[Denaturant gradient gel electrophoresis in the genetic diagnosis of hereditary multiple exostoses].

OBJECTIVE: To detect the mutations of EXT2 gene in hereditary multiple exostoses (HME) families and to investigate the sensitivity of denaturant gradient gel electrophoresis (DGGE) in screening the mutations in EXT2 gene. METHODS: Five HME families and 3 sporadic patients were screened for the mutation detection in all exons of EXT2 gene covering the coding sequence and the flanking intronic sequence by DGGE, and DNA sequencing was performed for products with abnormal conformation. RESULTS: Among these HME patients, we found 2 disease-causing mutations: A313T (nonsense mutation) and 319 insGT (frameshift mutation). CONCLUSION: Two mutations of EXT2 gene are identified in the sample. DGGE can be an ideal choice for gene diagnoses of HME.

Hydroxyapatite/tricalcium phosphate matrix scaffold as cell carriers in vitro.

OBJECTIVE: To explore the possibility of hydroxyapatite/tricalcium phosphate (HA/TCP) matrix scaffold as cell carriers in tissue engineering, and to provide a direct evidence for the resurfacing of matrix scaffold. METHODS: Human bone marrow stromal cells (hBMSCs) and umbilical vein endothelial cells (UVECs) were co-cultured on the surface of matrix scaffold. The morphologic characters during culture on HA/TCP matrix scaffold's surfaces were checked by light microscope, fluorescence microscope, and scanning electronic microscope. RESULTS: Both human bone marrow stromal cells and human umbilical vein endothelial cells showed good biocompatibility with HA/TCP matrix scaffold. The cell growth into the micropore of HA/TCP matrix scaffold could be seen under both fluorescence microscopy and scaning electron microscopy. CONCLUSION: HA/TCP matrix scaffold can be used as a vehicle for the cell transplantation.

[Operative results and prognosis of thoracic spinal stenosis].

OBJECTIVE: To discuss the clinical properties, operative results of thoracic spinal stenosis and factors correlating with prognosis. METHODS: From September 1992 to January 2001, 16 patients who suffered from thoracic spinal stenosis caused by degeneration, ossified ligamentum flavum, diffuse idiopathic hyperostosis and trauma, were decompressed by operation. The operative method was selected according to the compressed position of spinal cord. All patients were followed up 6 months to 9 years. The pain severity, ambulatory status and paraplegia index were compared between before operation and after operation. The correlation between prognosis and ages, the length of stenosis and the duration of disease was studied. RESULTS: The results of Wilcoxon Signed Ranks Test show significant difference in pain severity, ambulatory status and paraplegia index between before operation and after operation (P < 0.01). The results of partial correlation analyzing show that only the duration of disease was correlated with paraplegia index (P < 0.05). CONCLUSION: Thoracic spinal stenosis frequently develops in the lower-thoracic segments in middle and old aged men. Decompression by operation early can achieve a good clinical result. Duration of disease affects the prognosis.

[Effect of nicotine on the secretion of TNF of human peripheral blood mononuclear cells in vitro].

OBJECTIVE: To observe the effect of nicotine on the secretion of TNF of human peripheral blood mononuclear cells (PBMC) in vitro, and explore the possible mechanism of the high level of TNF caused by smoking. METHODS: Twenty-one non-smokers and 19 smokers were studied. The PBMCs isolated from each volunteer's blood sample were distributed to three groups, and cultured for 72 hours in supplemented RMPI 1640 alone or in supplemented RPMI 1640 containing 50 ng.ml-1 nicotine or 500 ng.ml-1 nicotine respectively. The level of TNF in supernate was quantified with the immuno-radioassay, and PBMC proliferation was observed. RESULTS: The level of TNF spontaneously secreted by PBMCs in smokers was significantly higher than that in non-smokers (P < 0.05). When the concentrations of nicotine were 50 ng.ml-1 and 500 ng.ml-1, the level of TNF increased significantly in non-smokers (P < 0.05, P < 0.01). In smokers, the level of TNF significantly increased when the concentration of nicotine was 50 ng.ml-1; however, the level of TNF significantly decreased (P < 0.05), and the proliferation of PBMCs was inhibited when the concentration of nicotine was 500 ng.ml-1 (P < 0.05, P < 0.05). The level of TNF was positively correlated to the multiple of PBMC proliferation (r = 0.93, P < 0.05). The linear regression equation was Y = 2.913X - 2.955. CONCLUSION: Nicotine can induce PBMCs to secrete more TNF, and the magnitude of the effect is strongly related to the dosage of nicotine, but a great dose of nicotine will inhibit the production of TNF.

[Osteogenic differentiation of murine yolk sac mesenchymal stem cells in vitro].

OBJECTIVE: To investigate the potential of yolk sac mesenchymal stem cells in osteogenic differentiation. METHODS: Murine yolk sacs were harvested on day 8.5 postcoitus, yolk sac cells were obtained after the yolk sacs were digested by 0.1% type I collagenase for 1 hour, the non-adherent cells were removed after being cultured for 1 hour. The adherent cells were cultured in DMEM containing of 5 ng/ml bFGF and 15% FBS, and passaged when they became subconfluent. The morphologic characteristics, and AKP, BMP-2, as well as type I, III collagen of the yolk sac adherent cells were observed and tested. The attached cells were treated with 10(-8) mol/L dexamethasone, 10 mmol/L beta-glycerophosphate, and 50 micrograms/ml vitamin C at passage 4. Alternations of morphological characteristic, AKP activity, collagen of type I, III and mineralization were detected. RESULTS: Pure mesenchymal stem cells which were of spindle shape, uniform in size, positive in type I, III collagen staining and weak positive in AKP activity could be induced to pleomorphism osteoblast-like cells in vitro. The cells were transformed from spindle shape to polygonal cells which were positive in type I collagen, negative in type III collagen, strong positive in BMP-2, and positive in Von Kossa's stain at week 8. The polygonal cells could form nodular structure and their AKP activity was increased. All these were coincidence with the characters of osteoblast. CONCLUSION: Yolk sac mesenchymal stem cell can be purified and induced to osteoblast in vitro.

[Clinical application of interbody cage systems in the lumbar spine].

OBJECTIVE: To study the clinical value of interbody cage systems in the surgical treatment of unstable lumbar segment, and evaluate the biomechanical properties. METHODS: Ninety-seven cases were selected for operation by posterior lumbar interbody fusion with the BAK-cage or TFC device. Of the patients, 18 were diagnosed as lumbar disc degeneration disease (DDD), 37 as lumbar stenosis, 23 as spondylolisthesis, and 19 as unstable traumatic lesion. Thirty-six of them received additional posterior pedicle devices; 13 cases with severe mechanical back pain underwent anterior interbody fusion with the BAK-cage or TFC or interbod spacer, 4 of them underwent surgery once again. A new anterior Mesh-cage was used in spinal reconstruction for some patients, including 8 patients with thoracolumbar tumor, 2 with lumbar burst fracture, and 1 with osteoportic late collapse of a vertebral body. RESULTS: In the follow-up for an average of 21 months, 63 patients were available for review. The clinical results were excellent and good in 91.2% of the patients who underwent posterior interbody cage fusion; the preoperative percentage of slip was corrected in 89% of the patients with spondlyolithsis and in 100% of the patients with traumatic dislocation. The satisfactory rate was 94.6% in the patients with severe back pain who underwent anterior interbody cage fusion. The rate of union in the grafted area was 95% at the 6th month after the operation. A preliminary experience showed that the clinical effect was obvious as the Mesh-cage was used in the patients with tumor, symptoms were relieved and motion ability was greatly improved in these patients. CONCLUSION: The interbody cage implant produces immediate stabilisation on unstable spinal segments, offers a conductive biomechanical environment for interbody graft healing by distraction properties and weight bearing function, and restores the normal interbody space and spinal column lordosis with satisfactory clinical effects on such diseases as DDD, traumatic lesion, osteoporotic late collapse of a vertebral body and spinal tumors.