Objective: To investigate the effects of novel targeted non-viral vector in gene therapy of human glioma. Methods: The EGF-R targeting gene delivery system GE7 was constructed. Human Glioma cell line U251 was transfected in vitro with ?－gal as reportor gene and p21 as therapeutic gene using this gene delivery system. By means of the assay of ?-galactosidase staining, Western blotting, in situ end labeling apoptosis cells and DNA ladder, the transferring of exogenous genes and the apoptosis of the tumor cells were examined.Results: It was showed that gene transfer efficiency is over 80%. When transfected with p21 gene, the growth of cells was inhibited significantly, and the apoptosis was detected in the transfected cell by the methods of in situ end labeling and DNA ladder. Conclusion: The GE7 gene delivery system has the ability to transfer exogenous gene to tumor cells and the expression of the therapeutic gene can inhibit the growth of the cells.

OBJECTIVE To evaluate whether the tumor promoting activity syste m based on the ex-pression of 22 tumor pro motion marker genes in Bhas 42 cell transformation assay can be developed to be a new screening technique for predicting tumor promoting potential of che mical.METHODS Chose the 12-O-tetradecanoylphorbol-13-acetate (TPA)as positive che mical and use the Bhas 42 cell lines as test syste m.Define the day of seeding cells as d 0.On d 4,medium in each well was changed with the medium DF5F containing 0.05 mg·L -1 TPA or 0.5 %DMSO,after treat for 24,48 and 72 hrs,collect the cell samples.Extract the RNA fro m the cell samples and detect the expression of Ccnb1 ,Rif1 , Mc m3,Chek1 ,Jun,Fosl1 ,Hells,Vegfa,St mn1 ,Prl2c3,Scarb1 ,Phex,Orm1 ,Orm2,Nup54, Slc2a1 ,Il1 rl1 ,Rad51 ap1 ,Tfrc,Ab1 ,Car13 and Pik3r5 at different ti mepoints by RT-PCR.RESULTS Co mpared to the negative group,the expression of 3 genes was increased after treat with TPA for 24 hr which are Vegfa,Il1 rl1 and Pik3r5;the expression of 12 genes was increased after treat with TPA for 48 hr which are Chek1 ,Hells,Mc m3,Rad51 ap1 ,Vegfa,Il1 rl1 ,Prl2c3,Slc2a1 ,Tfrc,Ab1 ,St mn1 and Rif1 ;the expression of 10 genes was increased after treat with TPA for 72 hr which are Chek1 ,Hells, Rad51 ap1 ,Vegfa,Il1 rl1 ,Prl2c3,Slc2a1 ,Tfrc,St mn1 and Ccnb1 .CONCLUSION The 22 tumor pro-motion marker genes showed different sensitivity to the positive control che mical at different ti mepoints after treat with TPA.The result was consistent with the pro motion activity of TPA.The 22 tumor pro motion marker genes combined with the Bhas 42 cell transformation assay can be developed to be a new screening technique for predicting tumor promoting potential of che mical.

Objective To investigate the expression of microRNA-21(miRNA-21)in two human malignant melanoma cell lines,A375 and M14,and its effect on the viability of these cells.Methods The expression of human miRNA-21 was assessed by quantitative fluorescent PCR in A375 and M14 cells.Then,three concentrations(90,180,270 nmol/L)of miRNA-21 inhibitor and were transfected both cells and a negative control were transfected a mixture of LipofectamineTM 2000 reagent,respectively.After another 3-day culture,the proliferation of cells was detected by cell counting kit-8,and R value was calculated to denote the relative activity of cells.Statistical analysis was carried out by SPSS13.0.Results The expression of miRNA-21 was higher on A375 cells than that on M14 cells with the average value of 2-deltaCT being 1.2928±0.1509 vs 0.1894±0.1803.With miRNA-21 inhibitor at the concentration of 90,180,270 nmol/L,the activity of A375 cells was significantly lowered in comparison with that in the control group,with the R value being 0.7362±0.1662.0.7248±0.3204 and 0.6767±0.2998 respectively(all P＜0.01).However,in the case of M14 cells,cell activity was only suppressed by miRNA-21 inhibitor at 90 nmol/L with the R value being 0.7295±0.1478.and no significant inhibition was observed with the inhibitor at 180 or 270 nmol/L (both P＞0.05).Conclusions miRNA-21 is expressed on human melanoma cell lines,A375 and M14,at different levels,with a promoting effect on the proliferation of both cells.Moreover,miRNA-21 may act as an oncogene-like gene via down-regulating the expression of some tumor-inhibiting factors.

The aim of the study is to investigate rat intestinal absorption behavior of three main active components, schisandrol A, schisandrin A and schisandrin B in Schisandra chinensis Baill extracts in intestine of rats. With phenol red as the indicator, in situ single pass intestinal perfusion (SPIP) model was used and the concentrations of three main active components in perfusion solution of different intestinal segments (duodenum, jejunum, ileum, and colon) were determined by HPLC in combination with diode array detection. The results showed that the absorption rate constant (Ka) and effective permeability values (Peff) of three main active components in Schisandra chinensis Baill extracts had significant difference (P < 0.05) at different concentrations of perfusion solution, the Ka and Peff first increased and then decreased with the increase of drug concentration, the middle concentration was higher than those of the other two concentrations. The saturate absorption phenomena were observed, and it suggested that the transport mechanisms of three main active components in vivo were similar to active transport or facilitated diffusion. Three active components can be well absorbed in all of the intestinal segments, while duodenum is the best absorption region. The Ka and Peff of three active components in jejunum and ileum had no significant difference (P > 0.05). The absorption of the three active components displayed significant difference (P < 0.05) at different intestinal segments of rats. Schisandrin A had the best absorption in duodenum. The Ka and Peff among three active components were sequenced as follows: schisandrin A > schisandrin B > schisandrol A in other intestinal segments, and there is significant difference (P < 0.05) between them.

Objective To analyze the gene expression of pathogenic factors in vaginal secretions of patients with vulvovaginal candidiasis by using Oligo chips. Methods RNA was extracted from vaginal secretions of 10 patients with vulvovaginal candidiasis and 3 asymptomatic carriers, and hybridized with oligonuscreened followed by a bioinformatic analysis. Results Comparing with the asymptomatic carriers, the patients showed a higher expression of 44 genes and lower expression of 17 genes. Of these differentially expressed (TLR) 4, HWP1, SAP2, SAP5, LIP4, EFG1 and CPH1 were highly expressed in more than 80% of the secretion samples from patients with an average ratio of 4.013, while LIP6 and WH11 were lowly expressed in more IFN-γ and TLR4 were associated with native immunity, HWP1 associated with hyphal adhesion and formation, SAP2, SAP5, LIP4 and LIP6 associated with extracellular hydrolysis, and EFG1, CPH1 and WH11 associated with phenotypic switching. Conclusions Both the host adaptive immunity deficiency and increased virulence of Candida species are involved in the pathogenesis of vulvovaginal candidiasis, and TLR4 possibly plays a certain role in the local immunity of patients with this entity.

Objective To investigate gene transfer efficiency of a novel target non-viral vector GE7 and effects of herpes simplex virus thymidine kinase (HSV 1-tk)/ganciclovir(GCV) mediated by it in vitro. Methods The epidermal growth factor receptor (EGF-R) target gene delivery system GE7 was constructed.Human ovarian cancer cell line CAOV3 was transfected in vitro with ?-galactosidase(?-gal) as reporter gene and HSV 1-tk gene as therapeutic gene using this gene delivery system.By means of the assay of X-gal staining, Northern blotting, cell growth-inhibiting curve and so on,the transferring efficiency of exogenous genes and killing effects are observed. Results It showed that gene transfer efficiency is over 80%.When 10 mg/L GCV was put into ovarian cells transfected with HSV 1-tk gene, 95% of cells were killed, and the apoptosis ratio reached up to 30. Conclusions The GE7 gene delivery system is an effective and safe delivery system.GE7/ HSV 1-tk /GCV therapeutic gene system is appraising for ovarian cancer.

OBJECTIVETo establish a high performance capillary electrophoresis method with diode array detection (HPCE-DAD) for simultaneous determination of rutin, isoquercitrin, hyperoside, quercitrin, kaempferol and quercetin in Hyperici Japonici Herba.

METHODBased on the mode of capillary zone electrophoresis, 40 mmol x L(-1) borax was used as buffer solution (pH 8.62), uncoated fused silica capillary (56 cm x 64.5 cm x 75 microm) was used, separation voltage was 25 kV, detection wavelength was at 206 nm, column temperature was maintained at 25 degrees C, and sample was injected at 50 mbar, 8 s.

RESULTSix flavonoids showed good linearity (r > 0.9953) in the range of the tested concentration, the average recoveries of the method were between 98.8%-102.9%.

CONCLUSIONThe method is simple, accurate and reproducible, and can be used for quality control of Hyperici Japonici Herba.

Electrophoresis, Capillary ; methods ; Flavonoids ; analysis ; Hydrogen-Ion Concentration ; Hypericum ; chemistry ; Temperature