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 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 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 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 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.

Objective:To evaluate the protective efficacy and safety of Brucella 104M against aerosol challenge in BALB/c mice and characterize its immunological effects. Methods:Female mice of 6-8 weeks old were immunized with Brucella abortus strain 104M by intratracheal aerosol delivery or intranasal instillation or subcutaneous injection route. Six mice of each group were sacrificed at 4, 8, 16, 24 weeks after immunization. At each time point, the clinical manifestations of mice were investigated, the serum, spleen and lung samples of mice were collected, body weight, spleen weight, bacteria loads in spleens, the anti- Brucella antibodies titers in serum and the cytokines concentrations of IFN-γ, IL-18 in serum or lung homogenate of the mice were detected. Twenty two weeks after immunization, all the mice were challenged with Brucella A19 through intratracheal aerosol delivery. Results:Compared with the control group, neither abnormal clinical symptoms nor significant changes in body weight were found in 104M immunization groups, at each time point when immunized through either nose dropping route, subcutaneous injection or aerosol routes; and the spleen weight of immunization groups were lower than control group after challenge ( P<0.05): *M1 (0.26±0.16)g

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 effect of different doses of rocuronium on the monitoring of recurrent laryngeal nerve during endoscopic thyroidectomy.Methods A total of 116 patients undergoing endoscopic thyroidectomy through areolar approach were selected from October 2021 to October 2022,30 males and 86 females,aged 18-64 years,BMI 18.5-30.0 kg/m2,ASA physical status Ⅰ or Ⅱ.All the patients were divided into three groups by random number table method:rocuronium 0.30 mg/kg group(group R1,n=39),rocuronium 0.45 mg/kg group(group R2,n=39),and rocuronium 0.60 mg/kg group(group R3,n=38).After induction of anesthesia,groups R1,R2,and R3 were injected intrave-nously with rocuronium 0.30,0.45,and 0.60 mg/kg,respectively.When the TOF value was 0,the nerve monitoring tracheal catheter was inserted,and the muscle relaxation was monitored throughout the operation.No muscle relaxants were added before the end of the nerve monitoring.The time and amplitude of recurrent laryngeal nerve electromyography(EMG)from intravenous rocuronium to the first occurrence were recorded.The time of intubation and quality of tracheal intubation(Cooper's score),intraoperative special conditions(hypotension,hypertension,bradycardia,tachycardia,intraoperative movement,etc.),postoperative throat pain,hoarseness,and muscle pain were recorded.Results There was no significant difference in the time of first occurrence of recurrent laryngeal nerve EMG among the three groups.Compared with group R1,the recurrent laryngeal nerve EMG amplitude in groups R2 and R3 was significantly decreased for the first occurrence(P＜0.05).Compared with group R1,the time of intubation in groups R2 and R3 was signifi-cantly shortened(P＜0.05).Compared with group R2,the time of intubation in group R3 was significantly shortened(P＜0.05).Compared with group R1,the quality of tracheal intubation in groups R2 and R3 was significantly higher(P＜0.05).Compared with group R1,the incidence of intraoperative and postop-erative laryngeal pain in groups R2 and R3 was significantly lower(P＜0.05).Conclusion During endo-scopic thyroidectomy,compared with rocuronium 0.30 mg/kg,rocuronium 0.45 and 0.60 mg/kg can not only provide good conditions for tracheal intubation,but also monitor recurrent laryngeal nerve signals,and rocuronium 0.60 mg/kg can be intubated for a shorter time.