Migraine is a common neurovascular disorder manifested by recurrent severe headaches on one or both sides， occasionally accompanied by nausea， vomiting， photophobia， and phonophobia. It has the characteristics of recurrent attacks and family inheritance. Traditional Chinese medicine （TCM） believes that migraine belongs to the category of "head wind"， which is mostly caused by external wind and is related to the internal stirring of liver wind. Sanpiantang comes from the Record of Syndorme Differentiation·Headache （Bianzhenglu·Toutongmen） created by the physician CHEN Shiduo of the Qing Dynasty. It is composed of Chuanxiong Rhizoma， Angelicae Dahuricae Radix， Pruni Semen， Cyperi Rhizoma， Bupleuri Radix， White Mustard Seed， and Glycyrrhizae Radix et Rhizoma， with the functions of moving Qi to release pain， activating blood and resolving stasis， which is commonly used for the treatment of migraine in clinic. Current clinical studies on the application of Sanpiantang to the treatment of migraine mostly used modified Sanpiantang， either alone or in combination with western medicine/acupuncture. The results of these clinical trials showed that Sanpiantang could significantly lower migraine score， pain visual analog scale and endothelin level， reduce the frequency of painkiller use， and remarkably alleviate migraine symptoms， with few side effects. The animal experiments focused on exploring the mechanism of action of modified Sanpiantang from different anatomical levels of migraine， which mainly included reducing nitric oxide （NO） and nitric oxide synthase （NOS）， reduceing the release of neurotransmitters such as 5 -hydroxyline (5-HT) and neurotipides (NPY)， suppressing neuronal excitation， and blocking the transmission of nociceptive pathways， thereby promoting cerebral blood flow， regulating neurotransmitters and preventing migraine. Based on the pathogenesis of migraine， this paper systematically reviewed the latest progress in clinical application and experimental research of modified Sanpiantang， and summarized its mechanism of action of preventing and treating migraine， which provided new ideas for clinical treatment of migraine.

The aim of this study is to determine the diversity of virulence genes carried by different vancomycin-resistant enterococci (VRE),which will provides a basis for studying pathogenic mechanism of VRE.Microdilution-based drug sensitivity test was applied to detect the vancomycin resistance of 490 Enterococcus faecium isolates and 862 Enterococcus faecalis isolates in Zhejiang area.The seven virulence genes (ace,asa1,cylA,efaA,esp,gelE and hyl) in the isolates of VRE were detected by PCR.According to the results of drug sensitivity test,10％ of the E.faecium isolates (49/490) and 0.8％ of the E.faecalis (7/862) were identified as VRE.In the vancomycin-resistant E.faecium isolates,five isolates were negative for any of the target genes and the other 44 isolates were positive for asa1,esp,gelE and hyl genes alone,in which the esp (73.5％,36/49) and hyl (53.1％,26/49) were the predominant genes and single or double virulence genes acted as the major carrying models.Except for the hyl gene,the vancomycin-resistant E.faecalis isolates were positive for the other six pathogenic genes,and the isolates could carry 3-6 pathogenic genes.All the data indicate that E.faeciurn is the major species of VRE in the local area,and the carrying rate,types and models of virulence genes in the vancomycin-resistant E.faecium and E.faecalis isolates are obviously different.

We investigated correlation between the level of miR-16 expression and the severity of Staphylococcus aureus sepsis,and further explored its potentially clinical significance.Blood samples were collected from 32 patients,including each 8 cases of septic shock,severe sepsis and general sepsis,as well as 8 cases of healthy volunteers.Blood samples from 24 cases of healthy subjects with different ages were measured,and additionally 8 cases of blood samples from gram negative bacteria sepsis were also determined in current study as a control.Trizol solution for the whole blood lysis was added into blood samples,and followed by the extraction of microRNA.The expression levels of miR-16 in different groups were measured by fluorescence quantitative PCR,in which 2-Delta Ct method was used.SPSS software (version 13.0) was used to analyze the statistical differences between the groups,and further analyze the correlation between miR-16 value and the corresponding CRP and PCT values.Results showed that the expression level of miR-16 was negatively correlated with the severity of Staphylococcus aureus sepsis.There were statistically significant differences in experimental groups when compared with the control (P＜0.001),and there was also a statistically significant difference between each experimental group (P＜0.01).We found that the expression level of miR-16 was negatively correlated with CRP and PCT,the correlation coefficients were-0.561 and-0.769 respectively,and trend analysis showed that there was a significantly negative correlation.A significantly negative correlation was found between the miR-16 expression level and severity of sepsis,suggesting that miR-16 may serve as a biomarker for the severity of Staphylococcus aureus sepsis.

Objective To construct a prokaryotic expression system for tlyA gene of Leptospira in-terrogans ( L.interrogans) strain and to investigate the effects of the expressed rTlyA protein on the hemolysis of sheep erythrocytes and the secretion of pro-inflammatory cytokines by human THP-1 cells and murine J774A.1 macrophages.Methods The fragment of tlyA gene of L.inetrrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai was amplified by PCR.The PCR product was sequenced after T-A cloning.A prokary-otic expression system for the tlyA gene was constructed by using pET-42a as the expression vector and E.coli BL21DE3 strain as the host strain.The expression of rTlyA protein was detected by SDS-PAGE.Ni-NTA af-finity chromatography was performed for purification.The lytic activity of the rTlyA protein on sheep erythro-cytes was determined by plate hemolytic test and spectrophotometry.Real-time fluorescence quantitative PCR and Western blot assay were performed to respectively detect the expression of tlyA gene in THP-1 and J774A.1 cells at mRNA and protein levels after infection with L.interrogans strain Lai.ELISA was per-formed to evaluate the effects of the rTlyA protein on the secretion of several pro-inflammatory cytokines ( IL-β, IL-6 and TNF-α) by THP-1 and J774A.1 cells.Results The nucleotide and amino acid sequences of the cloned tlyA gene were 99.2% and 99.8% identical to those corresponding sequences in GenBank, re-spectively.The constructed prokaryotic expression system for tlyA gene successfully expressed the rTlyA pro-tein.The rTlyA protein at the concentration of 10μg/ml showed stronger lytic activity on sheep erythrocytes. The transcription levels of tlyA gene (P<0.05) and the secretion of TlyA protein in THP-1 and J774A.1 cells were significantly increased after infecting with L.interrogans strain Lai for 1 to 8 hours.The rTlyA pro-tein at concentrations of 0.1, 1 and 10 μg/ml significantly enhanced the secretion of IL-1β, IL-6 and TNF-αby THP-1 and J774A.1 cells (P<0.05).Conclusion The TlyA protein, encoded by the tlyA gene of L.interrogans strain, was confirmed to be a hemolysin with the ability to induce macrophages to secret IL-1β, IL-6 and TNF-α.This study suggested that the TlyA protein played an important role in inflammatory responses during leptospirosis.

Objective To investigate the prognostic value of regulatory B cells (Bregs) in patients with acute pancreatitis .Methods Flow cytometry analysis was performed to detected the percentages of CD19+IL-10+and CD19+CD24hiCD27hi Bregs in peripheral blood samples collected from patients with acute pancreatitis (36 cases with mild acute pancreatitis and 15 cases with severe acute pancreatitis ) as well as the surface costimulatory molecules including CD80 and CD86 on CD19+CD24hiCD27hi Bregs.Their correlations with lymphocytes and C-reactive protein ( CRP) were further analyzed .Results The numbers of lympho-cytes, CD19+lymphocytes, CD19+IL-10+and CD19+CD24hi CD27hi Bregs in peripheral blood samples col-lected from patients with severe and mild acute pancreatitis as well as the mean fluorescence intensities ( MFI) of CD80 and CD86 were significantly lower than those from healthy subjects .Compared with patients with mild acute pancreatitis , the numbers of lymphocytes and CD 19+lymphocytes , the absolute numbers of CD19+IL-10+and CD19+CD24hiCD27hi Bregs as well as the mean fluorescence intensities (MFI) of CD80 and CD86 in patients with severe acute pancreatitis were significantly decreased .The percentages of CD19+IL-10+and CD19+CD24hiCD27hi Bregs in patients with mild acute pancreatitis were significantly increased af-ter an initial drop , but in patients with severe acute pancreatitis those values were continuously decreased along with the disease progression .The percentage of CD19+IL-10+Bregs was positively correlated with the percentage of CD19+CD24hiCD27hi Bregs and the absolute number of CD19+lymphocytes, but was negatively correlated with CRP .Conclusion The abnormal number and function of CD 19+IL-10+and CD19+CD24 hi CD27hi Bregs might be one of the important reasons causing immune dysfunction in patients with acute pan -creatitis.

Objective To investigate the prognostic value of CD 4+CD25+/high CD127 low/-and CD14+HLA-DRlow/-for evaluating the severity of acute pancreatitis .Methods The percentages of CD4+CD25+/highCD127low/-and CD14+HLA-DRlow/-and the CD64 index were measured by flow cytometry in pa-tients with acute pancreatitis ( including 43 cases of mild acute pancreatitis and 24 cases of severe acute pan-creatitis).Moreover, the levels of C-reactive protein (CRP), acute physiology and chronic health evaluationⅡ( APACHEⅡ) score and CT severity index ( CTSI ) were detected for a correlation analysis .Results The percentages of CD4+CD25+/highCD127low/-and CD14+HLA-DRlow/-and the CD64 index in patients with severe and mild acute pancreatitis were significantly higher than those in healthy subjects .Patients with se-vere acute pancreatitis showed higher percentages of CD 14+HLA-DRlow/-than patients with mild acute pan-creatitis.With the disease progression, the CD64 index and the levels of CD4+CD25+/highCD127low/-, CD14+HLA-DRlow/-and CRP were significantly dropped after an initial increase in patients with mild acute pancrea -titis, while these indexes were continuously elevated in patients with severe acute pancreatitis .The percent-age of CD14+HLA-DRlow/-was positively correlated with CD64 index, CRP level, APACHEⅡ score and CTSI.Conclusion CD14+HLA-DRlow/-level was closely related to the severity of acute pancreatitis , which could be used as immune parameter for the estimation of the clinical severity of acute pancreatitis .

Objective: To evaluate the effect of pure titanium modified by bioadhesive RGD peptide on the early attachment, growth and proliferation of osteoblasts. Methods: The titanium samples were hydroxylated by alkali/hot water aging and sol-gel layer-by-layer deposition technique. Afterwards, the terminal -NH_2 group was introduced to the titanium surface by organosilane APTMS self-assembled monolayers and the functional group -NH_2 was further reacted with EDC/NHS by which RGD peptides was covalently immobilized to titanium. The efficiency of this bioreactive surface in promoting cell attachment and the competitive inhibition effect of RGD peptide with different concentrations were observed by calculating the amount of osteoblasts attached on the modified titanium. The growth and proliferation were observed by MTT method and scanning electronic microscopy. Results: The cell adhesion percentage of the RGD modified titanium group was much higher than that of the other groups. The RGD peptide solutions with higher concentration had stronger inhibitory impact on the cell adhesion onto the titanium surface. The cell growth, morphology and proliferation on the RGD peptide modified titanium were better than other groups. Conclusion: Bioadhesive peptide can be chemically grafted onto the titanium surface by means of self-assembled monolayers technique. The cells′ biological behaviors on the surface of RGD immobilized titanium are greatly improved in vitro.

Objective To observe the in vitro inhibitory effects of brucea javanica oil on human washed platelet and explore the possible mechanism.Methods Human washed platelets were mixed withdifferent concentration of brucea javanica oil which were divided into four groups[untreated control group,negative control group,9.0% of brucea javanica oil group,and 22.5%of brueea javanica oil group].The maximal ratio of platelet aggregation induced by adenosine liphosphate(ADP),arachidonic acid(AA),collagen,and thrombin,respectively,was measured with platelet aggregation analyzer.The expressions of fibrinogen receptor(FIB-R)and P-selectin(CD_(62p))on external membrane of activated platelet were determinated with flow cytometry.The F-actin of cytoskeletal structure in activated platelet was detected by SDS-PAGE.Results At 9.0% of brucea javanica oil,the maximal ratio of platelet aggregation[(57.7±4.0)%,(62.2±3.9)%,(66.9±5.0)%and(71.8±5.1)%]induced by ADP,AA,collagen,and thrombin,was significantly lower than that[(75.3±4.1)%,(79.3±4.8)%,(80.6±5.4)%,(84.1±6.2)%]at negative control(0% of brucea javanica oil)(P<0.01),but makedly higher than that[(39.2±3.5)%,(45.8±3.4)%,(51.2±3.9)%and(56.7±4.8%)]at 22.5%,respectively(P<0.01).The inhibitory rate of platelet aggregation(47.9%,42.2%,36.5%and 32.6%)at 22.5%of brucea javanica oil was notably higher than that(23.4%,21.6%,17.0%and 14.6%)at 9.0%,respectively(P<0.001).There was a negative correlation between brucea javanica oil concentration and the aggregation ratio of platelet stimulated by the four agonists,respectively(r=-0.952,-0.961,-0.970,-0.975,P<0.001).At 9.0% of brucea javanica oil,the expression levels of FIB-R[(64.7±4.0)%]and CD_(62p) [(3.91±0.21)%] of platelet activated by ADP were significantly lower than that[(85.5±4.6)%and (5.05±0.27)%]at negative control,but remarkably higher than that[(36.2±3.9)%and(2.34±0.15)%]at 22.5%,respectively(P<0.01).There was a much higher inhibitory rate of platelet aggregation(57.7%)at 22.5%than that(24.3%)at 9.0%(P<0.01).The ratios(1.68±0.10 and 1.77±0.12)of F-actin photodensity at 22.5%and 9.0%to that in blank control were significantly lower than that(2.22±0.15)at negative control(P<0.01)but there was no statistical difference between the ratios in the group of 9.0%and 22.5%brucea javanica(P>0.05).Conclusions brucea javanica oil has special inhibitory effect on activated platelet and thrombosis in a dose-dependent manner.The mechanism is to inhibit the expression of fibrinogen receptor on external membrane of activated platelet,which is also related to the inhibition of F-actin and secretion of platelet.

Objective To observe the biological activities of human doppel (Dpl) protein transiently expressed and Dpl-like protein PrPΔ32-121 on a human neuroblastoma cell line SH-SY5Y. Methods Recombinant mammalian expression plasmids containing human PRND gene and truncated PrPΔ32-121 fragment were generated by PCR. The expression and location of Dpl and PrPΔ32-121 post-transfection were observed by IFA. The cytotoxicity was measured by MTT analysis. Cellular apoptosis was investigated by flow cytometry and Western blot. Results Both Dpl and PrPΔ32-121 protein were expressed and mainly located on the cell membrane. Remarkable cytotoxicity was detected on SH-SY5Y cells after 24 h transfection. Meanwhile, more Annexin V/PI positively-stained cells as well as lower levels of cellular pro-caspase-3 and Bel-2 were detected in the cells receiving Dpl and PrPΔ32-121 expressing plasmids. Conclusion Dpl protein transiently expressed and PrPΔ32-121 can lead to the similar neural cytotoxicity, probably triggering the cell apoptosis program.

Objective To explore the inhibition effects of CD4+CD25+/highCD127low on the EBV immortalized cells(CD23+)in children with infectious mononucleosis (IM). Methods The expression of CD3+, CD3+ CD4+ , CD3+ CD8+ , CD25+/highCD127low and CD19+ , CD19+ CD23+ were analyzed by flow cytometry in 23 children at the acute stage of IM and 10 children recovering from IM in comparison with the ones of 20 healthy controls. Results Compared with the following results in controls, CD3+, CD3+ CD8+ were significantly increased and CD3+ CD4+, CD4+/CD8+ ratio, CD4+ CD25+/highCD127low, CD19+, CD19+ CD23+ were significantly reduced in the IM patients. Compared with the ones in the IM pa-tients recovering from IM, CD3+ , CD3+ CD8+ were significantly increased and CD3+ CD4+, CD4+/CD8+ ratio, CD4+ CD25+/highCD127low, CD19+ , CD19+ CD23+ were significantly reduced in the IM patients at the acute stage. There was no obvious difference in all the marks except CD4+/CD8+ratio, CD8+ CD28+ , CD19+ , CD19+ CD23+ ratio between the children recovering from IM and the normal controls. There is a positive correlation between CD3+ and CD19+, CD19+ CD23+ during acute phase. There is a negative correlation between CD4+ CD25+/highCD127low and CD19+ , CD19+ CD23+ during acute phase. Conclusion The results indicate that the decreasing of CD4+ CD25+/highCD127low may play an important role in elimina-ting EBV immortalized cells.