Objective To explore the proper proportion of effective fractions in Shengmaisan(saponins of Radix ginseng,saponins of Radix ophiopogonis,Lignans of Fructus schisandrae)in different anoxia models.Methods Acute cerebral hypoxia was induced by sodium nitrite and decapitation in mice,and the orthogonal design was used in these two models to find the proper proportion of three effective fractions.The gasping time of the mice,which were decapitated was observed to compare the anti-anoxia effects of XZF with other clinical drugs,and cerebral ischemic reperfusion injury model of mice was also used to study the effect of XZF on related biochemical index.Results XZF(the proportion of saponins of Radix ginseng,saponins of Radix ophiopogonis,Lignans of Fructus schisandrae as 7 ∶ 2 ∶ 6)significantly prolonged the gasping time,and decreased brain nitrogen monoxidum(NO)content after reperfusion of the mice at dosages of 50 mg/kg and 150mg/kg.Meanwhile,XZF also reduced malondialdehyde(MDA)content and increased superoxide dismutase(SOD)activity at the higher dosage.Conclusion XZF obtained by experimental screening exerts a significant protective effect on cerebral ischemia injury in mice,which provide some pharmacological evidence for further development of new modern Chinese drug composed with effective fractions for cerebral vascular diseases.

Objective To investigate the effect of quinone oxidoreductase 1 (NQO1) and quinone oxidoreductase 2 (NQO2) expression in the prognosis of ovarian carcinoma.Methods The two online databases were used to perform the online analysis on the NQO1 and NQO2 mRNA levels and prognosis of ovarian cancer patients,1 306 cases of ovarian cancer were performed the Kaplan-Meier analysis by using the KaplanMeier plotter (K-M plotter);578 cases of ovarian cancer in the TCGA database were performed the univariate COX regression survival analysis.Results The K-M plotter analysis showed that the NQO1 expression level had no obvious correlation with the prognosis of ovarian cancer (P>0.05),the higher the NQO2 level,the better the prognosis (HR=0.83,P=0.006 2).The COX regression survival analysis showed that the NQO1 expression level had no obvious correlation with the prognosis of ovarian cancer(P>0.05),the higher the NQO2 level,the better the prognosis (P=0.038 29).Conclusion NQO2 expression level has no obvious correlation with the prognosis of ovarian cancer,moreover the higher the NQO2 expression level,the better the clinical prognosis of ovarian cancer.

Objective: To study the difference between the acid resistance of Streptococcus mulans Ingbritt C international standard strain and the acid resistance of LuxS mutant strain. Methods: Solutions of Streptococcus mulans standard strain and LuxS mutant strain with same density were prepared and cultured at pH 3. 5 to 7. 0 BH1 liquid for same period. Terminal growth situation was compared. After being acidized in pH 5.5 BHI liquid, the two strains were cultured at pH 3.0 BHI liquid. The acid tolerance responses of the two strains were compared. Results; (DAt pH 6.0 to 7. 0, the difference of growth between Streptococcus mulans standard strain and LuxS mutant strain was not significant at the same pH value, and the differences of bacterial growth situation under three different pH values were not significant. (1)At pH 4.5 to 5.5, the difference of growth between the two strains was significant. (2)At pH 3.0,the survival rate of LuxS mutant strain(0.006 5% )was significantly lower than the standard strain (0.078% ). (3)At pH 5.5, the survival rate of LuxS mutant strain(0.747% ) was lower than the standard strain(8.65% )by about 10 times after the pre-acidification. Conclusion; (4)At sub-lethal pH value, there is significant difference of aciduricity between Streptococcus mu-tans standard strain and LuxS mutant strain. The acid resistance of standard strain is stronger than that of LuxS mutant strain. The two strains both display the capability of acid tolerance responses. LuxS mutant strain is more sensitive to acid inactivation, but the capability of acid tolerance responses still exists.

Objective To investigate the effect of luxS inactivation on the oxidative stress of Streptococcus mutans and perform preliminary analysis of potential mechanism.Methods Strains were grown to mid-logarithmic phase and divided into three groups,one was used as control and inoculated into normal TPY medium,and the other two groups were experimental groups,and there were separately inoculated into TPY containing 58.8 mmol/L hydrogen peroxideor TPY containing 58.8 mmol/L hydrogen peroxide and 0.1 mmol/L 2,2'-dipyridyl.The survival rate of strain was calculated at 0.5,1,and 2 h.All the data were statistically analyzed.Results Compared with the control group,the survival rate of luxS mutation was always higher than standard strain at all pre-determined time inexperimental groups (P＜0.05),and compared with experimental group without iron chelator,the survival rate of strains was not raised with the added of iron chelator (P＞0.05).Conclusion luxS gene is involved in oxidative stress tolerance of Streptococcus mutans,and the oxidative stress tolerance is not achieved by avoiding the toxic effects of the Fenton reaction

Objective To construct markless gene deletion mutant at the clpP loci on the chromosome of Streptococcus mutans(S.mutans).Methods ASp resistance gene was amplified by PCR,to construct the Sp resistance cassette where the Sp resistance gene was flanked with two loxP site.After the clpP gene was cloned into the pGEM-T-Easy TA cloning vector,it was digested and linked with the Sp resistance cassette,yielding homologous recombination vector pIB △ clpP-Sp.The vector was linearized and used for the transformation of S.mutans UA159,with transformants selected on TPY plates containing Sp.The selected strain was transformed with the thermosensitive plasmid pCrePA to excise the Sp resistance gene.The pCre-PA was then easily eliminated at nonpermissive temperature,resulting in a markless mutant strain carrying a deletion at the clpP loci,which was verified by PCR and DNA sequencing.Results The result of the PCR analysis and DNA sequencing indicated that a part of the clpP gene was deleted.There was a loxP at this loci without the Sp resistance gene.Conclusion The markless clpP-deletion mutant of S.mutans was constructed successfully,which laid a foundation for further study of its biological function and its influence on the cariogenicity of S.mutans.

A new method using on-line cleanup technology combined with liquid chromatography tandem mass spectrometry ( UHPLC-MS/MS) was developed for the determination of seven kinds of β2-agonists residues, Formoterol, Salmeterol, Carbuterol, Clenisopenterol, Clenpenterol, Clencyclohexerol and Clenbuterol-hydroxymethyl in livestock manure. The sample was sufficiently extracted by acidic acetonitrile and diluted by 0. 2% formic acid. The extract was online purified on HyperSep Retain CX column where the sample matrix was washed away and the analytes were retained. The analytes were eluted into Hypersil Gold C18 column by 2% Ammonia-methanol solution. The seven β2-agonists were detected in selected reaction monitoring ( SRM) mode via positive electrospray ionization ( ESI+) by liquid chromatography-tandem mass spectrometry. The results showed good linearity correlation coefficients over 0 . 9928 for the seven kinds of β2-agonists in 0 . 5-100 μg/L concentration range. The LOD of seven kinds of β2-agonists in livestock mature is 1 μg/kg, while the LOQ is 5 μg/kg. When 5-50 μg/kg of the seven kinds ofβ2-agonists were added into the blank livestock manure, an average recovery of 67% -112% was obtained with the relative standard deviations of 2. 9%-10. 2%. The method is simple, rapid and has good reproducibility for quantitative and confirmatory analysis ofβ2-agonist residues.

To determine the residue of 14 sulfonamides in milk, a high performance liquid chromatography-tandem mass spectrometry ( HPLC-MS/MS ) method with on-line soild phase extraction ( SPE ) in cation exchange mode was established. 5 g of milk was extracted with 15 mL acetonitrile. Then the extraction was evaporated by 50 ℃ nitrogen and dissolved by 1. 00 mL 0. 2% formic acid. The dissolution was enriched and purified by MS/MS cation exchange on-line SPE column on a double ternary liquid chromatography, and eluted by the mixed solution of 2% ammonia methanol and 0. 2% formic acid (50:50, V/V). The compounds were separated by an octadecyl silica bonded column and determined by the tandem mass spectrometry. The results showed that the linearity of 14 sulfonamides was good in the range of 0 . 1–10 μg/kg ( r≥0 . 9995 ) .
The LOD of the method was 0. 05 μg/kg, while the LOQ was 0. 1 μg/kg. The recoveries of the 14 sulfonamides were in the range of 60 %-90 %, while the inter-batch and intra-batch RSDs were all lower than 10%. The method was proved to be more convenient, economical and stable than the traditional SPE column method.

Objective To construct the expression plasmid of human PAK4 gene and identify its recombinant protein expression.Methods Total RISA was extracted from human breast cancer MCF-7 cells.The hPAKA coding sequence was amplified by polymerase chain reaction (PCR) method and subcloned into pEBG vector.After the target region was sequenced.the plasmid was transfected into HEK293 cell line.The expression of the recombinant plasmid in HEK293 cells was proved by Western blot.Results hPAKA had been constructed into the expressing vector pEBG successfully.The length of the fragment was 1 800 bp,identified by restriction enzymes digestion.The expression of pEBG-hPAK4 fusion protein was detected by Western blot,with a molecular weight 94 KDa and was pulled down by Qutathione Sepharose 4B.Conclusion The recombinant plasmid was successfully cloned into eukaryotic expressing vector,and the expression of pEBG-hPAK4 fusion protein was identified and pulled down by Glutathione Sepharose 4B.

Objective:To determine the effects of Foxp3-overexpressing lung cancer cells on activated CD4+T lymphocyte.Methods: Stable Foxp3-overexpressing lung cancer cells NCIH-1299,NCIH-hFoxp3,was generated by transfection of NCIH-1299 cells with plasmid pcDNA3-hFoxp3 mediated by Lipofectamine 2000 and by selection with G418,and validated by quantitative PCR and Western blot.The expression levels of IL-8 and IL-10 secreted by NCIH-hFoxp3 and NCIH-control were measured by ELISA.IL-2 secrection by activated human CD4+T lymphocyte which was tested after stimulation with 20% conditioned medium of NCIH-hFoxp3 and NCIH-control cells.The proliferation of activated human CD4+ T lymphocytes was assessed by MTT after coculture with NCIH-hFoxp3 cells.The adhesive ability of activated human CD4+ T lymphocytes was probed with NCIH-hFoxp3 cells by immunocytochemistry.Results: Compared with NCIH-control cells,NCIH-hFoxp3 secreted high level of IL-10 and low level of IL-8.NCIH-hFoxp3 with Foxp3 overexpression significantly suppressed the proliferation,adhesive potential and IL-2 expression by activated CD4+ T cells.Conclusion: Suppression of immune activities of activated CD4+ T cells by Foxp3 overexpression in lung cancer cells may correlate with cytokine IL-8 and IL-10,which can contribute lung cancer progression.

Objective To explore the influence of Helicobacter pylori (Hp) infection in serum lipoprotein associated phospholipase A2 (Lp-PLA2), carotid intima-media thickness and stability of atherosclerotic plaques in atherosclerosis patients.Methods A total of 393 cases of patients with carotid artery arteriosclerosis confirmed by carotid color uhrasonography, who are informed consent, was selected as objects.The14C urea breath test was used to determine the infection situation of selected objects of helicobacter pylori.Meanwhile, enzyme-linked immunosorbent assay (ELISA) was used to determine the level of serum lipoprotein associated phospholipase A2 (Lp-PLA2).Results Serum Lp-PLA2 levels and carotid intimamedia thickness (IMT) of patients with carotid artery atherosclerosis in Hp infection group were higher than that of Hp non-infection group, and with the degree of Hp infection aggravating in the patients of carotid artery atherosclerosis, their serum Lp-PLA2 levels and carotid IMT were also increased accordingly.F test showed that the differences of serum Lp-PLA2 levels and carotid IMT in different degree of carotid artery atherosclerosis group were statistically significant (P ＜0.01).The incidence of unstable plaque of Hp infection group was obviously higher than that of the Hp non-infection group in the carotid atherosclerosis with plaques with statistical significance (chi square value =4.744, P =0.029).Multivariate linear regression analysis showed that the possibility of complication of unstable plaques in Hp infection group of carotid artery atherosclerosis was 1.82 times than that of non-infection group.With serum Lp-PLA2 every increasing 1 μg/L, the possibility of instability plaque increased by 2％.Conclusions Hp infection may promote the occurrence and development of carotid artery atherosclerosis by increasing serum level of Lp-PLA2 and changing the stability of atherosclerotic plaques.