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.

BACKGROUND:Extracorporeal shock wave has been shown to influence the physiological function of endothelial cel s via the activation of mechanoreceptors and specific signal transduction system, and gene expression regulation.
OBJECTIVE:To explore the impact of different energy flow densities and numbers of shots of extracorporeal shock waves on the new vessel formation ability, migration capability and apoptosis of bone microvascular endothelial cel s.
METHODS:Bone microvascular endothelial cel s isolated from the femoral head of patients undergoing arthroplasty were subcultured in vitro, and then were immunofluorescently evaluated with endothelial cel marker antibodies to CD31 and von Wil ebrand factor (vWF), and grouped according to different energy flow densities (low, 0.03 mJ/mm2;high, 0.11 mJ/mm2) and numbers of shots (400 and 800). Capil ary-like tube formation, migration capability and apoptosis of bone microvascular endothelial cel s were determined by 3-D culture in vitro, scratch test, and flow cytometry, respectively.
RESULTS AND CONCLUSION:vWF and CD31 were positively expressed in approximately 100%of bone microvascular endothelial cel s, which indicates the cultured cel s had characterization of microvascular endothelial cel s. Extracorporeal shock wave enhanced angiogenesis and migration capability of bone microvascular endothelial cel s derived from the femoral head, and especial y low-energy flow density of extracorporeal shock wave exerted more superior effects. Angiogenesis of bone microvascular endothelial cel s was decreased with the increased shot number in the low-energy flow density group. In addition, extracorporeal shock wave inhibited bone microvascular endothelial cel apoptosis induced by steroids. Our results suggest that energy flow density and number of shots of extracorporeal shock waves impact the physiological function of bone microvascular endothelial cel s derived from the femoral head.

This is designed to analyze and summarize medication rules for treating senile dementia with Chinese medicine in CNKI according to the traditional Chinese medicine (TCM) inheritance auxiliary system. Collect documents in CNKI that account treating senile dementia with Chinese formula; filter and establish a formula database, and then to search for medication rules on the TCM inheritance auxiliary system. It is filtered that 104 formulas are used for treating senile dementia screening treat senile dementia, involving 147 kinds of Chinese medicine. Tonic medicine are most frequently used, followed by the medicine of activating blood circulation and resuscitating; medicine pair most used is Ligusticum chuanxiong Hort-Acorus tatarinowii, accounting for 27.9% of all formula. And then 8 core pairs and 4 new formulas are evolved. Analysis on formulas for treating senile dementia filtered form CNKI by TCM inheritance auxiliary system shows prescription is mainly tonifying, activating blood circulation and resuscitating, that reveals prescription rules, to provide a reference for clinical treatment.
Databases, Factual ; Dementia ; drug therapy ; Drug Prescriptions ; Drug Therapy, Combination ; Drugs, Chinese Herbal ; chemistry ; therapeutic use ; Humans

OBJECTIVETo explore effective approaches of treating elderly patients with distal tibiofibular fractures.

METHODSFrom August 2008 to October 2012,175 elderly patients with distal tibiofibular fractures were treated with locking compression plate (LCP) through anterior tibial. There were 112 males and 63 females with an average of 71.3 (ranged 60 to 83) years old. Of them,89 cases were treated by anterior tibial tension reduced incision with LCP,including 62 males and 27 females with a mean age of (71.8 +/- 6.4) years old. Eighty-six patients were treated by distal tibial incision with LCP,including 58 males and 28 females with a mean age of (70.3 +/- 6.7) years old. Swelling time, operation time, intraoperative blood loss, hospital stay, healing time, complications and AOFAS scores were compared between two groups after operation.

RESULTSSwelling time in anterior tension reduced incision with LCP and distal tibial incision with LCP was (5.6 +/- 1.3) and (9.7 +/- 2.1) days, healing time was (4.2 +/- 1.4) and (5.4 +/- 1.9) months,and complications were found 3 in tension reduced incision and 10 in distak tibial incision respectively;and all data shown statistically significant differences between two groups (P < 0.05). At 12 months after operation,AOFAS score was 89.0 +/- 9.7, 87.9 +/- 9.4; and there was no statistically significant difference between two groups (P > 0.05).

CONCLUSIONTension reduced incision through anterior tibial combined with locking compression plate fixation in treating elderly patients with distal tibiofibular fractures can provide good clinical effects with quick fracture healing and low complications.

Adult ; Aged ; Aged, 80 and over ; Bone Plates ; Female ; Fibula ; injuries ; surgery ; Fracture Fixation, Internal ; instrumentation ; methods ; Humans ; Male ; Middle Aged ; Minimally Invasive Surgical Procedures ; Tibia ; injuries ; surgery ; Tibial Fractures ; surgery ; Treatment Outcome

BACKGROUND:Matrix metal oproteinase-13 is most active in the degradation of col agen type II in the extracel ular matrix of cartilage. Interleukin-1 (IL-1) is thought to be the inducer of matrix metal oproteinases, and participates in the degradation and degeneration of articular cartilage. OBJECTIVE:To study the influence of IL-1βon microRNA-27b (miR-27b) and matrix metal oproteinase-13 expression of chondrocytes in rats. METHODS:Chondrocytes isolated from seven male Wistar rats were cultured and divided into IL-1βstimulation group and control group. No stimulus was given in the control group;10μg/L of serum free medium was used to culture rat chondrocytes in the IL-1βstimulation group. Cel growth was observed at 0, 24, and 48 hours under an inverted microscope. miR-27b and matrix metal oproteinase-13 expression in the cultured chondrocytes were detected. RESULTS AND CONCLUSION:The relative expression of matrix metal oproteinase-13 in rat chondrocytes was gradual y increased when induced by IL-1βat 0, 24, and 48 hours (P<0.05). Expression of miR-27b and miR-31 in rat chondrocytes at 24 and 48 hours induced by IL-1βgradual y decreased (P<0.05);conversely, expression of MiR-26a, miR-26b, miR-23, and miR-204 gradual y increased (P<0.05). After 48 hours of IL-1βinduction, expression of miR-27b was the lowest in rat chondrocytes (P<0.05). These findings suggest that IL-1βinhibits miR-27b expression, strengthens the expression of matrix metal oproteinase-13, and damages chondrocytes, contributing to both the onset and progression of osteoarthritis.

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 the efficacy of laryngeal mask airway-Advance (ALMA) and laryngeal mask airway-Supreme (S-LMA) in patients undergoing laparoscopic cholecystectomy.Methods Two hundred and forty ASA Ⅰ or Ⅱ patients,aged 18-64 yr,weighing 50-70 kg,undergoing laparoscopic cholecystectomy,were randomly divided into2 groups (n=120 each):group S-LMA (group S) and group A-LMA (group A).LMA was inserted after induction of general anesthesia with propofol 2.0-2.5 mg/kg,sufentanil 0.2 μg/kg and rocuronium 0.6 mg/kg.After LMA was placed,the rim was immediately inflated and positive pressure was applied to the reservoir bag of anesthesia machine circuit to check for leaks.The plateau pressure at which leaks occurred was recorded.BP,HR,SpO2,PErCO2 and Ppeak were monitored during operation.Fiberoptic bronchoscopy was performed and the placement was scored (0 =unable to see the vocal cords and the ventilation of the lungs was inadequate,4=vocal cords were clearly seen and ventilation was adequate).The rate of successful placement,placement time,the incidence of leaks during operation,duration of anesthesia and surgery,extubation time,emergence time,blood stain on the LMA after being removed,backflow after extubation and postoperatve complacations including sore throat,hoarseness and dysphagia were recorded.The efficacy for airway management and the difficulty of placement were scored.Results There was no significant difference in the rate of successful placement,difficulty of placement scores,blood stain on the LMA after being removed,incidence of backflow,sore throat,hoarseness and dysphogia,anesthesia time,duration of surgery,extubation time and emergence time between the two groups.The placement time was shorter in group S than in group A,but the airway sealing pressure,FOB scores and efficacy for airway management scores were significantly higher and the incidence of leaks during operation was significantly lower in group A than in group S.Conclusion Both A-LMA and S-LMA can provide adequate ventilation during laparoscopic cholecystectomy.The efficacy of A-LMA is better.

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

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