Objective:To investigate the mechanism of endoplasmic reticulum (ER) stress-induced chemoresistance to cisplatin in gastric cancer cells. Methods:ER stress models were established in both BGC823 and SGC7901 gastric cancer cells. The expression of GRP78, an ER stress marker, was examined by Western blot analysis. Moreover, whether ER stress can decrease the sensitivity of gastric cancer cells to cisplatin and activate P38 was explored by flow cytometry and Western blot analysis, respectively. Whether ER stress-induced chemoresistance to cisplatin can be abrogated by blocking P38 activity in gastric cancer was also elucidated using flow cytometry. Results:GRP78 protein expression markedly increased after treating BGC823 and SGC7901 gastric cancer cells with tunica-mycin (TM) or thapsigargin (TG) for 8, 16, and 24 h (P<0.05), compared with that in the group treated for 0 h. The apoptotic rates of TM-(or TG)-, cisplatin-, and TM (or TG) plus cisplatin-treated groups significantly increased (P<0.05) in both BGC823 and SGC7901 gastric cancer cells compared with the rate in the control group. The apoptotic rate of TM (or TG) plus cisplatin-treated group signifi-cantly decreased (P<0.05) in both BGC823 and SGC7901 gastric cancer cells compared with that of the cisplatin-treated group. Com-pared with the group treated for 0 h, phospho-P38 expression markedly increased after treating BGC823 and SGC7901 gastric cancer cells with TM (or TG) for 8, 16, and 24 h (P<0.05). No difference in P38 protein expression was observed between each group in both BGC823 and SGC7901 gastric cancer cells (P>0.05). Both P38 inhibitors, either SB203580 or PD169316, can inhibit the activation of P38. The inhibition of P38 activity can overcome ER stress-induced chemoresistance to cisplatin in gastric cancer cells (P<0.05). Con-clusion:ER stress can trigger the chemoresistance to cisplatin by activating P38 in gastric cancer cells.

To explore the pathogenicity of VEG strain Toxoplasma gondii oocysts on China Kunming mice,T.gondii oocysts of ＜ 1 and 102-108 were chosen to feed the mice orally.And the modified agglutination test (MAT),H&E and IHC were used to check the infection of mice.The infection rate,the survival rate of mice,and number of cysts in brain were analyzed.Results showed that 100％ of the mice fed with ≥102 oocysts were infected,the minimum lethal dose was 102 oocysts and the 100％ lethal dose was 108 oocysts.The time of death in acute infection was 7 DPI to 14 DPI.T.gondii cysts formation rate was 32.14％ (9/28),and the number of cysts was 9 to 857 per mouse.The survival rate of infected mice was 67.44％ (29/43),and the longest survival time was more than 390 DPI.Accordingly,the virulence of T.gondii VEG strain is medium,and has a higher cysts formation rate.

Objective:To construct eukaryotic expression plasmids of pneumocystis carinii p55-v3 and p55-v0 antigenic genes and to identify their expression in COS-7 cells at mRNA level.Methods:Pneumocystis carinii total RNA was used as the template to amplify p55-v3 and p55-v0 antigenic gene by RT-PCR.The products were connected to pTA2 vector and then cloned in pVAX1 eukaryotic expression vector to construct recombinant plasmids as pVAX-p55-v3 and pVAX-p55-v0.After propagated in E.coli DH5α,the recombinant plasmids were transfected into COS-7 cells.After 24 h incubation,the RT-PCR was performed to identify the mRNA expression of p55-v3 and p55-v0 antigenic gene.Results:The recombinant plasmids were qualified by restrictive endonuclease digestion and sequencing.And when compared with that in GenBank,the homology of p55-v3 antigenic gene was 99.9% in nucleotides and 100% in amino acid.The homology between p55-v0 antigenic gene and the one reported previously in nucleotide and amino acid seguence were 99.8% and 100%.The results of RT-PCR confirmed that p55-v3 and p55-v0 antigenic genes were transfected into COS-7 cells successfully and the genes were expressed in the cells.Conclusion:In this study,the recombinant plasmids of pVAX-p55-v3 and pVAX-p55-v0 are conducted successfully and expressed in the COS-7 cells,which provide a basis for clarification of immunologic function of p55-v3 and study of DNA vaccine.

Objective To explore whether antisense-EGFR could enhance the radiosonsitivity of human lung cancer spc-a-1 cell line.Methods The spc-a-1 cells were transfected with antisenso.EGFR-pcDNA3 by lipofectamine 2000(pcDNA3 antiEGFR group).Two other groups were used for comparison:control group(spc-a-1 cell without transfection)and pcDNA3 group(spc-a-1 cell transfeeted with pcDNA3 which did not contain antisense EGFR).Cell clones that stable expressing antisense-EGFR wa8 selected with G41 8 and the suppression of the expression of EGFR mRNA and protein were detected by RT-PCR and Western blot.The influence of antisense-EGFR on cell cycle was testified by flow cytometry assay.The cell apoptosis was analyzed by flow cytometry after 8 Gy irradiation.Further,cells of each group were irradiated with X-rays at the dose of 0,2,4,6 and 8 Gy.Dose-survival curve of each group was established by colony-forming assay.Results The expression of EGFR mRNA and protein were significantly inhibited after antisense-EGFR-pcDNA3 transfection.The cells arrested at the G2/M phase in the pcDNA3 antiEGFR group,control group and pcDNA3 group were (29.53±1.91)%,(13.7±1.30)%and(12.40±1.34)%,respectively.The apoptosis index of spc-a-1 cells in the antisonse-EGFR combined with irradiation group was obviously higher than that of the comparable groups [(39.24±1.57)%,(13.79±0.63)%and(15.02±0.85%)].The values of D0,Dq,SF2 of pcDNA3 antiEGFR group declined obviously compared with the control group(2.11,2.49,0.84 vs 1.19,0.15,0.32).Conclusions Antisense-EGFR could induce the G2/M cell cycle arrest,promote cell apoptosis and inhibit the ability of sublethal cell damage repair induced by irradiation,80 that it could significantly improve the radiosensitivity of spc-a-1 cell in vitro.

AIM:To explore the expression level of microRNA-140 ( miR-140 ) in human gastric cancer and normal gastric tissues, and the regulatory effect of miR-140 expression on the function of SGC-7901 cells.METHODS:The expression levels of miR-140 in human gastric cancer and normal gastric tissues were detected by real-time PCR.miR-140 mimics ( miR-140 up-regulated expression) and miR-140 inhibitors ( miR-140 down-regulated expression) were trans-fected into human gastric cancer SGC-7901 cells by liposome method.At the same time, the untransfected control group ( control group) and miRNA nonsense sequence transfection group ( NC group) were set up .The expression of miR-140 in the cells after transfection was detected by real-time PCR.The cell viability and growth inhibition rate with DDP were meas-ured by MTT assay.The cell cycle and apoptotic rate of SGC-7901 cells were analyzed by flow cytometry.The invasion a-bility of SGC-7901 cells was measured by Transwell assay.The protein expression of histone deacetylase 4(HDAC4) in the cells was determined by Western blot.RESULTS:The expression level of miR-140 in human gastric cancer tissues was significantly lower than that in normal gastric tissues (P<0.05).Compared with control group and NC group, the viability and invasion ability of the SGC-7901 cells were decreased, the cell cycle was arrested, the cell growth inhibition rate and apoptotic rate with DDP treatment were increased, and the protein expression of HDAC4 was down-regulated ( P<0.05) in miR-140 mimics group.However, in miR-140 inhibitors group, the viability and invasion ability of the SGC-7901 cells were increased, the cell cycle was promoted, the cell growth inhibition rate and apoptotic rate with DDP treatment were de-creased, and the protein expression of HDAC4 was up-regulated ( P<0.05 ) .CONCLUSION:The expression level of miR-140 in the gastric cancer tissues is low.miR-140 serves as a tumor suppressor to regulate the viability, apoptosis and invasion ability of gastric cancer cells, and to play a role by down-regulating HDAC4 protein.miR-140 may serve as a new target for diagnosis and treatment of gastric cancer.

To investigate the pathogenic characteristic of ToxoDB # 17 Toxoplasma gondii in Kunming mice,the mice were fed with ＜1,1,101,102,103,104,105,106 oocysts,and the clinical symptoms of mice were observed.MAT,HE and IHC were used to research the infection rate of T.gondii,survival rate of mice and the number of cysts in brain.The results showed that the mice fed with ≥104 oocysts appeared transient depression,mice were arched,messy hair after infection.The mice fed with ≥102 oocysts were all infected,T.gondii cysts formation rate was 2.38％ (1/42),the survival rate of infected mice was 85.71％ (36/42),the survival time was greater than 360 days.In conclusion,the pathogenicity of ToxoDB# 17 T.gondii is weak,and cysts formation rate is also low.

Proteomics is an emerging discipline and has been widely used in a variety of fields despite of having very short history in comparison with other disciplines. In Chongqing Medical Univer-sity, the course contents were adjusted to fulfill the most effective integration of proteomics research with postgraduate training program for medical university. Diverse teaching was advocated here and af-ter-school communications were greatly encouraged in teaching. Traditional multimedia teaching plat-form remained the main teaching way and students were organized to visit the research platform as supplementing teaching way. The overall quality and effectiveness of teaching were effectively improved by successful implementation of the above initiatives.

Objective:To observe the changes in the expression of microRNAs in ventricular myocardium in a rat model of hypothermic ischemia-reperfusion (I/R).Methods:Healthy clean-grade male Sprague-Dawley rats, aged 2-3 months, weighing 300-400 g, were anesthetized with intraperitoneal chloral hydrate.Their hearts were excised and perfused in a Langendorff apparatus with K-H solution saturated with 95%O 2-5%CO 2.Sixteen Langendorff-perfused hearts were prepared and divided into 2 groups ( n=8 each) by a random number table method: control group (group C) and hypothermic I/R group (group I/R). The hearts were made globally ischemic for 60 min followed by 30-min hypothermic (4 ℃) reperfusion to establish the model of hypothermic I/R injury.The type and duration of arrhythmia and time of recovery of spontaneous heartbeats were recorded during reperfusion.The rats in group I/R were further divided into low-risk group (I/R-L group) and high-risk group (I/R-H group). The left ventricular myocardium was collected after the end of perfusion for high throughput sequencing to screen the differentially expressed microRNAs, and the reliability of the sequencing results was verified by quantitative real-time polymerase chain reaction.Gene Ontology and KEGG databases were used to analyze the biological regulatory pathways of differentially expressed target genes. Results:Compared with group C, there were 437 up-regulated microRNAs and 242 down-regulated microRNAs in group I/R-L and 419 up-regulated microRNAs and 260 down-regulated microRNAs in group I/R-H.Compared with group I/R-L, 392 microRNAs were up-regulated, and 287 microRNAs were down-regulated in group I/R-H.There were 84 microRNAs with absolute value of fold change ≥2 and significantly differential expression ( P<0.01) among the three groups.Subsequently, 4 microRNAs were randomly selected for validation using quantitative real-time polymerase chain reaction, confirming that the sequencing results were reliable.These differentially expressed target genes were involved in 11 biological processes and 6 KEGG pathways which were related to reperfusion arrhythmia.Potassium ion transmembrane transport and the adrenergic receptor signaling pathway in cardiomyocytes were enriched by the largest number of target genes. Conclusion:The expression of microRNAs in ventricular myocardium changes significantly after heart hypothermic I/R.These differentially expressed microRNAs regulate potassium ion transmembrane transport probably and mainly through the adrenergic receptor signaling pathway in the cardiomyocytes and thus are involved in the occurrence and development of hypothermic I/R arrhythmias.

Objective:To evaluate the changes in the electrical conduction of ventricular myocardium during hypothermic ischemia-reperfusion (I/R) in rats with arrhythmia.Methods:Healthy clean-grade adult male Sprague-Dawley rats, aged 2-3 months, weighing 200-300 g, were studied.The hearts were removed and retrogradely perfused with oxygenated K-H solution in a Langendorff apparatus. Sixteen isolated hearts were divided into 2 groups ( n=8 each) using a random number table method: normal control group (group C) and hypothermic I/R group (group I/R). In group C, the heart was perfused with K-H solution at 37 ℃ for 120 min.In group I/R, the heart was perfused with K-H solution at 37 ℃ for 30 min, and then perfusion was stopped, cardiac arrest was induced through injecting Thomas solution (4 ℃), the area around the heart was protected with low temperature (4 ℃) Thomas solution, and hearts were perfused with 4 ℃ Thomas solution at 30 min after cardiac arrest and with 37 ℃ K-H solution for 30 min staring from 60 min after cardiac arrest.The rats in group I/R were further divided into high-risk subgroup (I/R-H subgroup) and low-risk subgroup (I/R-L subgroup). The time of spontaneous recovery of heart beat and development of arrhythmia were recorded.At the end of reperfusion, the atrioventricular conduction 2∶1 block point (2∶1B) and ventricular electrical conduction velocity (CV) were measured and recorded by program-controlled electrical stimulation. Results:Compared with group C, CV and 2∶1B were significantly decreased in IR-L and IR-H subgroups ( P<0.05). Compared with IR-L subgroup, the time for restoration of spontaneous heart beat was significantly prolonged, the incidence of ventricular fibrillation and arrhythmia score were increased, and CV and 2∶1B were decreased in IR-H subgroup ( P<0.05). Conclusion:The electrical CV of ventricular myocardium is decreased during hypothermic I/R, which may be the mechanism of reperfusion-induced ventricular arrhythmia in rats with arrhythmia.

Objective:To determine the changes in the expression of myocardial miRNA and the target genes in the rats with hypothermic ischemia-reperfusion (I/R) arrhythmia.Methods:Clean-grade healthy male Sprague-Dawley rats, aged 2-3 months, weighing 300-400 g, were anesthetized, the chest was opened, and the heart was taken to establish an isolated heart perfusion model.Six successfully perfused isolated hearts were divided into 2 groups ( n=3 each) using a random number table method: control group (group C) and heart I/R group (IR group). The model of hypothermic global I/R injury was established by interrupting perfusion for 60-min followed by 30-min reperfusion in chloral hydrate-anesthetized rats.The arrhythmia score was recorded during reperfusion.High-throughput sequencing was used to identify the differentially expressed miRNAs in two groups.The RNAhybrid and miRanda databases were used to predict the target genes of mRNA regulated by the differentially expressed miRNAs, and the enrichment for target genes was performed by Gene Ontology and KEGG databases, and the miRNAs closely related to arrhythmia and with higher expression were selected to carry out the real-time polymerase chain reaction detection. Results:The results of high-throughput sequencing showed that there were 7 differentially expressed miRNAs (novel-miR-17, novel-miR-19, novel-miR-30, novel-miR-43, rno-miR-122-5p, novel-miR-16 and rno-miR-429) in group IR as compared with group C. There were 4 miRNAs that were closely related to arrhythmia and had higher expression: the expression of novel-miR-17, novel-miR-30 and rno-miR-122-5p was significantly up-regulated, and the expression of rno-miR-429 was down-regulated in group IR when compared with group C ( P<0.05). The miRNA-mRNA correlation analysis revealed that GJA1 gene was the target of novel-miR-17. Conclusion:Myocardial novel-miR-17 is involved in the occurrence of hypothermic I/R arrhythmia probably by acting on GJA1 gene in rats.