BADH-CMO double gene,CMO gene and DREB1A gene were transformed respectively to embryonic callus of Kentucky bluegrass by particle bombardment. Then the embryonic callas of kentucky bluegrass were put in meclium for subculture which is mixed with 100mg/L hygromycin and the meclium for plantlet regeneration which mixell with 50mg/L hy gromycin about one month. Thus,the hygromycin-selectecl plants were obtained and were transplantecl into tlowerpots. The results of the PCR and Southern blot analysis indicated that the DREB1A gene,CMO gene and BADH-CMO double-gene were integrated into the genomic DNA of Kentucky bluegrass.

Objective To evaluate the acute and delayed killing effect of high intensity focused ultrasound (HIFU) on Echinococcus granulosus(E. granulosus)protoscolices in vitro.Methods E. granulosus protoscolices were treated with different dosage of effective power(0,25,50,100,200,250 W)and time(5,10,20,30,40,50,60 s)of HIFU in vitro to obtain the dosage-effect curves.Then the survival pmtoscolices were incubated,and the mortality of each group was counted daily.The protoscolicidal effects were investigated by trypan blue exclusion assay.Results Compared with the untreated group,the Vitality of E.granulosus protoscolices significantly decreased immediately after treated by HIFU of different dosage(F=5201.59 vs 1865.65,P＜0.05),there were the interaction both different dosage and time(F=214.50,P＜0.05).The protoscolices were broken into pieces by HIFU of 250 W×40 s,whereas the growth of the surviving protoscolices after exposed to HIFU was obvious suppressed.Both the acute killing effect and the delayed inhibitory effect showed a dosage-dependant manner.The inhibitory effect increased along with the increased dosage of HIFU(P＜0.05).The inhibitory effect in 50 W×10 s group was stronger than 25 W×20 s group(P＜0.05).The mortality was increased in parallel with the increase of HIFU dosage.Conclusions HIFU show an effective immediately killing effect,as well as a growth-inhibiting effect on the E.granulosus protoscolices in vitro.

OBJECTIVETo study the betulonic acid on the cell cycle and related protein expressions on mice of bearing H22 tumor cells.

METHODFlow cytometray was used to observe the changes of betulonic acid on the cell cycle and P53 of H22 tumor cells. Immunohistochemistry was determined the espressions of PI3K and AKT.

RESULTIncreasing the doses of betulonic acid, the number of H22 cells in S phase and G2 phase was increasing gradually, it can speculate that when the betulonic acid act on cells, the cells were blocked in S and G2 phase and inhibited the protein expressions of PI3K and AKT.

CONCLUSIONBetulonic acid may be up-regulate the activity of P53 and inhibite the expressions of PI3K and AKT, so that it inhibited the survival pathway of tumor cells.

Animals ; Betula ; chemistry ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Survival ; drug effects ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Female ; Flow Cytometry ; G2 Phase ; drug effects ; Gene Expression Regulation ; drug effects ; Immunohistochemistry ; Male ; Mice ; Neoplasm Transplantation ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVETo study the effect of dexamethasone on airway morphology and on the expression of angiopoietin-1 (Ang-1) and its tyrosine kinase receptor Tie-2 in the airway of asthmatic rats.

METHODSForty-five Sprague-Dawley rats were randomly divided into control, asthmatic, and dexamethasone-treated asthmatic groups. Asthma was induced by repeated sensitization and challenge with ovalbumin in the latter two groups. The dexamethasone intervention group received an intraperitonea injection of dexamethasone (2 mg/kg) before asthma challenge. Immunohistochemistry was used to measure the expression of Ang-1 and Tie-2 in the airway. Airway thickness was estimated by a computerized digital image analyzer.

RESULTSAirway thickness in the asthmatic group (33.9333+/-8.3791 micro m2/micro m) increased significantly compared with that in the control group (21.1333+/-2.7740 micro m2/micro m) (P<0.01). The dexamethasone intervention group also showed increased thickness of the airway (27.4000 +/- 4.6105 micro m2/micro m) compared with the control group (P<0.01), but the airway thickness in the dexamethasone intervention group was significantly reduced compared with that in the untreated asthmatic group (P<0.01). The expression of Ang-1 (103.9487+/-8.2914 vs 76.0320+/-3.7728; P<0.01) and Tie-2 (99.2307+/-8.1913 vs 75.3153+/-3.7321; P<0.01) in the airway increased significantly in the asthmatic group compared to controls. The expression of Ang-1 and Tie-2 in the airway of the dexamethasone intervention group (90.6180+/-5.2339 and 86.6633+/-3.7321, respectively) was statistically higher than that in the control group (P<0.01) but statistically lower than that in the untreated asthmatic group (P<0.01). Ang-1 and Tie-2 expression in the airway was positively correlated with the thickness of airway (r(Ang)-1=0.719r(Tie)-2=0.746P<0.01). There was also a positive correlation between Ang-1 and Tie-2 expression (r=0.742P<0.01).

CONCLUSIONSThe expression of Ang-1 and Tie-2 in the airway increased in asthmatic rats and was positively correlated with the thickness of the airway. Ang-1 and Tie-2 may participate in the process of airway remodeling in asthma. Dexamethasone can decrease the expression of Ang-1 and Tie-2 in the airway and relieve the changes of airway morphology.

Angiopoietin-1 ; analysis ; physiology ; Animals ; Asthma ; metabolism ; pathology ; Female ; Lung ; chemistry ; pathology ; Rats ; Rats, Sprague-Dawley ; Receptor, TIE-2 ; analysis ; physiology

OBJECTIVETo evaluate the therapeutic effect of comprehensive therapeutic protocol of electroacupuncture combined with active-blood-and-dissolve-stasis herbs and rehabilitation training for cerebral infarction.

METHODSA multi-center randomized controlled trial was done, three hundred and twenty cases were divided into four groups: electroacupuncture combined with active-blood and dissolve-stasis herbs and rehabilitation training group (group A), electroacupuncture combined with rehabilitation training group (group B), herbs combined with rehabilitation training group (group C) and rehabilitation training group (group D), 80 cases in each group. The following two groups of acupoints were used alternatively in electroacupuncture treatment: the first group including Vasomotor Area, Jianyu (LI 15), Biguan (ST 31), Hegu (LI 4) and Taichong (LR 3); the second group including Motor Area, Quchi (LI 11), Yanglingquan (GB 34) and Shenshu (BL 23). 20 mL Xiangdan injection and 250 mL 5% glucose injection or 250 mL 0.9% sodium chloride injection were used by intravenous drip in herbs treatment once a day. The rehabilitation training was performed by the professional physical therapist. Each group was treated with corresponding treatment protocol. The therapeutic effect was evaluated by index of the mortality or disability rate 3 months after the onset of disease. The intention to treat analysis (ITT) was used in data.

RESULTSThe mortality or handicap rate 3 months after the onset of disease of four groups were 17.5% (14/80) in group A, 22.5% (18/80) in group B, 40. 0% (32/80) in group C, and 31.3% (25/80) in group D, respectively. The group A has a best therapeutic effect (vs group C, group D, both P<0.05), and there was no adverse event.

CONCLUSIONThe combined application of electroacupuncture, active-blood and dissolve-stasis herbs and rehabilitation training is a better treatment for cerebral infarction in clinic.

Adult ; Aged ; Cerebral Infarction ; drug therapy ; rehabilitation ; therapy ; Combined Modality Therapy ; Drugs, Chinese Herbal ; therapeutic use ; Electroacupuncture ; Female ; Humans ; Male ; Middle Aged

OBJECTIVETo generate an oncolytic herpes simplex virus (oHSV) permissive mouse melanoma cell line B16RHSV, preserving the tumorigenic ability in syngeneic mice.

METHODSThe herpes simplex virus entry mediator (HVEM) gene was amplified by PCR from human melanoma cell line A375, and cloned into pGEM-T Easy vector for sequencing. The HVEM gene was then cloned into pcDNA3 vector to generate pcDNA3-HVEM for transfection of mouse melanoma cell line B16-F10 cells. After that, the putative transfected cells were selected in full growth medium containing G418. The HVEM-expressing cells were isolated by immunomagnetic bead separation. The mouse melanoma cell line expressing oHSV receptor-HVEM, designated as B16RHSV, was generated. The permissibility of B16RHSV cells to oHSV infection was examined with green fluorescence protein (GFP)-expressing oHSV (oHSVGFP). To investigate the tumorigenic ability of both cells in vivo, 2×10(5) cells in 100 µl were subcutaneously inoculated into the right flanks of C57/BL mice.

RESULTSIn vitro, the B16RHSV mouse melanoma cells were shown by fluorescence microscopy capable of being infected by oHSVGFP. In vivo, the B16RHSV cells, like their wild type counterpart, grew to form melanoma in syngeneic mice.

CONCLUSIONA herpes simplex virus-permissive mouse melanoma cell line was established. Its tumorigenicity remained unchanged.

Animals ; Cell Line, Tumor ; Female ; Gene Amplification ; Genetic Vectors ; Herpesvirus 1, Human ; genetics ; physiology ; Humans ; Melanoma ; pathology ; virology ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Plasmids ; Receptors, Tumor Necrosis Factor, Member 14 ; genetics ; metabolism ; Transfection ; Tumor Burden

OBJECTIVEThe aim of this study was to construct a new oncolytic virus oHSV2hGM-CSF and evaluate its oncolytic activity in vitro and in vivo in parallel with oHSV1hGM-CSF.

METHODSoHSV2hGM-CSF was a replication-competent, attenuated HSV2 based on the HG52 virus (an HSV2 strain). It was engineered to be specific for cancer by deletion of the viral genes ICP34.5 and ICP47 and insertion of the gene encoding hGM-CSF. To measure the in vitro killing effect of the virus, 15 human tumor cell lines (HeLa, Eca-109, PG, HepG2, SK/FU, CNE-2Z, PC-3, SK-OV3, A-549, 786-0, MCF-7, Hep-2, HT-29, SK-Mel-28, U87-MG) and mouse melanoma (B16R) cell line were seeded into 24-well plates and infected with viruses at MOI = 1 (multiplicity of infection, MOI), or left uninfected. The cells were harvested 24 and 48 hours post infection, and observed under the microscope. For animal studies, the oncolytic viruses were administered intratumorally (at 3-day interval) at a dose of 2.3 x 10(6) PFU (plaque forming unit, PFU) for three times when the tumor volume reached 7-8 mm3. The tumor volume was measured at 3-day intervals and animal survival was recorded.

RESULTSBoth oHSV2hCM-CSFand oHSV1hGM-CSF induced widespread cytopathic effects at 24 h after infection. OHSV2hGM-CSF, by contrast, produced more plaques with a syncytial phenotype than oHSV1hGM-CSF. In the in vitro killing experiments for the cell lines HeLa, HepG2, SK-Mel-28, B16R and U87-MG, oHSV2hGM-CSF eradicated significantly more cells than oHSV1hGM-CSF under the same conditions. For the mouse experiments, it was observed that oHSV2hGM-CSF significantly inhibited the tumor growth. At 15 days after B16R tumor cells inoculation, the tumor volumes of the PBS, oHSV1hGCM-CSF and oHSV2hGM-CSF groups were (374.7 +/- 128.24) mm3, (128.23 +/- 45.32) mm3 (P < 0.05, vs. PBS group) or (10.06 +/- 5.1) mm3 (P < 0.01, vs. PBS group), respectively (mean +/- error). The long term therapeutic effect of oHSV2hGM-CSF on the B16R animal model was evaluated by recording animal survival over 110 days after tumor cells inoculation whereas all the mice in the PBS group died by day 22 (P < 0.01). The anti-tumor mechanism of the newly constructed oHSV2hGM-CSF against B16R cell tumor appeared to include the directly oncolytic activity and the induction of anti-tumor immunity to some degree.

CONCLUSIONThe findings of our study demonstrate that the newly constructed oHSV2hGM-CSF has potent anti-tumor activity in vitro to many tumor cell lines and in vive to the transplanted B16R tumor models.

Animals ; Cell Line, Tumor ; Female ; Gene Deletion ; Genetic Engineering ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; Herpesvirus 2, Human ; genetics ; immunology ; Humans ; Immediate-Early Proteins ; genetics ; metabolism ; Melanoma, Experimental ; pathology ; therapy ; virology ; Mice ; Mice, Inbred C57BL ; Oncolytic Virotherapy ; methods ; Oncolytic Viruses ; genetics ; physiology ; Random Allocation ; Tumor Burden ; Viral Proteins ; genetics ; metabolism ; Xenograft Model Antitumor Assays

Objective To evaluate video-assisted anal fistula treatment (VAAFT) for Parks type Ⅱ anal fistula.Methods 40 Parks type Ⅱ anal fistula patients underwent VAAFT procedure from June 2015 to June 2017.Results were compared with 40 cases treated by incision and thread drawing.Results There was no significant difference between the two groups for curative effect,postoperative urinary retention,wound edema,bleeding and recurrence rate after 6 months of operation (90％ vs.95％,x2 =0.722,P =0.697;5％ vs.8％,x2 =0.213,P=1.0;2％ vs.8％,x2 =1.053,P=0.615;0 vs.5％,x2 =2.051,P =0.494;10％ vs.5％,x2 =0.721,P =0.675).Pain on first day and one week after operation in the VAAFT was less [(1.9±0.6) vs.(3.7±1.0),t =9.438,P=0.001;(0.9±0.7) vs.(1.9±0.8),t=6.269,P=0.001],hospital stay was shorter [8.4 ±1.3) d vs.(9.2 ±2.2) d,t =2.030,P=0.047],wound healing was faster [(27 ±8) dm.(38 ±6) d,t =7.328,P =0.001].The Jorge-Wexner incontinence score [(0.5±0.7) vs.(1.2±1.3),t =2.951,P=0.005] and the fecal incontinence severity index [(1.1±1.6) vs.(5.1 ±3.2),t =7.097,P=0.001] were lower in patients receiving VAAFT procedure.Conclusion Video-assisted anal fistula treatment is a safe and effective surgical method with the advantages of less trauma,and pain,quicker recovery and no damage to the anal sphincter.

Animals ; Anthelmintics ; pharmacology ; therapeutic use ; Culture Media ; Echinococcosis ; drug therapy ; parasitology ; Echinococcus granulosus ; drug effects ; growth & development ; pathogenicity ; ultrastructure ; Parasitic Sensitivity Tests

BACKGROUNDLaparoscopic cholecystectomy (LC) has been a standard operation and replaced the open cholecystectomy (OC) rapidly because the technique resulted in less pain, smaller incision, and faster recovery. This study was to evaluate the value of blunt dissection in preventing bile duct injury (BDI) in laparoscopic cholecystectomy (LC).

METHODSFrom 2003 to 2015, LC was performed on 21,497 patients, 7470 males and 14,027 females, age 50.3 years (14-84 years). The Calot's triangle was bluntly dissected and each duct in Calot's triangle was identified before transecting the cystic duct.

RESULTSTwo hundred and thirty-nine patients (1.1%) were converted to open procedures. The postoperative hospital stay was 2.1 (0-158) days, and cases (46%) had hospitalization days of 1 day or less, and 92.8% had hospitalization days of 3 days or less; BDI was occurred in 20 cases (0.09%) including 6 cases of common BDI, 2 cases of common hepatic duct injury, 1 case of right hepatic duct injury, 1 case of accessory right hepatic duct, 1 case of aberrant BDI 1 case of biliary stricture, 1 case of biliary duct perforation, 3 cases of hemobilia, and 4 cases of bile leakage.

CONCLUSIONExposing Calot's triangle by blunt dissection in laparoscopic cholecystectomy could prevent intraoperative BDI.

Adolescent ; Adult ; Aged ; Bile Duct Diseases ; prevention & control ; Cholecystectomy, Laparoscopic ; methods ; Common Bile Duct ; injuries ; surgery ; Female ; Humans ; Male ; Middle Aged ; Retrospective Studies ; Young Adult