Objective To investigate and compare the killing effects of different prodrugs combined with suicide gene HSV-tk on laryngocacinoma cell, Hep-2 in vitro. Methods Retroviral expressing vector pL(tk)SN was constructed by recombinant DNA technology. Hep-2 cells were infected by the recombinant retrovirus. The positive cloning was obtained after G418 selection and were termed Hep/tk. The integration and expression of tk gene in Hep-2 cells were identified by RT-PCR and Southern blot. The growth state and prodrugs killing effect of tk gene modified cells were used to investigate the expression of tk gene and antitumour effect on Hep-2 cells. Results RT-PCR and Southern blot analysis confirmed the integration and expression of tk gene in Hep-2 cells. There was no significant difference in cell proliferation between the Hep/tk and Hep-2. After the treatment of GCV,the Hep/tk showed high sensitivity to GCV and bystander effects were observed siginificantly in vitro. However the efficiency of another two prodrugs ACV and BVdU was lower than that of GCV. Both tk-positive and tk-negative Hep-2 cells were relatively insensitive to ACV and BVdU.Treatment of tk gene modified cells mixed with different proportion parental cells shoewd obviously bystander effects.Conclusions The laryngocarcinoma cells Hep-2 have sensitive to HSV-tk/GCV system and have significant bystander effects, which might have therapeutic potential value for laryngocarcinoma.

BACKGROUND: Mesenchymal stem cells (MSCs) can differentiate into osteoblasts in the action of dexamethasone; Meanwhile, bone morphogenetic protein-2 (BMP-2) can promote the cell proliferation and differentiation, and matrix secretion in the process of bone repair. BMP-2 plays an important role in treating fracture and bone defects by inducing bone formation both in vivo and in vitro.OBJECTIVE: To analyze whether dexamethasone induction in vitro could enhance the ability of BMP-2 modified MSCs in osteogenic conversion.DESIGN: A randomized paired design.SETTING: Xijing Hospital of the Fourth Military Medical University of Chinese PLA.MATERIALS: The experiments were carried out in the Orthopaedic Oncology Institute of Chinese PLA, the Fourth Military Medical University of Chinese PLA from February to August in 2004. Twenty 20-month-old New Zealand rabbits were provided by the experimental animal center of the Fourth Military Medical University of Chinese PLA [Certificate number: 2005C00117]. The rabbits were raised normally at room temperature with normal humidity. The samples were collected bilaterally, the cells from the left limb were taken as the dexamethasone-induced group, and those from the right limb as the control group.METHODS: MSCs treated and untreated with dexamethasone were transfected with BMP-2 gene, then the BMP-2 expression was determined by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical staining. The growths of the MSCs were observed, and the activity of alkaline phosphatase (ALP) and osteocalcin of MSCs were determined with ALP kit and osteocalcin radioimmunoassay kit after Ad-BMP-2 transfection.MAIN OUTCOME MEASURES: ① BMP-2 expression in MSCs; ② Morphological changes of the MSCs; ③ ALP activity and osteocalcin content in the MSCs. RESULTS: ① Expressions of BMP-2 gene could be observed in MSCs both treated and untreated with dexamethasone after transfection. ② The forms were irregular in most of the MSCs treated with dexamethasone, appeared as triangular or polygonal changes.They grew more slowly than those cultured with basic medium. There were no obvious changes of cellular forms after gene transfection. ③ Five days after transgene, the ALP activity in the MSCs supernatant in the dexamethasone-induced group was higher than that in the control group [(134.36±8.84), (104.02±7.83) nkat/L, t =3.350 6, P ＜ 0.01, n =20]. The amount of osteocalcin secretion in MSCs in the dexamethasone-induced group was higher than that in the control group [(14.68±0.73), (6.52±1.21) μg/L, t =3.568 2, P ＜ 0.01, n =20].CONCLUSION: Dexamethasone induction before transgene can promote the proliferation of BMP-2 modified MSCs and the conversion into osteoblasts.

Objective To explore the possible mechanism of GABA B receptor subunit and epileptogenesis in absence seizure animal model Methods AY 9944 absence seizure model is developed by abdomenal injection of a cholesterol biosynthesis inhibitor Ay 9944 to neonatal Lon Evan hooded rats In situ hybridization is applied to detect GABA B receptor subunit GBR1b pan mRNA in rat brain of absence seizure Result There is increased hybridization signal in cortex, hippocampus and thalamus, especially in cortex (361?48 nCi/g) and hippocampus CA1 region (327?83 nCi/g)( P value 0 020 and 0 027 respectively) Conclusion The result of this study support the hypothesis that GABA B receptor subunit may be involved and induce seizures in absence epilepsy The finding of subunit change may direct the screen of new antiepileptic drug This finding deserves further study

Objective To investigate the clinical and pathological features of Miyoshi myopathy(MM) with dysferlin protein deficient. Methods The clinical and pathological data of the 3 patients with MM were analysed. Results 3 patients were onset at youngster.The clinical manifestation were myastheria and myoatrophy in distal of lower limbs.1 case combined myalgia and tumefaction in lower limbs at early stage of onset;1 case showed myathenia in proximal of lower limbs.The level of serum creatine phosphokinase (CK) was significantly ligher in the 3 cases (7543 IU/L, 5657 IU/L, 8721 IU/L respectively). The level of serum lactic dehydrogenase (LDH) was significantly higher in the 2 cases (456 IU/L ,636 IU/L respectively).The result of muscle pathology was showed myogenic damage in all the cases. The expression of dysferlin protain in membrane of muscle cells was completely deficient, although the expression of dystrophin was normal. Inflammatory cells infiltration was found in 1 case's muscle tissue. Conclusions The clinical characters of MM patient are onset at youngster,myasthenia and myoatrophy in lower limbs.The deficit of dysferlin protain can be found by pathology.

Adolescent ; Blood Glucose ; analysis ; Female ; Humans ; Kidney Transplantation ; Male ; Sodium ; blood ; Sodium Chloride ; administration & dosage

Objective To discuss the influences of irbesartan on high glucose-induced lipid oxidation effect and human umbilical vein endothelial cell (HUVEC) injury. Methods HUVECs of the logarithmic phase were divided into four groups:normal-concentration glucose control group (glucose final concentration of 5.5 mmol/L),high-glucose group (glucose final concentration of 33.3 mmol/L),irbesartan intervention group,and anti-oxidant N-acetyl-cysteinyl acid (NAC) intervention group. The intervention groups were first treated with irbesartan (10-5mmol/L) and NAC (10 mmol/L) for 1 h,respectively,then co-incubated with high glucose (glucose final concentration of 33.3 mmol/L) for 24 h,48 h and 72 h. Malondialdehyde (MDA) content and superoxide dismutase (SOD) activity of culture supernatant were determined by TBARS method and spectrophotometric assay,respectively. Results MDA content increased significantly (P0.05). Compared with that in high glucose group,MDA content significantly decreased (P

Objective To explore the effects of high glucose on release of endothelial microparticles (EMPs) and cell apoptosis in human umbilical vein endothelial cells (HUVECs). Methods HUVECs were incubated for 0h, 12h, 24h, 48h and 72h, respectively, with different concentrations of glucose (5.5mmol/L, 16.7mmol/L and 33.3mmol/L, respectively). The cells cultured in the media containing glucose 5.5mmol/L and mannitol 27.8mmol/L served as control groups. The survival rate of cells was examined by MTT assay. The level of EMPs release and percentage of cell apoptosis were detected by flow cytometry. Results ① According to MTT detection, the survival rate of HUVECs was 93% when incubated for 12h with 33.3mmol/L, 78% for 24h, 51% for 48h, and 40% for 72h. Compared with that for 0h, the survival rate for 12h did not decline significantly (P>0.05). the survival rate reduced significantly for 24, 48 and 72h, respectively, in the time-dependent manner (P<0.05). ② If the survival rate was 100% when incubated in 5.5mmol/L glucose for 48h, then it was 98% in mannitol 27.8mmol/L with no significant difference (P>0.05). It was 79% in 16.7mmol/L glucose and 51% in 33.3mmol/L glucose, obviously lower than that in 5.5mmol/L glucose, and the two groups differed significantly (P<0.05). ③ the level of EMPs release increased significantly in 33.3mmol/L glucose for 12, 24, 48 and 72h respectively, in the time-dependent manner. Compared with the normal glucose group, the level of EMPs release did not significantly differ in mannitol 27.8mmol/L for 12, 24, 48 and 72h , respectively (P>0.05). ④ There was a significantly positive correlation between EMPs release and cell apoptosis (r=0.659, P<0.05). Conclusion High glucose induces release of HUVECs endothelial miccroparticles and promotes cell apoptosis.

Acetamides ; metabolism ; toxicity

Objective To investigate the pathological features of sural nerve biopsy in patients with motor neuron disease （MND）. Methods 22 patients with amyotrophic lateral sclerosis (ALS) underwent sural nerve biopsy and routine electrophysiological examination. The transected images were captured and morphometrically analyzed.Results The myelinated fiber density decreased in ALS patients' sural nerves, and larger fibers were involved mostly. The thinly myelinated fibers increased. Conclusion The sural nerves of ALS patients shows mild but definite pathological changes.

Humans ; Protein Kinase Inhibitors ; therapeutic use ; Protein-Tyrosine Kinases ; antagonists & inhibitors