BACKGROUND: Gene therapy is a popular topic in domestic and overseas studies on biological therapy for brain tumor.OBJECTIVE: By using a newly constructed eukaryotic expression vector of pCR3-TK, the effect of the HSV-TK/ACV system on the proliferative activity of human glioma cells was investigated.DESIGN: Experimental study based on cells.SETTING: Department of neurosurgery and department of oncology in a university hospital.MATERIALS: The study was conducted at the National Key Laboratory of Veterinary Biotechnology of Harbin Veterinary Research Institute from January to April in 2004. The eukaryotic expression vector of pCR3-TK was constructed by the author. The TJ905 strain was a gift from professor Pu Pei-yu, who worked in the Neurology Institute of Tianjin city. The nontransfected cells and the cells transfected with pCR3-Uni vector were set as controls.METHODS: By using Lipofectamine(a cationic liposome), the pCR3-Uni vector and the recombinant pCR3-TK plasmid(inserted with HSV-TK gene)were transfected into the human glioma cell strain-TJ905. Then the positive clones were picked out and were given ACV(50 mg/L) . Totally 72 hours later, the cover slips were collected and silver staining for nucleolus organizer regions(AgNORs) was performed.MAIN OUTCOME MEASURES: After the ACV treatment and AgNORs staining, the numbers of silver-stained granules in TJ905 cells with or without transfections were counted respectively.RESULTS: In those cells transfected with HSV-TK gene, after ACV treatment, a significant decreasing in proliferative activity could be observed, and the average numbers of the silver-stained granules in cells transfected with pCR3-Uni or pCR3-TK were 14.33 and 6.67 respectively( P ＜ 0.01).CONCLUSION: As an easy-to-operate method, AgNOR counting is helpful for the studies on the proliferative activity of cells and the investigations into the potential anti-tumor mechanism of the HSV-TK/ACV system.

Objective To investigate the effect of lipid emulsion on mitochondrial energy metabolism during bupivacaine-induced myocardiotoxicity in rats.Methods H9c2 cells were inoculated in 96-well plates at a density of 1 × 105cells/ml, and were randomly divided into 4 groups (n =18 each) using a random number table: control group (group C) , bupivacaine group (group B) , lipid emulsion group (group LE) , and bupivacaine + lipid emulsion group (group B + LE).The cells were incubated in the normal culture medium in group C.In group LE, the cells were incubated in the culture medium containing 1％ lipid emulsion.In group B, the cells were incubated in the culture medium containing 1 mmol/L bupivacaine.In group B + LE, the cells were incubated in the culture medium containing 1 mmol/L bupivacaine and 1％ lipid emulsion.After 24 h of incubation, the contents of ATP, ADP, and AMP were measured by high-performance liquid chromatography, and apoptotic rate was calculated by Hoechst33342/ PI staining.Results Compared with group C, the contents of ATP, ADP and AMP were significantly decreased, and apoptotic rate was increased in group B (P＜0.05), and the contents of ATP and ADP in group LE and ATP content in group B + LE were increased, and no significant changes were found in apoptotic rate in LE and B+LE groups (P＞0.05).Compared with group C, the contents of ATP, ADP and AMP were significantly increased, and apoptotic rate was decreased in LE and B+LE groups (P＜ 0.05).Compared with group LE, the contents of ATP, ADP and AMP were significantly decreased (P＜ 0.05), and no significant change was found in apoptotic rate in group B+LE (P＞0.05).Conclusion The mechanism by which lipid emulsion reduces bupivacaine-induced myocardiotoxicity may be associated with improved mitochondrial energy metabolism in rats.

Objective To evaluate the effect of mitochondrial permeability transition pore (mPTP) in lipid emulsion-induced inversion of bupivacaine myocardiotoxicity in rats.Methods H9c2 cells were inoculated in 6-well plates at a density of 105 cells/ml, and were randomly divided into 4 groups (6 wells in each group, 2 ml/well) using a random number table: control group (group C) , bupivacaine group (group B) , lipid emusion + bupivacaine group (group LB) , and lipid emusion + bupivacaine + atractyloside group (group LBA).Phosphate buffer solution 100 μl was added to the culture medium in group C.In group B, bupivacaine was added to the culture medium with the final concentration of 1 mmol/L.In group LB, lipid emusion and bupivacaine were added to the culture medium with the final concentrations of 1％ and 1 mmol/L, respectively.In group LBA, lipid emusion, bupivacaine and atractyloside (an mPTP opener) were added to the culture medium with the final concentrations of 1％, 1 mmol/L and 30 μmol/L, respectively.All the cells were incubated for 24 h.After the end of incubation, the expression of Bcl-2, Bax, phosphorylated Bad (p-Bad) , caspase-3, activated caspase-3, caspase-9,activated caspase-9 and cytochrome c (Cyt c) was detected using Western blot.The expression of Bcl-2 mRNA, Bax mRNA, Bad mRNA, caspase-9 mRNA and Cyt c mRNA was detected using real-time reverse transcriptase polymerase chain reaction.The ratios of Bax/Bcl-2, activated caspase-3/caspase-3, activated caspase-9/caspase-9, and Bax mRNA/Bcl-2 mRNA were calculated.Results Compared with group C,the ratios of Bax/Bcl-2, activated caspase-3/caspase-3, activated caspase-9/caspase-9, and Bax mRNA/ Bcl-2 mRNA were significantly increased, the expression of p-Bad was down-regulated, and the expression of Cyt c, Bad mRNA, caspase-9 mRNA and Cyt c mRNA was up-regulated in group B (P＜0.05) , and no significant change was found in the parameters mentioned above in group LB (P＞0.05).Compared with group B, the ratios of Bax/Bcl-2, activated caspase-3/caspase-3, activated caspase-9/caspase-9, and Bax mRNA/Bcl-2 mRNA were significantly decreased, the expression of p-Bad was up-regulated, and the expression of Cyt c, Bad mRNA, caspase-9 mRNA and Cyt c mRNA was down-regulated in LB and LBA groups (P＜ 0.05).Compared with group LB, the ratios of Bax/Bcl-2, activated caspase-3/caspase-3, activated caspase-9/caspase-9, and Bax mRNA/Bcl-2 mRNA were significantly increased, the expression of p-Bad was down-regulated, and the expression of Cyt c, Bad mRNA, caspase-9 mRNA and Cyt c mRNA was up-regulated in group LBA (P ＜ 0.05).Conclusion The mechanism underlying lipid emulsioninduced inversion of bupivacaine myocardiotoxicity is related to inhibited mPTP opening in rats.

Objective To find time pint of the irreversible kidney injuru by observing renal pathological appearance and podocytes phenotype after complete ureteral obstruction.Methods The animal model of complete unilateral uretersal obstruction was consructed in 2-weekold and 2-month-old rats respectively.HE staining was employed to evaluate the degree of pathological changes,and immunofluorescence double staining to observe the transitional progression of podocyte.Results Grossly,there appeared pyelectasis at 2 h after obstruction and thinner renal cortex after 3 days of obstruction.Histologically,proximal tube display dilated and hydropic degeneration of tubular epithelial cells was observed after 12-day obstruction.The tubular epithelium showed distorted swelling after 3-day obstruction.Immunofluorescence double staining showed that podocytes decreased and the double-labeled macrophages was increased during 1 d to 2 d after unilateral ureteral obstruction operation.Conclusion Compared with control groups,the unilateral ureteral obstruction groups shows podocyte-to-macrophage transition at 1 d after the operation, while the pelvis shows obviously dilated.From the 1st day to the 7th day after unilateral ureteral obstruction,it shows increasing tendency of the podocytes transition.

Objective: To investigate the specific effects of resistant starch (RS) on zinc status in rats. Methods: (1)Zinc metabolism in normal rats: Three groups of Wistar rats were fed with basal diet (control), diet with 13% or 26% RS respectively, for 18 days. Urine, fecal and blood samples were collected for zinc measurement. Zinc apparent absorption was calculated.(2)Zinc status in rats fed with high sucrose (50%) diet: Three groups of Wistar rats were fed with basal diet, high sucrose digestible starch diet (S DS) or high sucrose resistant starch diet (S RS, containing 14% RS) respectively for 12 w. Samples of urine, blood, liver, pancreas and kidney were collected for measurement of zinc content. Results: (1)Zinc apparent absorption in normal rats was: control group 56.59%, 13%RS group 50.11%, and 26%RS group 54.40%. (2)High sucrose diet led to increased postprandial glucose and HbA 1C and depressed fast insulin level in S DS group rats, with decreased plasma and pancreas zinc level (compared with control group, P

Objective To determine the level of peripheral blood CD34 positive (CD34+) cells in patients with acute cerebral infarction (ACI),and to explore its clinical significance.Methods The level of peripheral blood CD34+ cells was determined by flow cytometry within 72 hours of onset of patients with acute cerebral infarction (infarct group,n=45),cerebrovascular risk factors in patients without cerebral infarction (high risk group,n=27) and healthy subjects (control group,n=20).The neural function defect score,infarction lesion volume and carotid artery intima-media thickness (IMT) were determined in patients with infarction group.Results The percentages of peripheral blood CD34 cells in infarction group (0.034 ±0.012)％ and the high risk group of patients (0.047±0.009)％ were lower than that of control group(0.063±0.009)％,and were lower in infarction group than in high-risk groups (all P＜0.05).The percentages of peripheral blood CD34+cells were significantly decreased compared with control group (P＜0.05) in infarction patients with mild [(0.047±0.009)％],moderate [(0.036±0.009)％],severe [(0.022±0.007)％] infarction nervous function defect score.Wherein,the percentages were lower in severe group than in the moderate group,moderate group was lower than in mild group (all P＜0.05).The percentages of peripheral blood CD34 cells in infarction patients with small,moderate,large infaret lesion volume were lower than in control group (P＜0.05),wherein,were lower in large group than in moderate group,lower in moderate group than in treatment group (all P＜0.05).Infarction patients were confirmed with carotid atherosclerosis (CAS) by carotid ultrasound.The extent of lesion were divided into carotid artery intimal thickening group [(0.043±0.010)％],carotid artery plaque group [(0.036±0.010)％],and carotid artery stenosis group [(0.023±0.009)％].The levels of peripheral blood CD34+ cells in three groups of patients were decreased compared with control group.The levels were lower in carotid artery stenosis group than in carotid artery plaque group,lower in carotid artery plaque group than in carotid artery intimal thickening group (all P＜0.05).Conclusions The level of peripheral blood CD34+ cells in acute cerebral ischemia is reduced,it can become a sensitive and early indicator of cerebral ischemia,and its level is related to neurologic impairment,infarction size and the degree of carotid artery atherosclerosis.

OBJECTIVETo investigate the changes of genetic damage in patients with arsenism caused by coal-burning in 9 years. To analyze the relationship between the changes of genetic damage and disease progression and provide a basis for condition monitoring.

METHODSOf 206 arsenism patients from the area with endemic arsenism in Guizhou province were tracking surveyed in February 1998 and divided into 4 groups, including suspicious, mild, moderate and severe poisoning group. Another 67 healthy residents from a neighbour township 12 km away where arsenic was not prevalent were surveyed. Over a 9-year follow-up, 131 arsenism patients and 45 controls with the complete biochemical indexes among them were selected as subjects in December 2006. Arsenic (As) concentration of urine and hair were detected by silver diethyldithiocarbamate spectrophotometry (Ag-DDC). Micronucleis (MN) and chromosome aberrations (CA) were analyzed by conventional methods. DNA single-strand breaks of peripheral blood were measured by single cell gel electrophoresis (SCGE), and the tail lengths of comet were used to measure DNA damage.

RESULTSAmong the control, suspicious, mild, moderate and severe arsenic poisoning group, the As contents of urine and hair were respectively (34.16 ± 10.25), (52.35 ± 22.41), (62.26 ± 31.13), (71.43 ± 49.92), (78.45 ± 50.64) µg/L and (1.37 ± 0.56), (3.69 ± 1.78), (4.88 ± 3.49), (5.21 ± 3.10), (6.25 ± 4.04) µg/g in 2006, which were lower than that 9 years before (urine as contents were (36.07 ± 20.70), (73.65 ± 41.33) , (90.92 ± 82.14) , (126.55 ± 107.31) and (139.44 ± 90.90) µg/L, and hair As contents were (1.41 ± 1.18), (4.85 ± 4.20), (5.72 ± 4.07) , (6.43 ± 4.32) and (7.19 ± 4.68) µg/g, respectively, F value was 10.63, 7.72, 14.66, 11.00 respectively, all P values were < 0.05). Except for suspicious poisoning group, the differences of urine As contents in the other groups all showed significance (P < 0.05). The incidences of MN were (0.238 ± 0.130) %, (0.268 ± 0.192) %, (0.283 ± 0.157) % and (0.391 ± 0.233)%; the incidences of CA were (14.36 ± 5.44) %, (18.09 ± 6.49) %, (19.38 ± 5.63)% and (19.83 ± 5.84) %; the tail lengths of comet were (29.88 ± 13.81) , (29.84 ± 12.80) , (34.50 ± 9.88) and (41.58 ± 12.98) µm respectively in 2006 for all poisoning groups; which were higher than that 9 years before(the incidences of MN were (0.163 ± 0.051) %, (0.186 ± 0.117) %, (0.196 ± 0.104) % and (0.273 ± 0.142) %; the incidences of CA were (13.18 ± 5.17)%, (14.48 ± 6.61)%, (15.67 ± 8.49) % and (16.90 ± 8.38) %; the tail lengths of comet were (15.07 ± 12.93) , (19.57 ± 8.80) , (27.03 ± 10.77) and (34.71 ± 14.95) µm) , except for the incidences of MN and CA in suspicious poisoning group and of MN in mild poisoning group , the differences of the three indexes in the other groups were significant (P < 0.05) . The state of illness of arsenic poisoning patients aggravated 9 years later. With the increase of urine and hair As contents and the development of arsenism, the incidences of MN, CA and the tail lengths of comet of all poisoning groups increased. There were positive correlations among them (r values were respectively 0.212, 0.316, 0.232, 0.263, 0.321, 0.654 and 0.760) (P < 0.05).

CONCLUSIONThe exacerbation of genetic damage was related to constantly high arsenic loads. The accumulation of genetic damage and its irreversibility might be one of the important reasons of the development of arsenism and cancer.

Background and Objectives: Acute myocardial infarction (AMI) often occurs suddenly and leads to fatal consequences. Ferroptosis is closely related to the progression of AMI.However, the specific mechanism of ferroptosis in AMI remains unclear. Methods: We constructed a cell model of AMI using AC16 cells under oxygen and glucose deprivation (OGD) conditions and a mice model of AMI using the left anterior descending (LAD) ligation. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide was employed to determine cell viability. The levels of lactate dehydrogenase, creatine kinase, reactive oxygen species (ROS), glutathione (GSH), malondialdehyde (MDA), and iron were measured using corresponding kits. Dual luciferase reporter gene assay, RNAbinding protein immunoprecipitation, and RNA pull-down were performed to validate the correlations among AC005332.7, miR-331-3p, and cyclin D2 (CCND2). Hematoxylin and eosin staining was employed to evaluate myocardial damage. Results: AC005332.7 and CCND2 were lowly expressed, while miR-331-3p was highly expressed in vivo and in vitro models of AMI. AC005332.7 sufficiency reduced ROS, MDA, iron, and ACSL4 while boosting the GSH and GPX4, indicating that AC005332.7 sufficiency impeded ferroptosis to improve cardiomyocyte injury in AMI. Mechanistically, AC005332.7 interacted with miR-331-3p, and miR-331-3p targeted CCND2. Additionally, miR-331-3p overexpression or CCND2 depletion abolished the suppressive impact of AC005332.7 on ferroptosis in OGD-induced AC16 cells. Moreover, AC005332.7 overexpression suppressed ferroptosis in mice models of AMI. Conclusions AC005332.7 suppressed ferroptosis in OGD-induced AC16 cells and LAD ligation-operated mice through modulating miR-331-3p/CCND2 axis, thereby mitigating the cardiomyocyte injury in AMI, which proposed novel targets for AMI treatment.

Objective: To investigate the differences of clinicopathological features, diagnosis, treatment and prognosis between patients with extra-gastrointestinal stromal tumors (EGIST) and duodenal gastrointestinal stromal tumors (DGIST). Methods: A retrospective case - control study was performed. Case inclusion criteria: (1) tumor confirmed by histology and pathology; (2) primary tumor locating in the extra - gastrointestinal tract or duodenum; (3) without other synchronous tumors; (4) complete clinical and pathological data. Clinical data of 20 EGIST patients and 32 DGIST patients from March 2011 to September 2016 at Department of Gastrointestinal Surgery, Ruijin Hospital, Shanghai Jiaotong University School of Medicine were retrospectively collected and analyzed. The observational parameters included clinicopathological characteristics, treatment and prognosis conditions. Continuous data of abnormal distribution were expressed as median (range) and compared using the Mann-Whitney U-test. Survival curves were drawn by the Kaplan-Meier method and compared with the Log-rank test. Results: Of the 20 EGIST patients, 8 were males and 12 were females with age of 61.0 (30.0 to 86.0) years and of the 32 DGIST patients, 12 were males and 20 were females with age of 55.5 (27.0 to 70.0) years. Compared with DGIST patients, EGIST patients were older (U=188.000, P=0.012], had larger tumor size [10.0 (3.0 to 29.0) cm vs. 4.0 (1.5 to 10.0) cm, U=98.500, P<0.001] and higher ratio of high risk classification [85.0% (17/20) vs. 12.5% (4/32), χ²=26.870, P<0.001]. Among the 20 EGIST patients, 5 were diagnosed with distal metastasis and received imatinib (400 mg/d), and the other 15 patients underwent radical resection who were included in survival analysis. All the 32 DGIST patients underwent radical resection. The median follow-up of whole group was 43 (14 to 76) months. The 3-year recurrence/metastasis-free survival rate of 15 cases undergoing radical resection in the EGIST group was 85.6%, which was lower than that of the DGIST group (88.6%), and the difference was not statistically significant (P=0.745). There was no significant difference in the 3-year overall survival rate between the EGIST group (92.9%) and the DGIST group (100%) (P=0.271). Conclusions As compared to DGIST, EGIST mostly occurs in those with older age, larger tumor size and higher risk grade. The prognosis of EGIST patients after radical resection is similar to that of DGIST patients.

Objective:To analyze the in vitro inhibitory effects of resveratrol on rabies virus. Methods:The challenge virus standard (CVS)-11 strain of rabies virus and BHK-21 cells were used to establish the infection model. In vitro inhibitory effects of resveratrol on rabies virus were analyzed at different stages of infection by direct immunofluorescence and cell fluorescence focus unit assay. Results:Without affecting cell growth, resveratrol could block the adsorption of virus, interfere with the entry of virus into cells and inhibit virus proliferation in a concentration-dependent manner. The inhibition rate could reach up to about 95%. The results of co-incubation experiment showed that 40 μmol/L resveratrol could directly kill the virus.Conclusions:This study indicated that resveratrol inhibited the activity of rabies virus in a concentration-dependent manner.