AIM: To investigate the immunoblot changeS of dystrophin expression in mdx mice after bone marrow stem cells transplantation. METHODS: The experiment was conducted in the laboratory of Department of Neurology, the First Affiliated Hospital, Sun Yat-sen University from September to December 2004. ①Twenty-five mdx mice of 7-8 weeks old were selected and randomly divided into transplantation 4, 8, 12 and 16 weeks groups and blank control group with 5 mice in each group. Meanwhile, 20 C57 mice of 4 to 6 weeks old were selected as donor mice, and 5 C57 mice aged 10 weeks served as positive control mice. ②All mice were preconditioned with 7 Gy ?-ray. After radiotherapy, 2?107 marrow stem cells per mouse (about 0.3-0.5 mL) were injected into the vena caudalis of mice; 0.3 mL PBS was injected into the blank control group and positive control group. ③The mice in 4 transplantation groups were killed at each time point, and the gastrocnemius was harvested to prepare dystrophin samples. The amount of dystrophin expression was detected by Western blot with GAPDH as control. RESULTS: All 25 mdx mice and 5 C57 mice were involved in the result analysis. Western Blot analysis of dystrophin after transplantation: No dystrophin was detected in the blank control group; in the 4 weeks after transplantation group, only few dystrophin expressions were detected, and the dystrophin/GAPDH was about 0.095?0.267; in the 12 weeks after transplantation group, dystrophin/GAPDH was about 0.218?0.338; dystrophin expressions were increased with time, at 16 weeks after transplantation, the expressions were much more than those at 8 weeks (dystrophin/GAPDH: 0.393?0.385, 0.173?0.284, t =6.062, P

AIM:To examine the relationship between the gene defect,change of protein hydrophobicity,spacial structure change and clinical phenotypes of Duchenne muscular dystrophy(DMD),and to explore the molecular pathogenesis of DMD.METHODS:The gene sequences of 59 cases of DMD/BMD patients with deletion from mutation were analyzed.The relationship between the protein hydrophobicity,3D-spacial structure and clinical phenotypes was examined by biological informatic technology.RESULTS:50 cases of frameshift mutation were all DMD.In other 5 cases with codon mutation that involved the 3rd hydrophobic region,4 cases were diagnosed as DMD and the rest one was BMD.The exon 3 deletion leaded to the intortion of dystrophin N-terminal,which in turn affected the combination of dystrophin and troponin resulting in the DMD pathopoiesis.CONCLUSION:The severity of clinical phenotypes of muscular dystrophy diseases is related to whether the deletion destroys the reading frame,involves the 3rd hydrophobic region or changes the protein special structure.The biological informatic technology provides a new potential research methodology for studying the pathogenesis of DMD.

BACKGROUND:The clinical research have found that the interbervebral disc herniation often occurs in several members or even al the members of a family, and the location, reason and symptom are basical y the same, indicating that genes play an important role in this kind of disease. OBJECTIVE:To analyze the apoptosis Fas gene expression characteristics of lumbar disc in the familial patients with intervertebral disc herniation. METHODS:Semi-quantitative reverse transcription-PCR was used to test Fas gene expression of vertebral pulp and cartilage endplate in the intervertebral disc among 15 familial patients, 21 ordinary patients and five fresh cadavers. RESULTS AND CONCLUSION:Fas gene expression level of endplate of familial and ordinary patients with intervertebral disc herniation was higher than that of fresh cadavers, and there was no significant difference (P<0.05);there was no significant difference in Fas gene expression in endplates between familial patients and ordinary patients with intervertebral disc herniation (P>0.05). Compared with the vertebral pulps of ordinary patients with intervertebral disc herniation and fresh cadavers, there was no significant difference in the Fas expression of vertebral pulps of familial patients with intervertebral disc herniation (P>0.05). The increasing Fas gene expression may be secondary in the endplates of familial patients with intervertebral disc herniation, which can prevent intervertebral disc degeneration through preventing the endplate degeneration.

AIM: To investigate the survival, migration and differentiation of human bone marrow mesenchymal stem cells (hMSC) in the middle cerebral artery occlusion (MACO) model and to observe the influence on motor function in the animal model. METHODS: hMSC with Hoeschst 33342 were injected into the animal model of MACO after Shenqiye induced for half an hour and their survival, migration, differentiation and the behavior changes in MACO rats were examined. RESULTS: hMSC had good homogeneousness and immunological reaction after implantation. The results showed that hMSC survived in rat brain for a long time over six weeks. As time going, hMSC were found in much more areas in the rat brain. Immunofluorescence staining suggested that implanted hMSC expressed human neuron specific enolase, neurofilament, and glial fibrillary acid protein. At the same time, improvements in abnormal behavior of MACO rats were observed. CONCLUSION: hMSC differentiate into neurons in the brain of rats, which means that hMSC is an ideal seed cells for the therapy of cerebral infarction.

AIM: To investigate the correlation between the expression of neuron-specific protein and apoptosis in the process of differentiation from rat bone marrow stromal cells into neuron with brain-derived neurotrophic factor (BDNF). METHODS: The 5th passage MSCs were induced by BDNF and 2-mercaptoethanol (?-ME), respectively. At 1 h, 6 h, 12 h and 24 h, nestin, neuron specific enolase (NSE), microtubulease associated protein (MAP)-2 and glail fibrillary acidic protein (GFAP) were detected by Western blotting. Cell cycle and apoptosis were examined by flow cytometry. RESULTS: Nestin and NSE of neuron-like cells induced by BDNF and ?-ME were all positive by Western blotting. At 12 h, nestin and NSE turned to negative and apoptosis was detected in ?-ME group, nestin and NSE still positive and apoptosis wasn't detected in BDNF group. Till 24 h, nestin and NSE in BDNF group were negative but apoptosis still not detected. Notably, GFAP (glial astrocyte marker) was detected and MAP-2 wasn't detected in the two induced groups. CONCLUSION: The down-expression of neuron-specific protein correspondingly with apoptosis in the process of differentiation from MSCs into neuron with ?-ME shows that apoptosis may be one of the causes of induced cell death. BDNF induction was not the cause of apoptosis. Other factors may include for the cell death in the presence of neuron-specific protein expression induced by BDNF.

OBJECTIVETo study the order and degree of muscular affection in patients with Duchenne muscular dystrophy (DMD) during the course of disease.

METHODSMultiplex ligation dependent probe amplification (MLPA) was used to detect potential mutation of dystrophin gene. Magnetic resonance imaging (MRI) was used to scan the anteromedial aspect of thigh muscles.

RESULTSAll of the 6 patients were found to have deletion or duplication mutations. The order of affection has been gluteus maximus, adductor magnus, quadriceps femoris, rectus femoris and biceps muscle of the thigh, while semimembranous muscle, semitendinosus, sartorius muscle and musculus gracilis are selectively affected and in a decreasing order.

CONCLUSIONMRI can reflect the order, extent and degree of skeletal muscle involvement in patients with DMD, and can reflect pathological changes of damaged skeletal muscle at each stage, which may provide an important means for patient examination and diagnosis. No apparent correlation between the severity of disease and the nature of mutations was noted.

Adolescent ; Child ; Child, Preschool ; Dystrophin ; genetics ; Female ; Humans ; Magnetic Resonance Imaging ; Male ; Muscle, Skeletal ; diagnostic imaging ; Muscular Dystrophy, Duchenne ; diagnosis ; diagnostic imaging ; genetics ; Mutation ; Radiography ; Sequence Deletion

OBJECTIVETo investigate the effect of shenqi fuzheng injection (SFI) in inducing differentiation of human mesenchymal stem cells (hMSCs) in brain stem and its effect on nervous function in model rats of cerebral infarction.

METHODSMiddle cerebral artery occlusion model rats were made, and hMSCs was injected into their brain after being amplified in vitro and incubated with SFI for 0.5 h, then the survival, migration and differentiation of hMSCs in brain stem as well as the change of nervous function in model rats were observed.

RESULTSThe post-transplantation reject reaction to hMSCs was low, it could survive as long as 6 weeks or more. No difference in area of infarction was shown before and after transplantation. Immunohistochemical staining showed that hMSCs expressed human neuron specific enolase (NSE), neurofilament (NF) and glial fibrillary acid protein (GFAP). The limb-kinetic function and tactile perception were improved in the model rats.

CONCLUSIONSFI can induce hMSCs differentiate into neurons in vivo, and hMSCs may be the ideal germinal cells for treating cerebral infarction.

Animals ; Brain ; metabolism ; Cell Differentiation ; drug effects ; Cell Division ; Cells, Cultured ; Cerebral Infarction ; etiology ; surgery ; Culture Media ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Male ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; cytology ; Neurofilament Proteins ; biosynthesis ; Neurons ; cytology ; Phosphopyruvate Hydratase ; biosynthesis ; Rats ; Rats, Sprague-Dawley ; Transplantation, Heterologous