Objective To study the effect of transfection of antisense MBD1 gene eukaryotic expression vector on the expression of MBD1 gene in human cholangiocarcinoma cell line QBC-939.Methods The(constructed) antisense MBD1 gene eukaryotic expression vector was transfected into the human(cholangiocarcinoma) cell line QBC-939 using lipofectamine transfection reagents,and positive cell clones were obtained using G418 selection after transfection.The constructed recombinant vector was transfected into(QBC-939) cells successfully and was confirmed by amplifying the exogenous neo~R gene with PCR method.The expression level of MBD1 gene mRNA and protein was detected by RT-PCR and FCM methods respectively.Results Following the transfection,the MBD1 gene mRNA level in human cholangiocarcinoma cell line QBC-939 decreased from 0.912?0.022 to 0.215?0.017,and the MBD1 gene protein level also(decreased) from(80.19?5.05)% to(35.11?4.05)%.There were very significant differences on the expression both at the transcription and post-transcription levels of MBD1 gene between non-tranfection group and the antisense MBD1 gene eukaryotic expression vector transfection group(P

Objective To investigate the effects of the tumor suppressor gene PTEN in growing inhibition and down-regulating mTOR in cholangiocarcinoma QBC939 cells in vitro.Methods QBC939 cells were transfected with plasmids wild-type PTEN and C124S-PTEN in vitro.After transfection,the expression of the PTEN and phosphorylation of AKT and mTOR was detected by Western blot.Flow cytometry was used to analyze apoptosis and cell cycle of the transfected cells.Results Compared with the control,the expression of phosphorylation AKT was decreased and mTOR were down-regulated respectively when transfected with the wild-type PTEN.However,after transfection with mutation-type PTEN,the level of PTEN in the cells by increased,but phosphorylation AKT level and mTOR expression had no significant change.Conclusions PTEN can be actived by phosphorylated AKT.Actived AKT decreased the mTOR which led to tumor cells apoptosis and regulation of the tumor cell cycle.In the pathway of signal transmission of PI3K/AKT/PTEN/mTOR,PTEN and mTOR are closely related through phosphorylation of AKT.

Hypermethylation of the promoter region is one of the major mechanism of tumor suppressor gene inactivation. In order to provide a research tool for the study on the function of MBD1 gene in DNA methylation and tumorigenesis, antisense MBD1 gene eukaryotic expression plasmid was constructed and transfected into human biliary tract carcinoma cell line QBC-939 to observe its effect on the expression of MBD1 mRNA and protein by using RT-PCR and FCM respectively. Following the transfection, the mRNA level of MBD1 gene decreased from 0. 912 +/- 0.022 to 0.215 +/- 0. 017, and the protein level of MBD1 gene also decreased from (80.19 +/- 5.05) % to (35.11 +/- 4.05) %. There were very significant differences in the expression both at the transcription and post-transcription levels of MBD1 gene between non-tranfection group and the antisense MBD1 gene eukaryotic expression plasmid transfection group (P < 0.01). It was suggested that transfection with the antisense MBD1 gene eukaryotic expression plasmid can significantly reduce the expression level of MBD1 gene in QBC-939, and this study may provide a valid tool for the investigation of the function of MBD1 gene and its role in biliary tract carcinoma.
Biliary Tract Neoplasms/*metabolism ; Biliary Tract Neoplasms/pathology ; Cell Line, Tumor ; DNA Methylation ; DNA-Binding Proteins/*biosynthesis ; DNA-Binding Proteins/genetics ; Eukaryotic Cells/metabolism ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; Oligonucleotides, Antisense/*genetics ; Plasmids/genetics ; Transcription Factors/*biosynthesis ; Transcription Factors/genetics ; Transfection

Hypermethylation in the promoter region is an important epigenetic mechanism for the transcriptional repression of a number of cancer-associated genes, and over-expression and/or increased activity of DNA methyltransferases are considered to be the main cause of promoter hypermethylation. In order to explore the roles of two methyltransferase members (DNMT1 and DNMT3b) in the cholangiocarcinoma tumorigenesis, antisense eukaryotic expression plasmid of DNMT1 and DNMT3b gene was constructed respectively, and were co-transfected into the human cholangiocarcinoma cell line QBC-939 to observe their biological effects on the cell growth and proliferation ability, apoptosis, cell cycle alteration, and the tumorigenesis ability in the subcutaneous tissue of nude mouse. The results demonstrated that co-transfection with antisense eukaryotic expression plasmid of DNMT1 and DNMT3b gene and single transfection with antisense eukaryotic expression plasmid of DNMT1 gene can suppress the growth and proliferation of QBC-939, block the cell cycle at G1 phase, increase the apoptosis rate, minimize the tumor size in the subcutaneous tissue of nude mouse. The suppressing biological effect of co-transfection is stronger than single transfection with antisense DNMT1. Meanwhile, single transfection with antisense eukaryotic expression plasmid of DNMT3b gene has no effects on the biological characteristics of QBC-939. This study suggests that DNMT1 gene plays a key role in DNA methylation and DNMT3b gene may act as an accessory to support its function in inactivation of tumor suppressor genes. Combination DNMT1 and DNMT3b will increase their biological effects and have the synergistic effect on suppressing the growth of human cholangiocarcinoma cell line QBC-939.
Apoptosis ; Biliary Tract Neoplasms/*metabolism ; Cell Line, Tumor ; Cell Proliferation ; Cholangiocarcinoma/*metabolism ; DNA (Cytosine-5-)-Methyltransferase/*biosynthesis ; DNA (Cytosine-5-)-Methyltransferase/genetics ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; Mice, Nude ; Neoplasm Transplantation

Objective: To explore the inhibitory effect of artesunate(Art)on human esophageal carcinoma cells and to study the related mechanism.Methods: Nude mice were inoculated with Eca109 cells subcutaneously on the left upper limbs to establish esophageal carcinoma model.The model mice were divided into 5 groups: first group received 100 mg/kg Art,second group 200 mg/kg Art,third group 300 mg/kg Art,forth group 3 mg/kg cisplatin(DDP),and the fifth group received normal saline.Mass and volume changes of transplant tumors in different groups were observed.Flow cytometry was used to detect the cell cycle,apoptosis,and the expression of CDC25A protein,Smad3 protein and TGF-?protein in the transplanted tumors in mouse model.RT-PCR was used to detect the expression of CDC25A,Smad3 and TGF-?mRNA in the transplanted tumors.Results: Nude mouse model bearing human esophageal carcinoma was success- fully created.Compared with the control group,the volume and mass of transplant tumors in Art groups were significantly smaller(P

OBJECTIVE: To investigate the process of apoptosis in lungs and liver induced by crushing hindlimbs of rat, and study the mechanism of crush injury. METHODS: The rat experimental model of hindlimbs crush injury was established. The cell apoptosis in lungs and liver was detected by TUNEL assay, and the expression of Bax, Bcl-2 and caspase-3 apoptin was examined by immunohistochemistry. RESULTS: Compared with the control group, the partial muscle injury of rat's hindlimbs was more serious with more apoptosis observed in lungs and liver (P < 0.05). The expression of Bax was up-regulated and Bcl-2 was down-regulated, whereas caspase-3 expression was activated (P < 0.05). CONCLUSION The cell apoptosis has increased significantly in lungs and liver after crush injury of hindlimbs in rat. The correlation factor released during tissue injury may mediate apoptosis process.
Animals ; Apoptosis/physiology* ; Caspase 3/metabolism* ; Genes, bcl-2 ; Hindlimb/injuries* ; Immunohistochemistry ; Liver/physiopathology* ; Lung/physiopathology* ; Rats ; Up-Regulation ; bcl-2-Associated X Protein

Objective To investigate the distribution of critical values of adults in Beijing, to provide the evidence for the formulation of the Standardized Management Guideline in Critical Values, in order to promote the accurate management of critical values.Methods A total of 110 398 data of critical values from the tertiary and above medical institutions during January 1 to May 31 in 2015 in Beijing were collected by the way of on-site inspection, covering the disciplines of hematology, clinical chemistry, coagulation and blood gas analysis.Fristly, the selected critical values were classified by the factor of admission departments and disease types,then were analyzed by using Kruskal-Wallis test, to compare the differences in each group.Secondly,the combined groups were classified by the factor of gender then were analyzed by using Mann-Whithey U test, to compare the differences in each group.Finally, the stratification thresholds of critical values were established.Results Except for the upper limits of Ca, pH, pCO2, Hb and the lower limits of Glu, pH, the rest of thresholds of critical values had significant differences due to different admission departments and disease types and/or gender.Conclusion Depending on the different admission departmentsces disease types and/or gender, hierarchical limit values on each critical value were formulated.

OBJECTIVETo investigate the ultrastructural variation of the facial nerve of rabbit with different dosage of (125)I seed brachytherapy.

METHODSFifty-four big ear rabbits were divided into 3 groups randomly and given 40 Gy, 80 Gy, 120 Gy respectively. Radioactive seeds were implanted in one side of parotid gland, the other side was implanted with vacant shell as a control group. The facial nerves were obtained 2, 4, 6 months respectively after operation and the histological ultrastructural changes observed by electromicroscope.

RESULTSIn the control group, epineurium was continuous, there was slight pitting edema under the epineurium, and axonal myelin was loose. In the test groups, there was slight pitting edema under the epineurium, and axonal myelin sheath was loose at 4th month. Macrophage and regenerated fibers were found in the 80 Gy group and myelin sheath lamellar separation, regeneration of nerve in the 120 Gy dosage. The myelin sheath lamellar was separated and axonal myelin loose in the test group at 6th month. Myelin sheath amellar separation and edema under the epineurium were found in the group of 80 Gy and 120 Gy.

CONCLUSIONSThe ultrastructure of the facial nerve is damaged by the dosage of 40 Gy, 80 Gy brachytherapy with (125)I seeds. The higher dosage the nerve receives, the more serious the damage will be. Both of the epineurium and axonal myelin sheath are integral and continuous 6 months after operation with dosage of 120 Gy.

Animals ; Brachytherapy ; Dose-Response Relationship, Radiation ; Facial Nerve ; radiation effects ; ultrastructure ; Female ; Iodine Radioisotopes ; administration & dosage ; radiation effects ; Male ; Rabbits ; Radiation Injuries, Experimental ; pathology ; Random Allocation

Objective: To investigate the value of ¹⁸F-fluorodeoxyglucose (FDG) PET/CT in differentiation of noninvasive and malignant pancreatic cystic lesions (PCL). Methods: A total of 125 PCL patients (66 males, 59 females, age range: 13-83 years), who had pathology or typical imaging performance with ≥12 months follow-up between August 2010 and December 2015, were enrolled in this retrospective study. The diagnostic effects of ¹⁸F-FDG PET/CT were calculated. The size, maximum standardized uptake value (SUV_max), delayed SUV_max and retention index (RI) of noninvasive and malignant PCL were analyzed. The results of pathological examination and follow-up were used as the gold standard, and Mann-Whitney u test was used to analyze the data. Receiver operating characteristic (ROC) curves of SUV_max, delayed SUV_max and RI were drawn and compared. Results: A total of 132 PCL were found in 125 patients, including 71 malignant PCL and 61 noninvasive PCL. The size, SUV_max, delayed SUV_max, RI of noninvasive and malignant PLC were 2.2 (1.4, 3.8) cm vs 3.9 (3.0, 5.0) cm, 1.5 (1.2, 1.8) vs 6.4 (4.4, 9.3), 1.5 (1.2, 1.9) vs 6.4 (5.4, 11.7), -6.3%(-19.4%, 5.6%) vs 17.5%(7.6%, 29.4%), respectively. All parameters were significantly different between 2 groups (z values: from -9.267 to -4.904, all P<0.05). The area under curve (AUC) and cut-off value of SUV_max were 0.969 and 2.5, and the corresponding accuracy, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) were 92.4%(122/132), 93.0%(66/71), 91.8%(56/61), 93.0%(66/71), 91.8%(56/61), respectively. The AUC and cut-off value of delayed SUV_max were 0.969 and 2.7, and the accuracy, sensitivity, specificity, PPV, NPV were 94.3%(99/105), 96.7%(58/60), 91.1%(41/45), 93.5%(58/62), 95.3%(41/43), respectively. ROC curves of SUV_max and delayed SUV_max were not significantly different (z=1.799, P>0.05). Conclusion ¹⁸F-FDG PET/CT has good performance in differentiation of noninvasive and malignant PCL, but the delayed scan cannot further improve the efficacy of differential diagnosis.

OBJECTIVETo investigate the role of sulfated tyrosine in regulating the activity of tyrosylprotein sulfotransferases (TPST) 1 and TPST2.

METHODSConstructs of TPST 1 and TPST2 were amplified by polymerase chain reaction (PCR), then fused into immunoglobulin G1 Fc region. All the variants in which sulfated tyrosines were mutated to phenylalanine were made by the PCR-based Quick Change method and confirmed by sequencing the entire reading frame. Small hairpin RNA (shRNA) constructs-targeting nucleotides 259-275 of TPST1 and nucleotides 73-94 of TPST2 were generated and subcloned into pBluescript. Human embryonic kidney (HEK) 293T cells were transfected with these plasmids. One day later, cells were split: one part was labeled with 35S-cysteine and methionine or 35S-Na2SO3 overnight, the second part was used for 125I labeled binding experiment, and the third part was retained for binding and flow cytometry.

RESULTSTyrosines at position 326 of TPST1 and position 325 of TPST2 were sulfated posttranslationally. Tyrosine sulfation of TPSTs was effectively inhibited by sulfation inhibitors, including specific shRNAs and non-specific NaCIO3. shRNAs reduced the sulfation of C3a receptor and C5a receptor, and partially blocked the binding of these two receptors to their respective ligands.

CONCLUSIONSThe activities of TPSTs were regulated by tyrosine sulfation. Inhibition of sulfotyrosine decreases the binding ability of C3a receptor and C5a receptor to their respective ligands.

Cell Line ; Complement C3a ; metabolism ; Complement C5a ; metabolism ; Humans ; Protein Binding ; Protein Processing, Post-Translational ; Receptor, Anaphylatoxin C5a ; metabolism ; Receptors, Complement ; metabolism ; Sulfotransferases ; genetics ; metabolism ; Transfection ; Tyrosine ; analogs & derivatives ; metabolism