Objective:To simultaneously determine the contents of ginsenoside Rb1 , ginsenoside Rb2 , ginsenoside Rb3 , ginsen-oside Re, ginsenoside Rg1 , ginsenoside Rf and ginsenoside Ro in Qipi pills by HPLC-QTOF-MS. Methods: The determination was performed on an Agilent Poroshell 120 EC-C18 column (2. 1 mm × 50 mm,2. 7 mm) with mobile phase consisting of acetonitrile( A)-water(B, containing 0. 1% formic acid) with gradient elution. The flow rate was 0. 21 ml·min-1. The column temperature was 30℃. The MS instrument was equipped with an ESI+ ion source. The exacted ion chromatograms were used to determine the quantities of different compounds in the samples while the mass spectra of product ions were used for confirming the compounds. Results:All the 7 kinds of ginsenoside showed good linearity (r>0. 9993). The RSDs of precision, repeatability and stability tests were all less than 5%. The average recoveries were within the range of 97. 11%-101. 98%. Conclusion:The method is simple, rapid and reliable with high specificity, which can be used for the quality control of Qipi pills.

With the development of imaging techniques, aphasia caused by subcortical impairment is increasingly found in recent years. This paper reviewed the literature about the subcortical aphasia and expolred the anatomical and clinical features and pathogenesis of subcortical aphasia.

We prepared gold nanostar (GNS) through seed growth method firstly,then formation of COX-2 siRNA(siCOX-2) and GNS composite modified with polyethylene glyco (PEG),2-amino-2-deoxy-D-glucos (DG) and 9-D-arginin (9R) was prepared.Mterwords,paclitaxel temperature sensitive liposome (PTX-TSL) was prepared by film dispersion method.Finally,siCOX-2 delivery systerm (PTX-TSL-(siCOX-2(9R/DG-GNS)))was obtained by hydrosulfuryl ligand reaction between siCOX-2 (9R/DG-GNS) and PTX-TSL The successful build of siCOX-2 (9R/DG-GNS) was vetified by nuclear magnetic resonance (NMR),sodium dodecyl sulfate polyacryl amide gel electrophoresis (SDS-PAGE),and ultraviolet spectrophotometry and agarose gel electrophoresis method.Particle size of PTX-TSL-(siCOX-2(9R/DG-GNS)) was (292 ± 14) nm and Zeta potential was about -(2.59 ± 0.12) mV,which were measured by Zetasizer Nano ZS90.The morphology of PTX-TSL-(siCOX-2 (9R/DG-GNS)) measured by transmissionelectronmicroscopy showed homogeneous star structure with phospholipid bilayer on the surface,and it showed good thermal conversion efficiency under radiation of 808 nm laser.Differential scanning calorimetry test showed that PTX-TSL phase transition temperature is about 42.6 ℃.The drug loading content(using dialysis method) and encapsulation efficiency of PTX-TSL were about 7.5％ and 95.4％,at the same time,the release process experiment of PTX-TSL showed that it had a good temperature sensitive release performance.It is hopeful that this siCOX-2 system can be used for reducing drug resistance of PTX and improving the treatment effect of PTX through the synergistic antitumor drug resistance effect of siCOX-2.

Objective To observe the effects of estrogen on behavior and 5-HT in periaqueductal gray (PAG)in migraine model rats. Methods Tewnty-four ovariectomized Wistar rats were divided randomly into four groups:control group (Group A),migraine group(Group B),low dose estradiol-treated ovariectomized group(Group C),high dose etradiol-treated ovariectomized group(Group D).After 1 week,the rats in Group B,C and D were injected with nitroglycerine 10 mg?kg-1 subcutaneously to make migraine rat models,the rats in Group A were given peanut oil alike,and the behavior changes were observed.2 h after injection,the rats were killed and the midbrains were separated and then 5-HT immunohistochemical staining was performed.Results Behavior: compared with Group B,the degrees of red-calws,red-ears and red-tail rats in Group D relieved obviously,the times of climbing hutch and scratching head were much fewer,while the rats in group C showed no significant difference;Immunohistochemical staining:compared with Group A,the 5-HT-positive neurons expression in PAG of Group B and C were more obviously(P

Objective: To investigate the hydrolysis conversion rate of alcohol amine-diterpene alkaloids from aconitum alkaloids, hydrolyze aconitum alkaloids reference substance, calculate the amount of alcohol amine-diterpene alkaloids in the hydrolysis solution by the hydrolysis conversion rate, which is used as the amount of alcohol amine-diterpene alkaloids reference substance, and establish a content determination method for aconine, hypaconitine and aconine in Aconiti radix cocta.Methods: Through controlling the hydrolysis conditions of aconitine, hypaconitine and mesaconitine, aconine, hypaconitine and aconine were obtained.The determination was performed on an Agilent ZORBAX Extend-C18 RRHT(2.1 mm×50 mm,1.8 μm) column with the mobile phase consisting of methanol(A)-water(B containing 0.1% formic acid and 2.5 mmol·L-1 ammonium acetate) with gradient elution by HPLC-QTOF-MS.The flow rate was 0.21 ml·min-1.The column temperature was 30 ℃.MS instrument was equipped with an ESI+ ion source.Results: Under the hydrolysis conditions of this study, the conversion rate of aconine from aconitine was 99.64%;the conversion rate of hypaconitine from hypaconine was 99.94%;the conversion rate of mesaconitine from mesaconine was 99.57%.The HPLC-QTOF-MS methodological investigation showed the 3 kinds of alcohol amine-diterpene alkaloids were with good linearity (r>0.999 1).The RSD of the precision, repeatability and stability tests were less than 5%.The average recoveries were within the range of 99.43%-100.10%.Conclusion: The validated method is simple, specific, reliable and reproducible.In the absence of reference substance, it can be used for the quality control of the herbs of Aconitum L.species.

Objective:To develop a method of quantitative analysis of multi-components by single marker( QAMS) for nine kinds of alkaloids in Xiaohuoluo pills. Methods: An HPLC-QTOF-MS method with an Agilent ZORBAX Extend-C18 RRHT(2. 1 mm × 50 mm,1. 8 μm) column was applied. The flow rate was 0. 21 ml·min-1 . The column temperature was 30 ℃. The mobile phase was methanol (A)-water (B;containing 0. 1％ formic acid and 2. 5 mmol·L-1 ammonium acetate) with gradient elution. The aconitine was used as the internal standard, and the relative correction factor ( RCFs) of hypaconitine, mesaconitine, benzoylaconine, benzoyl-hypaconine, benzoylmesaconine, aconine, hypaconine and mesaconine was respectively established, and the reproducibility inspection on the RCF was performed. The contents of the other 8 kinds of aconitum alkaloids were calculated according to the RCF. At the same time, an external standard method ( ESM) was performed for the content determination of the nine alkaloids. The results of the two methods were compared. The feasibility and accuracy of the QAMS method were verified. Results:Within a certain range,the RCF of hypacontine,mesacontine, benzoylaconine, benzoylhypaconine, benzoyl mesaconine, aconine, hypaconine and mesaconine to aconitine was 1. 736,1. 979,1. 0471,0. 9242,1. 2901,1. 3078,1. 2859,and 1. 0948,respectively. The QAMS method was established for determi-ning alkaloids. There were no significant differences between the results of the QAMS method and those of the external standard method ( ESM) . Conclusion:With the validation of methodology, the method established in our study can be used for the content determina-tion of aconitine, hypaconitine, mesaconitine, benzoylaconine, benzoylhypaconine, benzoylmesaconine, aconine, hypaconine and mesaconine in xiaohuoluo pills.

Objective To prepare the human bone sialoprotein (BSP) monoclonal antibodies (mAb)with high titer and specificity and identify its characterization,which is based on further studying BSP as clinical biomarker for breast cancer metastasizing to bone. Methods BALB/c mice were immunized with purified recombinant BSP protein.Cell fusion was performed between mouse splenic cells and myeloma cells (Sp2/0), and then the hybridoma cell lines secreting mAb against BSP antigen were screened and cloned. The ascites were prepared and purified with Protein G affinity chromatography.The titer and subtypes of mAb against BSP were identified and measured by ELISA and Western blotting analysis. ResultsNine hybridoma cell lines that stably secreted mAb against BSP were successfully obtained.Two of them,D001 and D002,were further identified, which belonged to the subtypes of IgG1 and κ light chain. The two antibodies titers in culture supernatant were 1∶5120 and 1∶10 240, respectively, and those in the ascites fluid were 1∶25 600 and 1∶51 200,respectively.Results of Western blotting analysis and immunohistochemistry showed that the two antibodies could specifically bind with BSP derived from human breast cancer cells.ConclusionNine mAb against BSP have been successfully prepared which can be used for further studying the biological properties of BSP and reveal its relationship with data from clinic patients.

Purpose　The aim is to prepare the extract of immunocompetent cell proliferation from human placenta and to try to find out a suitable method of preserving the extract.Methods　The extract was mainly prepared by heat-treating placenta homogenated fluid. Then the activity to stimulate murine splenic lymphocyte proliferation in vitro was done with MTT , exposing　the　extract to　radioisotope 60　Co.Results　The content of protein was 2～3mg per gram placenta measured by Lowry′s Method.The rate to promote cell proliferation was more than 80 percent.The activity lasted a few months after being exposed to radioisotope.Conclusion　The extract prepared by heat-treating not only had high activity, but also had the unique method and good repeatability.This prepared extract as a kind of stable,reliable and remarkably promoting lymphocyte proliferation reaction had the value of development production on broad scale as well as in clinical practice.

In order to culture the qualified clinicians, we should think about how to improve the quality of clinical practice of obstetrics and gynecology. It is of great importance to emphasize the teachers and students to value the teaching work together. Importance should be attached to the advantage of subject of academy, to make the clinical practice closer to practical, which has a perfect effect and will benefit our work.

Objective To express and purify recombinant and biologically active Clostridium difficile toxin B (rTcdB). Methods The genes of TcdB were amplified by polymerase chain reaction (PCR) using chromosomal DNA from a toxigenic strain, and cloned into a shuttle vector pHis1522.The sequences of TcdB genes in the vector were verified by DNA sequencing. The construction was transformed into Bacillus megaterium protoplasts and the protein expression was driven by a xylose promoter. The purified protein was tested for biological activity. Results rTcdB was successfully purified from bacterial crude extracts. Approximately 5-10 mg of highly purified recombinant toxin was obtained from one liter of bacterial culture. The expressed rTcdB had molecular mass similar to the native toxin, and its biological activity was proved to be similar to its native counterpart after an extensive examination. Conclusion rTcdB with biological activities is successfully expressed in Bacillus megaterium.