ObjectiveTo determine the effect of unfractionated heparin (UFH) on lipopolysaccharide (LPS)-induced expression of granulocyte colony-stimulating factor (G-CSF), and the role of Toll-like receptor 4 (TLR4) signaling pathway in this process.Methods Human pulmonary microvascular endothelial cells (HPMECs) were cultured in vitro, and the cells between passages 3 and 5 were used in the experiments. ExperimentⅠ: the cells were divided into four groups as follows: control group, LPS stimulation group (LPS 10μg/mL), LPS+ 0.1 U/mL UFH group, and LPS+ 1 U/mL UFH group. HPMECs in UFH groups were treated with 0.1 U/mL or 1 U/mL UFH 15 minutes before LPS stimulation, and HPMECs in control group were treated with an equal volume of phosphate-buffered saline (PBS) instead. The concentrations of interleukin-6 (IL-6) and G-CSF in cell culture supernatants were determined by enzyme linked immunosorbent assay (ELISA) 24 hours after LPS challenge to detect the effect of UFH on HPMECs. ExperimentⅡ: HPMECs were treated with 5μg/mL of rhodobacter sphaeroides LPS (LPS-RS, antagonist for TLR4) 4 hours before the addition of PBS or LPS. The concentrations of IL-6 and G-CSF in cell culture supernatants were determined 24 hours after LPS stimulation to detect the effect of TLR4 on LPS-induced HPMEC injury. ExperimentⅢ: HPMECs were divided into four groups as before: control group, LPS stimulation group, LPS+ 0.1 U/mL UFH group, LPS+ 1 U/mL UFH group. Treatments to cells were the same as experimentⅠ. The protein expression of TLR4 in HPMECs was determined by Western Blot 1 hour after LPS stimulation to detect the effect of UFH on TLR4.Results① Compared with control group, the levels of IL-6 and G-CSF in LPS stimulation group were increased [IL-6 (ng/L): 655.9±58.3 vs. 75.5±18.2, G-CSF (ng/L): 388.7±36.2 vs. 35.3±12.6, both P< 0.05]. Compared with those of LPS stimulation group, in LPS+ 0.1 U/mL UFH group and LPS+ 1 U/mL UFH group, the levels of IL-6 and G-CSF were significantly decreased [IL-6 (ng/L): 518.2±64.6, 489.1±75.6 vs. 655.9±58.3, G-CSF (ng/L): 298.8±41.0, 273.4±33.2 vs. 388.7±36.2, allP< 0.05]. The results indicated that 1 U/mL UFH had better results, though there was no statistical significance between the results of two UFH groups.② LPS-induced up-regulation of IL-6 and G-CSF levels was prevented by LPS-RS [IL-6 (ng/L): 139.1±37.6 vs. 655.9±58.3, G-CSF (ng/L): 73.7±19.7 vs. 388.7±36.2, bothP< 0.05]. LPS-RS alone had no effect on cytokines [IL-6 (ng/L):118.2±42.1 vs. 75.5±18.2, G-CSF (ng/L): 48.4±26.8 vs. 35.3±12.6, bothP> 0.05].③ Compared with control group, the protein expression of TLR4 (grey value) in LPS stimulation group was significantly upregulated after 1 hour (0.87±0.23 vs. 0.36±0.12,P< 0.05). UFH with 0.1 U/mL and 1 U/mL lowered TLR-4 protein expression induced by LPS (0.68±0.18, 0.62±0.26 vs. 0.87±0.23, bothP< 0.05).ConclusionsThe expressions of IL-6 and G-CSF were increased obviously in LPS treated HPMECs. UFH might take its therapeutic effect through TLR4-dependent pathway.

Objective To observe the immune regulatory effect of Marasmius androsaceus (MA) polysaccharide in normal mice. Methods The experimental mouse was injected with high,middle,and low dosages of marasmius androsaceus polysaccharide(5,10,20 mg?kg-1?d-1) in the abdomen (i.p.) ,and the comparison group was given the same volume of saline water i.p. once a day. After treatment for 14 consecutive days,the blood cell count instrument was used to measure the mice hemogram,the phagocytic function of peritoneal macrophage and the ability of carbon granular clearance were observed.,the ability of the spleen lymphocytes proliferation was tested by MTT,and the hemolysin production test was used to evaluate the influence of MA polysaccharide on specific humoral immunity in mice. Results Marasmius androsaceus polysaccharide at middle and high dosages can increase the number of lymphocytes and moncytes in normal reference,enhance the phagocytic function of peritoneal macrophage,promote the carbon granular clearance,accelerate the proliferation of T lymphocyte,enhance the specific humoral imnune function. Conclusion Marasmius androsaceus polysaccharide has certain potentiating effect on the phagocytic function and immunity regulation.

Objective To study the role of prostaglandin E1 in prevention of contrast-induced ne-phropathy (CIN)in elderly CHD patients undergoing PCI .Methods Three hundred elderly CHD patients who were going to undergo PCI in Tianjin Chest Hospital were divided into prostaglandin E1 treatment group (n=150) and conventional treatment group (n=150) .Patients in prostaglan-din E1 treatment group were treated with 20 μg prostaglandin E1 plus hydration therapy and those in conventional treatment group received simple hydration therapy .T heir serum levels of creatinine ,urea ,β2-microglobulin ,24 h proteinuria ,CRP ,IL-6 ,TNF-α,GPX ,SOD ,and creatinine clearance rate were measured before and 3 d after PCI .The incidence of CIN in two groups was analyzed .The hypotension events in prostaglandin E1 treatment group were recorded .Results The serum levels of CRP ,SOD ,GPX ,24 h proteinuria and the incidence of CIN were significantly lower while the creatinine clearance rate was significantly higher in prostaglandin E 1 treatment group than in conventional treatment group after PCI (P<0 .05) .The serum levels of CRP ,IL-6 , SOD ,GPX and 24 h proteinuria were significantly higher in two groups after PCI than before PCI (P<0 .05) .Conclusion Prostaglandin E1 can protect the renal function in CHD patients under-going PCI and play a certain role in preventing CIN .

Objective To investigate the correlation between TIP 30 and VEGF-C expression and clini-cophathological characteristics in resected small cell lung cancer ( SCLC) patients and to identify patients with in-creased risk of cancer recurrence and to provide a theoretical basis for the further clinical prevention of SCLC . Methods Sixty eight resected SCLC patients were included in this study .Paraffin-embedded specimens of pa-tients were used for the evaluation of TIP 30 and VEGF-C expression by immunohistochemistry .Results The expression of VEGF-C had positive correlation with lymph node metastasis .TIP30 expression was positively cor-related with VEGF-C expression.Patients with low TIP30 expression had shorter Overall survival (OS)and Dis-ease-Free survival(DFS)than those with high TIP30 expression.OS and DFS of the patients with VEGF -C-positive tumors were significantly lower than that of the patients with VEGF -C negative tumors .The multivariate Cox regression analysis showed that low TIP 30 and high VEGF-C expression were independent markers of poor OS(P<0.01)in operable SCLC patients.Conclusion The expression of VEGF -C shows positive correlation with lymph node metastasis .Low TIP30 and high VEGF -C expression are independent prognostic markers of poor overall survival in resected SCLC patients .

Objective To determine the expression of macrophage migration inhibitory factor (MIF)in different molecular subtypes of breast cancer and its clinical significance so as to detect the biological markers of different molecular subtypes of breast cancer.Methods We divided 100 breast cancer patients into four molecular subtypes by immunostaining:luminal subtype,HER-2(+)subtype,basal-like (BLs)subtype and normal breast-like (NBLs)subtype,and then compared the expression of MIF in the groups.We analyzed the associations of MIF-positive expression rate with age,menstruation,tumor size,auxiliary lymph node metastasis,histological type and grade,and clinical stage of the breast cancer patients.We also compared MVD level and 5-year overall survival rate between MIF-positive patients and MIF-negative ones.Results The positive expression of MIF was correlated with HER2(+)subtype breast cancer and auxiliary lymph node metastasis (P < 0.05 ).The patients with MIF-positive expression had a significantly higher level of MVD than those with MIF-negative expression (P < 0.05 ). Kaplan-Meier method showed that MIF-positive patients had a poor prognosis than MIF-negative ones (Log-rank=1 9.5 1 6,P = 0.000).Conclusion Breast cancer patients with MIF-positive expression may be mostly of HER2 (+)subtype,and tend to develop auxiliary lymph node metastasis.These patients have a significantly higher level of MVD and poor prognosis than those with MIF-negative expression.

Objective: To investigate the effect of chronic intermittent hypoxia (CIH) on liver injury and the expression of fractalkine in rats and explore its possible mechanism.
Methods: A CIH murine model was established to mimic the pathophysiology of obstructive sleep apnea-hypopnea syndrome (OSAHS) in humans. Thirty healthy male Spraque-Dawley rats were randomly assigned to 3 groups: a 5% CIH group, a 5% CIH+RH (removal of hypoxia) group and a control group ( 10 rats in each group). The 5% CIH and 5% CIH+RH groups were exposed to CIH for 3 weeks, 8 h/d, and the frequency of hypoxia was 20 times/h. The 5% CIH+RH group was then exposed to normal gaseous environment for another 3 weeks. After the experiment, liver sections were stained with hematoxylin-eosin (HE) and the liver pathology was observed. The expression of fractalkine in the liver tissues was detected by immunohistochemical method.
Results: 1) Compared with the control group, the hepatic steatosis and inflammatory activities in the 5% CIH and 5% CIH+RH groups were more severe (allP<0.01 ); compared with the 5% CIH group, the hepatic steatosis and inflammatory activity in the 5% CIH+RH group were dramatically reduced (P<0.01 ). 2) Compared with the control group, the fractalkine expression in the 5%CIH and 5% CIH+RH groups was increased (bothP<0.01). The fractalkine expression in the 5% CIH+RH group was dramatically downregulated compared with that in the 5% CIH group (P<0.01).
Conclusion: CIH can induce liver injury and high fractalkine expression in rat liver tissues.

Aim To examine the inhibitory effect of re-combinant cardiac troponin fusion protein composed of subunit I and artificial peptide which was called CIS on tumor growth. Methods The CIS ’ s effect on the growth of human umbilical vein endothelial cells ( HU-VEC) was examined using MTT assay in vitro. Chick chorioallantoic membrane model was applied to study the alteration of angiogenesis treated with purified re-combinant CIS protein. The effect of tumor growth trea-ted with CIS was observed using several in vivo mice xenograft models. Results There was a statistically significant reduction in HUVEC cell proliferative rate when the cells were treated with purified CIS fusion protein, which was also shown in a dose-dependent manner. A decreased amount of new blood vessel for-mation ( angiogenesis) on chick embryo chorioallantoic membranes was observed in recombinant CIS protein treated group compared to the untreated control group. A significant inhibition of tumor growth rate was a-chieved in CIS treated mice compared to CIS untreated control mice in 6 different mouse xenograft models. Conclusions The fusion protein CIS shows the inhibi-tory effect on the tumor growth in our in vivo mouse models, and such inhibition could be mediated by the mechanism of CIS’ s effect on the decrease of HUVEC cell proliferation and further the reduction of angiogen-esis in tumor tissues. This work could provide the foundation for the in-depth investigations on the phar-maceutical application of CIS targeting anti-tumor ther-apy.

Objective To investigate the binding protein of chlamydiaphage phiCPG1 capsid protein Vp1 on chlamydia trachomatis outer membrane.Methods The bacterium with recombinant plasmid Vp1/pet30a( + ) was induced.The expressed protein was purified by gel recycling.FarWestern blot was utilized to' investigate the binding protein of Vp1 on chlamydial outer membrane,including recombinant polymorphic outer membrane protein (rPmp) and major outer membrane protein (MOMP).Results The recombinant protein Vp1 was successfully expressed in E.coli.Monoclonal antibody against Vp1 was used as primary antibody in Western blot,and no specific band was present,which indicated that the monoclonal antibody did not specifically bind with any rPmp.Far-Western blot results showed that there was an obvious band for the rPmpI,but no specific band for other rPmp and MOMP,which suggested that Vp1 could specifically bind with rPmpI protein on the chlamydial outer membrane of serotype D.Conclusions There is a binding site of Vp1 on the chlamydia trachomatis outer membrane.Vp1 may play an important role in the interaction between the chlamydiaphage and the chlamydiae.

AIM: To observe the effect of phosphorylation protein kinase C delta (PKC?) on the procedure of PC12 cells apoptosis induced by 6-hydroxydopamine(6-OHDA) and to investigate the potential molecular pathogenesis of Parkinson disease.METHODS: TUNEL staining and transmission electron microscope were applied to measure apoptosis when dopaminergic PC12 cells exposed to the excitomotors and inhibitors of PKC before 6-OHDA for 18 hours. The expression of phosphorylation of PKC? was detected by Western blotting. RESULTS: PMA, an activating agent of PKC?, significantly increased PC12 cell apoptosis induced by 6-OHDA. Rottlerin, an inhibitor of PKC?, protected PC12 cells apparently. As contrast, bisindolylmaleimide I, an inhibitor of general PKC and G6976, the inhibitor of calcium-dependent PKC, did not show any protective role. CONCLUSION: The phosphorylation PKC? is one of the important links in the process of PC12 cell apoptosis induced by 6-OHDA. PKC? may directly participate in neurodegeneration process in parkinsonian.

Objective To study the effect of Aloe polysaccharides (AP) on the thymocytic apoptosis and cell cycle in ? - ray irradiated mice. Methods Single- cell thymocytes suspension was sampled at different time points to observe the thymocytic apoptosis and cell cycle by flow cytometry. DNA ladders were tested by 1.8 % agarose gel electrophoresis. Transmission electron microscopy was used to examine the ultrastructure of thymocytes. Results Pre- treating with AP (50 mg/kg,ip) 30 min before irradiation could significantly decrease the percentages of apoptotic thymocytes in? - ray irradiated mice 4 h, 8 h and 12 h after irradiation, increase the percentage of thymocytes at G0/G1 phase and reduce the percentage of thymocytes at G2/M phase. It could also lessen the DNA ladders and reduce the number of apoptotic bodies. Conclusion The protective effects of AP on the thymocytes in ? - ray irradiated mice is related with the alleviation of the disorder of cell cycle and the inhibition of the apoptosis of thymocytes.