Objective: To develop retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2 in mesenchymal stem cells. Methods: Mesenchymal stem cells from New Zealand white rabbits were transduced with retroviral pLEGFP-BMP2 vector by the optimized retroviral transduction protocol. Fluorescent microscopy's examination was to evaluate the results of the transduction, flow cytometers analysis was to evaluate the transduction efficiency and the Fluorescence-activated cell sorting method was to sort the transduced cells. Bioactivity test from C2C12K4 cells was to show the expression and bio-activity of the fusion gene. Results: Fluorescent microscopy showed the success of the transduction. By flow cytometer's analysis, the mean efficiency of the transduction with EGFP was (42. 8 ± 6. 1)% SD. Transduced cells were sorted efficiently by the fluorescence-activated cell sorting method and after sorting, almost of those showed the expression of BMP2. Fluorescently and strongly bioactivity test for C2C12 K4 cells demonstrated that fluorescent materials were located the surface of cells and the activity of luciferase increased compared with the control. Analysis of long-term expression showed there was no difference between 2 week-time point and 3 month-time point of culture post-sorting. Conclusion: Mesenchymal stem cells can be transduced efficiently by retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2, the highly pure transduced cells are obtained by the fluorescence-activated cell sorting technique, the expressed chimeric protein embraced the double bioactivity of EGFP and BMP2, and moreover, the expression had not attenuated over time.

Objective To investigate the effects of gene transfection with human growth/differentiation factor 5(hGDF5)on the differentiation and proliferation of rabbit bone marrow mesenchymal stem cells (BMSCs).Methods BMSCs were obtained from adult New zealand rabbits and purified by gradient centrifuge.Exogenous recombinant human GDF5 was transfected into BMSCs with liposome method.Then hGDF5 expression at mRNA and protein level was measured separately by reverse transcription-polymerase reaction and indirect immunofluorescence method.Activity of alkaline phosphates(ALP),expression of TypeⅡcollagen(ColⅡ),proteoglycan and growth of the cells were all measured by biological methods to evaluate the effects of hGDF5 gene transfer on the differentiation and proliferation of rabbit BMSCs.Results After hGDF5 gene transfection,BMSCs expressed hGDF5 mRNA and protein,and compared with the control groups,expression of proteoglycan and ColⅡ increased significantly,but no significant difference appeared in ALP activity and cell proliferation.Conclusion Gene transfer with hGDF5 is an effective way to enhance the expression of GDF5 at mRNA and protein.The expression of heterogenetic hGDF5 gene can induce BMSCs' differentiation to chondrogenic cells.But the gene transfection has no obvious effects on the proliferation and ALP activity of BMSCs.

Objective To establish an animal model of crush syndrome in rabbits and investigate the preventive effect of negative pressure drainage.Methods Twenty-four New Zealand rabbits were randomly divided into four groups:control group,traditional treatment group,negative-pressure treatment group and mixed treatment group,with six rabbits per group.Blood urea nitrogen (BUN),creatinine (Cr),K,creatine kinase (CK) and myoglobin (Myo) were detected before crush and at 6,12,24,48,72 hours after decompression.Edema and analgesia of extremities were determined and changes of kidney and muscular tissue were observed by light microscope.Results As time went on,K,Myo,BUN and CR had unobvious rise in each treatment group,without the presence of renal failure.The control group showed marvelous increases of K,Myo,BUN and Cr at 6 hours after decompression,from (4.61 ±0.98)mmol/L,(4.22±0.93) ng/ml,(7.76±1.40) mmol/L,(101.32±9.35)μmol/Lto (7.88±1.95)mmol/L,(11.34 ±3.86) ng/ml,(15.91 ± 1.76) mmol/L,(258.32 ±91.40) μmol/L separately,with renal failure and significant differences in comparison with other treatment group (P ＜0.05).The level of tissue edema and degree of analgesia in negative-pressure treatment group and mixed treatment group were lower than those in the traditional treatment group.Renal damage in all the treatment groups was milder than that in the control group by light microscopic observation,while there was no significant difference of muscle injury among groups.Conclusion Negative pressure drainage may prevent crush syndrome.

ObjectiveTo investigate the effect of operation treating spinal multi-level tuberculosis.Methods45 patients with multi-level spine tuberculosis were treated with debridement completely and anterior or lateral-anterior intervertebrae autograft. Of them, 5 patients added to anterior instrumentation.Results45 patients had been followed up for 12 to 40 months.The back pain of 40 cases were relieved within 5 weeks after operation, erythrocyte sedimentation rate decreased 3 weeks after operation. 38 patients who involved in kyphosis decreased their kyphosis mostly and only one patient lost 3 degrees in follow-up.15 patients who involved in neurological deficits improved one or two grades (Frankel). Grafts fused in 44 patients and there were no recurrent in follow-up.Conclusions Operative treatment is efficacious to multi-level body spine tuberculosis.

Objective To study the correlation between osteoporosis and degeneration of intervertebral disc in the aging rat.Methods Fifty healthy SD rats were fed for twenty-two months in order to build the model of aging rats,the histology of femoral bone,tibial bone and lumbal vertebra was used to observe,then we evaluated the indexes which reflect the degeneration of intervertebral disc including the quantity of collagen type Ⅱ,Ⅹ of intervertebral disc by the immunohistochemistry methods,the relative area of vascular bud,the thickness of calcified layer and un-calcified layer on cartilage endplate and the indexes which reflect the osteoporosis including the thickness ratio of cortical bone,the relative area of bone trabecula,the bone density,the max stress and straining of bone.Results There was moderate or strong negative correlation among the indexes of the degeneration of intervertebral disc including the relative area of vascular bud,the ratio of the un-calcified and the calcified layer,the quantitation of collagen type Ⅱand the indexes of osteoporosis including the thickness ratio of cortical bone,the relative area of bone trabecula,the density of femoral bone and vertebral body bone,the max stress and straining of bone.However,there was moderate or strong positive correlation with the quantitation of collagen type Ⅹ,of which the bone density of vertebral body had the strongest dependablity with collagen type Ⅹ.And the indexes of bone biomechanics including the max stress and straining had no correlation with the indexes of degeneration of intervertebral disc.Conclusion Osteoporosis has negative correlation with the degeneration of intervertebral disc,which is not distributed to the environment factors;but the indexes of bone biomechanics including the max stress and straining have no correlation with the indexes of degeneration of intervertebral disc.

Objective To determine the potential value of imaging for spinal tuberculosis. Methods 180 patients proved as spinal tuberculosis by operation or clinical follow who underwent X-ray film, CT and MRI were reviewed. They were classified A, B or C in term of imaging and clinical symptom. A was the normal of X- ray film and positive of CT or MRI. B was positive of X-ray film, CT and MRI. C was with the neurological symptoms. Results 40 patients ( 40/180 ) were categorized as A . They had short duration (

Objective To establish the animal model of cervical pain induced by formalin injection and explore the effect of cervical pain on cervical vertigo.Methods Formalin of 40 mL/L was injected near the nerve root of left side of neck to establish the model of cervical pain in rabbits.A total of 30 rabbits were divided randomly into two groups.Formalin-induced pain group(15 rabbits): indexes related to the vertebral artery were observed respectively before 2 mL of formalin injection and after it at 5,10 and 30 minutes.Lidocaine-blocking pain group(15 rabbits): pain induced by formalin was blocked by lidocaine of 10 g/L after 15-minute drug administration and the same indexes were observed at the same time and position of the neck.Results In formalin-induced pain group,vertebral artery systolic velocity,diastolic velocity and pulsatility indexes were decreased whereas resistance index was increased(P

Objective By exploring the effects of recombinant human bone morphogenetic protein (rhBMP)2 and rhBMP13 on chondrocytes proteoglycan production and phenotype expression to establish the theoritical mechanisms for the treatment of disc degeneration with chondrocytes transplantation plus BMPs.Methods The dose-dependent effects of rhBMP2 and rhBMP13 on PG protein synthesis and gene expression were detected under different concentrations (0,25,125,and 625 ng/ml).The sulfated-glycosaminoglycan (s-GAG) in the culture media and the pericellular matrix was measured with a 1,9-dimethyl-methylene blue (DMMB) colorimetric assay.Reverse transcriptase polymerase chain reaction (RT-PCR) was performed respectively to quantify the relative abundance of aggrecan Mrna.Cell proliferation was examined by Hoechst Dye assay.Results All rhBMP2 and rhBMP13 in different concentrations could significantly increase s-GAG synthesis and gene expression in chondrocytes (P＜0.05).And at the same concentration.rhBMP2 was more potent than rhBMP13 on s-GAG synthesis.Hoechst Dye assay showed neither rhBMP2 nor rhBMP13 had significant effect on cell proliferation.Conclusion rhBMP2 and rhBMP13 are able to upregulate s-GAG synthesis,in addition,rhBMP2 is more potent than rhBMP13 on aggrecan gene expression regulation,but rhBMP2 and rhBMP13 do not have significant effect on chondrocyte proliferation.

This study examined the correlation between osteoporosis and the degeneration of intervertebral discs. Sprague-Dawley rats were maintained up to 22 or 28 months. The femoral bone, tibial bone and lumbar vertebra were histologically studied and the expression of collagen type II and X in intervertebral discs was immunohistochemiscally determined. Several indices for the degeneration of intervertebral discs and osteoporosis and the correlation among them were then analyzed. Close correlations were found among the indices for the degeneration of intervertebral discs, including the relative area of the vascular bud, the ratio of the uncalcified and the calcified layers, the expression of collagen type II and X. The correlation with collagen type X was negative. There existed positive correlations among the indices for osteoporosis, including the thickness ratio of cortical bone, the relative area of bone trabecula, the density of femoral and vertebral body bones, and the maximum stress and strain on bone. Analysis on the relationship of osteoporosis and the disease on disc showed that the indices of osteoporosis were negatively correlated with the indices of the degeneration of intervertebral discs but the expression of collagen type X was positively correlated, with the density of vertebral body bones having the strongest dependence on collagen type X. The maximum stress and strain bore no correlation with the degeneration of intervertebral discs. These results suggest that osteoporosis was negatively correlated with the degeneration of intervertebral discs.

Objective To develop retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2 in mesenchymal stem cells. Methods Mesenchymal stem cells from New Zealand white rabbits were transduced with retroviral pLEGFP-BMP2 vector by the optimized retroviral transduction protocol. Fluorescent microscopy's examination was to evaluate the results of the transduction, flow cytometer's analysis was to evaluate the transduction efficiency and the Fluorescence-activated cell sorting method was to sort the transduced cells. Bioactivity test from C2C12K4 cells was to show the expression and bio-activity of the fusion gene. Results Fluorescent microscopy showed the success of the transduction. By flow cytometer's analysis, the mean efficiency of the transduction with EGFP was (42.8±6.1)% SD. Transduced cells were sorted efficiently by the fluorescence-activated cell sorting method and after sorting, almost of those showed the expression of BMP2. Fluorescently and strongly bioactivity test for C2C12K4 cells demonstrated that fluorescent materials were located the surface of cells and the activity of luciferase increased compared with the control. Analysis of long-term expression showed there was no difference between 2 week-time point and 3 month-time point of culture post-sorting. Conclusion Mesenchymal stem cells can be transduced efficiently by retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2, the highly pure transduced cells are obtained by the fluorescence-activated cell sorting technique, the expressed chimeric protein embraced the double bioactivity of EGFP and BMP2, and moreover, the expression had not attenuated over time.