Objective To investigate the drug resistance of side population cells in human gallbladder cancer cell line GBC-SD and explore its mechanism. Methods Drug sensitivity assays of 5chemotherapeutic agents were performed on side population cells (SP) and non-SP cells of GBC-SD.GBC-SD was cultured and then treated with the chemotherapeutic agent gemcitabine. The frequency of SP by FACS was measured. RT-PCR and Western blotting were used to detect the expression of AB-CG2 in both the SP and the corresponding non-SP subsets. Results After 1 d treatment with 4 chemotherapeutic agents (gemcitabine, cisplatin, 5-fluorouracil and mitoxantrone) in IC50 concentration to GBC-SD cell line, the reproductive ability of SP was higher than that of non-SP (P<0.05). However, statistical significance was not achieved when compared with epirubicin (P>0.05). The percentage of SP in GBC-SD treated with chemotherapeutic agent gemcitabine after 3 weeks was sharply elevated by FACS (8.02% ±0.13% vs 0.62% ±0.08%, P<0.05), and the expression of ABCG2mRNA and protein were increased in SP as compared with non-SP. Conclusion SP from human gallbladder cancer cell line GBC-SD, like stem cell, showed a heighten resistance to drugs. Increased expression of ABCG2 was largely responsible for the multi-drug resistance.

ObjectiveTo explore the tumor igenic property of side population cells (SP) from human gallbladder carcinoma cell line GBC-SD. Methods SP and non-SP cells were isolated from GBC-SD staining with Hoechst33342 dye by fluorescence-activated cell sorting (FACS). The soft agar clonal assay and xenograft assay were performed to characterize tumorigenic property of side population cells in vitro and in vivo, respectively. The percentage of SP cells was analyzed by FACS in 5 hu man gallbladder carcinoma specimens. ResultsThe percentage of SP cells accounted for approximately 0.87 ％ of GBC-SD cells. The clone-formed rates of SP was more frequent than that of non-SP cells (14.74％ ± 3.53％ vs 5.17％ ± 1.05％), there was statistically significant difference (t =2.75,P＜0. 05). SP cells could generate tumors with as few as 5 × 103 cells (four of seven animals), whereas at least 1 × 105 non-SP cells were needed to form a tumor (one of seven animals). Re-analysis of SPderived tumors by FACS showed that SP cells under in vivo conditions also have the capacity to regenerate the SP and non-SP fractions. Besides, analysis of Hoechst33342 revealed s small fraction of SP cells, ranging from 0. 27％ to 2.3％ in gallbladder carcinoma specimens. ConclusionSP cells from GBC-SD are highly tumorigenic similar as the cancer stem cells.

Objective:To discuss the factors related to the prognosis in the alpha fetoprotein(AFP)negative hepatocellular carcinoma(HCC)patients,and to construct the nomogram for predicting the survival time of the patients.Methods:The retrospective analysis on data of 2 064 cases of AFP negative HCC patients extracted from the Surveillance,Epidemiology,and End Results(SEER)Database was conducted,and all the patients were divided into training cohort and internal validation cohort at a ratio of 7∶3,and 101 AFP negative HCC patients from the Integrated Traditional Chinese and Western Medicine Hospital in Hunan Province were regarded as the external validation cohort.The univariate Cox regression analysis results were incorporated into the multivariate analysis,and the independent risk factors for the AFP negative HCC patients were obtained by multivariate Cox analysis to build a cancer specific survival(CSS)prognosis nomogram for the AFP negative HCC patients.The predictive efficacy and clinical utility of the nomogram were evaluated by time-dependent receiver operating characteristic curve(ROC),calibration plots,and decision curve analysis(DCA).The total score obtained from the nomogram was used for the risk stratification to compare the degree of risk discrimination between the nomogram and the American Joint Committee on Cancer(AJCC)staging system.Results:Ten independent risk factors were selected by multivariate Cox regression analysis to construct 3-year,4-year,and 5-year CSS prognostic nomograms for the AFP negative HCC patients,including the patient's age,pathological grade,surgical status,radiotherapy status,chemotherapy status,lung metastasis status,tumor size,tumor T stage,tumor M stage,and marital status.The area under curve(AUC)for the 3-year,4-year,and 5-year time-dependent ROC in the training cohort were 0.807(95%CI:0.786-0.828),0.804(95%CI:0.782-0.826),and 0.813(95%CI:0.790-0.835),respectively.In the internal validation cohort,they were 0.776(95%CI:0.743-0.810),0.772(95%CI:0.737-0.808),and 0.789(95%CI:0.752-0.826),and in the external validation cohort,they were 0.773(95%CI:0.677-0.868),0.746(95%CI:0.620-0.872),and 0.736(95%CI:0.577-0.895).The calibration plots verified that the nomogram fitted well with the perfect line.The DCA curve revealed that the net benefit of the nomogram was significatly higer than that of the AJCC staging system at certain probability thresholds compared with AJCC staging,the nomogram had a better ability to identify high-risk individuals.Conclusion:The serum AFP expression is one of the prognostic markers for the HCC patients.For those patients with AFP negative expression in serum,different considerations should be taken.The nomogram model based on multiple risk factors is a promising clinical tool for assessing the CSS in the AFP negative HCC patients.

Objective Ti study the applicatiin if the clinical nursing pathway in patients with sleep apnea hypipnea syndrime (OSAHS).Methods The patients were divided inti 2 griups.The cintril griup was applied riutine nursing interventiins after surgery while the ibser-vatiinal griup was cinducted by clinical nursing pathway.Results The average hispitalizatiin time and medical cists were much liwer in ibservatiinal griup while awareness if diseases if pa-tients and degree if satisfactiin were higher in the ibservatiinal griup.The difference was statisti-cally significant(P <0.01).Conclusion The standardized management based in clinical nursing pathway can imprive the quality if nursing and primite mental rehabilitatiin if patients with 0SAHS.

Objective Ti study the applicatiin if the clinical nursing pathway in patients with sleep apnea hypipnea syndrime (OSAHS).Methods The patients were divided inti 2 griups.The cintril griup was applied riutine nursing interventiins after surgery while the ibser-vatiinal griup was cinducted by clinical nursing pathway.Results The average hispitalizatiin time and medical cists were much liwer in ibservatiinal griup while awareness if diseases if pa-tients and degree if satisfactiin were higher in the ibservatiinal griup.The difference was statisti-cally significant(P <0.01).Conclusion The standardized management based in clinical nursing pathway can imprive the quality if nursing and primite mental rehabilitatiin if patients with 0SAHS.

Objective To identify lipid metabolism genes in cerebral infarction;To explore the intervention effect of Huoxue Rongluo Prescription.Methods Multi-chip combined differential analysis(GSE61616,GSE30655)was used to identify lipid metabolism genes in cerebral infarction in combination with Reactome database,and the expression differences of lipid metabolism genes in cerebral infarction were identified and verified in GSE97537 chip;Pearson correlation analysis was used to analyze the correlation of 51 cerebral infarction samples in GSE61616,GSE30655,GSE97537,GSE137595,GSE22255,GSE163614,and GSE78731 datasets;PPI,GO and KEGG analysis of lipid metabolism genes in cerebral infarction were performed through STRING database and R clusterProfiler package.SD rats were made to the model of cerebral infarction,and was administered with Huoxue Rongluo Prescription extract 11.7 g/kg by intragastric administration for 7 days.The symptoms of neurological deficit,the changes of Nissl bodies and the mRNA expressions of PLA2G4A,SPHK1,and PTGES key genes in lipid metabolism in cerebral infarction were observed.Results TSPO,CYP1B1,PLIN2,CH25H,PLA2G4A,ANGPTL4,PTGS1,SPHK1,and PTGES were identified as lipid metabolism genes in cerebral infarction,and were significantly highly expressed and positively correlated in cerebral infarction.Among them,PTGS1,PLA2G4A,and SPHK1 interacted with each other,which were the key genes of lipid metabolism in cerebral infarction;the lipid metabolism gene in cerebral infarction mainly exerted molecular functions such as oxidoreductase activity,iron ion binding,heme binding,etc.,mediating arachidonic acid metabolism,phospholipase D signaling pathway,VEGF signaling pathway,involved in regulation of lipid metabolism process,fatty acid metabolism process,fatty acid derivative metabolism process.The symptoms of neurological deficit in the model rats with cerebral infarction were severe(P<0.001),and Huoxue Rongluo Prescription could effectively improve the neurological deficit of model rats(P<0.001).The Nissl staining indicated that the neuronal structure was abnormal and the number was significantly reduced after cerebral infarction(P<0.001).Huoxue Rongluo Prescription could increase the number of neurons(P<0.001)and repair the neuronal structure.RT-qPCR showed that the key genes of lipid metabolism in cerebral infarction were significantly higher in cerebral infarction(P<0.001),corroborated with the bioinformatics results,and Huoxue Rongluo Prescription could reduce the expression of key lipid metabolism genes of PTGS1,PLA2G4A,and SPHK1(P<0.001,P<0.01,P<0.05).Conclusion Huoxue Rongluo Prescription can down-regulate the expressions of PTGS1,PLA2G4A,SPHK1,exert molecular functions such as oxidoreductase activity,iron ion binding,heme binding,and mediate arachidonic acid metabolism,phospholipase D signaling pathway,and VEGF signaling pathway.It participates in the process of lipid metabolism regulation,fatty acid metabolism,and fatty acid derivative metabolism,increases the number of Nissl bodies,improves the symptoms of neurological deficits,and exerts neuroprotective effects.

Objective To explore the expression of miR-548j-5p in pancreatic cancer and its effect on the invasion of pancreatic cancer. Methods Tissue samples of 16 patients with pancreatic cancer who received treatment in Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of Science &Technology between January 2016 and January 2017 were collected. There were 8 males and 8 females, aged from 41 to 62, including 7 cases with an age of ≤50 and 9 cases with an age of >50. The informed consents of all patients were obtained and the local ethical committee approval was received. The expression of miR-548j-5p in pancreatic cancer and adjacent tissues was detected by RT-PCR. PANC-1 cells were transfected by mimic and inhibitor of miR-548j-5p to construct PANC-1 cells with over-expression and low-expression of miR-548j-5p. And in control group, PANC-1 cells were transfected with empty plasmid Ctrl. The effect of miR-548j-5p on PANC-1 invasion was detected by Transwell assay, and its effect on PANC-1 metastasis was evaluated by wound scratch assay. Comparison of three groups was conducted by one-way analysis of variance and LSD-t test. Results The median relative expression of miR-548j-5p in pancreatic cancer was 0.35(0.03-4.47), significantly lower than 4.30 (0.04-65.55) in adjacent tissues (Z=-2.689, P<0.05).Transwell assay results showed that the average number of cells penetrating membrane was(58±34) in miR-548j-5p over-expression group, significantly lower than (231±61) in control group (LSD-t=-2.852, P<0.05);and it was(491±149)in miR-548j-5p low-expression group,significantly higher than that in control group (LSD-t=4.313, P<0.05). The wound scratch assay results showed that the wound scratch healing rate was (20±8)% in miR-548j-5p over-expression group, significantly lower than (38±6)% in control group (LSD-t=-3.759, P<0.05); and it was (53±6)% in miR-548j-5p low-expression group, significantly higher than that in control group (LSD-t=2.946, P<0.05). Conclusions Low expression of miR-548j-5p is observed in pancreatic cancer. Over expression of miR-548j-5p can decrease the invasive and metastatic ability of pancreatic cancer.