Objective To investigate the expression of cyclin D3 and proliferating cell nuclear antigen (PCNA) and their clinical significance in mal ignant melanoma. Methods The expression of cyclin D3 and PCNA was measured by streptavidin-peroxidase complex immunohistochemical technique in 57 cases of pri mary cutaneous malignant melanoma (CMM), 37 cases of metastatic melanoma and 20 cases of benign nevi. Results The positive expression rate of cyclin D3 in CM M and metastatic melanoma were 35.1% and 59.5% respectively, while the high expr ession rate of PCNA were 57.9% and 78.6% respectively. Compared with that in ben ign nevi, the expression of cyclin D3 and PCNA was significantly increased. The expression of cyclin D3 and PCNA in CMM was positively related to Clark′s grade and the metastasis to lymph nodes. The 3-year survival rate in patients with ne gative expression of cyclin D3 was significantly higher than that with positive expression in superficial melanomas. The 3-year survival rate of patients with l ow expression of PCNA was significantly higher than that with high expression in CMM. The expression of cyclin D3 was positively correlated with the high expres sion of PCNA in superficial and metastatic melanomas. Conclusions The expressi on of cyclin D3 and PCNA may be involved in the carcinogenesis process and progr ession of CMM and have important implications in the selection of therapeutic re gimens and prognostic assessment. The expression level of cyclin D3 may be regar ded as a prognostic factor for superficial melanoma.

AIM:To develop a method for determining cholic acid by HPLC-ELSD and GC was applied to determing muscone;in Jawei Xihuang Soft Capsule(Calculus Bovis,Moschus,Venenum Bufonis,Olibanum,Myrrha).METHODS:AC_(18) column(Kromasil C_(18),5 μm,4.6 mm×250 mm)was used as stationary phase,the mobile phase was methanol-0.01% glacial acetic acid(73:21)at a flow rate of 1.0 mL/min.The parameters of ELSD were set as follows:evaporation temperature was 40℃,carrier gas(N_2)pressure was 200 kPa.The GC system consisted of DB-1 capillary column(30 m×0.32 mm×0.25 μm)and FID as the detector.The programmed temperature-GC and internal standard method were employed to determine the content of muscone.RESULTS:The linear ranges of cholic acid and muscone were in the range of 45.2 ng-904 ng and 0.05 mg/mL-0.5 mg/mL respectively.The average recoveries were 99.06% and 99.40% with RSD of 1.56% and 0.95% respectively.CONCLUSION:The method is convenient and accurate,and it can be used for the quality evaluation of Jawei Xihuang Soft Capsule.

AIM:To develop a method for determining cholic acid by HPLC-ELSD and GC was applied to determing muscone;in Jawei Xihuang Soft Capsule(Calculus Bovis,Moschus,Venenum Bufonis,Olibanum,Myrrha).METHODS:A C18 column(Kromasil C18,5 ?m,4.6 mm?250 mm)was used as stationary phase,the mobile phase was methanol-0.01% glacial acetic acid(73:27) at a flow rate of 1.0 mL/min.The parameters of ELSD were set as follows:evaporation temperature was 40 ℃,carrier gas(N2) pressure was 200 kPa.The GC system consisted of DB-1 capillary column(30 m?0.32 mm?0.25 ?m) and FID as the detector.The programmed temperature-GC and internal standard method were employed to determine the content of muscone.RESULTS:The linear ranges of cholic acid and muscone were in the range of 45.2 ng-904 ng and 0.05 mg/mL—0.5 mg/mL respectively.The average recoveries were 99.06% and 99.40% with RSD of 1.56% and 0.95% respectively.CONCLUSION:The method is convenient and accurate,and it can be used for the quality evaluation of Jawei Xihuang Soft Capsule.

Objective To explore the basic medical mechanism of XinBao pill on electrophysiological characteristics of hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4), and illustrate the mechanism of its therapeutical effect on bradycardia.Methods Human HCN4 mRNA was injected into the Xenopus laevis oocytes, after incubated for 2 ~3 days, channel current properties of HCN4 perfused with 40 mg/L XinBao Pill were observed by double electrode voltage clamp technique.Results At-90mV test potential, compared with control group (no XinBao pill), HCN4 channel peak current and tail current in 40 mg/L XinBao pill group had obvious changes, and V1/2 from ( -103.61 ±3.57)mV to ( -106.42 ±5.33)mV in XinBao pill group, from( -81.11 ±4.26)mV to( -86.36 ±7.44)mV in control group.The values of k from (15.15 ±2.23)mV to (17.33 ±3.58) mV in XinBao pill group, from(11.78 ±0.85)mV to(12.39 ±1.51)mV in control group(n=10).At test potential -90 mV, 40 mg/L XinBao pill perfusion fluid decreased the instantaneous current of(0.15 ±0.24)%, the EC50 was (30.8 ±4.8)mg/L (n=8).At test potential-140 mV~-100 mV level, 40 mg/L XinBao pill group increased the channel activation time constant compared with control group[(226.73 ±31.36)ms vs(143.67 ± 21.44)ms;-140 mV,n=10,P<0.05].40 mg/L XinBao pill group increased the channel deactivation time constant compared with control group [(1293.53 ±95.02)ms vs (647.12 ±61.35)ms;-140 mV,n=10,P<0.05].Conclusion The XinBao pill enhances the instantaneous current of HCN4 in a concentration-dependent manner, and extents channel activation and deactivation processes.

Objective To investigate the expression of fragile histid ine triad (FHIT)gene and its relationship with the proliferation and apoptosis of tumor cells in cutaneous malignant melanoma (CMM).Methods The expression o f FHIT gene and PCNA were detected by streptavidin peroxidase method with skin s pecimens taken from 57 primary cutaneous melanoma and 20 normal controls.Apopto sis of tumor cells was detected by terminal deoxynuclneotidyl transferase mediat ed dUTP nick end labeling (TUNEL).Results The expression level of FHIT protei n was significantly lower in CMM than that in the normal skin tissue (P

OBJECTIVE:To analyze the differences of drug instructions between hospital directory and OTC standard model in-structions,and to provide reference for enhancing instruction management and reducing the safety risk of clinical drug use. METH-ODS:1 324 drugs of hospital directory in a hospital in 2014 were compared with OTC directory from CFDA websites. The instruc-tion of drug types included in OTC directory were compared OTC model instruction. According to the degree of risks which the dif-ferences may bring,differences were divided into four levels for analysis as negligible,general,important and severe. RESULTS:244 drugs belonged to OTC,of which 32.38%were different from standard model instructions. The four risk levels rates of negligi-ble,general,important and severe accounted for 29.11%,34.18%,7.59% and 29.11%,respectively. Among important risk,the difference of“indication limit”occupied the highest proportion,being 50.00%. Among severe risk,the difference of“forbidden for special disease”and“forbidden for pregnant women”accounted for 43.48% and 39.13%. CONCLUSIONS:There are problems, such as the absence of important medication information,statement conflicts. The hospital and administration departments should en-hance the standard management of drug instruction to guarantee safe and rational drug use in the clinic.

Objective To determine the effect of different types of Helicobacter pylori(H, pylori) on the gap junction intercellular communication (GJIC) in GES-1 cells, and investigate the types of H. pylorirelated to the dysfunction of GJIC. Methods Different types of H. pylori clinical strains were isolated and cultured, including the East Asian CagA-positive H. pylori( East Asian CagA +H. pylorl), Western CagA-positive H. pylori( Western CagA +H. pylori), and the CagA-negative H. pylori (CagA-H. pylori). We co-cultured these H. pyloristrains with GES-1 cells for 24 and 48h, respectively. The control group was cultured without any H. pylorifor 24 and 48 h. Change of the GJIC function in GES-1 cells was detected by the scrape-loading dye transfer (SLDT) technique. The cell proliferation of each group was examined by the methyl thiazolyl tetrazolium bromide (MTT) assay. Results The control group showed better GJIC function in the GES-1 cells, and the fluorescent dye migrated 4 - 5 rows to the adjacent cells at 24 and 48 h. Compared with the control group, the GJIC function of GES-1 cells in the CagA - H. pylori group decreased and the fluorescent dye migrated 3 rows to the adjacent cells. Compared with the control group and the CagA- H. pylori group, the GJIC function of GES-1 cells in the Western CagA + H. pylori group decreased and the fluorescent dye migrated 1 - 2 rows to the adjacent cells. The East Asian CagA * H. pylori group showed no GJIC function or weak GJIC function, and most of the fluorescent dye was confined to the area of scratched single row cells and only a few migrated 1 -2 rows to the adjacent cells. Difference in the cell proliferation between the CagA - H. pylorigroup and the control group was not significant. The cell proliferation of the Western CagA + H. pylori group and the East Asian CagA + H. pylori group at bacterium-to-cell ratio of 100:1 and 200:1 was higher than that of the control group. The cell proliferation of the East Asian CagA +H. pylori group at bacterium-to-cell ratio of 400:1 was significantly lower than that of the control group at 48 h. Conclusion H. pylorican inhibit the GJIC function in GES-1 cells, which may be associated with CagA +H. pylori, especially with East Asian CagA +H. pylori. The effect of H. pylori on the proliferation of GES-I cells is related to virulence factor CagA.

Objective:To establish the quality standard for Tongduhuoxue Tablets.Methods: Radix Salvia miltiorrhiza and Rhizoma Corydalis in this prescription were identified by TLC. Tanshinone Ⅱ A was determined by HPLC and astragaloside was determined by dual wavelength TLC Scanning.Results:Radix Salvia miltiorrhiza and Rhizoma Corydalis could be detected by TLC. Determination method of tanshinone Ⅱ A and astragaloside was linear at the range of 0.04 ～ 0.8 ?g( r= 0.99998 ,n=7 ) and 0.3 ～ 6.0 ?g( r= 0.9991 ,n=7 ),average recovery was 98.6% ( RSD= 1.1% ,n=5 ) and 97.7% ( RSD= 1.8% ,n=5 ),respectively. Conclusions: The established method is accurate and reproducible. This study provides a method for the quality control of Tongduhuoxue Tablets.

Objective To construct the eukaryotic expression vector harboring the fragment of Alia gene, and to investigate the effects of it on the signal of quorum sensing and virulence factors producted by Pseudomonas aeruginosa(Pa). Methods The plasmid pET-AiiA was cutted by Nhe Ⅰ and Xho Ⅰ , then the AiiA fragment was cloned into eukaryotic expression vector pEGFP-N2. After the plasmid was transfected into A549 cells, the protein was extracted and AiiA protein was found in it by Western blot. After the extrac- tion was admixed into the LB broth, from culture supernatant extracts of Pa, the N-acylhomoserine lactone (AHL) was detected by bioassay, and the expression of pyocyanin and elastase were assayed by RT-PCR and optical density. Results The fragment of AiiA gene was cutted and then cloned into pEGFP-N2. AiiA protein was found in the transfected cells. After admixed with the extract harboring AiiA protein, in Pa medium, the AHL was hydrolyzed, and the expression of pyocyanin and elastase were reduced. Conclusion The virulence factors synthesized by Pa were reduced by the AiiA protein expressed in eukaryotic cell.

OBJECTIVETo study the inhibition of berberine (BBR) against ECV-304 apoptosis induced by Staphylococcus aureus (S. aureus).

METHODSECV-304 cells were pre-treated with 128 microg/mL BBR for 2 h and then S. aureus was added (1:100). The viability of cells was detected by MTT (3-4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The morphological changes were observed by Hoechst 33258 staining. The protection of BBR for infected cells was detected by DNA Ladder.

RESULTSECV-304 cells' viability were not obviously affected by berberine. But S. aureus induced ECV-304 cells' viability could be significantly inhibited by pre-treatment of BBR (P < 0.05). Besides S. aureus-induced ECV-304 apoptosis could be reduced, with significantly lessened apoptotic body and unobvious DNA degradation.

CONCLUSIONBBR could significantly inhibit S. aureus induced ECV-304 apoptosis.

Apoptosis ; drug effects ; Berberine ; pharmacology ; Cell Line ; Human Umbilical Vein Endothelial Cells ; drug effects ; microbiology ; pathology ; Humans ; Staphylococcus aureus