Adult ; Female ; Humans ; Male ; Mercury Poisoning ; diagnosis ; therapy ; Middle Aged

Objective To investigate the effects of hypoxia-inducible factor (HIF)-1α on glycolysis of human esophageal squamous carcinoma cells and the possible mechanism.Methods TE13 and Eca109 cells were cultured under normal oxygen (20％O2) and hypoxia (1％O2) conditions.The hypoxia was duration 6 hours,12 hours,24 hours and 48 hours.HIF-1α gene was stable silented by RNA interference method and TE13/small interfering HIF cells and Eca109/siHIF cells were obtained.The cell culture condition and time was same as TE13 and Eca109 cells.The changes of HIF-1α expression were detected by Western-blot.The changes of lactic acid concentration in cell culture supernatant were determined by Spectrophotometry.The changes of glucose transporter-1 (GLUT-1) and lactic dehydrogenase A (LDHA) expression at mRNA level were examined by realtime polymerase chain reaction.The changes of GLUT-1 and LDHA expression at protein level were tested by Western blot.Using t or t' test to analyze the effects of hypoxia duration on HIF-1αexpression at protein level.One-way ANOVA was applied for the difference analysis between the groups.Results In TE13 and Eca109 cells,the HIF-1α expression significantly increased under hypoxia condition and reached the peak at 12 hour (t=6.11,8.31; both P＜0.05).The lactic acid secretion of TE13/siHIF cells and Eca109/siHIF cells was (1.24±0.33) and (1.28±0.37) mmol/L,which significantly decreased when compared with TE13 and Eca109 cells [(3.25±1.34) and (4.91±1.69) mmol/L,t=2.53,3.59,both P＜0.05].The lactic acid secretion of TE13 and Eca109 cells significantly increased after hypoxia [(6.48±1.73) and (8.02± 1.95) mmol/L,t=2.715,2.050,both P＜0.05].There was no significant lactic acid secretion in TE13/siHIF cells and Eca109/siHIF cells after hypoxia (P ＞ 0.05).The expressions of GLUT-1 and LDHA at mRNA level were significantly suppressed in TE13/siHIF cells and Eca109/siHIF cells (normal oxygen:t=6.98,3.92,7.25,3.67,all P＜0.05).The expression of GLUT-1 at protein level remarkably weaked (normal oxygen:t=4.57、16.56,hypoxia:t=6.19、6.09,all P＜0.05),while the expression of LDHA at protein level slightly decreased (P＞0.05).Conclusions The level of glycolysis can be lowered by suppression HIF-1α expression in human esophageal squamous carcinoma cells.The pathway may be involved in the suppression of GLUT-1 and LDHA expression.Except for HIF-1α,there may be other regulating factors in LDHA protein expression at same time.

Objective To investigate the expression of Ezrin in gastric cancer tissues and its clinical significance.Methods Gastric cancer tissues and adjacent normal gastric tissues from 60 patients with gastric cancer were collected from June 2008 to May 2009 at the Southwest Hospital.The mRNA and protein expressions of Ezrin were detected by using the reverse transcription polymerase chain reaction and western blot.The relationship between Ezrin and the gender and age of patients,and tumor differentiation,pathological staging,depth of invasion and lymph node metastasis was analyzed.All data were analyzed using the t test,chi-square test and Spearman rank correlation.Results The Ezrin mRNA expression level was increased in 33 (55％) cases of adjacent normal gastric tissues and 21 (35％) cases of gastric cancer tissues; the Ezrin protein expression level was increased in 45 (75％) cases of adjacent normal gastric tissues and 22 (37％) cases of gastric cancer tissues.The mRNA and protein expressions of Ezrin in the normal adjacent gastric tissues were 1.30 ± 0.04 and 3.57 ± 0.45,respectively,which were significantly higher than 0.53 ± 0.36 and 0.96 ± 0.18 in the gastric cancer tissues ( t =5.309,22.617,P ＜ 0.05 ).The mRNA expression of Ezrin was positively correlated with the protein expression of Ezrin (r =0.602,P ＜ 0.05 ).The mRNA and protein expressions of Ezrin were related to the pathological stages,depth of invasion and state of lymph node metastasis (x2 =6.41,6.49,4.62; 5.40,8.87,4.12,P ＜ 0.05),but not to the gender,age and tumor differentiation (x2 =0.50,0.07,1.07 ; 0.01,1.16,1.96,P ＞ 0.05).Conclusion The mRNA and protein expressions of Ezrin are significantly decreased in the gastric cancer tissue,which might be responsible for genesis,development and metastasis of gastric cancer.

Objective To investigate the influence of gene therapy using survivin as a gene target on biological behavior of pancreatic carcinoma cell line. Methods Chemically synthesized siRNA and shRNA in pGCSi vector were used to silence survivin expression of pancreatic carcinoma cell line PaTu8988. The therapeutical effects of survivin as a gene target were evaluated through determination of the down-regulation of survivin gene expression, cellular shape, cell apoptosis, cell viability and apoptosis signal pathway changes. Results After transfection of different arrays of siRNA and shRNA vectors to silence the survivin expression, survivin mRNA and protein levels were significantly decreased (P < 0.05) ; PI staining revealed the presence of karyopyknosis, the cell apoptosis index was more than 20%; hypodiploid DNA content before G0/G1 detected by flow cytometry ; cell viability measured by MTT assay was significantly decreased (P <0.05) ; the activity of caspase-3 remarkably increased (P < 0. 05). Conclusions The pancreatic carcinoma cell line PaTu8988 be induced to promote spontaneous apoptosis procedure through silencing survivin expression by RNAi, which could accelerate carcinoma cell apoptosis and improve therapeutic effect on pancreatic carcinoma.

Objective To analyze the differentially expressed genes in esophageal squamons cell carcinoma (ESCC), para-cancerous tissue (PCT) and normal esophagus tissue (NET) using oligomicroarray and to identify the target genes related to the development and progression of esophageal carcinoma. Methods The total RNAs isolated from ESCC, PCT or NET using one step Trizol method were purified and reversely transcribed into cRNAs. The cRNAs were then fluorescence labeled and hybridized with Agilent oligomicroarray (21 074 probes). The fluorescence intensity features were detected by Agilent scanner and quantified by feature extraction software. The selected candidate genes were confirmed by real time real time fluorescent quantitative RT-PCR immunohistochemistry andWestern blotting.Results ① The oligomicroarray demonstrated that there were 38 up-regulated genes and 61 down-regulated genes. ② The real time fluorescent quantitative RT-PCR revealed that five genes (CTHRC1, INHBA, SPP1 ,LUM, HRK)were more differentially expressed in up-regulated genes. Of which, CTHRC1 displayed more disparity.③ Immunohistochemistry examination showed that the higher expression of CTHRC1 (56.5 %, 26/46) was observed in ESCC. There was significantly difference in expression of CTHRC1 between patients with or without lymph node metastasis (P<0.05). ④ CTHRC1 protein was expressed in both TE-13 and Eca-109 cell lines. Conclusion CTHRC1 is probably one of the most significant biomolecules in ESCC.

Humans ; Male ; Middle Aged ; Silicosis ; surgery

Objective To investigate the changes of hypoxia-inducible factor(HIF)-1α and hexokinase-Ⅱ(HK-Ⅱ)expression in human esophageal squamous cell carcinoma and its effect in glycolysis.Methods TE13 cells and Eca109 cells were cultured under hypoxic condition(1 ％O2)for different hypoxic time(6,12,24 and 48 hours).Cells cultured under normal oxygen condition(20％O2)were set as control.The changes of HIF-1α and HK-Ⅱ expressions at protein level were detected by Western blot.HIF-1α genes were specifically silenced with RNA interference technology(RNAi),and then the changes of HIF-1α and HK-Ⅱ expression were determined by realtime PCR and Western blot.Under normal oxygen and hypoxic condition,the changes of lactic acid concentration in cell culture medium were detected by spectrophotometric method.Results Under hypoxic condition,the expression of HIF-1α and HK-Ⅱ gradually increased as hypoxic time extended(P＜0.05),reached a peak at 12h and then gradually decreased as time extended.Compared with that under normal oxygen condition,the expression of HK-Ⅱ in TE13 cells and Eca109 cells significantly increased under hypoxic condition(P＜0.05),which was more significant after 12 hours hypoxia.The result of realtime PCR indicated that under normal oxygen condition the expression of HIF-1α at RNA level in TE13/shRNA cells and Eca109/shRNA cells significantly decreased compared with TE13 cells and Eca109 cells without interference(P＜0.05).The expression of HK-Ⅱ at RNA level was consistent with the result of HIF-1α.Under normal and hypoxia condition,the expression of HK-Ⅱ at protein level in TE13/shRNA cells and Eca109/shRNA cells significantly decreased compared with TE13 cells and Eca109 cells without interference,and the difference was statistic significant(P＜0.05).The lactic acid secretion of TE13 cells and Eca109 cells under hypoxia condition(14.707 ± 3.594 and 15.062 ±3.901)was higher than that under normal oxygen condition(6.070±1.839 and 6.891±1.592,P＜0.05).The lactic acid secretion of TE13/shRNA cells and Eca109/shRNA cells significantly decreased compared with TE13 cells and Eca109 cells without interference,and the difference was statistic significant(P＜0.05).Conclusion The expressions of HIF-1α and HK-Ⅱ in human esophageal squamous cell carcinoma significantly increased under hypoxia conditions.The expression of HK-Ⅱ is closely correlated with lactic acid concentration and HIF-1α expression.HIF-1α may affect cell glycolysis through HK-Ⅱ.

Objective To investigate the inhibitory effect of blocking PI3K/AKT pathway by wortmannin on hypoxia-inducible factor 1α (HIF-1α) and the effect on the expression of glycolysis associated genes in human esophageal carcinoma cell lines TE1 and TE13,and to analyze the relation between PI3K/AKT-HIF-1α pathway and glycolysis in esophageal carcinoma cells. Methods Esophageal carcinoma cell lines TE1 and TE13 pretreated with wortmannin (2 μmol/L) were incubated under normoxic and hypoxic conditions.And each cell line was divided into four groups.The expression of HIF-1α and glycolysis associated genes GLUT-1,LDHA and HK-Ⅱ at protein level were measured by.Western blot.The expression of HIF-1α,GLUT-1,LDHA and HK-Ⅱ at mRNA level was determined by real-time PCR. The activities of LDH and HK-Ⅱ and lactic acid (LA)concentration in the culture supernatant were tested with spectrophotometer method.Results Under normoxic condition,HIF-1α was expressed in TE1 cells and the expression of HIF-1α was inhibited by wortmannin (2 μmol/L),the most significant inhibitory effect was at 12 hours,therefore 12 hours was selected for the subsequent hypoxia experiment.Compared with untreated cells,the expression of HIF-1α、HK-Ⅱ 、GLUT-1、LDH-A at protein level significantly decreased in TE1 and TE13 cells after pretreated with wortmannin (P ＜ 0.05),and the expression of HIF-1α、HK-Ⅱ at mRNA level significantly decreased (P＜ 0.05).Under normoxic and hypoxic conditions,the HK-Ⅱ and LDH activities in TE1 and Te13 esophageal carcinoma cells significantly decreased after treated with wortmannin compared with untreated cells (P＜0.05).Under hypoxia condition,the enzyme activity increased in untreated cells (P＜ 0.05). Under normoxic and hyp0xic conditions,the lactic acid concentration in the culture supernatant obviously decreased in cells treated with wortmannin compared with untreated cells (P＜ 0.05). Under hypoxia condition,lactic acid concentration increased in wortmannin treated cells (P ＜ 0.05). Conclusions Under normoxic and hypoxic conditions,wortmannin decrease lactic acid concentration through inhibiting the expression of HIF-1α and glycolysis associated genes, which indicate PI3K/AKT-HIF-1α pathway was closely related to glycolysis in esophageal carcinoma cells.

Objective To investigate the clinical value of a new anvil inserting method for esophagogastrostomy or esophagojejunostomy during laparoscopic radical proximal gastrectomy or radical total gastrectomy for gastric cancer.Methods The clinical data of 21 patients with gastric cancer who received laparoscopic radical proximal gastrectomy or radical total gastrectomy at the Southwest Hospital from March 2010 to February 2011 were retrospectively analyzed.Five trocars were inserted through the abdominal wall of the patients.After perigastric lymphadenectomy and mobilization of esophagus,an incision was made on the esophagus above the tumor,and then the anvil with drawn wire attached was inserted into the esophagus.An endo-cutter was applied to cut the esophagus adjacent to the incision left the drawn wire untouched,and then the stem of the anvil was pulled out by the drawn wire for laparoscopic anastomosis. Results The operations were successfully accomplished under the laparoscope with no conversion to open surgery.Fifteen patients received laparoscopic radical total gastrectomy and 6 received laparoscopic radical proximal gastrectomy. The mean operation time,volume of blood loss,time to off-bed activity,passage of flatus and postoperative duration of hospital stay were (257 ± 38) minutes,( 119 ± 32) ml,(2.5 ± 0.5 ) days,( 3.7 ± 0.8 ) days and (7.5 ± 2.6) days,respectively.No perioperative mortality,anastomotic bleeding or anastomotic fistula was detected.One patient was complicated with pulmonary infection + pleural effusion and was cured by conservative treatment; 1 was complicated with anastomotic stenosis which was alleviated by gastroscopic balloon dilation; 1 was complicated by incisional infection and was cured by medical treatment after drainage.No cancer cells were detected at the anastomotic ring or resection margin of the specimen.There were 4 patients with well-differentiated adenoma,8 with moderate-differentiated adenoma and 9 with poor-differentiated mucinous adenoma.There were 5 patients in stage Ⅰ,10 in stage Ⅱ and 6 in stage Ⅲ (UICC staging).Twenty-one patients were followed up for a mean period of (11 ±4) months (range,6-17 months ),no tumor recurrence or metastasis was detected. Conclusions The new technique for anvil insertion is safe,effective and easy for manipulation and learn.It offers a new approach for laparoscopic digestive tract reconstruction.

Advanced gastric cancer is usually dealt with D2 radical dissection. There are different opinions as to whether it is necessary to perform D3 radical lymphadenectomy.Some scholars thought that properly enlarged radical dissection can improve long-term outcomes for the treatment of advanced gastric cancer.In recent years,laparoscopic D1 and D2 radical dissection of gastric cancer could be carried out in many hospitals.However,the technique and related skills for performing D3 radical lymphadeneetomy through laparoscope remains to be explored.Based on our previous experiences,D3 radical lymphadeneetomy using artery suspension method and medial-to-lateral approach for advanced gastric cancer is proved to be safe and feasihle.