Objective Toinvestigatetheexpressionof SIRT1anditsassociationwith clinicopathologic features in breast carcinoma with type 2 diabetes mellitus.MethodsThe expression of SIRT1 in 30 breast cancer with type 2 diabetes mellitus,65 samples of breast cancer without diabetes mellitus and 18 samples of corresponding normal breast tissues was investigated using immunohistochemistry.ResultsThe positive rate of SIRT1 in breast cancer tissues was significantly higher than that breast cancer with type 2 diabetes mellitus and normal breast tissue (P ＜0.05). In breast cancer with type 2 diabetes mellitus group.The positive rate of SIRT1 was significantly higher than that normal breast tissue(P ＜0.05).The expression of SIRT1 was positively correlated with the number of lymph node(P =0.011),pTNM tumor stage (P =0.028), p53 (P =0.003) and Her-2 (P =0.031) in breast cancer with type 2 diabetes mellitus group. The expression level of SIRT1 in lymph node-positive group was higher than that in lymph node-negative group (P ＜0.05).The expression level of SIRT1 was in lymph node-positive group was higher than that in lymph node-negative group(P ＜0.05).ConclusionSIRTI was up-regulated in breast cancer with type 2 diabetes mellitus,but its expression was lower than that breast cancer without diabetes mellitus,and was associated with the progression of diabetes mellitus. SIRT1 was positively correlated with lymph node, pTNM tumor stage,p53 and Her-2, SIRT1 may be a novel biological parameter to evaluate the malignant degree of breast carcinoma and to predict prognosis of breast cancer.

Objective To investigate the protective effect of rosiglitazone on the rats with high altitude pulmonary edema.Methods Thirty-six SD rats were randomly (random number) divided into 6 groups (n =6 each):control group (Control),hypobaric hypoxia model group (HH),rosiglitazone groups (RSG) which were administered with 3 different doses [RSG-L:5 mg/ (kg · d),RSG-M:10 mg/ (kg·d),RSG-H:20 mg/ (kg· d)],dexamethasone group [Dex,4 mg/ (kg· d)].Rats were injected intraperitoneally with different doses of rosiglitazone (RSG),dexamethasone (Dex) or vehicle (Control and HH) for 3 days before placed in simulated altitude of 6 000 m hypobaric hypoxia animal chamber where the temperature and pressure were constant.After 72 h in the chamber,each rat was anesthetized.The water content of lung was determined with wet/dry weight ratio.Bronchoalveolar lavage fluid was measured by bradford method.The contents of GSH was measured by micro-ezymed labeled method.The contents of MDA was measured by TBA method.The enzymatic activities of SOD was measured by WST-1 method.The changes of the TNF-α,IL-6 and IL-10 in serum were determined by ELISA.Light microscope was used to observe the pathological changes of lung tissue.Results Compared with Control group,the wet/dry weight ratio of lung (5.08 ± 0.24) and total protein content of BALF (351.06 ± 44.55) μg/mL increased significantly (P ＜ 0.01) in HH group.There were red blood cells in the alveolar and interstitium,pink fluid exudation in the alveolar,the alveolar septum enhancement,and a large number of inflammatory cell infiltration;the SOD activity (10.65 ± 0.94) U/mgprot and the content of GSH (1.63 ±0.20) μmol/gprot in lung tissue were significantly decreased (P ＜ 0.01),the contents of MDA (2.1 5 ± 0.18) nmol/mgprot increased significantly (P ＜ 0.01),TNF-o (56.92 ± 2.87) pg/mL and IL-6 (217.80 ±48.01) pg/mL levels in serum were significantly increased (P ＜0.01),and IL-10 (76.85 ± 16.72) pg/mL level decreased (P ＜ 0.05).Compared with the HH group,the wet/dry ratio of lung and total protein content of BALF in different doses of rosiglitazone group significantly decreased (P ＜ 0.01),the pathological changes of the lung tissue was significantly improved,SOD activity and the content of GSH in lung tissue was significantly increased (P ＜ 0.01),the content of MDA decreased (P ＜ 0.01),The levels of TNF-α and IL-6 in serum were significantly decreased (P ＜ 0.01),while the IL-10 level was significantly increased (P ＜ 0.01).Conclusion Rosiglitazone could protect the high altitude pulmonary edema by alleviating the oxidative stress and inflammatory response.

Objective To investigate the effect of high altitude hypoxia on chronic inflammation in rats.Methods Forty SD rats were randomly divided into 4 groups: control group (Con),chronic inflammation group (CI),high altitude hypoxia group (HH),high altitude hypoxia+chronic inflammation group (HH+CI).Rats in CI group were injected with lipopolysaccharide (LPS) (0.5 mg/kg) through the caudal vein twice a week for 4 weeks.Rats in HH+CI group were treated just as CI group was,but together with HH group rats were settled in a hypoxic environment of 6000 m altitude for three days.Pathological changes in lung tissues were observed by hematoxylin eosin stain.The peripheral white blood cell count and classification were measured.The levels of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) in serum and lung tissues were detected by enzyme-linked immunosorbent assay (ELISA).Changes in IL-6 expression in rat lung tissues were observed by Western blotting.Results After LPS and high altitude hypoxia exposure,inflammatory cells infiltration and alveolar capillary expansion were observed in rats' lung tissue.Compared with Con group,not only the peripheral white blood cell count,but also the level of IL-6 and TNF-α in serum and lung tissue increased in CI and HH group(P<0.01).IL-6 expression levels observed by Western blotting were also increased in HH and CI group(P<0.01).High altitude hypoxia and chronic inflammation interacted(P<0.01).The peripheral white blood cell count was higher in HH+CI group than in other groups,and IL-6 and TNF-α expressions in lung tissue were increased(P<0.05).Conclusion An LPS-induced chronic inflammation model in rats is successfully obtained,and high altitude hypoxia could aggravate chronic inflammation.

Objective To prepare the anti-TLR4 C-terminal domain monoclonal antibody and investigate its effect in treatment of sepsis.Methods TLR4 C-terminal polypeptide (amino acid sequence:368-579,named as TLR4-C) was obtained through prokaryotic expression and Sephacryl S-100 gel purification,and then was used to immunize female Balb/c mice (6-8 weeks old).After cell fusion,antibody screening and purification,monoclonal antibody specific for the C terminal of TLR4 was obtained.Specificity of monoclonal antibody was detected by Western blot and cell immunofluorescence.In vitro antibody activity test,NR8383 was cultured for 1 h with adding antibody (100 μg/ml) and then 12 h after adding lipopolysaccharide (LPS) (10 ng/ml),and level of tumor growth factor (TNF)-α in the culture medium was tested by ELISA.In vivo septic animal experiment,40 SD rats were assigned to control antibody group (n =20) and anti-TLR4 monoclonal antibody group (n =20) according to the random number table.Each group was rejected 50 mg/kg corresponding antibodies via caudal vein for 1 h,and then LPS (10 mg/kg) via intraperitoneal injection for 4 h.Blood samples from caudal vein of ten rats in each group were collect to test the serum level of TNF-α.The rest rats in each group were used to measure the animal survival rate within 72 h.Results Three highly specific anti-TLR4 monoclonal antibodies were obtained and could combined with TLR4-C and TLR4 holoprotein.In vitro cell activity study indicated only one monoclonal antibody could obviously inhibit the release of TNF-α.In vivo animal experiment showed serum TNF-α level in anti-TLR4-C antibody group was (1.54 ± 0.18) ng/ml,significantly lower than (0.51 ± 0.10) ng/ml in antibody control group (P ＜ 0.01).Animal survival rate in anti-TLR4-C antibody group was 70％,higher than 30％ in antibody control group (P ＜ 0.05).Conclusion Anti-TLR4-C monoclonal antibodies have great capacity to neutralize TLR4 and good protective effect on LPS-induced sepsis.

Objective To obtain the recombinant corticotropin releasing hormone (CRH) protein with soluble, high purity protein through optimizing prokaryotic expression condition and purifying glutathione thiol transferase (GST)-CRH protein. Methods To detect the expression of soluble CRH protein through grope of the host strain GST-CRH temperature of induction expression, the host strain concentration (OD600), IPTG concentration and induction time, the purification of GST-CRH was performed by GST-CRH agarose gel. Western Blot assay was used for the expression identification of the target protein. Results The optimal conditions for the induction of CRH protein were determined: temperature of 30 ℃, IPTG induced concentration 0.1 mmol/L, bacteria density (OD600) 0.8, the induction time of 8 hours, purified GST-CRH>95% fusion protein was obtained. Conclusion The optimal expression conditions of GST-CRH are obtained, and the soluble protein of high purity GST-CRH is also obtained.

Objective To investigate the mechanism of oxidative damage caused by lipopolysaccharide (LPS)induced sepsis in rat brain.Methods The rats were randomly divided into control group and model group (low LPS group and high LPS group).Twenty-four hours after the modeling,the rats were sacrificed before their brain tissue was taken out and prepared for the test.The changes in malondialdehyde (MDA),superoxide dismutase (SOD),glutathione peroxidase (GSH-Px),total antioxidant capacity (T-AOC),hydrogen peroxide (H2 O2 )and succinate dehydrogenase (SDH)were detected.The expression level of JNK and Nrf2 protein in brain tissue was detected by qRT-PCR and Western blotting. Results Compared with the control group,the MDA,SOD,GSH-px,T-AOC,H2O2 and SDH level increased significantly in the model group,and the difference in expressions of JNK and Nrf2 was statistically significant (P <0.05). Conclusion The LPS induced septic oxidative brain damage model in rats is successfully established,and the process may be regulated through the Nrf2 and JNK signal pathways.

The mechanism of how stroma plays an important role in tumor carcinogenesis is now a hotspot. To delineate the features of stromal protein between nasopharyngeal carcinoma (NPC) and normal nasopharyngeal mucosa(NNM), laser capture microdissection (LCM) was performed to purify stromal cells from the NPC and NNM, respectively. The protein expressed profiles of the stroma of NPC and NNM were compared using fluorescent two-dimensional difference gel electrophoresis (2D-DIGE) and 34 differential protein spots between tumor stroma and normal stroma were chosen to be identified by mass spectrometry (MS). A total of 20 differential proteins were identified, and three differential proteins (CapG, L-plastin and S100A9) were selectively further validated by Western blotting and immunohistochemical analysis to confirm the results of 2D-DIGE. 2D-DIGE patterns of the stroma of NPC and NNM were established for the first time, the results suggested that differentially expressed proteins in the stroma of NPC and NNM may be useful for understanding the relationship between NPC cells and their surrounding microenvironment. Further studying of these proteins will be helpful to elucidate the mechanisms of NPC carcinogenesis and provide new thoughts on therapy of NPC through stroma.

Objective To explore the composition principles of prescriptions for simple obesity based on the analysis of medical literature issued in recently 30 years about Chinese medicine for simple obesity. Methods We collected literature of Chinese medicine in treating simple obesity from VIP, CNKI, Wanfang, CBM and CMCC/CMCI databases, and screened out the formulas for simple obesity to establish a formula database. And then the medication rules were figured out with the Traditional Chinese medicine Inheritance Support System version 2.5 (TCMISS 2.5) software. Results A total of 57 formulas were screened out. And the top 10 herbs with highest occurrence frequency in the prescriptions were Poria, Rhizoma Atractylodis Macrocephalae, Rhizoma Alismatis, Radix et Rhizoma Rhei, Fructus Crataegi, Radix Astragali, Radix Glycyrrhizae, Folium Nelumbinis, Pericarpium Citri Reticulatae and Semen Cassiae. After data mining, we got 13 high-frequency herb-pair combinations and 31 core combinations, and 8 new prescriptions were extracted. Conclusion The research has achieved the quantitative description of the relationship between the Chinese medicines, the extraction of the core combinations and the discovery of new Chinese medicine prescriptions for simple obesity. The results will provide references for the development of new Chinese medicines for the treatment of simple obesity .

The aging process of human colonic epithelium involves a slow decline in physiological vigor and an increasing susceptibility to age-related diseases, especially, colon cancer, but the molecular mechanisms of the aging and susceptibility of aged colonic epithelium to carcinogenesis is still unclear. Identification of aging related proteins in colonic epithelium will help to reveal the molecular mechanisms of colonic epithelial aging and age-related colonic diseases. Therefore, the total proteins of human normal colonic epithelial tissues from 10 young and 10 old men were separated by two-dimensional gel electrophoresis(2-DE), respectively. PDQuest software was applied to analyze 2-DE images, the differentially expressed protein spots of colonic epithelium between young and old groups were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS), and the expression levels of partial identified proteins were determined by real-time quantitative PCR and immunohistochemistry. Well-resolved, reproducible 2-DE maps of human colonic epithelial tissues from young and old men were established, 17 aging related proteins were identified by MALDI-TOF-MS, and the differential expression levels of partial identified proteins were confirmed by real-time quantitative PCR and immunohistochemistry. The results indicate that injury of mitochondrial function and decline of antioxidant capability are important reasons for the aging of human colonic epithelium, and four differential proteins (guanine nucleotide-binding protein beta subunit-like protein, stress-70 protein, 40 S ribosomal protein SA and chloride intracellular channel protein1) may be involved in susceptibility of aged colonic epithelium to carcinogenesis.

To optimize key enzymes, such as to explore the gene resources and to modify the expression level, can maximize metabolic pathways of target products. β-carotene is a terpenoid compound with important application value. Lycopene cyclase (CrtY) is the key enzyme in β-carotene biosynthesis pathway, catalyzing flavin adenine dinucleotide (FAD)-dependent cyclization reaction and β-carotene synthesis from lycopene precursor. We optimized lycopene cyclase (CrtY) to improve the synthesis of β-carotene and determined the effect of CrtY expression on metabolic pathways. Frist, we developed a β-carotene synthesis module by coexpressing the lycopene β-cyclase gene crtY with crtEBI module in Escherichia coli. Then we simultaneously optimized the ribosome-binding site (RBS) intensity and the species of crtY using oligo-linker mediated DNA assembly method (OLMA). Five strains with high β-carotene production capacity were screened out from the OLMA library. The β-carotene yields of these strains were up to 15.79-18.90 mg/g DCW (Dry cell weight), 65% higher than that of the original strain at shake flask level. The optimal strain CP12 was further identified and evaluated for β-carotene production at 5 L fermentation level. After process optimization, the final β-carotene yield could reach to 1.9 g/L. The results of RBS strength and metabolic intermediate analysis indicated that an appropriate expression level of CrtY could be beneficial for the function of the β-carotene synthesis module. The results of this study provide important insight into the optimization of β-carotene synthesis pathway in metabolic engineering.