Background and purpose: The morbidity of colorectal cancer in China increased year by year. This study aimed to explore the signiifcance of ADAMTS9 protein levels and promoter methylation in colorectal cancer onset and progression. Methods:ADAMTS9 promoter methylation status was detected by methylation speciifc PCR method in 162 colorectal cancer patients’ peripheral blood DNA samples; Plasmatic ADAMTS9 protein levels was detected by enzyme-linked immunosorbent assay method in 162 colorectal cancer patients and 150 healthy subjects. Results: Compared with healthy people, patients with colorectal cancer had a significant lower ADAMTS9 protein level in plasma [(65.25±9.70)μg vs (50.28±9.66)μg, P<0.001];ADAMTS9 gene promoter methylation was observed in 66 among 162 colorectal cancer patients (40.7%);The plasma level of ADAMTS9 protein in patients with methylated ADAMTS9 gene had signiifcantly reduced (P<0.001), while the plasma level of ADAMTS9 protein in patients with low ADAMTS9 protein had signiifcantly increased (P=0.007);ADAMTS9 methylation is closely related to tumor size (larger, P=0.017) and tumor differentiation degree (P=0.029), ADAMTS9 protein low expression is closely related to invasion depth (P=0.020), lymph node metastasis (P=0.019) and Dukes staging (P=0.002).Conclusion: ADAMTS9 protein downregulation induced by DNA promoter methylation may be involved in the pathogenesis, invasion and metastasis, and promote the progression in colorectal cancer.

Objective To investigate the value of single photon emission computed tomography (CT) imaging and transmis‐sion CT imaging (microSPECT‐CT ) bremsstrahlung imaging for the solid tumor mesenchymal implantaion of 32 P‐chromic phos‐phate‐paclitaxel‐poly‐L‐lactic acid (32 P‐CP‐PSP‐PLLA) sustained release seeds and to investigate the 32 P in vivo biodistribution and degradation sustained release churacteristics .Methods The animal model of prostate cancer subcutaneously transplanted tumor was established .32 P‐CP‐PSP‐PLLA sustained‐release seeds were intratumorally implanted by the mediation of microSPECT‐CT brems‐strahlung imaging and the 32 P distribution in bearing tumor mouse was verified by the imaging and biological distrubtion tests .The ultrastructural changes of 32 P seeds were observed by the scanning electron microscope .Results The MicroSPECT/CT brems‐strahlung imaging could effectively guide the intratumoral implantation operation of the 32 P sustained‐release seeds with clear visu‐alization .Partial sustained‐release 32 P was remained in the tumor tissues with little distribution in important organs of spleen and liver ,which was proved by the biodistribution results .The particle surface and inside micropores and tunnels formation ,their pro‐gressive increase ,fusion and connection were found by the electronic microscope after the 32 P sustained‐release seeds intratumoral implantation .Conclusion The MicroSPECT/CT bremsstrahlung imaging can effectively monitor the 32 P sustained‐release seeds and their in vivo biodistribution and lays a foundation for the sustained‐release seeds prostatic targeted implantation .

Objective To study the effect of specific hammerhead ribozyme (Rz) to hepatitis C virus (HCV) in vitro. Methods Rz 1 Rz 2 were designed to cleavage at 5′-NCR nucleotide positions under 136～160 and 313～337, Rz 3 was designed to cleavage at C region nucleotide position under 373～388. As a control, cleavage deficient Rzm that have A→G point mutations in the catalytic loop of the hammerhead domain. 32P-labeled transcript of target HCV RNA was incubated with gel-purified Rz ( Rz 1, Rz 2, Rz 3 and Rzm ) respectively at different concentration based on specified condition and autoradiographed after denaturing gel-electrophoresis. Results Except Rzm, Rz 1 Rz 2 Rz 3 were active at 37 ℃ and more so at higher concentration, and more so with cleavage site nearly to the HCV initial code. Conclusions The HCV specific hammerhead ribozyme can be designed in vitro, further study about cleavage in vitro and in vivo will continue.

BACKGROUND: Previous studies have found that nerve growth factors play an important role in the process of wound healing, but there is less research for the low-affinity nerve growth factor receptor p75 and sortilin in fibroblasts, and no reports on whether there are differences in expression of p75 and sortilin in the scar fibroblasts and normal skin fibroblasts. OBJECTIVE: To study the expression of low-affility nerve growth factor receptor p75 and sortilin in the normal human skin fibroblasts and the human keloid fibroblasts. METHODS: The keloid fibroblasts and normal hunman skin fibroblasts were cultured in vitro, and the immortalized epithelial cells HaCaT were used as the positive control. The real-time PCR was used to detect the mRNA expression of the p75 and sortilin in the keloid fibroblasts and normal human skin fibroblasts, and western blot and immunocytochemical staining were used to detect the protein expression of p75 and sortilin. RESULTS AND CONCLUSION: The real-time PCR and western blot results showed that in the protein and mRNA levels, p75 and sortilin showed positive expression in the keloid fibroblasts and normal human skin fibroblasts, and there was no significant difference in the expression of p75 between keloid fibroblasts and normal human skin fibroblasts, and the expressions of p75 and sortilin in the keloid fibroblasts and normal human skin fibroblasts were significantly lower than those in HaCaT. There was no significant difference of p75 expression between keloid fibroblasts and normal human skin fibroblasts, and the expression of sortilin in the keloid fibroblasts was significantly lower than that in the normal human skin fibroblasts (P < 0.05). Immunocytochemical staining result showed that the expression of p75 and sortilin in the keloid fibroblasts and normal human skin fibroblasts were distributed in the membrane and cytoplasm. Precursor nerve growth factor combined with high-affinity p75 receptor could promote the apoptosis of the cells with the help of sortilin, and the expression of sortilin in the keloid fibroblasts was significantly lower than that in the normal human skin fibroblasts, which may associated with the high proliferation of the keloid fibroblasts. The results provide a new target for the prevention and treatment of pathological scars.

BACKGROUND: Nitric oxide in the central nervous system is involved in controlling the sympathetic outflow. The authors' recent data show that the reduction of nitric oxide in the rostral ventrolateral medulla (RVLM)enhanced the cardiac sympathetic afferent reflex (CSAR) evoked by stimulating the cardiac sympathetic afferent nerves in rats with chronic heart failure (CHF).OBJECTIVE: To further investigate the effect of nitric oxide in the RVLM on modulating the CSAR evoked by epicardial chemical stimulation in rats with CHF.DESIGN: Randomized controlled experiment.SETTING: Department of Physiology, Nanjing Medical University, and Department of Cellular and Integrative Physiology, University of Nebraska College of Medicine.MATERIALS: This study was carried out in the Department of Physiology, Nanjing Medical University from July 2003 to May 2004. A total of 52male Sprague-Dawley rats weighing 360-420 g were used, and were randomly divided into chronic heart failure group and control group with 23 in each group.METHODS: The rats were carried out either sham surgery or the left coronary artery ligation. Six to eight weeks later, all rats were anesthetized with α-chloralose and urethane and baroreceptor denervated and vagotomized. The CSAR was evoked by epicardial application of bradykinin (BK, 0.04 μg and 0.4 μg in 2.0 μL) to mimic the effect of chemical stimulation on the heart in the CHF state. The renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP) and heart rate (HR) were recorded at baseline and during elicitation of the CSAR. Cannulae were inserted into the RVLM for microinjections.croinjection of MeTC, a nitric oxide synthase inhibitor, into the RVLM on Effects of epicardial pretreatment with lidocaine on the CSAR in CHF rats.infarction of (30.6±2.0) % of the left ventricular (LV) surface. The systolic arterial pressure, pulse pressure, left ventricle peak systolic pressure and maximum of the first differentiation of left ventricular pressure were decreased and the left ventricular end-diastolic pressure was significantly ininto the RVLM had no significant effects on the CSAR in rats with CHF,of SNAP (50 nmol) into the RVLM inhibited the CSAR in both sham rats ventricle abolished the CSAR evoked by epicardial application ofBK on the same area.CONCLUSION: Nitric oxide in the RVLM inhibits the CSAR evoked by epicardial application of BK in normal rats and CHF rats, and the reduction of nitric oxide in the RVLM led to the augmentation of the CSAR in CHF rats.

Objective To prepare and characterize monoclonal antibody against a malaria vaccine candidate, PfCP-2.9 chimeric protein of Plasmodium falciparum. Methods BALB/c mice were immunized with PfCP-2.9,and the spleen cells were used for fusion with SP2/0 cells. The monoclonal antibodies were analyzed by ELISA,Western blotting as well as growth inhibition assay. Result A monoclonal antibody was obtained. It interacted with the PfCP-2.9 recombinant protein by ELISA and Western blotting. The interaction of the monoclonal antibody with the protein was reduction-sensitive,indicating that the antibody recognized a conformational epitope. Moreover,the antibody also recognized the cultured parasites of P.falciparum by indirect immunofluorescent antibody test(IFA). When tested by growth inhibition assay,the antibody significantly inhibited parasite growth in vitro of 56% inhibition rate at the antibody concentration of 0.3 mg/ml. Conclusion A monoclonal antibody against PfCP-2.9 malaria vaccine candidate has been obtained,which recognizes a conformational epitope of the protein and natural protein.

Objective Study the relationship between HLA antibodies production and the number of Gravidity.Methods A total of 856 pregnant women of first pretanal in Xiasha Hospital of Sir Run Run Shaw Hospital of Zhejiang University from Nov 2016 to Mar 2017 were recruited.The blood samples were collected during 24-28 weeks of pregnancy,and the expression of platelet-specific alloantibody was detected by solid-phase red cell adherence assay.Results There were 67 positive HLA antibody in pregnant women,and the antibody positive rate was 7.8％.There were 15 positive HLA antibody in 334 first pregnant women,14 in 214 two or more pregnancy pregnant women without abortion,10 in 107 pregnant women with once abortion,28 in 223 pregnant women with two or more abortion.The antibody positive rate respectively is 4.5％,6.5％,9.3％ and 12.6％.The production of HLA antibody was significantly associated with the numberof gravidity.Conclusion The antibody positive rate of two or more pregnancy is significantly higher than the fisrt pregnancy.

ObjectiveTo investigate the effect of immunoglobulin G (IgG) extravasated from blood circulation on the expression of toll-like receptor 4 (TLR4) induced by peripheral lipopolysaccharide (LPS) in rat brain. Methods The rats were divided into four groups in random, 5 rats in each. Group one received LPS 100μg/kg by intraperitoneal administration, normal saline was given by intravenous injection 6 hours later; group two was injected with adrenalin (AD) 15μg/kg intravenously; group three was treated with LPS intraperitoneally, AD was injected 6 hours later; group four was injected normal saline intravenously as control. For all groups, the animals were sacrificed 30 min after the last injection, and the brains were taken for investigation of the TLR4 expressions by immunofluorescence staining and RT-PCR. Result Immunofluorescence staining showed that IgG immunoreactive product was patch-like, distributed in the brain parenchyma in all the animals that received AD. In the LPS+normal saline group, IgG was found merely around the blood vessels. Meanwhile, in LPS+AD animals, TLR4 immunoreactive product coexisted with microglia marker Iba-1 within the IgG extravasated area. The double-labeled cells dispersed in the brain parenchyma and near to the cerebral vessels. In the LPS+saline group, TLR4 positive cells were endothelial-like. RT-PCR results indicated that the expression level of TLR4 in the LPS+AD group were significantly higher than that in the LPS+saline group or AD group or the saline control (P<0.01). Conclusion Extravasated circulating IgG may enhance the TLR4 expression in the rat brain induced by peripheral LPS.

Amino Acids ; urine ; Animals ; Biogenic Monoamines ; metabolism ; urine ; Male ; Microwaves ; Rats ; Rats, Wistar