To optimize the solvent system of high-speed counter-current chromatography (HSCCC) for diterpenoidtanshinone separation and define it's antitumor activity in vitro. Total diterpenoidtanshinone was made by CO2 supercritical fluid extraction (SFE), UPLC was applied to determining the peak area of tanshinone IIA and cryptotanshinone in different solvents up or under phase, and the partition coefficient K values of them were calculated. CKK-8 was used to observe the inhibitory effects of diterpenoidtanshinone on human liver cancer (QGY-7703), lung cancer (PC9, A549), gastric cancer (MKN-45, HGC-27), colon cancer (HCT116), myeloma (U266, RPMI8226), and breast cancer (MCF-7). The best solvent system for HSCCC was petroleum ether-ethyl acetate-methanol-aqua (12∶8∶ 13∶7). The yield of diterpenoidtanshinone was 8.65%. Diterpenoidtanshinone has good effect of antitumor in vitro especially on human PC9 cell. The selected solvent system is suitable for diterpenoidtanshinone separation by HSCCC, and the established HSCCC method is reliable and easy for operating. Diterpenoidtanshinone has good antitumor effect in vitro.

Objective To observe the positive rate of plasma cytomegalovirus DNA(CMV‐DNA) level after allogenic hema‐topoietic stem cell transplantation (allo‐HSCT) ,analysis and explore the risk factors related to CMV infection .Methods Choose 30 patients who had performed allo‐HSCT in our department from July 2012 to September 2014 .PCR were used regularly to detect the plasma CMV‐DNA levels in these patients .The regular monitoring times were as follow :the first month(once a week) ,the second to third month(twice a week) ,the fourth to sixth month(once a month) after allo‐HSCT respectively .The positive rates were coun‐ted in every period .Results Thirteen patients had CMV infection ,and the infection rate were 43 .3% .In the first month ,there were 4 cases (13 .3% )whose plasma CMV‐DNA levels were positive ,however ,the positive cases in the second month ,the third month , the fourth month ,the fifth month and the sixth month were 11(36 .7% ) ,2(6 .7% ) ,0 ,2(6 .7% ) and 0 respectively .Statistical data showed that it was in the second month after allo‐HSCT that the CMV‐DNA positive rate was higher than other periods .The anal‐ysis suggested that the positive rate of CMV‐DNA related to the administration of rabbit anti‐human thymocyte globulin(ATG) ,ba‐siliximab ,and the occurrence of acute graft versus host disease(GVHD) ,there were no relationship among gender ,age ,risk stratifi‐cation of primary disease ,HLA condition ,preparative project ,recovery time of neutrophile granulocyte .Conclusion It is necessary and beneficial to monitor blood CMV‐DNA level regularly and take treatment early to avoid CMV related comobidity after allo‐HSCT .

Objective To determine the effects of Che A and CheY proteins of Campylobacter jejuni regulating the bacterial chemotaxis in vitro and colonization in vivo. Methods By using pET42a plasmid and E. coli BL21DE3 as expression vector and expression strain, respectively, prokaryotic expression systems of cheA and cheY genes of C. jejuni strain NCTC11168 was constructed. Rabbits were immunized with the target recombinant expression proteins, rCheA and rCheY, to prepare the antisera. rCheA-IgG and rCheY-IgG in the antisera were separated using DEAE-52 ion exchange column. pBluescript- II -SK was applied to construct suicide plasmid which used to generate cheA gene knock-out mutant (cheA-). A chemotaxis model in vitro of C. jejuni based on DOC-HAP, the chemotactic ability of cheA' mutant as well as the effect of rCheA-IgG and closantel inhibiting the bacterial chemotaxis were demonstrated. The phosphorylation levels of CheA and CheY after DOC treatment were examined by using either rCheA-IgG or CheY-IgG capture method. The difference of colonization ability between cheA- mutant and wild-type of C. jejuni in mice were checked and then compared. Results The constructed prokaryotic expression systems could efficiently express rCheA and rCheY. The data from PCR and sequencing confirmed the cheA gene knock out from cheA- mutant chromosome. cheA- mutant lost its chemotactic ability towards DOC. Both the rCheA-IgG and closantel could inhibit the chemotaxis of wild-type of C. jejuni (P < 0.05 ). When treatment of DOC, the phosphorylation levels of CheA and CheY in wild-type of C. jejuni rapidly decreased (P < 0. 05 ). The colonization ability in murine jejunum of cheA- mutant was also lower than that of the wild-type ( P<0.05 ) . Conclusion Chemotaxis-associated two-component signaling system (Che-TCSS) of C. jejuni are composed of CheA and CheY, and the two proteins are activated by dephosphorylation. CheA in the Che-TCSS play a critical role in chemotaxis in vitro and colonization in vivo of C. jejuni, and this protein can be used as a target for developing novel anti- C. jejuni drugs.

Aged ; Female ; Fibroma ; pathology ; Fibroma, Ossifying ; pathology ; Gingival Neoplasms ; pathology ; Humans

Objective To explore the influence of montelukast on plasma nitric oxide in preschool children with asthma.Methods Forty-four preschool children with asthma aged 2-5 years who firstly met a criterion of asthma and treated 4 weeks with montelukast were investigated;and nitric oxide levels of plasma were inspected respectively before treatment and after treatment 1 week,4 weeks.Results The level of nitric oxide in the plasma of asthmatic children was obviously higher than that in normal control group(P

Objective: To observe the effect of acupoint massage plus Vitalstim electrical stimulation on deglutition function and surface electromyography (SEMG) of deglutition muscle groups. Methods: A total of 60 patients with deglutition disorder after stroke were selected and divided into an electrical stimulation group, a massage group and an integrated group according to the random number table method, with 20 cases in each group. Patients in these three groups were given the same routine rehabilitation training for deglutition. In addition, patients in the electrical stimulation group were given extra Vitalstim electrical stimulation, patients in the massage group were given extra acupoint massage on the head, face and neck, and patients in the integrated group were given extra acupoint massage plus Vitalstim electrical stimulation. Fujishima Ichiro food intake level scale (FILS) was scored before and after treatment. The swallowing duration and maximal amplitude of masseter muscle in SEMG were evaluated before and after treatment. Results: After treatment, the FILS score and the maximal amplitude of recruitment potential generated by muscular contraction of masseter muscle group in the three groups were higher than those before treatment (all P<0.05), and the swallowing duration of masseter muscle group was shortened compared with that in the same group before treatment (all P<0.05). After treatment, the FILS score in the integrated group was higher than that in the electrical stimulation group and the massage group (both P<0.05). The swallowing duration of masseter muscle group measured by SEMG was lower than that in the electrical stimulation group and the massage group (both P<0.05), while the maximal amplitude was higher than that of the electrical stimulation group and the massage group (P<0.05). After treatment, there were no significant differences in the FILS score, swallowing duration and maximal amplitude of masseter muscle group between the electrical stimulation group and the massage group (all P>0.05). Conclusion: Both acupoint massage and electrical stimulation can improve the deglutition function in patients with deglutition disorder after stroke, and improve the coordination and flexibility of masseter muscle. The integration of the two is more effective.

A growing number of researches have shown that ouabain can regulate mammalian sperm function and male reproduction by modulating the sperm motility, capacitation and acrosome reaction in vitro. This study further examined the relationship between ouabain and asthenozoospermia. In this study, the rat was intraperitoneally injected with ouabain at different concentrations (low-dose ouabain group: 12.5 μg/kg body weight per day, and high-dose ouabain group: 25 μg/kg body weight per day) for 30 days to establish the asthenozoospermia model. The sperms from 60 males with normal fertility were incubated with ouabain of gradient concentrations (10(-7)-10(-2) mol/L) for 4 h. The sperm motility was evaluated under a microscope. Moreover, the endogenous ouabain (EO) level was determined in seminal plasma of mild or severe asthenozoospermia patients and males with normal fertility by competitive inhibition ELISA. The results showed that the sperm motility was significantly diminished in the rats treated with different concentrations of ouabain. The number of motile sperms (grades a and b) was decreased greatly in a time- and dose-dependent manner in 10(-5)-10(-2) mol/L ouabain groups (P<0.01), while no obvious change in sperm motility was observed in 10(-7)-10(-6)mol/L groups even for 4-h incubation (P>0.05). Furthermore, the EO level was significantly increased in asthenozoospermia patients as compared with that in males with normal fertility (25.27±1.71 μg/L in mild asthenozoospermia patients, 26.52±1.82 μg/L in severe asthenozoospermia patients, 19.31±1.45 μg/L in normal fertility men) (P<0.01). In conclusion, rat asthenozoospermia was successfully established by intraperitoneal injection of ouabain, and 10(-5) mol/L ouabain was sufficient enough to inhibit sperm motility in vitro. Moreover, EO, a normal constituent of seminal plasma, was highly expressed in asthenozoospermia males as compared with normal fertility ones.
Animals ; Asthenozoospermia ; chemically induced ; metabolism ; physiopathology ; Disease Models, Animal ; Dose-Response Relationship, Drug ; Humans ; Injections, Intraperitoneal ; Male ; Ouabain ; metabolism ; pharmacology ; toxicity ; Rats ; Rats, Sprague-Dawley ; Semen ; metabolism ; Sperm Motility ; drug effects ; physiology ; Spermatozoa ; drug effects ; physiology ; Time Factors

OBJECTIVETo develop a RP-HPLC method for simultaneous determination of orientin, isorientin, vitexin and isovitexin in Lophatherum gracile from different habitat and harvesting time.

METHODThe HPLC method was applied and the chromatographic column was a Waters XBridge C18 column (4.6 mm x 250 mm, 5 microm). The mobile phase consisted of methanol-0.05% acetic acid (35:65). The flow rate was 1.0 mL min(-1) and the detection wavelength was set at 340 nm. The column temperature was set at 25 degrees C.

RESULTFour components were isolated well, the linear relationships were excellent. The mean recoveries and RSD values of orientin, isorientin, vitexin and isovitexin were 103.2%, 2.1%; 101.6%, 2.7%; 98.4%, 2.3%; 99.2%, 1.8%, respectively.

CONCLUSIONThe HPLC method is simple, sensitive and reliable, and can be used for the quality control of the medicinal material.

Apigenin ; chemistry ; Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; chemistry ; Flavonoids ; chemistry ; Glucosides ; chemistry ; Glycosides ; chemistry ; Poaceae ; chemistry ; Reproducibility of Results

Objective To explore the role of long noncoding RNA-MGC (lnc-MGC) on the trans-differentiation of human peritoneal mesothelial cells (HPMCs) induced by high glucose.Methods The immortalized HPMCs were used to establish control group and high glucose group (60mmol/L) respectively.Cells in control group were cultured with ordinary cell medium,and in high glucose group were stimulated with high glucose medium for 72h.Changes of lnc-MGC expression in the both groups were measured by RT-PCR,and the changes of mRNA and protein expression of E-Cadherin,α smooth muscle actin (α-SMA),connective tissue growth factor (CTGF),type Ⅰ collagen (COL-1) and type Ⅲ collagen (COL-3) in epithelial cells of both groups were measured by RT-PCR and Western blotting.The HPMCs were transfected with lentivirus,and then the changes of the above indexes were observed after up-and down-regulation of lnc-MGC.Results By high glucose stimulation of HPMCs for 72h,RTPCR results showed that the expressions oflnc-MGC,α-SMA,CTGF,COL-1 and COL-3 mRNA increased obviously (P＜0.05),and the expression of E-Cadherin mRNA decreased markedly (P＜0.05) in high glucose group than in control group;Western-blotting results indicated that the expression of protein was consistent with that of mRNA,and the differences were statistically significant (P＜0.05).After lentivirus transfection and down-regulation oflnc-MGC,RT-PCR results showed that the expressions of lnc-MGC,α-SMA,CTGF,COL-1 and COL-3 mRNA decreased obviously (P＜0.05),and the expression of E-Cadherin mRNA increased markedly (P＜0.05) in high glucose group than in control group;Western-blotting results showed that the expression of protein was consistent with that of mRNA,and the differences were statistically significant (P＜0.05).After lentivirus transfection and upregulation of lnc-MGC,RT-PCR results showed that the expressions of lnc-MGC,α-SMA,CTGF,COL-1 and COL-3 mRNA increased significantly (P＜0.05),and the expression of E-Cadherin mRNA decreased obviously (P＜0.05) in high glucose group than in control group;Western blotting results showed that the expression of protein was consistent with that of mRNA,and the differences were statistically significant (P＜0.05).The downstream target was predicted as miRNA126-3p,and compared with the control group,the expression ofmiRNA126-3p increased (P＜0.05) after high glucose stimulation,and after transfection with down regulated lnc-MGC lentivirus,the expression of miRNA126-3p decreased obviously (P＜0.05),and transfection with up regulated lnc-MGC lentivirus,the expression ofmiRNA126-3p increased obviously (P＜0.05).Conclusions lnc-MGC participates in the process of HPMCs transdifferentiation through regulating miRNA126-3p.Regulation oflnc-MGC expression level may control the phenotype transition of HPMCs,and delay the development of peritoneal fibrosis.

Objective:To investigate the effects of circ_0000285/miR-127/VAMP2 axis on proliferation and invasion of ovarian cancer (OC) cells.Methods:Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of circ_0000285, miR-127 and VAMP2 in tissue with OC and normal tissue respectively, and the correlation circ_0000285/miR-127/VAMP2 was analyzed by correlation analysis. Dual luciferase reporter assay was used to verify the targeting relationship between miR-127 and circ_0000285/VAMP2. Cell proliferation and invasion were subsequently detected by MTT and Transwell assay.Results:Compared with the paracancerous tissue, the expression of miR-127 was significantly decreased, while the expression of circ_0000285 and VAMP2 was increased in OC tissue. miR-127 was negatively correlated with circ_0000285 and VAMP2 ( r=-0.534 8, P<0.000 1; r=-0.376 6, P<0.000 1) . Compared with proliferative activity at 24 h, 48 h, 72 h (0.47±0.03) (0.79±0.05) (1.16±0.09) and invasion (100.00±12.33) in si-NC group, knocking down of circ_0000285 inhibited the proliferation (0.28±0.02) (0.51±0.04) (0.78±0.06) and invasion (49.22±5.08) of OC cells (all P<0.05) . Overexpression of miR-127 could also restrain proliferation, invasion of OC cells, but this effect can be partly saved by circ_0000285. The regulation circ_0000285 on OC may be realized through miR-127/VAMP2. Conclusions:circ_0000285 participates in the progression of ovarian cancer via regulating miR-127/VAMP2 axis. Down-regulating the expression of circ_0000285 inhibits the proliferation and invasion of ovarian cancer cells, circ_0000285 is expected to play a role in the targeted therapies of OC.