OBJECTIVETo explore cell death and apoptosis in rat hippocampal neurons at different time points after ischemia, hypoxia and reperfusion injury and to elucidate time window characteristics in ischemia neuronal injury.

METHODSHippocampal neurons were obtained from rat embryo and were cultured in vitro. The ischemia and reperfusion of cultured rat hippocampal neurons were simulated by oxygen-glucose deprivation (OGD) and recovery. OGD at different time points (0.25 h to 3.0 h) and then the same recovery (24 h) were prepared. Annexin V-PI staining and flow cytometry examined neuron death and apoptosis at different time after injury.

RESULTSAfter OGD and recovery, both necrosis and apoptosis were observed. At different times after OGD, there were statistically significant differences in neuron necrosis rate (P < 0.05), but not in apoptosis rate (P > 0.05). At recovery, survival rate of hippocampal neurons further decreased while apoptosis rate increased. Furthermore, apoptosis rates of different time differed greatly (P < 0.05). Apoptosis rate gradually increased with significant difference among those of different time points (P < 0.05). However, 2 h after ischemia, apoptosis rate decreased markedly.

CONCLUSIONSApoptosis is an important pathway of delayed neuron death. The therapeutic time window should be within 2 h after cerebral ischemia and hypoxia.

Animals ; Apoptosis ; physiology ; Brain Ischemia ; pathology ; Cell Death ; physiology ; Cell Hypoxia ; Cells, Cultured ; Disease Models, Animal ; Female ; Fetus ; cytology ; Flow Cytometry ; Hippocampus ; pathology ; Neurons ; pathology ; Pregnancy ; Pregnancy, Animal ; Probability ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; pathology ; Sensitivity and Specificity ; Time Factors

OBJECTIVETo construct an eukaryotic expression vector carrying rat brain-derived neurotrophic factor receptor trkB gene.

METHODSUsing the total RNA isolated from rat brain as template, the trkB gene was amplified by reverse-transcription-polymerase chain reaction (RT-PCR) with a pair of specific primers which contained the restrictive sites of EcoR I and BamH I. The amplified fragment of trkB gene was digested with EcoR I and BamH I, and then subcloned into cloning vector pMD18-T and expression vector pEGFP-C2 respectively. The recombinant plasmids were identified by restriction endonuclease enzyme analysis and PCR.

RESULTSThe amplified DNA fragment was about 1461 bp in length. Enzyme digestion and PCR analysis showed that the gene of trkB had been successfully cloned into vector pMD18-T and pEGFP-C2.

CONCLUSIONSThe trkB gene of rat has been amplified and cloned into the eukaryotic expression vector pEGFP-C2.

Animals ; Brain-Derived Neurotrophic Factor ; genetics ; pharmacology ; Cloning, Molecular ; methods ; Eukaryotic Cells ; Female ; Gene Expression Regulation ; Genetic Therapy ; methods ; Genetic Vectors ; Male ; Models, Animal ; RNA ; analysis ; Rats ; Rats, Wistar ; Receptor, trkB ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Schwann Cells ; cytology ; Sensitivity and Specificity ; Templates, Genetic ; Transfection

OBJECTIVETo investigate the effect of 5-aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) on human gastric cancer xenografts in vivo and to explore its potential tumoricidal mechanism.

METHODSCultured MGC-803 human gastric cancer cells were injected below the skins of the nude mice to develop the tumor model. The tumor-bearing nude mice were examined under the Leica LT-9 MACIMSYSPULS to detect the fluorescence. The tumor volume of day 1, 3, 7, 14, 21 after treatment were measured, and its histological changes were also studied. The tissues of the tumors in nude mice of the control group, light group, 5-ALA group and PDT group were examined with the electron microscope and apoptosis was detected by TUNEL assay.

RESULTSThe tumor model was successfully developed. The tumor in the nude mice emitted the red fluorescence under the Leica LT-9 MACIMSYSPULS. The tumor volumes were (0.189+/-0.010) cm(3), (0.183+/-0.011) cm(3), (0.185+/-0.019)cm(3), (0.182+/-0.015)cm(3) for the control group, light group, 5-ALA group, PDT group, respectively at day 1 after treatment, while at day 3, (0.294+/-0.010) cm(3), (0.280+/-0.013) cm(3), (0.278+/-0.016) cm(3), (0.183+/-0.014) cm(3); at day 7, (0.409+/-0.016) cm(3), (0.411+/-0.009) cm(3), (0.407+/-0.015) cm(3), (0.221+/-0.008) cm(3); at day 14, (0.970+/-0.055) cm(3), (0.976+/-0.054) cm(3), (0.981+/-0.032)cm(3), (0.318+/-0.005) cm(3); at day 21, (1.495+/-0.059) cm(3), (1.513+/-0.057) cm(3), (1.524+/-0.063) cm(3), (0.446+/-0.042) cm(3) (F=1003.086, P=0.000). The histology demonstrated that most tumor blood vessels were congested and necrosis developed after PDT while not in the control group, light group and 5-ALA group. Necrosis and apoptosis were observed in the cells of the tumors of the PDT group examined by TUNEL and electron microscope while not in the cells of the tumors of the other groups.

CONCLUSIONS5-aminolevulinic acid-mediated photodynamic therapy (PDT) can induce injury to human gastric cancer xenografts and inhibit the tumor growth while light only and 5-ALA only can not. 5-aminolevulinic acid-mediated photodynamic therapy (ALA- PDT) appears to be a promising therapy for human gastric cancer, whose mechanism involves in the destruction of the tumors partly by apoptosis other than necrosis.

Aminolevulinic Acid ; therapeutic use ; Animals ; Cell Line, Tumor ; Female ; Humans ; Male ; Mice ; Mice, Nude ; Neoplasm Transplantation ; Neoplasms, Experimental ; Photochemotherapy ; Stomach Neoplasms ; therapy ; Xenograft Model Antitumor Assays

OBJECTIVETo construct lentivector carrying Tie2-Small interfering RNA (SiRNA), so as to study its influence on malignant melanoma cells.

METHODSRecombinant plasmid pSilencer 1.0-U6-Tie2-siRNA and plasmid pNL-EGFP were digested with XbaI, ligated a target lentiviral transfer plasmid of pNL-EGFP-U6-Tie2-I or pNL-EGFP-U6-Tie2-II, and then the electrophoresis clones was sequenced. Plasmids of pNL-EGFP-U6-Tie2-I and pNL-EGFP-U6-Tie2-II were constructed and combined with pVSVG and pHelper, respectively, to constitute lentiviral vector system of three plasmids. The Lentiviral vector system was transfected into 293T cell to produce pNL-EGFP-U6-Tie2- I and pNL-EGFP-U6-Tie2-II lentivirus. Then the supernatant was collected to determine the titer. Malignant melanoma cells were infected by both lentiviruses and identified by Realtime RT-PCR to assess inhibitory efficiency.

RESULTSThe recombinant lentiviral vectors of Tie2-RNAi were constructed successfully which were analyzed with restriction enzyme digestion and identified by sequencing. And the titer of lentiviral vector was 8.8 x 10(3)/ml, which was determined by 293T cell. The results of Realtime RT-PCR demonstrated that the lentiviral vectors of Tie2-RNAi could infect malignant melanoma cells and inhibit the expression of Tie2 genes in malignant melanoma cells (P<0.01). There was no significant difference in the expression level (P>0.05) between the two lentiviral vectors of Tie2-RNAi.

CONCLUSIONSLentivector carrying Tie2-SiRNA can be constructed successfully and inhibit the expression of Tie2 gene in vitro significantly. The study will supply the theory basis for the further research on the inhibition of tumor growth in vivo.

Cell Line, Tumor ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; Melanoma ; genetics ; Plasmids ; RNA Interference ; RNA, Small Interfering ; genetics ; Receptor, TIE-2 ; genetics ; Transfection

To investigate the efficacy and feasibility of parent non-T cell depleted haploidentical bone marrow transplants (haploidentical BMT) for children with leukemia, the efficacy of haploidentical BMT was evaluated in 8 leukemia children (1.9-9 years) received hematopoietic stem cell transplantation, donors were their parents with HLA-mismatched for two or three loci. Five children were pre-conditioned with a myeloablative regimen consisting of high-dose cytarabine (Ara-C), cyclophosphamide (CY) and total body irradiation. Busulfan (BU), Ara-C and CY were used for preconditioning regimen in other three children. The donors were given G-CSF prior to marrow harvest and the non-T-cell depleted grafts were used. A combination of CsA, MTX, ATG, mycophenolate mofetil (MMF) and CD25 monoclonal antibody were used for GVHD prophylaxis. The results showed that rapid engraftment was observed in all cases after transplantation by cytogenetic evidence. The mean time of neutrophil count exceeded 0.5 x 10(9)/L and the mean time of platelet count exceeded 20 x 10(9)/L were 16 and 17 days after transplantation respectively. Incidence of lethal aGVHD was lower, II-III acute aGVHD was found only in one out of eight patients. Chronic GVHD was observed in five patients, 4 from which showed local cGVHD, one developed extensive cGVHD. During the follow-up of 33 months (range 7-56 months), two patients died from relapsed leukemia, including one relapsed as donor-origin leukemia. Disease-free survival was achieved in the remaining six patients. No death occurred during the follow-up of 6 months. It is concluded that above-mentioned preconditioning regimen and GVHD prophylaxis procedure in non-T-cell depleted bone marrow transplantation from HLA-mismatched parents are effective approaches and safe strategy for the treatment of children leukemia.
Bone Marrow Transplantation ; adverse effects ; methods ; Child ; Child, Preschool ; Female ; Graft vs Host Disease ; prevention & control ; Haploidy ; Humans ; Infant ; Leukemia ; therapy ; Leukemia, Myeloid, Acute ; therapy ; Male ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; therapy ; T-Lymphocytes ; cytology

OBJECTIVETo investigate the effect of 5-aminolevulinic acid (5-ALA)-mediated photodynamic therapy (PDT) on MGC-803 human gastric cancer cells in vitro.

METHODSMGC-803 human gastric cancer cells were treated with 5-ALA at various concentrations followed by laser irradiation. The cells were also treated with 5-ALA at the same concentration before laser exposure at various doses. PDT-induced phototoxicity of the cells was determined by MTT assay.

RESULTSAfter laser exposure of the cells at the same dose (25.0 J/cm(2)), the cell survival rates decreased significantly with incubation of the cells with 5-ALA at 0.25, 0.5, 1.0, 2.0 and 4.0 mmol/L, respectively (F=266.39, P<0.001), but 2.0 and 4.0 mmol/L ALA showed no significant difference in lowering the cell survival rates (P>0.05). Following treatment with the same 5-ALA concentration (1 mmol/L), the cell survival rates decreased in response to increased laser doses (at 6.25, 12.5, 25.0, 50.0, and 100 J/cm(2), respectively, F=226.31, P<0.0001). Without laser exposure, the survival rate of the cells did not significantly change for different 5-ALA concentrations (F=0.79, P=0.5383), nor did it undergo obvious variation in response to different laser doses without 5-ALA incubation (F=0.61, P=0.6551).

CONCLUSIONSThe damage of MGC-803 cells by PDT increases with 5-ALA concentration within a relative lower range and is proportional to the laser doses delivered. Without 5-ALA treatment, the laser at the chosen dose cannot produce photodynamic effect and ALA itself is nontoxic. ALA-mediated PDT appears to be a promising therapy for gastric cancer.

Aminolevulinic Acid ; pharmacology ; Cell Line, Tumor ; Cell Survival ; drug effects ; radiation effects ; Dose-Response Relationship, Drug ; Dose-Response Relationship, Radiation ; Humans ; Lasers ; Photochemotherapy ; Photosensitizing Agents ; pharmacology ; Stomach Neoplasms ; drug therapy ; pathology

UNLABELLEDTo explore the occurrence patterns of neurological complications following haploidentical bone marrow transplantation, a series of clinical data as the onset time, etiology, choices of therapies and prognosis in 10 patients with neurological disorders were summarized. The results showed that complications occurred in 10 out of 74 patients after bone marrow transplantation, which include 3 with encephalorrhagia, 3 infection, 1 epilepsy, 1 Guillian-Barr's syndrome, 1 encephalopathy and 1 schizophrenia. 4 patients died of neurological complications.

IN CONCLUSIONneurological complications commonly occurred in haploidentical bone marrow transplantation, and encephalorrhagia might be the main indication that needs intensive care. Moreover, central nerve system infection and peripheral nerve diseases associated with slow immune reconstitution should have clinical interests.

Adolescent ; Adult ; Bone Marrow Transplantation ; adverse effects ; immunology ; Child ; Female ; Histocompatibility ; Humans ; Intracranial Hemorrhages ; etiology ; Leukemia ; surgery ; Male ; Nervous System Diseases ; etiology

In order to evaluate the diagnostic value of fibrotic bronchoscopy (FB) in the pulmonary infiltration following bone marrow transplantation (BMT), 18 patients with pulmonary complications after BMT from November 2003 to March 2006 were performed with FB. Bronchoalveolar lavage (BAL) and brushing were performed in patients who had received short-term empirical therapy without good response, and transbronchial lung biopsy (TBLB) was carried out in 3 cases. The results showed that 9 out of 10 cases with pulmonary infection, including bacterial pneumonia (n = 3), aspergillosis (n = 2), pneumocystis carinii pneumonia (n = 3) and viral infection (n = 1) were diagnosed by using FB. One case was diagnosed as tuberculosis after open lung biopsy following negative results from twice BAL. 2 out of 8 cases were diagnosed by TBLB as noninfectious pulmonary complications. In conclusion, FB, especially with BAL, is a safe and useful procedure for the evaluation of pulmonary complications, which is particularly suitable for diagnosis of pulmonary infection after BMT. Furthermore, TBLB should be recommended in order to avoid open lung biopsy, if the patients tolerate the operation.
Adolescent ; Adult ; Bone Marrow Transplantation ; adverse effects ; Bronchoalveolar Lavage Fluid ; microbiology ; parasitology ; Bronchoscopy ; Child ; Female ; Humans ; Lung Diseases ; diagnosis ; etiology ; Male ; Middle Aged ; Pneumonia ; diagnosis ; etiology ; microbiology ; Pneumonia, Pneumocystis ; diagnosis ; etiology ; microbiology ; Young Adult

Age Distribution ; Aged ; Aged, 80 and over ; Calcium ; blood ; Female ; Herpes Zoster ; blood ; epidemiology ; Humans ; Incidence ; Male ; Middle Aged ; Neuralgia, Postherpetic ; blood ; epidemiology

The objective of study was to investigate the effect of low-dose antithymocyte globulin (ATG) on steroid-resistant severe acute graft versus host disease (aGVHD). Six patients with steroid-resistant severe aGVHD after haploidentical bone marrow transplantation (BMT) received the treatment with ATG at a low dose of 1.25 mg/kg for 3 - 5 doses every other day. The results showed that 3 out of 6 patients got completely remission (CR), among them 2 patients have still been in disease-free survival, 1 patient died from leukemia relapse. 1 out of the other 3 patients got partial remissin (PR), 2 patients were aggravated. The other 3 patients all died from GVHD. The major complications observed in these patients were infections. In conclusion, low-dose ATG is effective for some patients with steroid-resistant severe aGVHD, and has not severe side effect. To strengthen environmental protection should be considered as important for prevention of infection.
Adolescent ; Adult ; Antilymphocyte Serum ; administration & dosage ; Bone Marrow Transplantation ; adverse effects ; Child ; Female ; Graft vs Host Disease ; drug therapy ; etiology ; HLA Antigens ; immunology ; Haplotypes ; immunology ; Humans ; Male ; Young Adult