AIM:To construct eukaryotic expression vector expressing double shRNA sections targeting Survivin gene.METHODS: Eukaryotic expression vector expressing double shRNA sections targeting Survivin gene were designed and chemically synthesized.They were directionally inserted into plasmid pGenesil-1 with respectively U6 promoter and termination code,the common green fluorescence protein(EGFP) gene and Neo gene. In this way,the vector of pGenesil-1 shRNA containing 2 sections of Survivin shRNA were constructed and they were transfected into the pancreatic cancer cell Bx-PC3.Transfection was detected by fluorescence microscope.The inhibition expression of Survivin mRNA was measured by RT-PCR.RESULTS: HE1 and HE2 plasmids were identified by the biocatalyst cut which confirmed the exactitude and were analyzed by the sequence analysis which verified the perfect clone plasmid inserted by them.CONCLUSION: A eukaryotic expression vector of double short hairpin RNA for Survivin gene is successfully constructed.The pancreatic cancer cells Bx-PC3 succeed to be transfected and expression of Survivin mRNA is inhibited obviously.

Aim To explore the effects of curcumin on inflammatory reaction and blood-brain barrier permeability in rats following cerebral ischemic injury,and to further investigate its potential mechanisms.Methods SD rats underwent the cerebral ischemia/reperfusion injury by the sutrure occlusion model were randomly divided into sham-control,cerebral ischemia and curcumin-treatment groups.Neurological deficit scores,cerebral infarction volume,brain water content and blood-brain barrier (BBB) permeability were measured,myeloperoxidase(MPO)activities in rat brain were measured as an index of neutrophil infiltration;content of tumor necrosis factor-α(TNF-α)in rat brain was detected by ELISA;expression of matrix metalloproteinase-9(MMP-9) in rat brain was determined by Western blot.Results Neurological deficit and cerebral infarction volume was decreased in curcumin-treatment group.The degree of neutrophilicgranulo cyte infiltration in cerebral tissues was decreased and the integrity of BBB was improved.Curcumin could also inhibit TNF-α and MMP-9 expression.Conclusion Curcumin exerts the neuroprotective effect on cerebral ischemia/reperfusion injury in rats through inhibiting the inflammatory reaction and improving BBB integrity,which may be associated with the inhibiton of TNF-α and MMP-9.

Aim To detect the role of sirtuin1 ( SIRT1 ) in hepatotoxity caused by valproic acid ( VPA) . Methods The changes of SIRT1 expression of HepG2 cells were detected by Western blot. And then SIRT1 plasmid or siRNA was transfected to con-struct SIRT1 overexpressed or knocked-down HepG2 cells. Furthermore, SRB assays were taken to observe the changes of viability of these cells exposed to VPA. Results VPA suppressed SIRT1 expression in a time and dose-dependent manner. SIRT1 overexpression showed a protective effect to the cytotoxicity caused by VPA, and the IC50 before and after transfection was (4. 025 ± 0. 47) and (10. 87 ± 1. 50) mmol·L-1 re-spectively. Moreover, transfection of SIRT1 siRNA sensitized HepG2 cells to VPA, and the IC50 before and after transfection was (1. 938 ± 0. 16) and (0. 663 ± 0. 05) mmol·L-1 respectively. Conclusion VPA suppressed SIRT1 expression in HepG2 cells and over-expression of SIRT1 could reduce cytotoxicity induced by VPA.

Purpose To evaluate the tumor tropism of cytokine-induced killer ( CIK) cells, the movement track in nude mice bearing breast carcinoma and the influence on major organs of nude mice. Methods Separated and prepared CIK cells using human peripheral blood. The transwell migration assay was used to study the migratory response of CIK cells to human MDA-MB-435 breast carcinoma cells. A nude mouse xenograft model ( BALB/c) was established by injection of human MDA-MB-435 breast carcinoma cells. CIK cells labelled with DiI were injected into caudal vein of the nude mice bearing transplantation tumor. Movement track of CIK cells in vi-vo and influence on major organs were observed by living imaging technology, histopathology and immunohistopathology. Results When cultured in vitro during 14 ~20 days, CIK cells reached the peak level in proliferating stage with the maximum proportion of CD3 +CD56 + T cells. Transwell migration assay showed that the migrating number of CIK cells was increasing along with the increasing concentration of tumor cell cultural supernatants. Living imaging technology showed that the fluorescence signal began to appear 24 hours after injection of CIK cells and was strongest at 48 hours. Immunohistochemical technique and hematoxylin-eosin stain showed CIK cells tended to gather around tumor tissue 6 hours after injection, the most at 48 hours, and with some of the remaining cells on 14 day. In the meantime, no pathological damage caused by CIK cells was observed. Conclusion CIK cells have good tropism to the tumor tissue and safety to the normal tissue, and could be used as a promising cell vector for targeted therapy of cancer.

BACKGROUND:Previous studies discovered that pRST98, original y isolated from Salmonel a enterica serovar typhimurium (S.typhimurium) could promote bacterial biofilm formation. In addition, bacterial harboring pRST98 can promote the secretion and expression of interleukin-10 after infection in cells and animals.
OBJECTIVE:In vitro studies have discovered the effects of interleukin-10 at varying concentrations and conjugative plasmid pRST98 on the biofilm formation of S.typhimurium.
METHODS:S.typhimurium wild-type strainχ3306, virulence plasmid-deletion S.typhimurium strainχ3337 and pRST98-transconjugant S.typhimuriumχ3337/pRST98 were established in vitro and cultured for biofilm formation. 1, 10, 100 μg/L interleukin-10 were added during the biofilm formation. 0 μg/L interleukin-10 was set as a control. Crystal violet staining method, semi-quantitative method, confocal laser scanning microscopy and scanning electron microscopy were used to determine the effects of interleukin-10 on the biofilm formation and compare the effects of S.typhimurium with or without pRST98.
RESULTS AND CONCLUSION:Intra-group comparison showed that, compared with the control group, S.typhimurium gathered together and formed thicker biofilm in concentration of 1 and 10 μg/L of interleukin-10. The promotion effects of S.typhimurium on biofilm formation were greatly improved in 10 μg/L. Interleukin-10 in 100 μg/L inhibited S.typhimurium biofilm formation. Inter-group comparison showed that, A570 inχ3337/pRST98 was greatly higher than that inχ3306 andχ3337 under the same concentration of interleukin-10. The results indicate that both 1 and 10 μg/L of interleukin-10 promote biofilm formation, especial y bacteria harboring pRST98.

OBJECTIVE:To investigate in vitro antibacterial effect of extracts from Miao medicine Rubus multibracteatus leaves. METHODS:The aqueous extract and 80% ethanol extracts from R. multibracteatus leaves were used to prepare solution with mass concentration of 200 mg/mL. Using ampicillin and fluconazol as positive control (50 mg/mL),cup plate method was used to determine antibacterial effects of the extracts from R. multibracteatus leaves to Staphylococcus aureus,Staphylococcus epi-dermidis,Escherichia coli,Shigella dysenteriae,Proteus vulgaris,Candida albicans and Cryptococcus neoformans. The petroleum ether,acetic ether and n-butyl alcohol were used to extract 80% ethanol extracts in turns. After obtaining relevant extracts (50 mg/mL),cup plate method was used to investigate antibacterial effects of them to above 7 bacterial strains. The parts with antibacte-rial effects and bacterial strains sensitive to drug were screened. Micro-broth dilution method and agar culture medium plate method were used to determine minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC). RESULTS:The aqueous extract of R. multibracteatus leaves almost had no inhibitory effect while 80% ethanol extract showed different degrees of antibacterial effect to tested bacterial strains,and it also had higher activity against 5 bacterial stains than 2 fungus. The ethyl ace-tate and n-butyl alcohol fractions of 80% ethanol extracts showed good effect while the petroleum ether and water layer fractions had no antibacterial effect,and all the fractions of 80% ethanol extracts showed no inhibitory effect on fungus. To 5 bacterial stains,MIC and MBC of 80% ethanol extract were 6.25-12.5,12.5-25 mg/mL,those of ethyl acetate fractions were 3.13,6.25 mg/mL and those of n-butyl alcohol fraction were 3.13-6.25,6.25-12.5 mg/mL,respectively. CONCLUSIONS:80% ethanol ex-tract of Miao medicine R. multibracteatus leaves and its ethyl acetate and n-butyl alcohol fractions have obvious in vitro antibacteri-al effect to bacteria.

OBJECTIVE:To study the pharmacokinetics and absolute bioavailability of oleanolic acid and its derivative di-hydrooleanolic acid in rats in vivo. METHODS:12 SD rats were randomly divided into oleanolic acid administration group and di-hydrooleanolic acid administration group,6 in each group. All rats were intragastrically given related medicine(50 mg/kg),then in-travenously injected related medicine(2 mg/kg)in tail vein after 1 week. Sample blood 0.3 mL was taken from tail vein before ad-ministration and after administration(0.1,0.25,0.5,0.75,1,1.5,2,3,5,7,9,12 h for ig;0.05,0.15,0.25,0.5,0.75,1, 2,3,5,7,9,12 h for iv). UPLC-QTOF was conducted to determine the plasma concentration,and DAS 2.0 pharmacokinetic software was used to calculate pharmacokinetic parameters and absolute bioavailability. RESULTS:After ig and iv oleanolic acid, the AUC0-∞ were(232.10±7.17),(1203.99±19.65)ng·h/mL,t1/2 were(1.75±0.10),(1.41±0.04)h respectively;after ig,cmax was (121.3 ± 18.92) ng/mL,tmax was (0.54 ± 0.10) h,absolute bioavailability was 0.77%. After ig and iv dihydrooleanolic acid, AUC0-∞ were(382.03±23.73),(386.14±10.65)ng·h/mL,t1/2 were(2.47±0.45),(1.44±0.03)h;after ig,cmax was(124.52± 12.28)ng/mL,tmax was(0.63±0.14)h,absolute bioavailability was 3.96%. CONCLUSIONS:The absolute bioavailability of di-hydrooleanolic acid is significantly higher than oleanolic acid in rats.

Objective:To investigate the effect of upregulated and downregulated PSMA7 on the cell cycle and Cyclin D1,CDK4,P16,Rb of RB pathway in A549 cells.Methods:Transfected upregulated pcDNA3.1-PSMA7 vecter and downregulated pGPU6/Hygro-PSMA7-265 vecter into A549 cells,and then tested the effect of PSMA7 on the cell cycle of A549 cells by flow cytometry,and detected the protein level of Cyclin D1,CDK4,P16,Rb by Western blot.Results:Compared with the control group,the cell cycle of the A549 cells did not change significantly,and the expression of Cyclin D1,CDK4 decreased but P16,Rb increased when PSMA7 was upregulated.Compared with the control group,the proportion of phase G0/G1,G2/M of the A549 cells decreased and phase S increased,and the expression of Cyclin D1,CDK4 increased but P16,Rb decreased when PSMA7 was downregulated.There was statistical significance for those results.Conclusion:PSMA7 could affect the expression of Cyclin D1,CDK4,P16,Rb protein level of RB pathway in A549 and promoted the A549 cells into phase S when it′s downregulated.

OBJECTIVETo investigate development of a cell extraction process for preparing human meniscus acellular matrix, and morphology and biomechanical properties.

METHODSHuman meniscus were subjected to modified eight-step detergent, then, the specimens were assessed by staining with haematoxylin-eosin, toluidine blue, sirius red, saffron O, alcain blue and hoechst-33258, et al. The ultrastructure of the specimens was observed with scanning electron microscope. Transient recovery rate of deformation, maximal recovery rate of deformation and maximal compressive strength were tested to determine the biomechanical properties of the scaffold.

RESULTSEvery stain confirmed that the celluar constituents of the specimens were removed. The specimens stained positively by staining with sirius red. Lacuna were found irregularly not only on the surface of the meniscus,but also in the meniscus with scanning electron microscope. Pores in the specinmens were large, the diameter of pores was 80 to 760 microm, porosity was over 67%. The transient recovery rate of deformation was (89.62 +/- 1.04)%, the maximal recovery rate of deformation was 100% and the maximal compressive strength was (3.04 +/- 0.13)N, when the specimens were compressed 30%.

CONCLUSIONThe modified eight-step detergent can remove the immunogenic cell components from human meniscus, in addition, 3D extracellular matrix can be retained. The scaffold has good biomechanical properties. This scaffold stands a good chance to be an implant for future tissue engineering of the human meniscus.

Adult ; Cell Separation ; methods ; Cells ; chemistry ; cytology ; Cells, Cultured ; Humans ; Male ; Menisci, Tibial ; cytology ; Staining and Labeling

Objective:To investigate the effect of scutellarin on P-gp protein expression and activity in Caco-2 cells. Methods:Scutellarin(25,50 and 100 μmol·L-1 )was incubated with Caco-2 cells respectively for 24 h,48 h and 72 h. The expression of P-gp was determined by western blot assay and the activity of P-gp was determined by Rhodamine-123 assay. Results:P-gp protein ex-pression levels were significantly increased by scutelarin. After the incubation for 24 h with scutellarin,P-gp protein expression was up-regulated 2. 34-,2. 65-and 2. 00-fold in Caco-2 cells. After the incubation with scutellarin for 48 h,P-gp protein expression was up-regulated 2. 70-,4. 66-and 3. 13-fold. After the incubation with scutellarin for 72 h,P-gp protein expression was up-regulated 2. 82-, 2. 62-and 1. 84-fold. The intracellular accumulation of rhodamine-123 was significantly decreased by scutellarin,indicating that the ef-flux transport activity of P-gp was increased by scutellarin in Caco-2 cells. Conclusion:Scutellarin can significantly up-regulate P-gp protein expression and increase the efflux transport activity of P-gp in Caco-2 cells.