Natural biocompatible nanomaterials such as self-assembled triterpene natural small molecule products with favorable anticancer activity show great potential for biomedical application. However, the mechanisms of their molecular self-assembled structures have not been investigated systematically, while there are still few reports of the natural active carrier for drug delivery. Herein, in this work, we further explored the molecular assembly mechanism and common regularity of tetracyclic triterpenes ergosterol, stigmasterol as well as pentacyclic triterpenes glycyrrhetinic acid and ursolic acid, which suggested that the coplanarity and orderliness of molecular arrangements which are speculated to be responsible for their self-assembly into the spherical, rod-like or lamellar nanostructure. Besides, ergosterol (ET) with better anticancer activity was chosen as a representative substance for construction of the synergistic antitumor nanodrug. By intermolecular hydrogen bonding and π-π stacking, chemotherapeutic drug paclitaxel (PTX) was encapsulated into ET-PTX NPs successfully. Then, the anti-cancer efficacy of the tumor-bearing mice was evaluated according to the protocol approved by the Experimental Animal Research Center of Harbin Medical University. The resulting nanodrug exhibited excellent biosafety and enhanced in vivo anticancer activity efficiency of 52.3%, higher than free PTX (29.4%) or ET NPs (32.5%) alone, further verifying the potential medical application value of triterpene natural products. This work provides not only a theoretical basis for exploring the self-assembly behavior of small molecule natural products, but also a promising perspective for the fabrication of active natural biocompatible nanodrug delivery systems for synergistic antitumor therapy and other biomedical applications.

Protein-protein binding is the basis of virus and host cell interactions. With the application of technology of studying protein interactions, more knowledge of replication and pathogenesis of hepatitis C virus (HCV) was acquired. Non-structure protein 5B(NS5B) of HCV is a kind of viral protein, which plays an important role in replication of HCV. However, the effect of NS5B is not clear. To investigate the biological function of NS5B, we performed yeast two hybrid to look for proteins in hepatocytes interacting with NS5B. We constructed NS5B bait plasmid by cloning the gene of NS5B into pGBKT7, then transformed it into yeast AH109(a type). The transformed yeast was mated with yeast Y187(? type)containing liver cDNA library plasmid in 2?YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-?-gal for screening. Thirty-three colonies were selected and sequenced. Among them, two colonies were new genes with unknown function. The preliminary successful cloning of gene of protein interacting with NS5B paved the way for the study of the physiological function of NS5B and its associated protein.

Objective To study immunological property of chimeric BPVL1 / HPV16 E7 virus-like particles (VLPs) in mice. Methods HPV16 E7 gene was cut into three fragments, ligated to BPVL1 sequence, respectively, and then expressed as chimeric VLPs. The ability of the chimeric VLPs to stimulate in vivo cytotoxic response was analysed with lymphocytes taken from mice C57BL/6J lymph nodes. Results A strong CTL response against C-2 cells (HPV16 E7 aa47-59 transfected EL-4 cell) was induced in mice immunised with chimeric VLPs BPVL1 / HPV16 E7(b). Furthermore, no CTL response was detected against EL-4 cells in immunized mice. Conclusions Chimeric BPVL1/HPV16 E7 VLPs, serving as antigens, can activate mouse T lymphocytes and elicit a strong antigen-specific CTL response. Chimeric BPVL1/HPV16 E7 VLPs could be used as an efficient antigen delivery system, and might provide a novel strategy for HPV16 vaccine design.

To try to find out the possible use of histochemistry in the prenatal diagnosis in first trimester of pregnancy, 20 kinds of enzymes in chorionic villi from 6th to 9th week of gestation which are related to inborn errors of metabolism were investigated and graded according to their histochemical reactive intensity; the enzymes listed below: (1). ?-glucuronidase, (2). ?-galactosidase, (3). arylsulfatase, (4). ?-N-acetylglucosaminidase, (5). 3?-hydroxysteroid dehydrogenase, (6). NADH dehydrogenase, (7). adenosine triphosphatase, (8). alkaline phosphatase,(9). acid phosphatase, (10). creatine phosphokinase, (11). glutamic dehydrogenase, (12). aldolase, (13). xylitol dehydrogenase, (14). alcohol dehydrogenase, (15). xanthine oxidase, (16). catalase, (17). ornithine carbamoyl transferase, (18). lipase, (19). cholinesterase, (20). ?-glutamyl transpeptidase. The experimental results show that the last 6 kinds of enzymes present negative reactions. Since the his- tochemical reactions of the first 14 kinds of enzymes are positive, it is possible that they can be used for the prenatal diagnosis of inborn errors of metabolism caused by dificiency of corresponding enzymes. The results also show that compared with biochemical method, histochemical method has the advantages of requiring smaller amount of chorionic villi, being without contamination by maternal cells, simplicity and rapidity.

Objective Through analyzing and summarizing the main pathogens of bacterial infections of children in Sichuan Province and trends of drug resistance in 2013 to provide a reference for the clinical use of antibiotics.Methods The pediatric pathogen were collected by member of Sichuan province in china antimicrobial resistance surveillance system.Results A total of 22 470 clinical bacterial were isolated from the members,in which Staphylococci,Escherichia coli,Streptococcus pneumoniae,Kleb-siella pneumoniae,Hemophilus influenza,Moraxella catarrhalis were the most common Bacteria.The resistance rates of Staphylo-coccus aureus and Coagulase-negative staphylococci to oxacillin were 1 7.7% and 71.1%.4 strains of Staphylococcus aureus and 20 strains of coagulase-negative staphylococci were teicoplanin resistance.Escherichia coli and Klebsiella pneumoniae were clearly re-sistant to the third generation cephalosporins,ampicillin and ampicillin/sulbactam,with the exception of ceftazidime.Carbapenems remained highly active against all the target bacteria.The resistance rate of Streptococcus pneumoniae to Penicillin was 3.9% .All antibiotics excepted cotrimoxazole remained highly active against the haemophilus influenzae.All antibiotics except macrolide anti-biotics remained highly active against the moraxella catarrhalis.Conclusion Penicillin-insensitive Streptococcus pneumoniae,mac-rolides-resistant gram-positive cocci,cephalosporin-resistant Enterobacteriaceae ,Oxacillin-resistance coagulase-negative staphylo-cocci were revealed to be the most serious problems in terms of bacteria resistance for children in Sichuan province.

Objective To screen proteins binding with interferon?(IFN?)from human hepatic cDNA libraty by yeast-two hybrid technique.Methods The IFN?gene was amplified by polymerase chain reaction(PCR)and constructed into pGBKT7 vector as the bait plasmid in yeast-two hybrid system3,pGBKT7-IFN?was then transfected into yeast AH109.The transfected yeast were mated with yeast Y187 containing liver cDNA library plasmid in 2?YPDA medium.Diploid yeast was plated on synthetic dropout nutrient medium(SD/-Trp Leu-His-Ade)and synthetic dropout nutrient medium(SD/-Trp-Leu-His-Ade)containing X-?-gal for selecting.After plasmid extracting and en- zyme cutting analysis,the blue colonies were performed sequence analysis,the results were analyzed by bioinformatics.Results IFN?gene was successfully cloned and expressed in yeast cells.Thirty- four positive colonies were obtained using yeast-two hybrid technique.After sequence analysis,eight clones were found may have a binding effect with IFN protein.Conclusions IFN?genes was success- ful cloned and eight proteins that could bind with IFN?protein were also screened.

Objective To investigate the effect of glucose-based peritoneal dialysis fluids and icodextrin-based peritoneal dial-ysis fluids on the expression of TLR2 and TLR4 on huamn peritoneal mesothelial cells .Methods Human peritoneal mesothelial cell line 5 - 10 generations(HMrSV5) was cultured in DMEM /F12 medium supplemented with 10% (v/v) fetal calf serum (FCS) .Cell viability and cell proliferation were assessed using M TT method .The experiment were divided into 5 different groups :group A (control group) ,1 .5% dextrose group ,2 .5% dextrose group ,4 .25% dextrose group and 7 .5% Lcodextrin group .Icodextrin group (aikau dextrin) ,TLR2 and TLR4 expression were detected by Western blot .Results Treatment with different concentrations of glucose-based peritoneal dialysis fluids for 24 h did not affect the expression of TLR2 and TLR4 protein .In addition ,after stimula-tion for 48 h ,1 .5% dextrose ,2 .5% dextrose ,4 .25% dextrose decreased TLR2 expression by (5 .5 ± 2 .8)% ,(31 .4 ± 7 .5)% , (54 .9 ± 1 .9)% respectively ,TLR4 expression by (32 .9 ± 17 .6)% ,(47 .7 ± 13 .5)% ,(66 .4 ± 13 .5)% respectively .Stimulation for 72 h ,decreased TLR2 expression by (29 .4 ± 14 .7)% ,(38 .9 ± 9 .9)% ,(63 .5 ± 16 .5)% respectively ,TLR4 expression by(59 .5 ± 16 .8)% ,(63 .1 ± 9 .5)% ,(79 .2 ± 14 .0)% respectively .There was no significant change in TLR2 and TLR4 protein expression on 7 .5% icodextrin group .Conclusion Glucose-based peritoneal dialysis fluids ,but not icodextrin-based peritoneal dialysis fluids downregulates expression of TLR2 and TLR4 by HM rSV5 .

A strain named as WJ44 with capability of producing branched-chain aminotransferase was isolated from the soil, the enzyme activity is especially high when used leucine as a substrate. According to the physiological characteristics, biochemical characteristics and 16S rRNA sequence of WJ44, the strain was identified as Bacillus cereus. The optimal carbon source and nitrogen source were also determined as 20 g/L glucose, 15 g/L tryptone and 5 g/L beef extract, respectively. The optimal proportion of other component in the medium is corn steep liquor 15 g/L, KH2PO4 3 g/L, MgSO4?7H2O 0.5 g/L. The optimum original pH value is 7.0, the cultural temperature is 37℃, liquid volume is 20 mL/250 mL, the shaker’s rotating speed is 200 r/min. The highest leucine transferase activity of 45.787 U/mL was achieved after 10 h under the opti- mal condition, and the dry weight of cell is 8.643 g/L, increased by 54% and 10% respectively. This result shows that WJ44 is a branched-chain aminotransferase-production strain with fine capability.

OBJECTIVETo discuss that pathogenesis evolvement regularity of Chinese miniature swine with phlege-stasis cementation syndrome of coronary heart disease.

METHODEighteen Chinese miniature swine were randomly divided to the normal control group, the model group and the Danlou tablet group, with six swine in each group. Except for the normal control group, all of the other groups were fed with high fat diet for two weeks. The coronary heart disease model with phlegm-stasis cementation syndrome was established by injuring left anterior descending artery with interventional balloons and continuously feeding with high fat diet for eight weeks. The levels of BMI, hemorheological parameters, lipids in serum and inflammatory cytokines were observed at the 0th (before the experiment), 2nd (before operation or drug administration), 6th (four weeks after drug administration) and 10th week (eight weeks after drug administration) of study. The levels of TG and TC in liver and the pathological changes in coronary artery tissues were also observed at the end of study.

RESULTCompared with the normal control group, the model group had showed significant increase in the levels of TC, TG, LDL-C and VLDL-C in serum (P < 0.01) from the second week to the end of the experiment, with notable rise in the whole blood viscosity under the shear rates of 5 s(-1) and 60 s(-1). At the 6th week, the levels of BMI and TG and TNF-alpha in serum significantly increased. At the 10th week, the levels of BMI and hs-CRP, IL-6 and TNF-alpha in serum significantly increased as well, with remarkable increase in coronary stenosis, intimal thickness and the ratio between intimal thickness and media thickness (P < 0.05 and P < 0.01), and significant rise in TC and TG in livers (P < 0.01). Compared with the model group, the Danlou tablet group showed obvious reduction in severity of coronary artery lesion, intimal thickness and lumen stenosis ratio and ratio between intimal thickness and media thickness (P < 0.01), BMI, TC, TG, LDL-C and VLDL-C in serum, TC and TG in liver, as well as hs-CRP, IL-6 and TNF-alpha levels in serum (P < 0.05 and P < 0.01), with notable decline in the whole blood viscosity under the shear rates of 5 s(-1) and 60 s(-1).

CONCLUSIONThe interaction of phlegm, blood stasis and toxin syndromes helps promote the progress and development of AS plaques, which is the key pathogenesis of phlegm-stasis cementation syndrome in coronary heart disease.

Animals ; Body Weight ; Coronary Disease ; blood ; physiopathology ; Female ; Hemodynamics ; Inflammation Mediators ; metabolism ; Lipids ; blood ; Liver ; metabolism ; Male ; Medicine, Chinese Traditional ; Swine ; Swine, Miniature

A cell model is desired for adipocyte differentiation investigation and for high-throughput screening of anti-obesity and anti-diabetes molecules from chemical resources due to the world wide epidemic of obesity and diabetes. In order to establish such a cell model, a plasmid of pPPARgamma2-promoter-EGFP was constructed by inserting a 660bp sequence of mouse PPARgamma2 promoter into the Ase I and Kpn I sites of pEGFP-N3 and transferred into 3T3-L1 preadipocyte cells. The cells were induced to differentiate and the expression of PPARgamma2 was detected by the microscopic observation of EGFP and by RT-PCR assays. The results showed that the EGFP gene expression patterns were similar to that of pPPARgamma2's, which indicated that the EGFP gene was transferred into the mouse 3T3-L1 preadipocyte cells, and its expression was under the control of pPPARgamma2 promoter. RT-PCR assays showed that the EGFP expression authentically represented the stable expression of PPARgamma2. In conclusion, a preadipocyte cell line expressing EGFP under the control of the promoter of adipocyte-specific expression gene PPARgamma2 was generated. The cell line provides a powerful approach for the research of adipocyte differentiation and for the high-throughput screening of anti-obesity and anti-diabetes chemicals.
3T3-L1 Cells ; Adipocytes ; cytology ; Animals ; Cell Differentiation ; Green Fluorescent Proteins ; genetics ; Mice ; PPAR gamma ; genetics ; Promoter Regions, Genetic ; Stem Cells ; cytology