Objective To compare the difficult degree,differences in gastric pouch volume,the complications,and postoperative weight loss effect of two gastric pouch practices in laparoscopic gastric bypass for bariatrics.Methods We retrospectively analyzed laparoscopic gastric bypass surgery in 76 bariatrics cases from May 2010 to May 2012.Two methods were used to create gastric pouch,among which 39 cases were operated with the dissection landmark method (called dissection),and 37 cases with bougie airbags 30 ml (called the bougie).Parameters were compared between the 2 groups including operation time for gastric pouch,the amount of staple cartridges,intraoperative complications,blood loss,and postoperative excess weight loss percentage (EWL ％),the postoperative bleeding,obstruction,fistula and other complications.Results All patients were followed up for more than one month.The dissection group used longer time in creating gastric pouch than that in the bougie group [(58 ± 27) min,(42 ±21) min,P ＜ 0.01].The number of staple cartridges used were significantly different between the two groups [(6.2 ±3.0),(4.3 ± 2.0),P ＜ 0.01].There were 10 cases of complications in the dissection group (26％),and 3 cases in the bougie group (8％) (P ＜0.05).One month after the surgery,one each patient had transient hematochezia.There was no gastric pouch-related complications in the two groups postoperation.There was no difference in excess weight loss (P ＞ 0.05).Conclusions The two methods can achieve the same effect on excess weight loss.The bougie method used less time in creating the gastric pouch,less consumable materials,and less complications occurred in creating gastric pouch.

Aim To study the mechanism of synergistic antitumor of EBB and doxorubicin in doxorubicin-resistant MCF-7/ADR breast carcinoma cells.Methods The antitumor activity of doxorubiein alone and its combination with EBB were measured by MTT assay in MCF-7/ADR and MCF-7cells. The rate of doxorubicin-induced apoptosis and the protein and mRNA levels of P-glycoprotein(P-gp) were determined in MCF-7/ADR treated with EBB by flow cytometry (FACS), respectively.Laser scanning confocal microscopy was used to detect the intracellular accumulation of drug in EBB-treated MCF-7 and MCF-7/ADR cells.Results EBB had antitumor effects for MCF-7 and MCF-7/ADR.It could potentiate the antitumor effect of dororubicin with CDI of 0.73 and 0.49 for MCF-7 and MCF-7/ADR,respectively.EBB and doxorubicin acted synergistically in elevating apoptosis of MCF-7/ADR and downregulating the expression of P-gp in a dose-dependent manner in MCF-7/ADR.EBB restored the intracellular accumulation of doxorubicin in MCF-7/ADR cells in a dose-dependent manner.After pretreatment with EBB for 24 h and 48 h,the intracellular accumulation of doxorubicin and Rh123 was obviousely restored in MCF-7/ADR cells compared with control in a time-dependent manner.Conclusion EBB is a potential agent which has strong inhibitory effect on both multidrug resistant cells and their parental cells.EBB can significantly potentiate the antitumor effects of dororubicin in MCF-7/ADR cells by blocking the function of P-glycoprotein and inhibiting the expression of P-glycoprotein.

Background and purpose:Bispecific antibody is a special pattern of genetic engineering antibody.It possesses 2 antigen binding sites, one directed at the effecter cells and the other at the target cells which are always the tumor cells.This enables the effecter cells to recruit to the targets efficiently and exert cytotoxic effect.The experiment was aimed to construct the expression vector of anti-CD3?anti-CD19 bispecific antibody, purification and analyzing the activity of the diabody.Methods:Applyed PCR and overlap PCR to construct the expression vector of anti-CD3?anti-CD19 bispecific antibodies, transformed it into E.coli 16C9 for expression.Purified the diabody by His-tag affinity chromatography and confirmed by 12% SDS-PAGE and Western blot.Antigen binding activity of the diabody was analyzed by FACS.Results:The DNA sequence of the expression vector containing anti-CD3?anti-CD19 fragment was comfirmed.The yield of purified diabody was up to 5 mg/L.The molecular weight of diabody was correctly indicated by SDS-PAGE and Western blot.The diabody could specifically bind to CD19+ Raji cells and CD3+ Jurkat cells while competitively inhibit binding of HIT3a and HIT19a to the cells.Conclusion:Successfully constructed the expression vector of anti-CD3?anti-CD19 bispecific antibodies, the diabody acquired by the method could be produced with high level expression and also had high specific affinity with CD19+ Raji cells and CD3+ Jurkat cells.

Background and purpose:Multidrug resistance(MDR)is one of the major causes of progressive breast cancer chemotherapy failure.One of the major mechanisms of MDR is the overexpression of P-glycoprotein (P-gp).Therefore,the identification of novel agents which can inhibit the drug transporter function of P-gp or its expression is of utmost interest in cancer research.The aim of this study was to explore the antitumor and reversal effect of PHⅡ-7,natural products from traditional Chinese medicine(TCM).Methods:The cytotoxicity of PHⅡ7 alone and combined application of PHⅡ-7 and adriamycin(ADR)on breast cancer cells were determined using MTT assay.Annexin V–FITC/PI apoptosis detection kit was used to observe the apoptosis-inducing effect of PHⅡ-7 in MCF-7 and MCF-7/ADR cells.The effect of PHⅡ-7 on mdr1 mRNA was determined by reverse transcription PCR and real time PCR,flow cytometer was used to measure the intracellular ADR accumulation.Results:PHⅡ-7 alone inhibited cell growth of MCF-7 and MCF-7/ADR cells with the IC 50 (6.07?0.85),(5.51?1.22)?mol/L,respectively when combined with ADR,PHⅡ-7 enhanced the cytotoxicity of ADR toward MCF-7/ADR cells.In addition,PHⅡ-7 induced apoptosis both on MCF-7 and MCF-7/ADR cells;PHⅡ-7 reversed the drug resistance to ADR in MCF-7/ADR cells by inhibiting mdr1 mRNA transcription and increasing the intracellular ADR accumulation.Conclusion:PHⅡ-7 displayed significant anti-proliferative and apoptosis-inducing effect on sensitive and multidrug resistant breast cells in vitro.PHⅡ-7 reversed effectively MDR by blocking the drugs to be pumped out by inhibiting P-gp expression and function pathway.

Objective:To prepare and characterize specific and discrepant mouse hybridoma antibodies on membrane of HL60 and HL60/ADR cell lines.Methods:BALB/c mice were immunized by subtractive immunization induced Cp(Cyclophosphamide).McAbs were prepared by hybridoma technique,screened and detected by FACS and LSCM.Results:51 candidates and discrepant antibodies were found,and one of them (5F6) was purified and identified.Conclusion:Combination of SI with discrepant screening method should facilitate the preparing and identifying discrepant McAbs for identifying antibodies that can distinguish the differences in proteins expressed in HL60 and HL60/ADR,which is a significative and potential method in the research and target therapy associated drug-resistance.

Objective To establish a multiplex loop-mediated isothermal amplification(mLAMP)method for simultaneous detection of Salmonella,Vibrio parahaemolyticus (VPH)and Listeria monocytogenes (LM).Methods Three sets of mLAMP primers were designed to specifically target bcfD of Salmonella and tlh of VPH and iap of LM.The respective single LAMP assay of the three kinds of bacteria was developed,and the ratio of primer concentration was optimized to develop a multiplex LAMP system.The specificity and sensitivity of multiplex LAMP were observed.Results Turbidity monitoring results in real time suggests that the mLAMP was highly specific and amplification could be obtained within 45 min under isothermal conditions.The sensitivity of this mLAMP was found to be 300 fg/μl genomic DNAs for Salmonella and 4.2 pg/μl for VPH and 4.5 pg/μl for LM,which was consistent with conventional PCR.Conclusion The mLAMP described can potentially facilitate simultaneous detection of three kinds of bacteria in a large number of food samples, which could be used as a primary screening method and as a supplement to classical detection methods.

Aim To determine whether membrane cytokeratin 8(CK8 )and BCRP expression cooperatively contributed to multidrug resistance(MDR)in MCF-7/MX cells.Methods MCF-7/MX cells were transfected with specific anti CK8-siRNAs and anti BCRP-siRNAs via LipofectAMINE2000.The expression of CK8 and BCRP was determined using Western blot,and membrane staining was observed by laser confocal microscopy.Sensitivity to chemical drugs was examined by Sulforhodamine B method.Results The expression levels of cell surface CK8 and BCRP were obviously reduced by siRNAs,and inhibition of CK8 and BCRP expression could effectively restore the sensitivity to drugs and reverse MDR phenotype of MCF-7/MX cells.Conclusions CK8 together with BCRP may play significant roles in conferring the multifactorial MDR phenotype of MCF-7/MX cells,but may act independently via potentially different mechanisms.Combinational approaches that target multiple drug-resistance-related molecules/pathways in cancer cells may represent more efficacious strategies to overcome MDR.

Objective To establish a detection system of ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for everolimus concentration in whole blood of liver transplant recipients. Methods The proteins of samples were precipitated with methanol and zinc sulfate, and everolimus-D4 was used as the internal standard. Phenomenex Kinetex PFP column was used. The mobile phase A was water (containing 2 mmol/Lammonium formate and 0.1% formic acid), and the mobile phase B was methanol (containing 2 mmol/L ammonium formate and 0.1% formic acid). The gradient elution was performed with the flow rate of 1 mL/min, the column temperature of 50 ℃ and the injection volume of 1 μL. The multi-reaction monitoring mode was used to quantitatively analyze with electrospray positive ionization. The UPLC-MS/MS detection system required only 100 μL of whole blood, and could achieve a sufficient lower limit of quantification without complicated sample preparation. The total running time was within 4.5 min. Linear regression (1/x²) analysis was performed using peak area of everolimus / peak area of everolimus-D4 (y) and concentration of everolimus/concentration of everolimus-D4 (x) to calculate the calibration function and analyze its accuracy and linear relationship. UPLC-MS/MS was used to detect the trough blood concentration of everolimus in blood samples of 5 recipients after liver transplantation. Results The accuracy of quality control was within 15%, and the linear relationship of everolimus was good in the blood concentration range of 1-100 ng /mL(R² > 0.990). Trough blood concentration of everolimus measured in blood samples of 5 liver transplant recipients ranged from 3.77 to 9.27 ng/mL. Conclusions The detection system of UPLC-MS/MS in this study is suitable for monitoring the concentration of everolimus in whole blood of liver transplant recipients because of its high accuracy, simple sample processing method and short detection time.

After ischemic stroke, the key to reduce the mortality and disability rate is to restore the blood supply of brain tissue as soon as possible. However, the cerebral ischemia-reperfusion injury (CIRI) caused by blood flow restoration is also an important cause of brain tissue structural damage and dysfunction. Studies in recent years have shown that the activation of mitophagy at CIRI stage can reduce the volume of cerebral infarction and protect neurons from CIRI, while excessive or insufficient mitophagy can aggravate CIRI. This suggests that inducing moderate mitophagy may be a potential therapeutic target for neuroprotection after stroke. However, the neuroprotective mechanism of mitophagy has not yet been fully elucidated. This article reviews the neuroprotective mechanism and potential application of mitophagy in stroke, and discusses some problems of mitophagy as a therapeutic target for stroke.