Objective To investigate the expression and clinicopathological significance of matrix metallo-proteinase(MMP-9) and matrix metalloproteinase tissue inhibiting factor-1 (TIMP-1) in colorectal carcinomas.Methods Immunohistochemistry (PowerVision method) in the paraffin embedded tissue samples was used to deter-mine MMP-9 and TIMP-1 in 42 colorectal carcinomas and its adjacent normal colorectal mucosa.The relationship of their expression with some clinicopathological characteristics was analyzed.Results MMP-9 and TIMP-1 were both significantly overexpressed in colorectal carcinomas compared with its adjacent normal colorectal mucosa(69.1% and 61.9% vs.45.2% and 40.5% ,P =0.009 and P =0.004) ,and MMP-9 was positively associated with the re-gional lymph node metastasis,bowel wall invasion and Dukes stage(rs=0.372,P =0.015;rs =0.372,P =0.015;rs = 0.429,P = 0.005).TIMP-1 was positively associated with Dukes stage, bowel wall invasion and regional lymph node metastasis (rs = 0.394, P = 0.010;rs= 0.382,P = 0.013rs = 0.382, P = 0.013 ).There was a positive corre-lafion between the expression of MMP-9 and TIMP-1 (rs=0.641, P <0.001).Conclusion MMP-9 and TIMP-1 may play a key role in colorectal carcinogenesis.Examination of MMP-9 and TIMP-1 expression may have an impor-tant significance for evaluating prognosis, predicting of invasion and metastasis and comprehensive therapy.

Objective To investigate the expression of MMP-2 and MMP-7 and their elinieopathologieal significance in eoloreetal carcinomas. Methods The expression of MMP-2 mRNA, MMP-7 mRNA and MMP-2, MMP-7 in 42 samples of eoloreetal carcinomas and its adjacent normal eoloreetal mueosa were examined using fluo-rescence quantitative RT-PCR (FQ-RT-PCR) and immunohistoehemistry. The relationship of their expression with some elinieopathologieal characteristics was analyzed. Results MMP-2 mRNA, MMP-7 mRNA and MMP-2, MMP-7 were significantly over-expressed in eoloreetal carcinomas compared with its adjacent normal eoloreetal mueosa( P < 0.05 ) , and they were positively associated with bowel wall invasion, the regional lymph node metastasis and Dukes stage. The expression of MMP-2 mRNA and MMP-7 mRNA was positively correlated with MMP-2 and MMP-7 in eoloreetal eareinornas. Conclusions MMP-2 and MMP-7 may play a key role in eoloreetal carcinogenesis,tumor in-vasion and metastasis. Examination of combined MMP-2 and MMP-7 expression may have an important significance to judge the malignant degree and biological behavior of human eoloreetal carcinoma and for evaluating the progno-sis.

Objective To research the antiviral activity of artesunate (ART) in vitro fighting against both standard laboratory strains and ganciclovir(GCV)-resistance strains of human cytomegalovims(HCMV) and to explore whether fractionation dosage method can obviously enhance the antiviral effect of ART.Methods 1.Cytotoxicity assay to ART was performed by the use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetry.The 0％ toxic concentration (TC0) were determined,and median cytotoxic concentration (TC50) was calculated with Probit regression method.2.Antiviral activity assays of ART against HCMV:human embryonic lung fibroblast cells (HELs) were infected with standard laboratory strains and GCV-resistance strains of HCMV,respectively,after which virus was removed and overlays of dulbecco's modified eagle medium(MEM) containing different antiviral drugs were added to the wells.All cells were cultured continuously at 37 ℃ in a 50 mL/L CO2 humidified atmosphere for 7-10 days and the cytopathic effect (CPE) was observed under a microscope.When the degree of CPE was clear (+ + +-+ + + +),the values of absorbency at 490 nm of all cell wells were measured by MTT colorimetry.The cell survival rate (CSR)and drug inhibitory rate (IR) for HCMV were calculated.By Probit regression method,the median inhibitory concentration (IC50) of 2 drugs was calculated respectively.3.To explore whether fractionation dosage method could obviously enhance the antiviral effect of ART against HCMV,the experiment was divided into 3 groups and compared with GCV group,respectively:Group 1:ART antiviral compounds were added to cell layers by one dosage.Group 2:Total drug dosage was divided into 3 parts,and each part was added to cell layers once a day for 3 days.Group 3:Total antiviral compounds were divided into 6 and delivery 2 times a day.The values of absorbency at 490 nm of all cell wells were measured by MTT colorimetry.The CSR and viral inhibitory rates were calculated.All data were statistically analyzed by One-Way ANOVA analyzing using SPSS 18.0 statistical software.P value of ＜0.05 was considered to indicate statistical significance.Results 1.Cytotoxicity assay showed that cytotoxicity was not found in the relevant range of ART concentrations under 62.5 μmol/L.TC0 and TC50 value of ART were 62.5 μmol/L and 171.7 μmol/L.2.In concentration of 5 μmol/L,15 μmol/L and 30 μmol/L,ART and GCV could obviously inhibit growth of HCMV AD169 strains.There was no significant difference between them.The value of GCV IC50 was 3.49μmol/L,and the value of ART IC50 was 2.17 μmol/L.Treatment index (TI) of ART was 28.8,and GCV was 716.3.ART could still obviously inhibit growth of HCMV resistant strains,but GCV couldn't.Differences between them were statistically significant.The value of GCV IC50 to HCMV resistant strains was 44.4 μmol/L,and the value of ART IC50 was 2.5 μmol/L.3.Fractionation dosage method (2 times a day) of ART could improve the inhibition rate of virus significantly compared to that used once a day and single dose method.Difference was statistically significant(P ＜ 0.01).GCV delivered as the same method had little different changes in virus suppression ratio(P ＞ 0.05).Conclusions 1.Cytotoxicity was not found in the relevant range of ART concentrations under 62.5 μmol/L.2.ART could obviously inhibit growth of HCMV resistant strains and standard laboratory strains.3.Fractionation dosage method (2 times a day) of ART could improve the inhibition rate of virus significantly compared to that used once a day and single dose method.4.Because the action mode of ART is different from other anti-HCMV drugs,and ART has a high biological activity and fewer side effects,it is expected to become a kind of new antiviral drugs for HCMV infections.

Objective To assess the relationship between BRAF gene mutations and clinical phenotype of malignant melanoma.Methods Tissue specimens were collected from the lesions of 80 patients with malignant melanoma,and from the normal skin of 30 patients with trauma in the Department of Plastic Surgery or General Surgery,and subjected to paraffin embedding and DNA extraction.PCR was performed to amplify the exon 11 and 15 of BRAF gene followed by DNA sequencing.Chi-square test and Fisher's exact test were carried out to assess the relationship between BRAF gene mutations and clinical phenotypes of malignant melanoma.Results BRAF gene mutations were found in 19 (23.8％) of the 80 malignant melanoma specimens.Among the 19 mutationpositive specimens,17 (88.2％) carried mutations in exon 15 of BRAF gene with V600E as the most frequent (88.2％,15/17) mutation type,and 2 (10.5％) carried mutations in exon 11.No mutation was found in any of the normal skin tissue specimens.The average age at onset was 57.5 years in these patients.The frequency of BRAF gene mutation was significantly higher in patients younger than 60 years than in those older than 60 years (37.1％ vs.13.3％,x2=6.613,P ＜ 0.05).A significant difference was observed in the frequency of BRAF gene mutation among tissue specimens of mueosal,acral and non-aeral malignant melanoma (18.2％ (4/21) vs.14.7％(5/34) vs.41.7％ (10/24),x2=6.167,P ＜ 0.05).There was no significant association between BRAF gene mutation and gender,race or lymph node metastasis (all P ＞ 0.05).Conclusions BRAF gene is a hot spot for mutations in patients with malignant melanoma in Xinjiang Uygur Autonomous Region,with V600E point mutation in exon 15 as the most frequent mutation type.BRAF gene mutations appear to be closely correlated with the age at onset of and lesional sites in,but uncorrelated with gender and race of or lymph node metastasis in,patients with malignant melanoma.

Objective To monitor human cytomegalovirus (HCMV) drug resistance in recipients of hematopoietic stem cell transplantation by phenotypic and genotypic methods. Methods HCMV clinical isolates was isolated from the urine of hematopoietic stem cell transplantation recipients treated with GCV. Tissue cell infection median dose (TCID50) of the isolates was calculated using Reed-Muench method, and their drug susceptibility was determined by plaque reduction assay. We amplified the UL97 DNA fragment of the virus by nested PCR followed by automated DNA sequencing. Results HCMV clinical strain isolated from the urine samples of the recipients using a human fibroblast cell line showed a TCID50 value of 10-4.618/0.1ml and a 50%inhibitory concentration (IC50) to GCV of 5.847 μmol/L, suggesting its sensitivity to GCV. Alignment with the AD169 DNA reference sequence identified 4 point mutations of the virus at 1509 (T-C), 1575 (C-T), 1794 (T-C), and 1815 (C-G), and only the last mutation resulted in one amino acid mutation to D605E. No gene mutation was found in relation to GCV resistance. Conclusions Phenotypic and genotypic assays were established to examine antiviral drug resistance of HCMV in recipients of hematopoietic stem cell transplantation. We did not find any drug resistance of the clinical HCMV isolate.

Objective To monitor human cytomegalovirus (HCMV) drug resistance in recipients of hematopoietic stem cell transplantation by phenotypic and genotypic methods. Methods HCMV clinical isolates was isolated from the urine of hematopoietic stem cell transplantation recipients treated with GCV. Tissue cell infection median dose (TCID50) of the isolates was calculated using Reed-Muench method, and their drug susceptibility was determined by plaque reduction assay. We amplified the UL97 DNA fragment of the virus by nested PCR followed by automated DNA sequencing. Results HCMV clinical strain isolated from the urine samples of the recipients using a human fibroblast cell line showed a TCID50 value of 10-4.618/0.1ml and a 50%inhibitory concentration (IC50) to GCV of 5.847 μmol/L, suggesting its sensitivity to GCV. Alignment with the AD169 DNA reference sequence identified 4 point mutations of the virus at 1509 (T-C), 1575 (C-T), 1794 (T-C), and 1815 (C-G), and only the last mutation resulted in one amino acid mutation to D605E. No gene mutation was found in relation to GCV resistance. Conclusions Phenotypic and genotypic assays were established to examine antiviral drug resistance of HCMV in recipients of hematopoietic stem cell transplantation. We did not find any drug resistance of the clinical HCMV isolate.

Objective To investigate the impact of micro ribonucleic acid -214 (miR-214) on the proliferation of hepatocellular carcinoma (HCC) and its mechanism. Methods MHCC97L cells were respectively transfected using miR-214 mimics and negative control mimics to establish M214 group, negative control (NC214) group. And untransfected control (Ctrl) group was established. The contents of miR-214 of MHCC97L cells in three groups were detected by fluorescence quantitative polymerase chain reaction (PCR). Cell counting kit (CCK)-8 assay was used to define the cell proliferation inhibition rate. Plate cloning formation assay was used to define the cell clonality. Cell apoptosis rate was detected by flow cytometery. The expressions of protein c-myc, Bax and B-cell lymphoma (Bcl)-2 were detected by Western blot assay. The comparison of three groups was conducted using one way analysis of variance and pairwise comparison using LSD-t test. Results The miR-214 content of MHCC97L cells in M214 group was (2 536±7) times of that in Ctrl group, where significant difference was observed (LSD-t=58.75, P<0.05). The cell proliferation inhibition rates were (15.33±0.62) %, (24.07±0.75) %, (41.02±0.91) % at the 24, 48, 72 h in M214 group, and significant difference was observed compared with that in Ctrl group (LSD-t =14.64, 19.87, 31.86; P<0.05). The clone formation quantity was (5.3±0.7) /well in M214 group, which was significantly lower compared with that in Ctrl group [(37.1±1.0)/well] (LSD-t =-12.51, P<0.05). The cell apoptosis rate was (42.1±3.2)% in M214 group, which was significantly higher compared with that in Ctrl group [(7.0±0.7) % ] (LSD-t=35.66, P<0.05). In M214 group, the expressions of protein c-myc, Bcl-2 decreased and Bax increased. Conclusion The miR-214 can inhibit the proliferation of human HCC cells through down-regulating the protein c-myc expression and Bcl-2/Bax ratio.

Objective To investigate the impact of purinergic receptors P2Y12 gene inhibited by small interference RNA (siRNA) on the angiogenesis of hepatocellular carcinoma (HCC). Methods Human HCC cell line MHCC97H was infected using P2Y12-siRNA lentiviral expression vector and empty negative control vector to establish siRNA group and control group. The expression of P2Y12 protein in two groups was detected by Western blot assay. angiogenesis assay was used to deifne the angiogenesis potential of HCC cell. The microvessel density (MVD) of nude mice subcutaneous tumors was observed. The experimental data between two groups were compared using t test. Results The average relative expression of P2Y12 protein were 0.07±0.01 and 0.26±0.02 respectively in siRNA group and control group. The expression of P2Y12 protein in siRNA group decreased significantly compared with that in control group (t=-35.64, P<0.05). No continuous closed small tubular structure was observed in the human umbilical vein endothelial cell (HUVEC) stimulated by the supernatant of siRNA group, while large number of closed tubules was observed in the HUVEC stimulated by the supernatant of control group. A few microvessels of nude mice subcutaneous tumors were observed in siRNA group. The MVD was 5±1, which was signiifcantly lower than that in control group (23±6) (t=-17.01, P<0.05). Conclusion Purinergic receptors P2Y12 gene inhibited by siRNA can suppress the angiogenesis of HCC.

OBJECTIVETo monitor human cytomegalovirus (HCMV) drug resistance in recipients of hematopoietic stem cell transplantation by phenotypic and genotypic methods.

METHODSHCMV clinical isolates was isolated from the urine of hematopoietic stem cell transplantation recipients treated with GCV. Tissue cell infection median dose (TCID50) of the isolates was calculated using Reed-Muench method, and their drug susceptibility was determined by plaque reduction assay. We amplified the UL97 DNA fragment of the virus by nested PCR followed by automated DNA sequencing.

RESULTSHCMV clinical strain isolated from the urine samples of the recipients using a human fibroblast cell line showed a TCID50 value of 10(-4.618)/0.1 ml and a 50% inhibitory concentration (IC50) to GCV of 5.847 µmol/L, suggesting its sensitivity to GCV. Alignment with the AD169 DNA reference sequence identified 4 point mutations of the virus at 1509 (T-C), 1575 (C-T), 1794 (T-C), and 1815 (C-G), and only the last mutation resulted in one amino acid mutation to D605E. No gene mutation was found in relation to GCV resistance.

CONCLUSIONSPhenotypic and genotypic assays were established to examine antiviral drug resistance of HCMV in recipients of hematopoietic stem cell transplantation. We did not find any drug resistance of the clinical HCMV isolate.

Antiviral Agents ; pharmacology ; Cell Line ; Cytomegalovirus ; drug effects ; genetics ; isolation & purification ; Drug Resistance, Viral ; genetics ; Ganciclovir ; pharmacology ; Genes, Viral ; Genotype ; Hematopoietic Stem Cell Transplantation ; Humans ; Mutation ; Phosphotransferases (Alcohol Group Acceptor) ; genetics

Objective Permanent epicardial pacemaker is seldom used clinically and it is even less likely to be used for the treatment of seriously ill pacing-dependent patients with cardiac electronic device related endocarditis.Method We retrospectively analyzed the feasibility and efficacy of permanent epicardial pacing for the treatment of 3 pacing-dependent patients with cardiac electronic device related endocarditis,who were treated by removal of all pacemaker devices and reimplantation of permanent epicardial pacing system combined with antibiotics.The reason of using epicardial pacing system was as follows:uncontrolled sepsis (case 1) ; big vegetation on the electrode of pacemaker and tricuspid valve but not a candidate for open heart surgery because of high operative risk (case 2) ; occlusion of superior vena cava (case 3).Results All 3 patients were cured with the treatment of extraction of infected pacing system,re-implanted permanent epicardial pacing system and antibiotics.The permanent epicardial pacemaker worked well during the 2-12 months follow-up period and there was no recurrence of infection.Conclusions Permanent epicardial pacing is useful and efficient in treatment of seriously ill and high risk pacing-dependent patients with cardiac device related endocarditis.