intervertebral disc degeneration was evaluated and graded histologicallyResults:All the animals survived after puncture, One rabbit suffered from local infection and improved after antibody treatment. DHI decreased at 2 weeks post-operation and became lower at 4 weeks post-operation. X-rays showed lumbar stenosis at 8 weeks post-operation. MRI showed the onset of intervertebral disc degeneration. And signal intensity decreased at 2 weeks post-operation. DHI also continued to decrease. It became black discs at 8 weeks post-operation. Histology results showed that puncture site of intervertebral disc remodeled. There was reduction in the volume of nucleus pulposus and decreased number of cells. Annulus fibrosus was deranged, shrined of nucleus and the nucleus pulposus were gradually separated from annulus fibrosus. Intervertebral disc slice grade of annulus punctured group at 2,4,8 weeks post-operation were increased significantly compared with the control group. There was significant difference (P< 0.01) and showed that the annulus fibrosus puncture can induce intervertebral disc degeneration.Conclusion:Rabbit intervertebral disc degeneration model could be induced by annulus fibrosus puncture, which can provide a reliable animal model.Part Ⅴ CXCR4-MSCs regeneration of intervertebral discObjective:To evaluate the role of high expression of CXCR4 in MSCs in the enhancing regeneration of intervertebral disc and CXCR4-MSCs migration in the disc.Methods:In vitro cultured MSCs, lentiviral vector mediated CXCR4 gene transduction to MSCs. CXCR4-MSCs labeling with SPIO were transplanted to the degenerated intervertebral disc. After 8 weeks and 16 weeks post-operation, DHI was measured by x-ray image. Distribution of CXCR4-MSCs in the disc was tracked by 3.0 MR. Prussian blue staining were used to compare to MR finding. Type Ⅱ collagen and aggrecan mRNA expression were measured by Real time PCR.Results:A low signal area were found in the core of disc by MR in MSCs group and CXCR4-MSCs group, but not in the PBS groups immediately after transplantation. Low signal area was gradually dispersed after 8 and 16 weeks post transplantation. Low signal area was becoming smaller as time gone on. Transplanted CXCR4-MSCs that remained in the disc were also confirmed by the prussian blue stain. Compared with MSCs group, CXCR4-MSCs groups showed more cells rested in the intervertebral disc. Aggrecan and Col2 mRNA expression at MSCs group were more that of PBS groups at 8 weeks post-transplantation and the difference is statistically significant. Aggrecan and Col2 mRNA expression at CXCR4-MSCs group were more that of PBS groups at 16 weeks post-transplantation and the difference is statistically significant;Conclusion:Transducted MSCs high expressed of CXCR4 and improved the ability of stem cells to regenerate intervertebral disc, which promote the intradiscal residence and migration.