Objective To investigate the possible mechanism of recombinant human erythropoietin (rhEPO) neuroprotection by studying the effect of rhEPO on expressions of matrix metalloproteinase-9 (MMP-9) and BCL-2 following focal cerebral ischemia-reperfusion in rats. Methods A rat middle cerebral artery occlusion/reperfusion (MCAO/R) model was induced by the intraluminal filament method, and intraperitoneal injection of rhEPO was used for intervention. Histopathological changes were observed by HE staining, and the expressions of MMP-9 and BCL-2 in the cerebral cortex of ischemic side were detected with immunohisto-chemistry. Results HE staining: At all time points, the numbers of surviving nerve cells were significantly higher in the rhEPO group, and their injury degree was significantly lower. MMP-9 immunohistochemistry staining: The positive cells were observed occasionally in the normal control group and the sham-operation group; the MMP-9 positive cells at the ischemic side of brain tissue in a normal saline control group began to appear at 6 hours after reperfusion, it reached the peak at 24 hours and began to decrease at 72 hours; the change trend of MMP-9 positive cells in the rhEPO group was similar to that in the normal saline control group, but it was significantly lower than that in the normal saline control group at the same time points (t were 12. 023 6, 12. 635 0, 12. 779 6, respectively, all P <0. 01). BCL-2 immunohistochemistry staining: No positive cells were found in the normal control group and sham-operation group. The numbers of BCL-2 positive cells reached the peak at the ischemic side of brain tissue in the normal saline control group at 6 hours after reperfusion, it reached the peak at 24 hours and further decreased at 72 hours; the change trend of BCL-2 positive cells in the rhEPO group was similar to that in the normal saline control group, but it was significantly higher than that in the normal saline control group at the same time points (t were 5. 763 1,8. 110 1, and 5. 798 7, respectively, all P <0. 01). Conclusions rhEPO may inhibit cortical neuronal apop-tosis at the ischemic side by inhibiting MMP-9 expression and up-regulating BCL-2 expression so as to play a neuroprotective effect.

At present, the assessment of the same hospital at the same time by different means may lead to different assessment results, viz. there may be a variety (inconsistency) of assessment results. This can be chiefly attributed to the variety of assessment systems, with different assessment indexes and assessment methods, and to the variety of people making the assessments and assessing environments. Integrated measures can be taken in order to render assessment results more accurate: ① refusing to have blind faith in any particular method; ② seeing clearly assessment goals and features; ③ properly selecting index systems for the assessment; ④ correctly fixing the weights for indexes; ⑤ appropriately selecting assessment methods; ⑥ making combined use of various methods; ⑦ pooling the results from different assessment methods.

Objective To explore the protective effect of erythropoietin(EPO)on inflammatory injury induced by focal cerebral ischemia/reperfusion in rats.Methods 54 male Sprague-Dawley rats were randomly divided into 3 groups:sham operation group,normal saline control group and EPO group.The focal cerebral ischemia-reperfusion injury model was made by suture-occluded method.The content of interleukin-1 β(IL-1β)and the activity of myeloperoxidease(MPO)were measured by radioimmunoassay and chromatoptometry at 6 h,24 h and 48 h reperfusion following ischemia 2 h.The scores of behavior obstacle in rats were assessed at 24 h.Results Compared with the normal saline control group,EPO group got better score of behavior obstacle(all P<0.05).The content of IL-1β and the activity of MPO in brain in the EPO group were significantly lower than those in the normal saline control group(all P<0.01).Compared with two reperfusion groups,the sham operation group had no obvious abnormal change in each measurement item.Conclusion EPO can reduce the content of IL-1β and inhibit the infiltration of leukoeyte,which can provide protective effect on cerebral ischemic-repefusion injury in rats.

Intellectual property protection is an indispensable management measure in the process of knowledge innovation in the National Traditional Chinese Medicine (TCM) Clinical Research Bases. Longhua Hospital, a National TCM Clinical Research Base , has actively participated in studies and construction of TCM intellectual property policy , laws and regulations , and endeavored to enhance international research cooperation and broaden the international repercussions of TCM . Longhua Hospital made great efforts to strengthen management on intel-lectual property management team and measures, advocacy training, property rights formation and transactions. Intellectual property protection played an important role in the construction of the National TCM Clinical Re-search Base in Longhua Hospital .

Many gene therapy protocols can induce antitumor immunity, however, the ex vivo approach restricts their uses. This sutydy intended to induce antitumor immunity by direct transfer of MHC class Ⅱ gene in vivo. Methods: MHC class Ⅱ gene cDNA was introduced directly into two tumors: P815, (a murine weak immunogenic mas-tocytoma) and B16 (a murine nonirnmunogenic melanoma) to observe the survival rate of the mice. Results: Tumori-genicity of P815 was reduced when MHC class Ⅱ gene was introduced directly into tumors in vivo. Further more, most vaccinated mice could survive after second challenge of P815. Co-injection of MHC class Ⅱ and B7 genes in the B16 also resulted in the tumor grow slowly, while the injection of MHC class Ⅱ gene was not enough to induce effective antitumor responses. Conclusion: The results showed the potential applications of direct transfer of MHC class Ⅱ gene in the treatment of tumor.

Objective To study the effect of transfection with antisense DNMT3b gene eukaryotic expression plasmid on the growth of human cholangiocarcinoma cell line QBC939,and to explore the role of DNMT3b in the cholangiocarcinoma tumorigenesis.Methods The constructed antisense DNMT3b gene eukaryotic expression plasmid was transfected into QBC939 cells using liposome.The expression level of DNMT3b protein was detected by Western blot after stable transfection.The growth curves of transfected cells and un-transfected cells were observed by MTT method respectively.The cell proliferation ability was also observed by the test of colony formation in soft agar.The alterations of the cell cycle and the apoptosis rate were detected by FCM.Results Following the transfection,the protein level of DNMT3b decreased significantly;transfection with antisense DNMT3b gene eukaryotic expression plasmid did not affect the cell growth curve of QBC939,and did not decrease the cell colony formation rate(P=0.717);transfection with antisense DNMT3b gene also did not result in cell cycle alterations or induce cell apoptosis(P=0.089).Conclusions Transfection with antisense DNMT3b gene eukaryotic expression plasmid can down-regulate the expression level of DNMT3b in QBC939.It can not affect the growth and proliferation of human cholangiocarcinoma cell line QBC939,nor alter the cell cycle and induce cell apoptosis.

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.

Objective To explore the relationship of plasma NT-proBNP level and severity of chronic congestive heart failure (CHF) and investigate the curative effect and security of meglumine adenosine cyclophosphate (MAC) combined with perindopril on patients with CHF.Methods From June 2011 to June 2013,126 inpatients with chronic congestive heart failure were randomly divided into A group (42 cases,routine therapy),B group (41 cases,routine therapy and perindopril) and C group (43 cases,routine therapy and perindopril plus MAC),all cases treated for 14 days.The left ventricular ejection fraction (LVEF) and left ventricular end-diastolic diameter (LVEDD) by echocardiography and plasma NT-proBNP levels were evaluated before and after 14 days therapy.Results The plasma NT-proBNP levels in NYHA Ⅱ ～ Ⅳ classes were significantly difference compared each other between any two classes (P ＜0.05) and the levels was positively correlated with NYHA cardiac function class and LVEDD (r =0.617,P ＜ 0.01 ; r =0.412,P ＜ 0.01),negatively correlated with LVEF (r =-0.372,P ＜ 0.01).After 14 days therapy,compared with A group,the LVEF and LVEDD significantly improved (P ＜ 0.05) and NT-proBNP level significantly decreased (P ＜ 0.05) in B,C groups; Compared with B group,C group had lower NT-proBNP level (P ＜ 0.05) although no further improvement in cardiac function.Conclusions The plasma NT-proBNP level is correlated closely with the severity of CHF and it is a good examination of diagnose,therapy and evaluating prognosis of CHF.Perindopril may significantly decline plasma NT-proBNP level and improve cardiac function of CHF patients,combined with MAC may further decline plasma NT-proBNP level although not further improved LVEF.Giving MAC and perindopril to patients with CHF was secure and patients tolerated it well.

Objective To investigate the pathogens,drug resistance and outcomes of continuous ambulatory peritoneal dialysis(CAPD) patients with peritoneal dialysis-related peritonitis in our peritoneal dialysis(PD) centers. Method Data including clinical manifestations,pathogens,treatment,outcome of 93 CAPD cases with peritoneal dialysis-related peritonitis in our peritoneal dialysis(PD) centers were retrospectively analyzed.Results Dialysate culture of 75cases was positive with a positive rate of 80.2％,including 45 cases of gram-positive cocci,21cases of gram-negative bacilli,2 cases of fungi and 5 cases of mixed infection.Coagulase-negative staphylococci were the most common gram-positive cocci.All the gram-positive cocci were sensitive to vancomycin,but the resistance rate to cefazolin was 60.0％ with an increasing tendence year by year.Resistance rate of gram-negative bacilli to ceftazidime was 46.1％.All the gram-negative bacilli were sensitive to imipenem.The withdraw rate of CAPD was 17.2％(16/93) because of peritonitis. Noobviousside-effectofperitonealadministrationofvancomycinwasfound.Conclusions Gram-positive cocci are major pathogens in CAPD-related peritonitis.Now cefasolin is not suitable for the empiric initial treatment.Peritoneal administration of vancomycin should be recommended for peritonitis caused by gram-positive cocci.

Objective The aim of this study is to examine the expressions of Toll like receptor (TLR) 7 and TLR9 in the peripheral blood B lymphocytes of SLE patients and to analyze the correlation between TLR7/9 and clinical parameters. Methods lntracellular expression of TLR7/9 in the peripheral blood CD19+Blymphocytes was analyzed in 50 SLE patients and 30 healthy controls by flow cytometry. The difference of intracellular TLR7/9 expression levels in two groups was compared. Furthermore,the correlation between TLR7/9 expression and clinical parameters such as ESR, CRP, complement 3 (C3), complement 4 (CA), the level of serum IgG, anti-double stranded DNA antibody, anti-nuclear antibodies, SLEDAI score and urine protein excretion level, were analyzed. Results Compared with healthy subjects, the proportion of B cells expressing TLR7 and TLR9 was higher among SLE patients. Positive correlation was observed between TLR7 expression levels and clinical measurement of the SLEDAI and ESR. Negative correlation was observed between TLR7 expression levels and serum C3 levels. Positive correlation was observed between TLR9 expression levels and SLEDAI scores. Negative correlation was observed between TLR9 expression levels and serum C3 levels. Conclusion TLR7 and TLR9 expression is increased in the peripheral blood B cells of SLE patients, and correlates well with clinical parameters.