Objective:To investigate the effects of human glycolipid transfer protein(GLTP)on proliferation,migration and invasion of pancreatic cancer(PC)cells,and to elucidate their mechanisms.Methods:The difference in the expression levels of GLTP proteins between PC tissue and normal pancreas tissue was analyzed by University of Alabama at Birmingham Cancer Data Analysis Platform(UALCAN)Database,as well as the difference in the expression levels of GLTP protein between PC tissue and normal pancreas tissue of the PC patients with different clinicopathological characteristics.The PANC-1 cells were cultured in vitro and divided into control group(transfected with pFLAG-CMV4 plasmid)and GLTP-overexpression(GLTP-OE)group(transfected with pFLAG-GLTP plasmid).The stably GLTP transfected cells were established using the antibiotic screening method.Knock-down experiments were performed using non-specific siRNA transfected PANC-1 cells as control group and si-GLTP transfected PANC-1 cells as si-GLTP group.Western blotting method was used to detect the expression of GLTP protein in the cells in various groups,cell counting kit-8(CCK-8)method was used to detect the proliferation activities of PANC-1 cells,clone formation assay was used to detect the number of clone formation,and Transwell chamber assay were used to detect the numbers of migration and invasion cells in various groups.Transcriptomics sequencing analyses were conducted to assess the possible mechanism of GLTP in the PANC-1 cells.Western blotting method was employed to detect the expression levels of phosphatidylinositol 3-kinase(PI3K),phosphorylated PI3K(p-PI3K),protein kinase B(Akt),and phosphorylated Akt(p-Akt)proteins in the PANC-1 cells in various groups;Real-time fluorescence quantitative PCR(RT-qPCR)was used to assess the expression levels of amphiregulin(AREG)and kinase insertion domain receptor(KDR)mRNA in the cells in various groups.The mice were randomly divided into control group(injected with pFLAG-GMV4 transfected PANC-1 cells)and experimental group(injected with pFLAG-GLTP stably transfected PANC-1 cells),and the subcutaneously transplanted tumor models were prepared;the volumes and weights of the transplanted tumors of the mice in two groups were measured.Results:UALCAN database analysis showed that the expression level of GLTP protein in PC tissue was lower than that in normal pancreas tissue(P＜0.01),and there were statistically significant differences in the GLTP protein expression between PC tissue and normal pancreas tissue of the PC patients with different cancer stages(P＜0.05),tumor grades(P＜0.05),ages(P＜0.001),and genders(P＜0.05).Compared with control group,the proliferation activity(P＜0.01)and the number of clone formation(P＜0.001)of the cells in GLTP-OE group were decreased,while the numbers of migration cells(P＜0.001)and invasion cells(P＜0.01)were decreased.In the knock-down experiment,compared with control group,the proliferation activity(P＜0.01)and the number of clone formation(P＜0.05)of the cells in GLTP-OE group were increased,while the numbers of migration cells(P＜0.001)and invasion cells(P＜0.001)were increased.Compared with control group,the tumor weight and volume of the mice in experimental group were decreased(P＜0.01),following the injection of tumor cells for a period of four weeks.In the over-expression experiment,compared with control group,the expression levels of p-PI3K(P＜0.01),p-Akt-S473(P＜0.01),and p-Akt-T308(P＜0.05)proteins in the cells in GLTP-OE group were decreased;the expression levels of AREG(P＜0.01)and KDR(P＜0.01)mRNA were decreased.In the knock-down experiment,compared with control group,the expression levels of p-PI3K(P＜0.01),p-Akt-S473(P＜0.01),and p-Akt-T308(P＜0.01)in the cells in si-GLPT group were increased,and the expression levels of AREG(P＜0.01)and KDR(P＜0.05)mRNA were increased.Conclusion:Low expression levels of GLTP in PC tissue are present.The over-expression of GLTP can inhibit the proliferation,migration and invasion of PANC-1 cells,as well as the growth of subcutaneously transplanted tumors in the nude mice;its possible mechanism may be related to decreasing the activity of the PI3K/Akt signaling pathway.

Objective:To investigate the mechanism of Compound Mufurong Nasal Ointment in interfering with allergic rhinitis in young rats based on the Th1/Th2 cytokines and c-jun N-terminal kinase (JNK) signaling pathway.Methods:Totally 60 young SD male rats were divided into normal group, model group, experimental group, Western medicine group, and combination group according to random number table method, with 12 rats in each group. Except for the normal group, allergic rhinitis rat models were prepared by intraperitoneal injection of ovalbumin and nasal instillation of ovalbumin sensitization in all other groups of rats. After successful modeling, the normal group and the model group were given 0.9% sodium chloride injection 50 μl nasal drip, and the experimental group, the Western medicine group and the combination group were given compound Mufurong Nasal Ointment, fluticasone propionate nasal spray, compound Mufurong Nasal Ointment and fluticasone propionate nasal spray respectively. HE staining was used to observe the pathological morphology of nasal mucosal tissue; ELISA method was used to detect the levels of total IgE, interferon - γ (IFN - γ), IL-1 β, and IL-4 in the serum and nasal lavage fluid of rats in each group; Western blot was used to detect the expressions of JNK and p-JNK proteins in nasal tissues of rats in each group; Immunohistochemistry was used to detect the expressions of p-JNK and p-c-Jun proteins in nasal mucosal tissue.Results:Compared with model group, the levels of IgE, IL-1β and IL-4 in serum and nasal lavage solution of experimental group, western medicine group and combination group decreased ( P<0.05), IFN-γ level increased ( P<0.05); the expressions of JNK and p-JNK protein decreased ( P<0.05), and the expressions of p-JNK and p-c-Jun decreased ( P<0.05). Conclusion:Compound Mufurong Nasal Ointment may regulate Th1/Th2 cytokines through the JNK signaling pathway, inhibit Th2 over-expression, thereby reducing IgE, IL-1 β, IL-4 levels in serum and nasal lavage fluid, up-regulating IFN - γ levels, and intervening in the occurrence of allergic rhinitis.

OBJECTIVE To establish HPLC methods with different separation principles to analyze the relevant impurities in the APIs of bivalirudin from seven enterprises, to provide a basis for the comprehensive control of related substances of bivalirudin. METHODS Reversed-phase high performance liquid chromatography(RP-HPLC) was used to separate and analyze 11 kinds of impurities. Hydrophilic chromatography(HILIC)-HPLC was used to control four process impurities. Polymers were determined by size exclusion chromatography(SEC)-HPLC. RESULTS The established RP-HPLC could effectively separate the principal component and 11 impurities, the correction factors of 11 impurities were between 0.8−1.2, the detection concentration of bivalirudin was 0.1 μg·mL−1, and the detection limit was 0.004%. The established HILIC-HPLC could effectively separate the principal components and four process impurities, and the detection concentration of bivalirudin was 0.3 μg·mL−1, and the detection limit was 0.01%. Under SEC-HPLC conditions, the polymer and bivalirudin peaked sequentially, the resolution of the two was 2.9, the detection concentration of bivalirudin was 6 ng·mL−1, and the detection limit was 0.000 6%. Fifteen kinds of known impurities and polymers in 15 batches of samples from 7 enterprises were calculated by the self-control method of principal components, and the impurity contents from different enterprises had a certain correlation with their production processes. CONCLUSION The three different principles of the method have good specificity, high sensitivity, good durability, and reliable results, and can be used for quality control of substances related to bivalirudin.

Objective:To determine the relative correction factor of pre-vitamin D and simplify the calculation method of vitamin D assay.Methods:By studying the calculation method of vitamin D content in drug standards of various countries,HPLC was used to determine the relative correction factor of pre-vitamin D,and the influencing factors of determination were investigated.Results:The relative correction factors of pre-vitamin D at 254 nm and 265nm wavelength were determined by statistical analysis of 7 laboratories in China.Conclusion:Using the pre-vi-tamin D relative correction factor method to calculate the total amount of vitamin D simplified the experimental steps can be simplified by the pre-vitamin D relative correction factor method to calculate the total amount of vitamin D and the random operating errors can be avoided.The method is rapid and accurate,and lay a solid foundation for further improving the standard of vitamin D preparations.

Host cell residue DNA is one of the most common impurity which can affect the safety of biological products,therefore,domestic and international regulatory agencies have required the limit for host cell residue DNA in different biological products,either at the final product qualification or the appropriate intermediate control stage.The removal effect is verified by monitoring the residue DNA of products in different production stages,which is beneficial for assuring the scientificity and stability of the production process.In order to strengthen the understanding of control strategy about host cell residual DNA,the paper reviews progress in host cell residual DNA in biological products by authors'work experience and other's research,which provides reference for future work.

Abstract OBJECTIVE To establish a detection method for fingerprints and content determination of free sugar and hydrolyzed monosaccharide of intracellular glycopeptides in Coriolus versicolor. METHODS PMP derivatization after ultrasonic extraction with water as solvent was determined for free sugars, and PMP derivatization after acid hydrolysis was determined for hydrolyzed monosaccharides. The free sugars and hydrolyzed monosaccharides of raw materials and intermediates of intracellular glycopeptides from different manufacturers were analyzed by HPLC qualitatively and quantitatively. The fingerprints were analyzed by chemometrics analysis, and the differences between intracellular glycopeptides produced by different manufacturers were discussed. RESULTS Both two methods were validated by methodology. The samples from different manufacturers were clustered separately, and three free sugars and two hydrolyzed monosaccharides contributed greatly to the quality difference of intracellular glycopeptides of Coriolus versicolor. CONCLUSION The established analytical method can be effectively used for the determination of free sugars and hydrolyzed monosaccharides in the intracellular glycopeptide of Coriolus versicolor and the study of fingerprints. The adopted chemometrics research method provides guidance for the quality control of the intracellular glycopeptide of Coriolus versicolor.

Humans ; HIV Infections/drug therapy* ; HIV-1/genetics* ; Tenofovir/therapeutic use* ; Anti-HIV Agents/therapeutic use* ; RNA ; Drug Combinations

Objective:To investigate the c-Jun N-terminal kinase (JNK), CCAAT/enhancer-binding protein homologous protein (CHOP) pathway apoptosis and the changes of cytokine levels in immune-related organs and tissues of sepsis mice at different time points.Methods:Twenty-seven male BALB/c mice were divided into normal group, sepsis 6 hours group and sepsis 12 hours group by the block randomization method, with 9 mice in each group. The sepsis model was made by cecal ligation and puncture (CLP). Blood sample was collected from each group at the corresponding time point, and the serum levels of tumor necrosis factor-α (TNF-α), interleukin (IL-1β, IL-10) were measured by enzyme linked immunosorbent assay (ELISA). The spleen, thymus and appendix tissues were taken from the mice to detect the expressions of phosphorylation-JNK (p-JNK), JNK1, CHOP and cleaved caspase-3 protein by Western Blot.Results:The level of cytokines, p-JNK/JNK1 ratio, CHOP and caspase-3 in spleen tissues, and the CHOP, caspase-3 in thymus and appendix tissue in the sepsis 6 hours group were significantly higher than those in the normal group [serum TNF-α (ng/L): 24.29±3.09 vs. 2.93±2.09, serum IL-1β (ng/L): 5.00±3.19 vs. 3.54±1.53, serum IL-10 (ng/L): 1 963.93±270.20 vs. 275.09±45.21, spleen p-JNK/JNK1 ratio: 0.257±0.126 vs. 0.154±0.068, spleen CHOP/β-actin: 0.201±0.131 vs. 0.142±0.068, spleen caspase-3/β-actin: 0.215±0.126 vs. 0.098±0.088, thymus CHOP/β-actin: 0.122±0.071 vs. 0.089±0.067, thymus caspase-3/β-actin: 0.258±0.145 vs. 0.108±0.045, appendix CHOP/β-actin: 0.361±0.134 vs. 0.215±0.112, appendix caspase-3/β-actin: 0.439±0.211 vs. 0.321±0.145, all P < 0.05]. However, there were no significant difference in the p-JNK/JNK1 ratio in thymus and appendix (thymus p-JNK/JNK1 ratio: 1.221±0.776 vs. 1.168±0.475, appendix p-JNK/JNK1 ratio: 2.014±1.227 vs. 1.828±0.915, both P > 0.05). Cytokine levels and the p-JNK/JNK1 ratio, CHOP, caspase-3 in spleen, thymus, and appendix in the sepsis 12 hours group were further increased when compared with those in the sepsis 6 hours group, except for a significant decrease in IL-10 level [serum IL-10 (ng/L): 1 698.98±210.52 vs. 1 963.93±270.20, serum TNF-α (ng/L): 41.66±6.57 vs. 24.29±3.09, serum IL-1β (ng/L): 10.37±4.14 vs. 5.00±3.19, spleen p-JNK/JNK1 ratio: 0.399±0.135 vs. 0.257±0.126, spleen CHOP/β-actin: 0.298±0.145 vs. 0.201±0.131, spleen caspase-3/β-actin: 0.353±0.145 vs. 0.215±0.126, thymus p-JNK/JNK1 ratio: 1.667±0.891 vs. 1.221±0.776, thymus CHOP/β-actin: 0.207±0.133 vs. 0.122±0.071, thymus caspase-3/β-actin: 0.416±0.179 vs. 0.258±0.145, appendix p-JNK/JNK1 ratio: 2.425±1.361 vs. 2.014±1.227, appendix CHOP/β-actin: 0.456±0.189 vs. 0.361±0.134, appendix caspase-3/β-actin: 0.635±0.289 vs. 0.439±0.211, all P < 0.05]. Conclusions:The endoplasmic reticulum pathway JNK and CHOP pathways are involved in immune-related cell apoptosis and cytokine expression in mice with sepsis. Apoptosis is more obvious at 12 hours than at 6 hours, and the inflammatory response is stronger.

Objective To explore the etiology and treatment of acute intestinal obstruction.Methods Clinical data of patients who underwent operation for acute intestinal obstruction in Zhongshan Hospital from May 2012 to May 2017 were collected and retrospectively analyzed.Results 721 patients were included and the ratio of males to females was 1.55 ∶ 1.There were 48.8％ in old-aged group and 51.1％ in young-middle-aged group.The most common causes of ileus included tumor in 376 cases (51.5％),adhesion in 168 cases (23.3％),hernia in 70 cases (9.7％),intraluminal obstruction in 42 cases (5.8％) and others in 79 cases.There was a significant difference between incarcerated hernia in elderly group and middle-young-aged group (4.6％ vs.15.1％,x2 =22.4,P ＜ 0.01).The length of hospitalized days in patients with tumor and incarcerated hernia in elderly group were significantly longer than young-middle-aged patients [(15.3±8.6)d vs.(13.4±6.3)d,t =-2.5,P＜0.05;(10.1 ± 6.7) d vs.(6.4 ± 2.9) d,t =-2.2,P ＜ 0.05].The length of hospitalized days by limited operation in patients with tumor were significantly shorter than those by emergency operation [(16 ± 12)d vs.(18 ± 24) d,t =-0.3,P ＞ 0.05].Conclusion Tumor and adhesion are the main causes of acute intestinal obstruction.Neoplastic bowel obstruction from small intestine or proximal colon requires emergency surgery.