Objective To develop a reversed-phase high-performance liquid chromatography(RP-HPLC) method for the determination of residual phenylmethanesulfonyl fluoride(PMSF) content in recombinant virus-like particle(VLP) vaccine stock solution,and to verify the method for the determination of PMSF residues in recombinant VLP vaccine stock solution.Methods A RP-HPLC method for the determination of PMSF residues was developed by screening detection wavelength,flow rate,injection amount and chromatographic column.The specificity,linearity and range,limit of quantitation(LOQ),limit of detection(LOD),accuracy,precision and durability of the method were verified.The developed method was used to detect PMSF residues in three batches of recombinant hepatitis E vaccine stock solution.Results The RP-HPLC method was developed,with ChromCore 300 C18(4.6 mm × 250 mm,5 μm) as the chromatographic column at the column temperature of 25 ℃,the detection wavelength of 210 nm,the injection amount of 100 μL,0.1% trifluoroacetic acid(TFA)/water and0.1% TFA/acetonitrile as mobile phase at a flow rate of 1 mL/min,and the detection time of 18 min.The absorption peaks of PMSF control and vaccine stock solution containing PMSF appeared around 5.0 min,while the vaccine stock solution and stock solution buffer showed no absorption peak.There was a good linear relationship between the concentration of PMSF control and peak area in the range of 0-5.0 μg/mL,with R~2 of 0.999.The LOQ and LOD of this method was 0.250 μg/mL and 0.125 μg/mL,respectively.The recovery rates of PMSF control with high,medium and low concentrations were all between 90% and 110%.The relative standard deviations(RSDs) of retention time,peak height and peak area for the determination of instrument repeatability,sample repeatability and intermediate precision were all less than 5%.The RSDs of retention time,peak height and peak area were all less than 5% under the TFA concentration of 0.09%,0.1% and 0.11% at column temperature of 20,25 and 30 ℃.PMSF was not detected in the three batches of recombinant hepatitis E vaccine stock solution.Conclusion The developed RP-HPLC method has good specificity,linearity and durability,high accuracy and precision,and can be used to detect residual PMSF content in recombinant VLP vaccine stock solution.
Phenylmethanesulfonyl fluoride(PMSF) ; Reversed-phase high-performance liquid chromatography(RP-HPLC) ; Virus-like particle(VLP) vaccine

0.05).Conclusions:Occlusal splint is effective in the control of TMD pain,the treatment mechanism is related to the quick improvement of masticatory muscles.

OBJECTIVE:To investigate the protective effect of Compound ginkgo leaf granules on liver function damage of acute pancreatitis. METHODS:Totally 82 patients with severe acute pancreatitis(SAP)complicated with liver function damage se-lected from our hospital during Sept. 2013-Sept. 2015 were randomized into control group and observation group according to ran-dom number table,with 41 cases in each group. Control group was given SAP related routine therapy. On the basis of the control group,observation group was additionally given Compound ginkgo leaf granules 5 g added into warm boiled water 80 mL via naso-gastric tube,tid,one week as a treatment course,for 2 courses. The remission time of abdominal pain,serum amylase recovery time,length of hospital stay and adverse drug reactions were observed in 2 groups. The levels of serum amylase,ALT,TBIL, AST,γ-GT,ALB,IL-6,TNF-α,creotoxin concentration,MDA,SOD,GSH-Px,total antioxidant capacity(TAC)and peripher-al blood PMN NF-κB levels were determined in 2 groups before and after treatment. RESULTS:In control group,3 patients with-drew from the study and 38 patients completed the study;in observation group,6 patients withdrew from the study and 35 patients completed the study. The remission time of abdominal pain,recovery time of serum amylase and the length of hospital stay in obser-vation group were significantly shorter than control group,with statistical significance (P<0.05). Before treatment,there was no statistical significance in the levels of serum amylase,ALT,TBIL,AST,γ-GT,ALB,IL-6,TNF-α,creotoxin concentration, MDA,SOD,GSH-Px,TAC and NF-κB between 2 groups(P>0.05). After treatment,the levels of serum amylase,ALT,TBIL, AST,γ-GT,IL-6,TNF-α,creotoxin concentration,NF-κB and MDA in 2 groups were decreased significantly,while the levels of ALB,SOD,GSH-Px and TAC were increased significantly;the observation group was significantly lower or higher than the con-trol group,with statistically significant (P<0.05). There was no statistical significance in the incidence of ADR between 2 groups (P>0.05). CONCLUSIONS:Compound ginkgo leaf granules can significantly decrease serum amylase,protect liver function, with good safety. The mechanismmay be inhibiting the release of inflammatory factors,improving oxidative stress reactions,reduce the concentration of creotoxin and inhibiting the activity of NF-κB.

Objective To observe the baseline characteristics and visual outcomes after two years follow-up of exudative age-related macular degeneration (AMD) patients treated with ranibizumb. Methods Forty-four eyes of 44 patients with exudative AMD were enrolled into this retrospective study, 19 were men and 25 were women. The mean age was 78 years (range 64–92 years). All patients were underwent best corrected visual acuity (BCVA, Early Treatment of Diabetic Retinopathy Study), fundus color photography, fundus fluorescein angiography (FFA), indocyanine green angiography (ICGA) and optical coherence tomography (OCT). The mean BCVA was (50.36±14.43) letters, the mean central foveal thickness (CFT) was (291.95± 82.19)μm, and the fluorescence leakage area of choroidal neovascularization (CNV) was (7.61±5.84) mm2. All patients received three initial intravitreous injection of ranibizumb (IVR) and were retreated with monthly IVR when needed. The mean follow up time was 25.6 months (range 24–29 months). On 1, 2, 3, 6, 12, 18 and 24 months after treatment, BCVA and OCT were repeated. On 3, 6, 12, 18 and 24 months after treatment, FFA and ICGA were repeated. The change of BCVA, CFT and fluorescence leakage area of CNV were observed. The association of baseline characteristics and two year visual outcomes were analyzed. Results On 1, 2, 3, 6, 12, 18 and 24 months after treatment, the BCVA were improved significantly (t=?1.89,?3.51,?4.61,?4.04,?5.77,?4.69;P<0.05), the CFT were decreased significantly (t=1.51, 2.30, 3.40, 3.28, 3.54, 3.88, 3.73;P<0.05). On 3, 6, 12, 18 and 24 months after treatment, the fluorescence leakage area of CNV were reduced significantly (t=2.12, 2.90, 3.51, 4.12, 4.06;P<0.05). The lower baseline BCVA, the more improved after treatment. The BCVA improvement degree has a negative relationship with baseline BCVA and fluorescence leakage area of CNV (r=0.505,?0.550;P<0.05), but no correlation with baseline CFT (r=0.210, P>0.05). Conclusion Two year visual outcomes of exudative AMD patients treated with ranibizumb is negative correlated with baseline BCVA and fluorescence leakage area of CNV, but not correlated with baseline CFT.

Smoking and exposure to second hand smoke are the deadly killer of human health. Tobacco kills more than one million people each year in China. According to 2015 Chinese adult tobacco survey, the number of smokers in China increased by 15 million during five years, from 301 million in 2010 to 316 million in 2015. Smoking addiction is a chronic recurrent disease, and its pathogenesis is associated with the mesolimbic dopamine system. In recent years, domestic and overseas scholars studied the brain function of smoking addicts through resting state functional magnetic resonance imaging (fMRI), and found that the brain functional activity of smoking addicts has changed. fMRI can help us understand the mechanism of tobacco dependence, and provide the evidence of diagnosis, treatment and health management of tobacco dependence. In this paper, the present study situation and the application in the field of the treatment and management of smoking cessation of fMRI in smoking addicts are reviewed.

In the modernization course of Chinese materia medica(CMM),two misunderstanding mistakes which are out of line with traditional Chinese medicine(TCM)theory and excessively emphasize the research of CMM compound prescription and two difficult points which are the establishment of CMM quality control system and the establishment of CMM evaluation system were pointed out.Because the main means of TCM syndrome differentiation treatment and TCM combination therapy is CMM processing pieces and the starting point of the research of CMM efficacy substantial basis should be CMM processing pieces,the priority of the modernization of CMM should be the research on modern CMM processing pieces and the conditions have been mature now.The research on modern CMM pieces were supposed to take three steps:the research on "standardized extract",the research on "effective fraction" and the research on the CMM nature of "effective fraction".

Objective To evaluate the clinical utility of CMV pp65 antigenemia by CMV brite Kit for predicting active/reactive CMV infection S as well as of CMV diseases in bone marrow or peripheral stem cell transplant patients. We also investigated the efficacy of preemptive therapy guided by detection of CMV antigenemia. Methods A total of 210 EDTA anticoagulant plasma samples from 36 bone marrow or Peripheral Stem Cell Transplant Patients were prospectively collected from September 1999 to April 2000. The specific CMV antibody IgG/IgM of all patients were detected by ELISA. We detected CMV pp65 antigenemia by indirect immunofluorescence assay using CMV Brite Kit. All blood samples were detected weekly from week 3 after bone marrow transplantation until 100 days or antigenemia turning negative/dischage or death. Ganciclovir preemptive therapy was initiated at first positive pp65 antigenemia. Results Of 36 bone marrow or Peripheral Stem Cell Transplant Patients, 16 patients occurred positive pp65 antigenemia, 15 patients suffered from symptomatic CMV infections or CMV diseases. In 14 patients of positive pp65 antigenemia receiving gaciclovir therapy at first antigenemia, 2 patients died (mortality rate 14.2%), 12 patients of pp65 antigenemia became negative. Otherwise, 2 untreated cases died. The study showed a significant difference in mortality rate between treated and untreated patients (P

Cartilage is vulnerable to traumatic injury and unable to facilitate a satisfactory healing response due to its poor vascularity and inability to access mesenchymal stem cells.Continuous defects in the joint surfaces cause pain,swelling,and mechanical symptoms that result in functional impairment and limitation of athletic participation.Commonly used repair techniques include marrow stimulation,structural osteo-articular autografts or chondrocyte implantation.Platelet-rich plasma (PRP) is a concentrate extract of platelets from autologous blood,which is rich in growth factors and other cytokines and provides local environment for tissue regeneration and lends a possible option for the stimulation and acceleration of cartilage regeneration.This review gives summarization on the current state of the use of PRP for cartilage regeneration.

AIM To establish an HPLC method for the simultaneous content determination of saikosaponin a,saikosaponin c,saikosaponin d,rutin,quercetin,isorhmnetin and kaempferol in the aerial and underground parts of “Zhongchai No.2” (Bupleurum chinense DC.from Sichuan Province).METHODS The analysis of “Zhongchai No.2” methanol extract was performed on a 30 ℃ thermostatic Hypersil GOLD column (250 mm × 4.6mm,5.0 μm),with the mobile phase comprising of acetonitrile-0.1％ phosphoric acid flowing at 0.8 mL/min in a gradient elution manner,and the detection wavelength was set at 210 nm.RESULTS Seven constituents showed good linear relationships within their own ranges (r≥0.999 1),whose average recoveries were 96.30％-102.7％ with the RSDs of 0.95％-3.8％.There were a lot of flavonoids in the aerial parts of samples,while a large number of saponins existed in the underground parts,but almost no flavonoids were found.CONCLUSION The aerial parts (stems and leaves) of “Zhongchai No.2” also have medicinal value,to which we should pay attention.

Objective To investigate the effect of construct the Notch1 (NICD) eukaryotic expression vector on the proliferation and differentiation of rat bone marrow mesenchymal stem cells (BMSCs) in vitro. Methods Rat BMSCs were experimented as the object. NICD eukaryotic expression vector was constructed. pEGFP-N1-NICD expressing plasmids were used to transfect BMSCs. The study included control group (CON group), empty vector group (VEC group) and the trans-fection group (TRA group). After 48-hour transfection, BMSCs were observed for general morphology. The protein expres-sions of NSE, GFAP and Notch1 were detected by real-time PCR and Western blotting assay respectively. The apoptosis, cy-cle distribution and cell proliferation were evaluated by flow cytometry and MTT assay. Results The DNA sequencing con-firmed that the pEGFP-N1-NICD recombinant plasmid was successfully constructed, and both VEC group and TRA group expressed green fluorescence after 48-hour transfection. The relative expression levels of Notch1 and GFAP mRNA and pro-tein were significantly higher in TRA group than those in VEC group and CON group (P<0.05), and there was no significant difference between VEC group and CON group. After 48-hour transfection, the ratio of living cells was significantly lower in TRA group than that of CON group and VEC group, and the early apoptotic rate and late apoptotic rate were significantly higher in TRA group than those of CON group and VEC group (P<0.05). The late apoptotic rate was significantly higher in VEC group than that of CON group. The proportion of G1/G0 cells was significantly higher in TRA group than that of CON group and VEC group, but S and G2/M cells were significantly lower (P<0.05). The value of growth curve was gradually de-creased in TRA group than that of CON group and VEC group (P<0.05). Conclusion The high expression of NICD gene might induce apoptosis of BMSCs, inhibit the proliferation in part, and induce into glial-like cell differentiation.