Objective To evaluate the effects of metformin on transforming growth factor-betal (TGF-β 1)-induced epithelial-mesenchymal transition (EMT) in and invasion of the human melanoma cell line 1205Lu.Methods In vitro cultured 1205Lu cells were divided into 3 groups to be treated with serumfree culture medium (blank control group),serum-free culture medium containing 5 ng/ml TGF-β 1 (TGF-β 1 group) and serum-free culture medium containing 5 ng/ml TGF-β 1 and 1 mmol/L metformin (TGF-β1 + metformin group),respectively.After 48-hour treatment,morphological changes of 1205Lu cells in the above groups were observed by using a microscope,and photos were taken at the same time.Transwell invasion assay was performed to estimate cellular invasive activity,Western blot analysis and real-time fluorescence-based quantitative RT-PCR were conducted to determine the mRNA and protein expression of N-cadherin and matrix metalloproteinase-9 (MMP-9),respectively.Statistical analysis was carried out by one-way analysis of variance (ANOVA) and least significant difference (LSD) test.Results Compared with the blank control group,the stimulation of TGF-β 1 could induce the epithelial-mesenchymal transition (EMT)-like morphological changes in 1205Lu cells,while TGF-β 1 combined with metformin could reverse the EMT-like morpho-logical changes.The number of 1205Lu cells crossing the transwell Matrigel per high-power field (× 200) was significantly higher in the TGF-β1 group (412.2 ± 13.427) than in the blank control group (194.1 ± 8.295) and TGF-β1 + metformin group (175.3 ± 8.693).Compared with the blank control group and TGF-β1 + metformin group,the TGF-β1 group also showed significantly increased mRNA and protein expression of N-cadherin (mRNA:6.678 ± 0.043 vs.1.000 ± 0.000,1.035 ± 0.015;protein:1.963 ± 0.016 vs.0.603 ± 0.029,0.207 ± 0.009) and MMP-9 (mRNA:5.351 ± 0.604 vs.1.000 ± 0.000,0.930 ±0.130;protein:1.243 ± 0.027 vs.0.575 ± 0.009,0.629 ± 0.008).Conclusion Metformin can obviously inhibit TGF-β1-induced EMT-like morphological changes in,the capacity to penetrate Matrigel-coated transwell chambers of and the mRNA and protein expression of N-cadherin and MMP-9 in 1205Lu cells.

Objective To evaluate the impact of small interference RNA (siRNA)-induced silencing of adrenomedullin (ADM) gene on the growth of and apoptosis in a malignant melanoma cell line A375.Methods Three siRNAs targeting ADM gene were constructed and transfected into A375 cells.Real-time quantitative reverse transcription PCR (qRT-PCR) was performed to measure the expression of ADM to select the most efficient siRNA for the next experiment.A375 cells were classified into 3 groups to be transfected with the selected siRNA (test group),non-specific sequences (negative control group),or remain untransfected (blank control group).Then,an immunohistochemical SP method was used to determine the expression of ADM at 24hours,methyl thiazolyl tetrazolium (MTT) assay to detect the proliferation of A375 cells at 24 and 48 hours,flow cytometry to observe cell apoptosis at 48 hours.Results qRT-PCR showed that ADM-siRNA-2 was the most efficient.The expression of ADM was significantly lower in the test group than in the other two groups.The proliferation level (represented as the absorbance value at 490 nm) of A375 cells was 0.389 ± 0.0375 and 0.469 ± 0.0330 in the test group at 24 and 48 hours respectively,significantly lower than that in the negative control group (0.548 ± 0.0376,0.676 ± 0.0374,both P＜ 0.05) and blank control group (0.574 ± 0.0733,0.685 ± 0.0154,both P ＜ 0.05).Flow cytometry revealed a statistical increase in early apoptosis rate in the test group compared with the blank control group and negative control group ((20.200 ± 6.046)％ vs.(1.667 ± 0.340)％ and (4.587 ± 1.294)％,both P ＜ 0.05).No significant differences were observed in the proliferation level or early apoptosis rate between the negative control group and blank control group (both P ＜ 0.05).Conclusion The siRNA-induced silencing of ADM gene can inhibit the proliferation of,but promote the apoptosis in,A375 cells.

Objective To evaluate the value of modified calcofluor white fluorescent staining in the histopathological diagnosis of subcutaneous mycosis,in order to provide a new method for histopathological diagnosis of subcutaneous mycosis.Methods A total of 21 lesional skin tissues were collected from patients with subcutaneous mycosis in the Affiliated Hospital of Chengde Medical University between 1987 and 2017,and embedded in paraffin.Then,each paraffin-embedded tissue section was cut into 4 4-μm-thick serial sections,and subjected to modified calcofluor white fluorescent staining,hematoxylin and eosin (HE) staining,periodic acid Schiff (PAS) staining and Gomori methenamine silver nitrate (GMS) staining respectively.Positive rates and staining outcomes were compared among the above staining methods.Statistical analysis was carried out with SPSS 19.0 software by using chi-square test for comparing the positive rates among the above 4 staining methods.Results Of 21 patients with fungal infections,14 (66.67％) were positive for modified calcofluor white fluorescent staining,5 (23.80％) for HE staining,6 (28.57％) for PAS staining,and 11 (52.38％) for GMS staining.The positive rate by modified calcofluor white fluorescent staining was significantly higher than that by HE staining and PAS staining (x2 =6.718,5.200,respectively,both P ＜ 0.05),while no significant difference was observed between the modified calcofluor white fluorescent staining and GMS staining (x2 =0.693,P =0.530).Conclusion The modified calcofluor white fluorescent staining is an accurate method for detecting fungi,and has a certain application value in the histopathological diagnosis of subcutaneous mycosis.

To establish cisplatin (CDDP)-resistant melanoma B16F10 (CDDP-R B16F10) cell line with stable expression of T-cadherin, and to study its biological characteristics.
 Methods: CDDP-R B16F10 cell line was exposure to high and gradually increased dose of CDDP. 3-(4.5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was used to test the proliferation of CDDP-R B16F10 cell line, and the sensitivity of CDDP-R B16F10 cell line to CDDP and paclitaxel was examined. The pEGFP-N1-T-cadherin, a plasmid vector encoding human T-cadherin, was generated by inserting T-cadherin cDNA into a pEGFP-N1 vector. The pEGFP-N1-T-cadherin was transfected into CDDP-R B16F10 cell line. The expression of T-cadherin mRNA and protein were measured by reverse transcription polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry SP method, respectively. The effect of T-cadherin combined with CDDP on proliferation of CDDP-R B16F10 cell line was determined by MTT assay. The sensitivity of CDDP-R B16F10 cell line with stably transfected T-cadherin to paclitaxel was examined by MTT assay.
 Results: The CDDP-R B16F10 cell line was established successfully. There was no difference in proliferation between the CDDP-R B16F10 cell line and B16F10 cell line (P>0.05). The IC50 of CDDP-R B16F10 cell line and B16F10 cell line to CDDP were 268.706 and 19.748 mg/L, respectively, and the resistance index was 13.61. The IC50 of CDDP-R B16F10 cell line and B16F10 cells to paclitaxel were 11.415 and 7.799 mg/L, respectively, and the resistance index was 1.46. The expression vector pEGFP-N1-T-cadherin was constructed successfully. RT-PCR, Western blotting and immunohistochemistry SP method showed that T-cadherin could be transcribed and expressed. MTT assay showed that T-cadherin combined with CDDP could inhibit the proliferation of CDDP-R B16F10 cell line (P<0.05). Factorial analysis showed that there was interaction between T-cadherin and CDDP in inhibiting the proliferation of CDDP-R B16F10 cell line (P<0.05). T-cadherin combined with paclitaxel could inhibit the proliferation of CDDP-R B16F10 cell line (P<0.05). Factorial analysis showed that there was no interaction between T-cadherin and paclitaxel in inhibiting the proliferation of CDDP-R B16F10 cell line (P>0.05). 
 Conclusion: The CDDP-R B16F10 cell line with stable expression of T-cadherin is established successfully. T-cadherin can reverse the CDDP resistance to CDDP-R B16F10 cell line, and promote its sensitivity to paclitaxel.
Antineoplastic Agents ; Cadherins ; Cell Line, Tumor ; Cell Proliferation ; Cisplatin ; Drug Resistance, Neoplasm ; Humans ; Melanoma

Objective: To evaluate the impact of psoriasis on spatial learning and memory abilities in mouse models by using Morris water maze. Methods: Twenty healthy male C57BL/6J mice aged 10 months were randomly and equally divided into 2 groups: psoriasis group topically treated with imiquimod 5% cream on the back once a day for a week, and control group topically treated with vaseline once a day for a week. After successful establishment of mouse models, the Morris water maze (MWM) test was used to assess the learning and memory abilities in the mice in the 2 groups. Results: In the place navigation experiment, the escape latency was significantly longer in the psoriasis group (38.24 ± 13.59 s) than in the control group (14.28 ± 3.80 s, t = 5.37, P < 0.01) . In the spatial probe test, the number of times passing through the platform (1.70 ± 0.95 vs. 5.00 ± 1.76, t = 5.21, P < 0.01) , the duration of stay in the target quadrant (t = 2.80, P < 0.05) and the swimming distance (t = 5.74, P < 0.01) were all significantly lower in the psoriasis group than in the control group. The psoriasis group showed significantly decreased swimming distance in the second quadrant (t = 2.49, P < 0.05) , but significantly longer duration of stay in the fourth quadrant compared with the control group (t = 2.46, P < 0.05) . There were no significant differences in swimming distance or duration of stay in other quadrants between the psoriasis group and control group (all P > 0.05) . Conclusion The spatial learning and memory abilities were impaired in the mouse model of psoriasis.