Risk factors analysis of breast cancer-related lymphedema based on the proportion of the arm lymph flow above and below the axillary vein / 中华普通外科杂志

Objective:To develop and validate an clinical prediction model for the risk of breast cancer-related lymphedema (BCRL).Methods:Breast cancer patients who were prepared to undergo axillary lymph node dissection were propsectively enrolled, indocyanine green combined with Photodynamic Eye (PDE) was applied to reveal the arm lymphatic flow . The arm lymphatic fluorescence images were collected to calculate the proportion of arm lymph flow above and below the axilla vein. Volumetric measurements of both arms and subjective questionnaire were performed to evaluate the occurrence of lymphedema. A difference in volume between the arms >10% was defined as lymphedema. Univariate logistic regression analysis was used to analyze the relationship between each factor and BCRL. The stepwise forward method was used to include multiple factors in the logistic regression analysis to establish the prediction model.Results:Three hundred and twelve patients were enrolled. Fourty-five (14.4%) patients developed BCRL. Using the coefficients obtained from multivariate analysis, BMI ( OR 95% CI: 1.34 (1.25-1.77), P<0.05), chemotherapy ( OR 95% CI: 2.26 (1.97-2.63), P<0.05), regional lymph node radiotherapy ( OR 95% CI: 1.59 (1.05-2.41), P<0.05) and the proportion of arm lymph flow above the level of the axillary vein ( OR 95% CI: 0.70 (0.68-0.81), P<0.05) were identified as independent predictive factors for BCRL, and the following prediction equation was derived: Y=0.369×(BMI at surgery)+0.713×(taxane-based chemotherapy)+0.862×(radiotherapy)-9.058×(proportion of the arm lymph above the axillary vein)-6.859 8. The ROC curve was screened to the optimal boundary value of 0.118 6 by the Youden's index. The sensitivity, specificity, positive predictive value and negative predictive value of prediction of this model were 93.3%, 79.4%, 73.3%, 98.6%, respectively. Conclusion:With the guidance of the predictive model, particular patients who need the preservation of axillary lymphatic system can be identified, and timely intervention can be carried out.

A prospective study on transcatheter arterial chemoembolization combined with sorafenib and sequential microwave ablation versus transcatheter arterial chemoembolization combined with sorafenib in the treatment of hepatocellular carcinoma with tumor diameter over 5 cm / 中华消化外科杂志

Objective:To investigate the efficacy and safety of transcatheter arterial chemoembolization (TACE) combined with sorafenib and sequential microwave ablation (MWA) versus TACE combined with sorafenib in the treatment of hepatocellular carcinoma (HCC) with tumor diameter over 5 cm, and to analyze risk factors affecting the prognosis of patients.Methods:The prospective cohort study was conducted. The clinicopathological data of 61 HCC patients with tumor diameter over 5 cm who were admitted to two medical centers (30 in the Laiyang Central Hospital of Yantai City Affiliated to Weifang Medical College and 31 in the Qingdao Central Hospital Affiliated to Qingdao University) between July 2012 and November 2013 were collected. Patients who were treated with TACE combined with sorafenib and sequential MWA were allocated into observation group, and patients who were treated with TACE combined with sorafenib were allocated into control group. Observation indicators: (1) treatment, complications and adverse drug reactions; (2) short-term efficacies; (3) follow-up and survival situations; (4) analysis of prognostic factors. Follow-up was performed by inpatient, outpatient examinations or telephone interview once a month within the first 6 months after treatment and once every 3 months thereafter up to November 2018. The follow-up included laboratory indicators, tumor markers, abdominal enhanced computed tomography or magnetic resonance imaging examinations. The survival of patients and disease progression were fully documented. Measurement data with normal distribution were expressed as Mean± SD, and comparison between groups was performed by the t test. Measurement data with skewed distribution were described as M (range), and comparison between groups was performed using the Wilcoxon rank sum test. Count data were expressed as absolute numbers or percentages, and comparison between groups was performed using the chi-square test or pearson-corrected chi-square test. Ranked data were analyzed using the Wilcoxon rank sum test. The Kaplan-Meier method was used to calculate survival rates and draw survival curves. The Log-rank test was used for survival analysis. Comparison of survival rates between time points was performed using the Bonferroni method to adjust the test level. Univariate and multivariate analyses were performed using the multiple COX proportional hazard model. Results:A total of 61 HCC patients were selected for eligibility, including 36 males and 25 females, aged (58±8)years, with a range from 43 to 73 years. Of the 61 patients, 31 were in the observation group and 30 in the control group. (1) Treatment, complications and adverse drug reactions: ① treatment information. The treatment times of TACE, treatment times of MWA, time from the first TACE to the first sorafenib medication, and duration of sorafenib medication in the observation group were 1 time (range, 1-5 times), 2 times (range, 1-4 times), 5 days (range, 5-9 days), 24 months (range, 6-72 months), respectively. The above indicators of patients in the control group were 3 times (range, 1-5 times), 0, 6 days (range, 5-9 days), and 16 months (range, 6-60 months). There were significant differences in the treatment times of TACE, treatment times of MWA, and duration of sorafenib medication between the two groups ( Z=4.701, -7.213, -2.614, P<0.05). There was no significant difference in the time from the first TACE to the first sorafenib medication between the two groups ( Z=0.573, P>0.05). ② Complications: there was no TACE related complications in the two groups. There were 3 patients with MWA related complications in the observation group, including 2 cases of minor hemorrhage under the liver capsule and 1 case of pleural effusion, and they were relieved after conservative treatment. ③ Adverse reactions to sorafenib: after 2 months of sorafenib medication, patients in the observation group and control group had at least one kind of sorafenib related stage Ⅰ-Ⅲ adverse reaction, without stage Ⅳ adverse reaction. The numbers of cases with hand-foot skin reaction, rash, pruritus, loss of skin pigmentation, diarrhea, decreased appetite, nausea and vomiting, pain in the liver area, fever, fatigue, liver dysfunction, bone marrow suppression were 8, 3, 4, 3, 10, 18, 20, 20, 20, 15, 3, 2 in the observation group, and 9, 3, 3, 2, 13, 19, 23, 12, 21, 12, 6, 2 in the control group, respectively, showing no significant difference in the above indices between the two groups ( χ2=0.133, 0.000, 0.000, 0.000, 0.796, 0.177, 1.082, 3.674, 0.208, 0.435, 0.601, 0.000, P>0.05). Patients with adverse reactions to sorafenib were relieved by symptomatic treatment, reducing the dose of sorafenib or intermittent drug withdrawal. (2) Short-term efficacies: the level of alpha fetoprotein was 16 μg/L (range, 3-538 μg/L) in the observation group and 292 μg/L (range, 9-642 μg/L) in the control group after one month of treatment, showing a significant difference between the two groups ( Z=3.744, P<0.05). After 3 months of treatment, cases with tumor complete remission, cases with tumor partial remission, cases with stable disease, cases with progressive disease, objective response rate, and disease control rate in the observation group were 14, 11, 6, 0, 80.6%(25/31) , 100.0%(31/31), respectively. The above indicators in the control group were 2, 13, 12, 3, 50.0%(15/30), 90.0%(27/30). There was a significant difference in the objective response rate between the two groups ( χ2=6.343, P<0.05), but no significant difference in the disease control rate between the two groups ( χ2= 1.473, P>0.05). (3) Follow-up and survival situations: 61 HCC patients were followed up for 9.0-75.0 months, with a median follow-up time of 22.0 months. During the follow-up, 28 patients in the observation group had progressive disease, including 8 cases of local tumor progression, 4 of portal vein tumor thrombi, 11 of intrahepatic metastasis, and 5 of pulmonary metastasis. Thirty patients in the control group had progressive disease, including 13 cases of local tumor progression, 6 of portal vein tumor thrombi, 6 of intrahepatic metastasis, and 5 of pulmonary metastasis. Among the 61 patients, 28 patients in the observation group and 29 patients in the control group died. The median overall survival time and median progression-free survival time of the observation group was 28.0 months and 18.0 months, versus 19.5 months and 11.5 months of the control group, showing significant differences between the two groups ( χ2=8.021, 10.506, P<0.05). The 1-, 3-, 5-year overall survival rates of the observation group were 97%, 37% and 20%, respectively, versus 83%, 13% and 7% of the control group, showing significant differences in the above indicators between the two groups ( Z=23.635, 4.623, 3.139, P<0.0167). The 1-, 2-, 3-year progression-free survival rates of the observation group were 77%, 40%, and 27%, respectively, versus 43%, 13%, and 7% of the control group, showing significant differences in the above indicators between the two groups ( Z=9.965, 4.900, 3.684, P<0.0167). (4) Analysis of prognostic factors: results of univariate analysis showed that treatment method, maximum tumor diameter, Barcelona clinical liver cancer (BCLC) stage, liver cirrhosis, hepatitis B virus(HBV) infection, and Child-Pugh classification were related factors for overall survival time [ hazard ratio ( HR)=0.483, 6.196, 12.646, 5.049, 2.950, 4.791, 95% confidence interval ( CI): 0.284-0.823, 3.198-12.003, 5.031-31.785, 2.586-9.858, 1.366-6.369, 2.507-9.155, P<0.05] and progression-free survival time ( HR=0.427, 5.804, 7.032, 5.405, 2.925, 4.410, 95% CI: 0.248-0.735, 3.043-11.070, 3.071-16.101, 2.685-10.881, 1.364-6.270, 2.331-8.342, P<0.05). Results of multivariate analysis showed that treatment method, maximum tumor diameter, BCLC stage, liver cirrhosis, and HBV infection were independent influencing factors for overall survival time ( HR=0.183, 5.886, 17.544, 4.702, 3.801, 95% CI: 0.090-0.370, 2.648-13.083, 5.740-53.622, 1.928-11.470, 1.368-10.562, P<0.05) and progression-free survival time ( HR=0.201, 3.850, 3.843, 3.598, 3.726, 95% CI: 0.098-0.411, 1.761-8.414, 1.526-9.682, 1.444-8.963, 1.396-9.947, P<0.05). Conclusions:Compared with TACE combined with sorafenib, TACE combined with sorafenib and sequential MWA is safe and effective in the treatment of HCC with tumor diameter over 5 cm. The treatment method, maximum tumor diameter, BCLC stage, liver cirrhosis, and HBV infection are independent influencing factors for overall survival time and progression-free survival time of patients.

Re-understanding of surgical treatment of intrahepatic bile duct stones / 临床肝胆病杂志

The incidence of intrahepatic bile duct stones as primary cholelithiasis varies in different regions,though it decreases to some ex-tent.Minimally invasive hepatectomy is the correct procedure for treating early intrahepatic bile duct stones.However,there are still many problems concerning the surgical treatment of end-stage liver disease caused by complex intrahepatic bile duct stones.The multidisciplinary mode should be recommended to provide comprehensive treatment,and studies on the prevention,etiology,and pathogenesis of intrahepatic bile duct stones should be strengthened,so as to improve the treatment of intrahepatic bile duct stones.

The effect and mechanism of decitabine on the proliferation of human cholangiocarcinoma CCLP1 cells / 中华肝胆外科杂志

Objective To investigate the effect and mechanism of decitabine (DAC) on the proliferation of human cholangiocarcinoma CCLP1 cells in vitro and in vivo.Methods After treated with various concentrations of DAC,cell growth inhibition rates were determined by CCK-8 assay.Cell cycle and cell apoptosis were analyzed by flow cytometry.Cell autophagy was observed under fluorescence microscope.The effect of DAC on the growth of cholangiocarcinoma in vivo was determined in a CCLP1 mice xenograft model.Results The proliferation rate of CCLP1 cells in the DAC-treated group decreased in a time-concentrated dependent manner.After treatment with DAC,the cell cycle of CCLP1 cells was arrested at the G2/M phase.The apoptosis rate was significantly higher in the treatment group over the control group.Cell autophagy was observed after treatment with DAC in CCLP1 cells.The tumor growth of implanted CCLP1 cells significantly slowed down after the mice were treated with 0.8 mg/kg DAC,6 times weekly for 2 weeks.Conclusion DAC can inhibit the proliferation of cholangiocarcinoma in vitro and in vivo.

Comparison of the effect of epigenetical treatment in 2D and 3D cholangiocarcinoma tumor models / 中华肝胆外科杂志

Objective To investigate the effects of epigenetic drugs on 2D- and 3D-cultured cholangiocarcinoma cells in vitro.Methods In this study,we have built compact and round TFK-1 spheroid in poly-HEMA coated 96-well plate and determined the effects of epigenetical drugs on 2D and 3D cultured cholangiocarcinoma cells:TFK-1.Viability of 2D and 3D cells model was examined by WST assay and FDA/PI staining. Using methylation-specific PCR analysis,we demonstrated the changes of methylation status of promoters regarding three tumor suppressor genes APC,E-Cadherin,and p16 INK4A.Results The average diameters of compact and round TFK-1 spheroids were in the range of 350-400 μm.The TFK-1 spheroid cells were more resistant to the epigenetic drugs and demonstrated a 11.2155-fold higher IC50 values for hydralazine and valproic acid than the same cells grown as monolayer. Higher doses of epigenetic drugs were needed to reverse the hypermethylation status in 3D cultured cells than 2D cells; however,the parallel dosage - dependent characteristic did not show in the 3D spheroid group.Conclusions Taken together,we established a 3D culture model of human cholangiocarcinoma epithelial spheroid.The 3D spheroid cells are more complex than the 2D monolayer cells and their unique characteristics are able to affect the consequences of epigenetic therapy.The 3D spheroid is a promising model for the research of epigenetic therapy.

In vitro and in vivo inhibitory effects and mechanism of 9-nitrocamptothecin liposomes on HepG2 cell / 中华肝胆外科杂志

Objective To observe the inhibitory effect and mechanism of 9-nitrocamptothecin liposomes on HepG2 liver carcinoma cells. Methods HepG2 cells were incubated with 9-nitrocampto-thecin(9NC) or with 9-nitrocamptothecin liposomes(9NC-LP) for 24 h, 48 h and 72 h. Cell viability was then measured by the MTT assay. Cell cycle and apoptosis were evaluated by flow cytometry.Western Blot was used to determine the expression of cell cycle and apoptosis related proteins. HepG2tumor-bearing mouse models were then established. The HepG2 tumor-bearing mice were randomly divided into control group, free liposomes group, DMSO group, 9NC low dose group, 9NC high dose group, 9NC-LP low dose group and 9NC-LP high dose group. There were 10 mice in each group.Drugs were administered by tail vein and tumor volume and body weight were observed 28 days after administration. Then animals were sacrificed and the expression of proteins from tumor homogenates was analyzed by Western blotting. Results In vitro, HepG2 cell viability was apparently inhibited by 9NC and 9NC-LP, and the inhibitory effect increased in a time-dependent and dose-dependent manner.Both S and G2/M phase arrests were observed after incubation with drugs. HepG2 cells were completely arrested in S phase with 9NC concentration over than 0.1 μmol/L after incubation for 24 h,while more than 95% of cells arrested in G2/M phase when 9NC concentration was 0.1 μmol/L after incubation for 72 h. In vivo, compared with the control group, the average tumor volume was reduced in both the 9NC and 9NC-LP group (P50% of animals died 14 days after drug administration. Conclusion 9NC and 9NC-LP can inhibit HepG2 cell growth via cell cycle arrest and apoptosis induction. 9NC-LP has a more potent anti-tumor effect and fewer side effects in vivo,which means 9NC-LP is a promising compound for cancer therapy via intravenous administration.

Modulating effects of 9-nitrocamptothecin and its liposomes to induce apoptosis and inhibit cell cycle in HepG2 and L02 cell lines / 中华肝胆外科杂志

Objective To investigate the modulating effects and explore their mechanism of 9-nitrocamptothecin and its liposomes to induce apoptosis and inhibit cell cycle in HepG2 and L02 cell lines. Methods Cells were incubated with 9-nitrocamptothecin(9NC) or with 9-nitrocamptothecin liposomes for 24 h, 48 h and 72 h, then, the cell viability was measured via MTT assay; cell cycle and apoptosis was evaluated by flow cytometry after stained by PI and Annexin V-PE/7AAD. Additional, Western Blot was used to evaluate the expression of cell cycle and apoptosis related protein. Results Both cells viability were apparently inhibited by the 9-nitrocamptothecin and 9-nitrocamptothecin liposomes, the inhibitory effect showed a time-dependent and dose-dependent manner. Both S and G2/M phases arrest were observed after incubated with drugs. HepG2 cell was completely arrested in S phase when 9NC concentration over than 0. 1 μmol/L after incubation for 24 h, while more than 95% cells arrested in G2/M phase when 9NC concentration is 0. 1 μmol/L after incubation for 72 h. Apoptosis induction effect also showed a time-dependent and dose-dependent manner. Western Blot results showed the expression of Bax and Caspase-3 were upregulated while Cyclin A, Cdk2, Cyclin E and Bcl-2 were downregulated. More importantly, the compounds were more cytotoxic to the cancer cell lines than to the normal liver cell. Conclusions 9-nitrocamptothecin and 9-nitrocamptothecin liposomes can potently inhibit cell growth via regulation of cell cycle and induction of apoptosis, and this effect was preferentially in cancer cell. Inhibitory of 9-nitrocamptothecin liposomes was slightly better than the 9-nitrocamptothecin.

Identification of differentially expressed biliary proteins induced by cholangiocarcinoma using 2D-DIGE / 中华肝胆外科杂志

Objective To determine the probability of identification of differential expression of biliary proteins induced by cholangiocarcinoma using 2D-DIGE. Methods Bile was obtained from 12patients with obstructive jaundice (including 6 cases of cholangiocarcinoma and 6 of cholelithiasis).Each sample was labeled with three different CyDyes (y3,Cy5,Cy2) including one internal standard,pooled from all the samples, and separated with 2-D DIGE in triplicate experiments. MALDI-TOF-MS and bioinformatics were adopted to identify and elucidate the significance of differentially expressed proteins in bile induced by cholangiocarcinoma. Results 55 matched protein spots differences in abundance were detected with statistical variance of two groups(Average Volum Ratio ≥1.5, t-test, P＜0. 05). Among these proteins, 13 PMF were obtained by MALDI-TOF-MS analysis. Eight proteins were identified by searching a protein database. Conclusion The differentially displayed proteomes between the pathological bile obtained from benign and malignant obstructive jaundice indicates the potential application of 2D-DIGE to identify the biomarker of cholangiocarcinoma.

Value of serum SDF-1a half-life in predicting postoperative recurrence and metastasis in patients of breast cancer / 中华普通外科杂志

Objective To investigate the plasma level of SDF-1a in patients with breast cancer after modified radical mastectomy and chemotherapy, and to evaluate the value of serum SDF-1a half-life for predicting breast cancer recurrence and metastasis. The correlation of SDF-1 a half-life and breast cancer recurrence and metastasis after treatment were retrospectively analyzed. Methods Serum chemokine SDF-1a of 112 cases of breast cancer were detected before and after modified radical surgery, and 1 day beforeeach chemotherapy session. SDF-1a levels and the dynamic changes in the process were observed and calculated. Results In 85 cases with no recurrence and metastasis the plasma level of SDF-1 a decreased rapidly to normal level ,while that in 27 cases with recurrence and metastasis was on high level with the halflife of SDF-1a being longer than that in no recurrence group(P ＜0. 01 ). Taking SDF-1a half-life ≥14 d as cut off point to predict breast cancer recurrence and metastasis after treatment, the sensitivity is 81.5%,specificity is 70. 6 %, and accuracy is 73.2%. Conclusions Serum SDF-1a half-life is a valuable marker in predicting postoperative breast cancer recurrence and metastasis.

Expression of Survlvin and p53 in human biiiary tract carcinoma cell line regulated by nanochemotherapy drug / 中国医师进修杂志

Objective To determine the effect of nanochemotherapy drug on Survivin and p53 ex-pressed by human biliary traet carcinoma cell line QBC939.Methods Culturing the human biliary tract carcinoma cell line QBC939 in vitro and it was divided into five groups including saline,nanochemotherapy drug,nanopartiele withoul nanochemotherapy drug,5-FU and gemcitabine.Using the methods of MTT and flow cytometry to observe the growth of QBC939 which was dealt with different drugs.In addition,RT-PCR and Western blot were used to delect the expression of mRNA and protein by Survivin or p53.Results The expression of mRNA and protein by Survivin decreased in the following set:saline,nanoparlicle withoul nanochemotherapy drug,5-FU,gemcitabine and nanochemotherapy drug,respeclively.However,the ex-pression of mRNA and protein by p53 were in reverse order.The inhibiting action to QBC939 was obvious in nanochenmtherapy drng and the apoplotic rate was higher than others except for gemcitabine(P＜0.05). Conclusion Nanochemotherapy drug has significant effect on treatment cholangiocarcinoma in vilro,which may attribute to the down regulation of Survivin and up regulation of p53.

Effect of immune enteral nutrition support on the patients in early stage after severe multiple injury / 中华急诊医学杂志

Objective To explore the recuperative effect of immunological function and nutritional status on the patients treated by immune enteral nutrition in early stage after severe multiple injury (SMI). Method The patients with SMI,in department of Trauma Surgery,Tongji Hospital,Tongji Medical College, Huazhong University of Science and Technology,between January 2006 to May 2007 were randomly divided into 2 groups: immune enteral nutrition group (IEN group, 20 cases), enteral nutrition group (EN group, 20 cases). The health persons served as the control group(15 cases) .Since 1st postinjury day, all patients were treated with nutritional support. The T-cell subgroup in periphera blood were detected by FCM and the level of PA, RBP, IL-2 and IL-4 in blood serum were detected by ELASA on the 1st, 3rd,5th, 8th postinjury day. Results After the treatment of IEN and EN,the serum levels of PA, RBP and the proportion of T-cell subgroup were significantly increased on the 8th postinjury day compared with on 1st postinjury day (P ＜ 0.01), but there were no differences between IEN group and EN group. The level of IL-4 were significantly decreased and the level of IL-2 were significantly increased in each group on 8th postinjury day, at same time, the level of IL-2 were significantly increased in IEN group compared with EN group (P ＜ 0.05), and the level of IL-4 were significantly decreased in IEN group compared with EN group (P ＜ 0.05). The duration of SIRS was transient and the infected complication was low on the patients treatment by IEN than EN. Conclusions On the patients with severe multiple injury, IEN was most ascendant than EN to improve the immunosuppression and clinical prognosis.

Suppressing effects of down-regulating DNMT1 and DNMT3b expression on the growth of human cholangiocarcinoma cell line / 华中科技大学学报（医学）（英德文版）

Hypermethylation in the promoter region is an important epigenetic mechanism for the transcriptional repression of a number of cancer-associated genes, and over-expression and/or increased activity of DNA methyltransferases are considered to be the main cause of promoter hypermethylation. In order to explore the roles of two methyltransferase members (DNMT1 and DNMT3b) in the cholangiocarcinoma tumorigenesis, antisense eukaryotic expression plasmid of DNMT1 and DNMT3b gene was constructed respectively, and were co-transfected into the human cholangiocarcinoma cell line QBC-939 to observe their biological effects on the cell growth and proliferation ability, apoptosis, cell cycle alteration, and the tumorigenesis ability in the subcutaneous tissue of nude mouse. The results demonstrated that co-transfection with antisense eukaryotic expression plasmid of DNMT1 and DNMT3b gene and single transfection with antisense eukaryotic expression plasmid of DNMT1 gene can suppress the growth and proliferation of QBC-939, block the cell cycle at G1 phase, increase the apoptosis rate, minimize the tumor size in the subcutaneous tissue of nude mouse. The suppressing biological effect of co-transfection is stronger than single transfection with antisense DNMT1. Meanwhile, single transfection with antisense eukaryotic expression plasmid of DNMT3b gene has no effects on the biological characteristics of QBC-939. This study suggests that DNMT1 gene plays a key role in DNA methylation and DNMT3b gene may act as an accessory to support its function in inactivation of tumor suppressor genes. Combination DNMT1 and DNMT3b will increase their biological effects and have the synergistic effect on suppressing the growth of human cholangiocarcinoma cell line QBC-939.

The effect of targeted magnetic nanopaticles on hepatoma and the expression of bcl-2/bax protein / 华中科技大学学报（医学）（英德文版）

The effect of targeted magnetic nanoparticles on hepatoma and the underlying mechanism were examined. Nude mice transplanted with a human hepatoma cell line (HepG2 cells) were randomized into 5 groups, including: (1) group A, receiving normal saline, (2) group B, receiving 5-fluorouracil (5-Fu), (3) group C, receiving magnetic nanoparticles containing 5-Fu, (4) group D, consisting of treatment with magnetic nanoparticles containing 5-Fu and inside magnetic field and (5) group E, receiving pure magnetic nanoparticles and inside magnetic field. Morphological features of transplanted tumors in mice in each group were observed under transmission electron microscope (TEM). The expression of bcl-2/bax protein was immunohistochemically detected by SABC method. The results showed that a large number of apoptotic tumor cells were found in group B and group D under TEM. The expression of bcl-2 protein was significantly decreased and the expression of bax protein increased significantly in both group B and D as compared with those in group A, C and E (P<0.01 for all). The decrease in bcl-2 and the increase in bax were more in group D as compared with group B (P<0.01). It is concluded that the targeted magnetic nanoparticles containing 5-Fu can improve the chemotherapeutic effect of 5-Fu by decreasing bcl-2 expression, increasing bax expression and inducing apoptosis of the liver cancer cells.

Re-expression of RASSF1A by 5-Aza-CdR induced demethylation of the promoter region in human biliary tract carcinoma cells / 华中科技大学学报（医学）（英德文版）

Hypermethylation of the promoter region is an important mean for the transcriptional repression of a number of cancer-associated genes, and over-expression and/or increased activity of DNA methyltransferase are considered to be the main cause of promoter hypermethylation. In order to further explore the epigenetic mechanism of tumor suppressor gene RASSF1A inactivation, 5-aza-2'-deoxycytidine (5-Aza-CdR), a DNA methyltransferase inhibitor, was used to treat the human biliary tract carcinoma cell line QBC-939 at the concentration of 5 micromol/L for 24 h in this study. After the chemical intervention with 5-Aza-CdR, the methylation status in the promoter region of RASSF1A gene was detected by methylation specific PCR (MS-PCR), and the expression alteration of RASSF1A mRNA and protein were observed by RT-PCR and Western Blot respectively. Following the treatment with 5-Aza-CdR, methylation status in the promoter region of RASSF1A gene was reversed from methylation to unmethylation. A 280 bp DNA band which represented RASS1FA expression at transcriptional level and a 40 kDa (1 kDa=0.9921 ku) protein band which represented RASSF1A expression at protein level were detected by RT-PCR and Western Blot respectively in the experimental group cells and there were no corresponding bands in the control group cells. The experimental results suggest that 5-Aza-CdR can induce demethylation in the promoter region of RASSF1A. It can also reverse epigenetic transcriptional silencing caused by DNA methylation and induce the re-expression of RASSF1A in QBC-939. This study also suggest that the mechanism of RASSF1A inactivation is very closely related to the methylation of the promoter region, which may provide a new epigenetic understanding for tumor related gene inactivation and the pathogenesis of biliary tract carcinoma.

Re-expression of RASSF1A by 5-Aza-CdR Induced Demethylation of the Promoter Region in Human Biliary Tract Carcinoma Cells / 华中科技大学学报（医学）（英德文版）

Hypermethylation of the promoter region is an important mean for the transcriptional repression of a number of cancer-associated genes, and over-expression and/or increased activity of DNA methyltransferase are considered to be the main cause of promoter hypermethylation. In order to further explore the epigenetic mechanism of tumor suppressor gene RASSF1A inactivation,5-aza-2'-deoxycytidine (5-Aza-CdR), a DNA methyltransferase inhibitor, was used to treat the human biliary tract carcinoma cell line QBC-939 at the concentration of 5 μmol/L for 24 h in this study. After the chemical intervention with 5-Aza-CdR, the methylation status in the promoter region of RASSF1A gene was detected by methylation specific PCR (MS-PCR), and the expression alteration of RASSF1A mRNA and protein were observed by RT-PCR and Western Blot respectively. Following the treatment with 5-Aza-CdR, methylaiton status in the promoter region of RASSF1A gene was reversed from methylation to unmethylation. A 280 bp DNA band which represented RASS1FA expression at transcriptional level and a 40 kDa (1kDa=0.9921 ku) protein band which represented RASSF1A expression at protein level were detected by RT-PCR and Western Blot respectively in the experimental group cells and there were no corresponding bands in the control group cells. The experimental results suggest that 5-Aza-CdR can induce demethylation in the promoter region of RASSF1A. It can also reverse epigenetic transcriptional silencing caused by DNA methylation and induce the re-expression of RASSF1A in QBC-939. This study also suggest that the mechanism of RASSF1A inactivation is very closely related to the methylation of the promoter region, which may provide a new epigenetic understanding for tumor related gene inactivation and the pathogenesis of biliary tract carcinoma.

Effect of antisense MBD1 gene eukaryotic expression plasmid on expression of MBD1 gene in human biliary tract carcinoma cells / 华中科技大学学报（医学）（英德文版）

Hypermethylation of the promoter region is one of the major mechanism of tumor suppressor gene inactivation. In order to provide a research tool for the study on the function of MBD1 gene in DNA methylation and tumorigenesis, antisense MBD1 gene eukaryotic expression plasmid was constructed and transfected into human biliary tract carcinoma cell line QBC-939 to observe its effect on the expression of MBD1 mRNA and protein by using RT-PCR and FCM respectively. Following the transfection, the mRNA level of MBD1 gene decreased from 0. 912 +/- 0.022 to 0.215 +/- 0. 017, and the protein level of MBD1 gene also decreased from (80.19 +/- 5.05) % to (35.11 +/- 4.05) %. There were very significant differences in the expression both at the transcription and post-transcription levels of MBD1 gene between non-tranfection group and the antisense MBD1 gene eukaryotic expression plasmid transfection group (P < 0.01). It was suggested that transfection with the antisense MBD1 gene eukaryotic expression plasmid can significantly reduce the expression level of MBD1 gene in QBC-939, and this study may provide a valid tool for the investigation of the function of MBD1 gene and its role in biliary tract carcinoma.

Celecoxib Inhibits Proliferation and Induces Apoptosis via Cyclooxygen-ase-2 Pathway in Human Pancreatic Carcinoma Cells / 华中科技大学学报（医学）（英德文版）

In order to evaluate the effects and mechanisms of celecoxib in inhibiting proliferation and inducing apoptosis on human pancreatic carcinoma cells, the anti-proliferative effect was measured by using methabenzthiazuron (MTT) assay. Cell cycle and apoptosis were analyzed by using flow cytometry (FCM), and the PGE2 levels in the supernatant of cultured pancreatic carcinoma cells were quantitated by enzyme-linked immunoabsordent assay (ELISA). Our results showed that celecoxib suppressed the production of PGE2 and inhibited the growth of JF-305 cells, and the anti-proliferative effect of celecoxib could be abolished by addition of PGE2. FCM revealed that celecoxib could inhibit proliferation and induce apoptosis by G1-S cell cycle arrest. It was concluded that cyclooxygenase-2 specific inhibitor celecoxib could inhibit proliferation and induced apoptosis of human pancreatic carcinoma cells via suppression of PGE2 production in vitro.

Roles of Protein Kinase C and Fructose in Hepatic Injury Caused by Obstructive Jaundice / 华中科技大学学报（医学）（英德文版）

The regulating mechanism in hepatic injury caused by obstructive jaundice (OJ) was examined in this study. Rat hepatocytes were harvestedby in situ collagenase perfusion and subjected to primary culture. The heptocytes were pre-treated with various concentrations of protein kinase C(PKC) agonist PMA and its inhibitor chelerythrine and cultured for 20 min. After the treatment,50 μmol/L glycochenodeoxycholate (GCDC) was added and the cells were cultured for an additional24 h. Cells were then detected by flow cytometry (FCM) and TUNEL. After hepatocytes were treated with different concentrations of fructose and 100 μM GCDC, the cells were examined by FCM and TUNEL. Experimental obstructive jaundice (BDL) was induced by double ligation of the bile duct. After BDL, the rats were fed with or without fructos and sacrificed 3, 7, 14 and 21 days after the ligation. The apoptotic status was observed in liver of all rats with TUNEL and PKC protein in liver of OJ was studied by immunohistochemical method. Our results showed that PMA increased GCDC-induced apoptosis and chelerythrine decreased GCDC-induced apoptosis in a concentration-dependent manner. After the treatment with fructose of different concentrations, 100 μM GCDC decreased the apoptotic rate and the apoptotic rate decreased with the increase of fructose concentration. The apoptotic rate of liver was related to the time of OJ. Without the treatment of fructose, PKC and apoptosis index (AI) were highest 14 days after the bile duct ligation. With the treatment of fructose, apoptosis index (AI) and PKC were decreased from the 14th day after the bile duct ligation. It is concluded that PKC is involved in the regulation of apoptosis in the liver cells with OJ and plays important roles in the development and progression of liver injury caused by OJ.Fructose can protect hepatocytes in the bile salt-induced apoptosis by regulating PKC.

Effect of Antisense MBD1 Gene Eukaryotic Expression Plasmid on Expression of MBD1 Gene in Human Biliary Tract Carcinoma Cells / 华中科技大学学报（医学）（英德文版）

Hypermethylation of the promoter region is one of the major mechanism of tumor suppressor gene inactivation. In order to provide a research tool for the study on the function of MBD1gene in DNA methylation and tumorigenesis, antisense MBD1 gene eukaryotic expression plasmid was constructed and transfected into human biliary tract carcinoma cell line QBC-939 to observe its effect on the expression of MBD1 mRNA and protein by using RT-PCR and FCM respectively. Following the transfection, the mRNA level of MBD1 gene decreased from 0. 912±0. 022 to 0. 215±0.017, and the protein level of MBD1 gene also decreased from (80.19±5.05) % to (35.11±4.05)%. There were very significant differences in the expression both at the transcription and post-transcription levels of MBD1 gene between non-tranfection group and the antisense MBD1 gene eukary otic expression plasmid transfection group (P＜0.01). It was suggested that transfection with the antisense MBD1 gene eukaryotic expression plasmid can significantly reduce the expression level of MBD1 gene in QBC-939, and this study may provide a valid tool for the investigation of the function of MBD1 gene and its role in biliary tract carcinoma.

Analysis of etiology and drug resistance of biliary infections / 华中科技大学学报（医学）（英德文版）

The bile was collected from fro patients with biliary infections, with the bacterium isolated to study the sensitivity of each kind of the bacterium to several antibiotics in common use. Except G- bacterium, we also found some kinds of G+ bacterium in infection bile. G- bacterium were not sensitive to Clindamycin, G+ bacterium were sensitive to Ciprofloxacin. Escherichia coli, Xanthomonas maltophilia, Enterobacter cloacae, Pseudomonas aeruginosa were sensitive to Ampicillin. G+ bacterium were not sensitive to Azactam. Enterococcus faecalis, Enterococcus faecium, Enterobacter cloacae were not sensitive to Ceftazidime. Enterococcus faecalis, Staphylococcus coagulase negative, Staphylococcus epidermidis, Pseudomonas aeruginosa were not sensitive to Ceftriaxone Sodium. We didn't found any bacterium resistance Imipenem. The possibility of the existence of G+ bacterium as well as drug resistance should be considered n patients with biliary infections. The value of susceptibility test should be respected to avoid drug abuse of antibiotics.