Objective: To construct siRNA plasmid expression vector in order to knockdown E2F-3 activity. Methods: Sixty-four base-pair oligos for hairpin RNA expression, which targeted E2F-3 gene, were chemically synthesized and annealed. The pRNAT-U6.1/Neo vector was linearized with Bam HI and HindⅢ. Finally, the annealed oligos were inserted into the lined pRNAT-U6.1/Neo to construct RNAi plasmid(pRNAT-U6.1-E2F-3/Neo). The reconstructed RNAi plasmids were i-dentified by electrophoresis after digestion with BamHI and Hind Ⅲ, and were confirmed by sequencing analysis. Results: The recombinant pRNAT-U6.1-E2F-3/Neo vector was identified by polymerase chain reaction, and confirmed by sequencing analysis. The results demonstrated that 64 bp had been inserted into the expected site. Furthermore, the insertion sequence was exactly correct and no mutation site was found. Conclusion: The pRNAT-U6.1-E2F-3/Neo RNAi system was constructed successfully. This will facilitate the study of E2F-3 in bladder cancer cell lines.

Objective To investigate the effects of Berberine on growth of Immorto-Min colonic epithelial cell line (IMCE) and explore its possible mechanisms. Methods IMCE cells were treated with Berberine in the absence or presence of epidermal growth factor (EGF) and TNFα. Ki-67 staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay were used to identify the cell proliferation and apoptosis respectively. Furthermore, Western blot analysis was performed to detect the epidermal growth factor receptor ( EGFR), protein kinase B (Akt) and their phosphorylation.Results ( 1 ) Proliferating activity of IMCE cells was increased after adding EGF and the proportion of cell proliferation was ( 10. 64 ± 1.41 ) %. The proportion was significantly lowed in EGF plus Berberine group [(1.81 ±0. 85)%] compared to the EGF group(P ＜0. 01 ), while the lowest was the Berberine group [(0.49 ± 0.42) %]. (2) The proportions of cell apoptosis were ( 8.47 ± 2. 52 ) % and (9. 39 ± 2. 13 ) %in the Berberine group and TNFt group respectively which were significantly higher compared to the normal control [(0. 27 ± 0. 30)%], both P ＜ 0. 01. (3) The phosphorylation of EGFR was significantly increased after adding EGF and p-EGFR was decreased in EGF plus Berberine group at a concentration-dependent manner. (4) Moreover, the phosphorylation of Akt was enhanced after addition of TNFα , while the phosphorylation in the TNFα and Berberine group was inhibited compared to the TNFα group. Conclusions Berberine may suppress the proliferation and promote the apoptosis of IMCE cells. The mechanisms may relate to the inhibition of the phosphorylation of EGFR and Akt.

Objective To study the effects of supplemental Dangguibeimukushen pill prescription on the expressions of MMP13 and bFGF in tumor tissues on H22 hepatoma bearing mice.Methods H22 hepatoma bearing mice was employed to carry out anti-tumor experiment in vivo in this study.A total of 60 mice were randomly divided into model group,DDP positive control group,low and high dosage of supplemental Dangguibeimukushen pill treatment group,supplemental Dangguibeimukushen pill combined with DDP group.In each gruop,gastric infusion was performed continuously 14 d.The tumor growth and the general conditions of mice were recorded.After the last administration of gavages orally treatment,all mice were anaesthetized and killed by cervical disloca-tion method to obtain completely tumor tissue for further HE staining measure and detection of MMP13 and bFGF in tumor tissue with the method of RT-qPCR and immunohistochemistry.Results The tumor volume of model group and low dosage of supple-mental Dangguibeimukushen pill treatment group were larger than that of other groups,futhermore,ulceration were found inside tumor,and basic integrity were observed of tumor capsule.However,in all the treatment groups,the tumor volume was relatively smaller,and the capsule of tumor was incomplete,ulceration of different degrees and bleeding when tumor was peeled off.In combi-nation groups,the tumor volume was much smaller,and the tumor had incomplete capsule and less ulceration inside.Pathological observation showed that connective tissue was found inside tumor,cells were sparsely spread and the number of necrosis cancer cells were increased in all treatment groups.RT-qPCR detection showed that the mRNA expression of MMP13 and bFGF in each treat-ment group were decreased comparing with the model group,and the difference was statistically significant(P <0.05).Immunohis-tochemical detection showed that the expression of MMP13 and bFGF in the combined group were less colored and weakly positive expression comparing with the positive group or the pure Chinese medicine treatment group,the difference was statistically signifi-cant(P <0.05).Conclusion Dangguibeimukushen pill could downregulate the expression of MMP13 and bFGF of tumor tissue in H22 hepatocarcinoma xenograft mice at mRNA and protein levels,and played a vital role in inhibiting cancer invasion and angiogen-esis,reducing toxics and side effects and enchancing its efficiency.

Objective: To acquire the expression of E2F3 protein and mRNA in bladder transitional cell carcinoma (BTCC) tissue and normal bladder epithelial tissue, and the relationship between E2F3 expression and the biological behaviors of BTCC thereof. Methods: Immunohistochemistry was used to detect the expression of E2F3 in BTCC(n = 64) and normal bladder mucosa(n = 10). Immunohistochemistry result was analysed by Image-pro Plus software and the expression result was indicated by integrated optical density (IOD). The expression of E2F3 mRNA was investigated using RT-PCR analysis in fresh bladder tumor tissues and normal bladder mucosa. Results: The expression rate of E2F3 in BTCC (32.8%) was higher than that of normal bladder mucosa(P < 0.01). The expression rate of E2F3 was strongly correlated with the pathological grade and clinical stage (P < 0.05;P < 0.01). Immunohistochemistry result indicated that the IOD of E2F3 was significantly higher in BTCC than that of normal bladder mucosa (P < 0.01). The expression level of E2F3 was strongly correlated with pathological grade (P < 0.01). Conclusion: E2F3 was the diagnostic and prognostic index of BTCC, and provided theory basis about the gene target therapy in BTCC.

Objective To investigate the imaging features of splenic littoral cell angioma (LCA)and correlate with pathological findings.Methods Ten patients of LCA with pathologically confirmed diagnosis were included in this study.A retrospective review of clinical data and imaging findings on CT and MRI was performed,along with review of the literature.Results Splenic littory cell angiomas presented with multiple nodules of varying sizes with a predoninance of small ones.Nine of 10 patients had clinical symptoms of splenomegalia and hypersplenia.MR T2WI and DWI showed masses with high-signalintensity.The CT and MRI enhancing pattern of LCA was similar to splenic hemangioma.There were many mammiliform structures pointing inside in the wall of the vascular channels,a hallmark feature allowing its differentiation from splenic hemangioma pathologically.Conclusion The CT and MRI findings of LCA can show some of its characteristic signs,especially on DWI,which can assist to identify LCA in clinical practice.

The patterns of efficient components recognition and quality control for Chinese materia medica (CMM) have been the difficult and hot topics for CMM modernization.To get a radical and significant breakthrough in the investigation on the efficient component recognition and quality control standard for CMM,a tentative idea about efficient component recognition and quality control pattern for CMM based on constituent knock-out/knock-in is initially proposed in this article on the foundation of retrospective and prospective analyses.And its main aim is to provide some creative and practical ideas and methods to recognize the key efficient components of CMM precisely and quickly,and to meet with the requirements that the quality control standards for CMM will be effectiveness-related,quantitative and accurate,controllable and assessable.

Background Dry eye is the most common ocular surface disease.Anterior segment pattern of optical coherent tomography (OCT) is able to be used to assess the tear meniscus directly and accurately.Objective This study was to evaluate the correlation between measuring outcomes of tear meniscus by anterior segment module of OCT with signs and symptoms of dry eye.Methods A retrospective serial cases-observational study was designed.Thirty-six patients who completed Ocular Surface Disease Index (OSDI) and got a score more than 20 were enrolled in the First Hospital of Peking University from September to October,2010 under the informed consent.The upper and lower tear meniscus height (U/LTMH,μm),depth (U/LTMD,μm) and cross sectional area (U/LMA,mm2) were measured with the anterior segment module of OCT (RTVue.pitch).A series of clinical tests were performed,including tear film interferometry,break-up time (BUT),fluorescein corneal staining,lissamine green conjunctival staining and Schirmer Ⅰ test (S Ⅰ t).The correlations of measuring outcomes of tear meniscus by anterior segment module of OCT with dry eye-related examinations were analyzed with Spearman correlation coefficients.Results The values of UTMH,UTMD,UMA were (232±56) μm,(156±53) μm,(0.021 ±0.008) mm2,respectively,and the values of LTMH,LTMD,LMA were (222 ± 72) μm,(163 ± 65) μm,(0.020 ± 0.006) mm2,respectively.No significant differences were found in the measuring values between the upper and the lower tear meniscus (all at P＞0.05).The quantitative values of tear meniscus were not significantly correlated with the OSDI scores or tear film interferometry grade.Significant correlations were seen between UTMH,UTMD,UMA and BUT (r=0.46,P=0.01 ;r=0.40,P=0.02;r=0.43,P=0.01).LTMH,LTMD,LMA were positively correlated with BUT (r=0.61,P=0.00;r=0.64,P=0.00;r=0.64,P=0.00) and S Ⅰ t (r=0.54,P=0.02 ;r=0.46,P=0.01;r=0.55,P=0.01) as well as negatively correlated with fluorescein cornealstaining (r=-0.37,P=0.04;r=-0.37,P=0.04;r=-0.41,P =0.05) and lissamine green conjunctival staining (r =-0.56,P =0.01 ; r =-0.37,P =0.04 ; r =-0.47,P =0.00).Conclusions Anterior segment module of OCT is an effective approach to the measurement of tear meniscus.The measuring outcomes of anterior segment of OCT are associated with dry eye-related signs.

Objective To initially explore the feasibility and quality control of producing fecal-derived microbiota enteric capsules.Methods Fecal-derived microbiota was put into double layered enteric capsules.The bacteria colony numbers of fresh prepared glycerol containing fecal-derived microbiota liquid,glycerol containing and glycerol free fecal-derived microbiota after stored at -80 ℃ for 72 h were counted with standard plate count methods in order to investigate the stability after frozen.Methylene blue was taken as the standard,resistance to acid and release rate of capsules was evaluated.The t test was performed for statistical analysis.Results The preparation process of double layered microbiota capsules was simple and practicable.The data of 12 plates of each microbiota were acquired.The number of bacteria colony of fresh prepared glycerol containing fecal-derived microbiota ((5.08 ±1.37)×107 colony-forming units (cfu)/mL)was significantly more than that of the group without glycerin ((1.73±0.64)×107 cfu/mL)at -80 ℃for 72 h (t = 7.621 ,P <0.01).There was no significant difference in the number of bacteria colony between glycerin containing frozen fecal-derived microbiota ((4.67±1 .56)×10 7 cfu/mL)and fresh fecal-derived microbiota (t = 0.694,P = 0.495).Regression equations were achieved with fecal-derived microbiota containing methylene blue,and there was a good linear relation between 0.5 μg/mL and 8.0 μg/mL.Three fecal-derived microbiota enteric capsules containing methylene blue were prepared.Their resistance to acid was 96.0%,99.1 %,and 95 .5 %,while the release rate was 88.6%,95 .1 % and 86.5 %, respectively.All met the requirement of Chinese Pharmacopoeia to enteric capsules.Conclusion The preparation of double layered fecal-derived microbiota enteric capsules had feasible technology and stable quality,which could provide reference in prevention and treatment of diseases related with colonic microbiota imbalance.

BACKGROUND: Cerebral infarction is commonly associated with blood stasis syndrome. Abnormal alternation of blood rheology is generally manifested as increased blood viscosity and hematocrit (HCT). In isometric hemodilution, a certain amount of red blood cell (RBC) is shifted by bleeding and simultaneously, isometric diluter is supplemented to reduce whole blood viscosity.OBJECTIVE: To observe the improvement of astragalus injection, the Chinese herb for qi tonification and isometric hemodilution on blood rheology in blood stasis syndrome of cerebral infarction.DESIGN: Randomized controlled experiment and case-control analysis were designed.SETTING: Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology. PARTICIPANTS: In cerebral infarction group (infarction group), 64 inpatients of senile ischemic cerebral vascular disease were collected from Union Hospital Affiliated to Huazhong University of Science and Technology from March 2002 to March 2004. Al l of cases were aged over 60 years and were in conformity with the diagnostic criteria on blood stasis syndrome. According to random number table, routine treatment group (routine group) and the group of integrative therapy of Chinese and western medicine (experimental group) were divided, 32 cases in each one. 47 healthy people of similar age and diagnosed with routine physical examination were selected in normal control. METHODS: In routine group, cerebral infarction was treated with routine therapy, including extending capacity, reducing viscosity, resisting coagulation, blocking aggregation of platelet and dehydration and general symp tomatic supporting treatment. In experimental group, on basis of routine treatment, isometric hemodilution and astragalus injection, the Chinese herb for qi tonification were used. 10% of total blood amount (about 450-650 mL) was collected from vein, and colloid solution of same volume was injected intravenously. The treatment was applied once every 5 days, continuously for 3 times. Astragalus injection 50 mL mixed with physical saline 250 mL was intravenous dropped, once per day, continuously for 3 weeks. MAIN OUTCOME MEASURES: ① Comparison of indexes in bloodrheology before and after treatment in routine group and experimental group. ② Comparison of indexes in blood rheology between normal control and infarction group. RESULTS: According to intention management, 64 patients and 47 normal persons all entered result analysis. ① Comparison between infarction group and normal control: RVB, HCT and PFC (fibrinogen) were higher than normal control [(3.90±0.73), (3.40±0.28) mPa·s; (46.39±6.03) %,(42.61±2.91)%; (3.25±0.75), (3.08±0.46) g/L, P < 0.01, 0.05], MTIE (de formity index of RBC) was lower than normal control (0.958±0.006, 0.961 Shen H,Lu YD.Study on quantitative messurement of immunohistochemical ±0.004, P < 0.05). ② Comparison between routine group and experimental group: Difference in some indexes presented before the treatment. After treatment, RVB, HCT and PFC in experimental grou p were all lower than routine group [(3.90±0.52), (4.21±0.68) mPa·s; (43.80±3.29)%, (48.47±4.50)%; (3.31±0.60), (3.68±0.67) g/L, P < 0.01, 0.05]. CONCLUSION: Isometric hemodilution therapy and astragalus injection reduces blood viscosity, improves blood rheology and alleviates clinical svmptoms of blood stasis syndrome in senile cerebral infarction.

Objective To explore the inhibitory effect of curcumin on LPS-induced inflammation and the activation of Toll-like receptor 4 (TLR4 )/NADPH oxidase/reactive oxygen species (ROS)signaling pathway in vascular smooth muscle cells (VSMCs).Methods Primary VSMCs were cultured and divided into control group, LPS group,LPS + curcumin 5 μmol/L group,LPS + curcumin 10 μmol/L group and LPS + curcumin 30 μmol/L group.Cell activity was observed by MTT assay.The secretion of tumor necrosis factor-α(TNF-α)and interleukin-1 (IL-1)was measured by enzyme linked immunosorbent assay (ELISA)kits.The mRNA expressions of TLR4 and p22phox were detected by real-time PCR.Expression of intracellular ROS was measured by flow cytometry. Results The activities of VSMCs were not significantly affected by curcumin at the concentration between 0 and 80 μmol/L.Curcumin (5,10 and 30 μmol/L)significantly inhibited LPS-induced oversecretion of TNF-αand IL-1, as well as overexpression of TLR4 and p22phox at the mRNA and protein levels,and ROS production in VSMCs in a concentration-dependent manner.Conclusion Curcumin has a concentration-dependent inhibitory effect on the secretion of inflammatory cytokine,overexpressions of TLR4 and p22phox,and production of ROS in VSMCs stimulated by LPS.Furthermore,curcumin may partly depend on TLR4/NADPH oxidase/ROS signaling pathways to inhibit inflammation in LPS-induced VSMCs.