Objective To construct eukaryotic expression vector of antisense MBD1 gene fragment and to provide a tool for studying MBD1 gene function. Methods PCR primers were designed according to the coding sequence of MBD1 gene. Xba I and Kpn I recognition sequences and cutting sites were added to the 5' end of the sense and antisense primer respectively. The 342 bp specific PCR fragment was obtained from the cDNA of biliary tract carcinoma cell line QBC-939 using RT-PCR, the purified PCR fragment was then inserted reversely into the multiple cloning site of eukaryotic expression vector pcDNA3. 1 ( + ). The constructed recombinant plasmid was identified by PCR confirmation, Xba I and Kpn I double enzyme digestion and DNA sequencing. Results The 322 bp specific DNA band was obtained by PCR, Xba I and Kpn I double digestion produced a 327 bp and a 5. 4 kb DNA band which represent the inserted target gene fragment and the vector respectively. The sequencing result confirmed that the sequence of inserted fragment was correct. Conclusion The eukaryotic expression vector of antisense MBD1 gene fragment was constructed successfully by using gene cloning technique. It will be a useful tool for studying MBD1 gene function in DNA methylation and tumorigenesis.

Objective To discuss the pathogenesis,diagnosis and treatment of delayed traumatic intracerebral hematoma.Methods The clinic data of traumatic delayed intracranial hematoma patients in this hospital were retro- spectively analyzed.According to clinic observation and CT re-examination,47 cases were diagnosed as delayed trau- matic intracranial hematoma(45 cases by operative treatment,and the other 2 by conservative treatment).Results There were 21 cases of recovery,10 cases of slight disability,8 cases of severe disability,8 cases of death.The total mortality rate was 17 %.Conclusion Brain contusion,subarachnoid hemorrhage and skull base fracture were impor- tant factors of DTICH.Fine-observation and prompt CT re-examination offered excellent results for DTICH.

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

To study the transactivation of a novel thyroid hormone receptor isoform, TR?. pcDNA3. 1-TR? and pGL3-Promoter/thyroid hormone responsive element were co-transfected into COS-7 cells. The expression of reporter gene was detected. It could be increased up to 45 times by T3. TR? and showed the characteristics of a transcription factor.

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.

Objective To compare and analyze the phylogenetic tree and sequence variant characteristics of Klebsiella species between 16S rDNA and rpoB. Methods Eighteen isolates identified as genus Klebsiella (with 15 of K. Pneumoniae and 3 of K. Oxytoca) by automated biochemical tests were selected. DNA were extracted, 16S rDNA and rpoB genes were amplified and sequenced with Klebsiella 16S rDNA and rpoB primers. Together with already published 8 species of Klebsiella and 9 species of Enterobacteriaceae 16S rDNA and rpoB sequences from GenBank, totally 35 sequences of 16S rDNA and rpoB respectively, phylogenetic trees were constructed with MEGA 4.0 to the analysis of groups. DNAStar/MegAlign was used for comparison of variable regions of 16S rDNA and rpoB, with analysis of degree of divergent at the same time. Results As for all 35 sequences, both 16S rDNA and rpoB phylogenetic trees divided Klebsiella species into three groups, 15 of K. Pneumoniae in this study and 6 of K. Pneumoniae from GenBank (except for K. Oxytoca and K. Mobilis) cluster to group Ⅰ, K. Oxytoca and K. Mobilis were cluster to group Ⅱ and Ⅲ, respectively. In rpoB phylogenetic tree, no matter group Ⅰ and group Ⅱ, or subgroup within group Ⅰ, the bootstrap values in each node of rpoB phylogenetic tree is obviously higher than that of 16S rDNA. Moreover, as for cluster to K. Oxytoca, rpoB is better than 16S rDNA. Analysis nucleic acid sequences of Klebsiella species, with 41 variable regions and 4 most significant regions were found within the Klebsiella 16S rDNA, while rpoB with 63 variable regions, and 1 most significant region. The similarity of 16S rDNA and rpoB within Klebsiella were 95.9%-100% and 90.2%-100% respectively. Further analysis divergent degree of 16S rDNA and rpoB within Klebsiella, the divergent value of rpoB (0-10.6) is higher than that of the 16S rDNA(0-4.0). Conclusion As for molecular classification and identification within KlebsieUa species, rpoB has more advantages than 16S rDNA.

OBJECTIVE: To observe cardiac ultrastructure and the expression of heat shock protein 70 (HSP70) and hypoxia inducible factor-lα (HIF-lα) in electric shock death rats and to explore the application of these indexes as the basis of medical identification in electric shock death. METHODS: Seventy-two SD rats were randomly divided into electric shock death group, postmortem electric shock group and the control group. The changes of myocardial ultrastructure were observed by transmission electron microscope, and the expressions of myocardial HSP70 and HIF-1α were observed by immunohistochemical technology. RESULTS: Myocardial myofibril fracture, mitochondrial cristae and membrane dissolution, and disordered arrangement of Z lines and M lines were observed in electric shock rats. HSP70 and HIF-lα were strong positive expressions in the electric shock death group, significantly compared with the control and postmortem electric shock groups (P < 0.05). CONCLUSION The expressions of HSP70 and HIF-lα were obviously increased in electric shock death group, which may be used as the diagnostic indicator of electric shock death.
Animals ; Death ; HSP70 Heat-Shock Proteins/metabolism* ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism* ; Myocardium/pathology* ; Rats ; Rats, Sprague-Dawley

Objective:To examine the potential influence factors of abdominal aortic aneurysm (AAA).Methods:A 1∶2 pair-matched, case-control study was conducted from July 2011 to December 2012 .A pair was composed of one AAA patient recruited from the Vascular Surgery Department , Chinese PLA General Hospital and two gender-and age-matched non-AAA subjects , one from the same hospital and the other from the community in Fangshan District in Beijing .Demographic data , medical history and the lifestyle of each subject were collected .Moreover , all the participants underwent abdominal ultra-sound or computed tomography ( CT ) and peripheral venous blood samples were obtained .Results:There were 155 case/control pairs .The multivariate conditional logistic regression model confirmed that suffering from hypertension conferred a 1.98-fold (95%CI 1.12-3.18) increased likelihood of AAA. Smoking was a strong independent risk factor of AAA , with odds ratios ( 95% confidence intervals ) of 5.23 (2.44-11.23).Dyslipidemia(OR=2.61,95%CI 1.45-4.70), a higher level of serum hs-CRP (OR=2.43,95%CI 1.37-4.31) and homocysteine (OR=2.73,95%CI 1.61-4.65) were all asso-ciated with AAA.Conclusion: Hypertension and smoking are the risk factors of AAA .Dyslipidemia, hsCRP and Hcy are associated with AAA .