Via activating G-protein receptor transduction pathways,nitric oxide (NO),an important signal molecule,has a complex and diverse role which is closely related with activity and gene expression of nitric oxide synthetase (NOS).NO is involved in tumor related events such as cellular proliferation,migration,especially the angiogenic process,and it is closely related with the occurrence and progression of prostate cancer.NO donors and NOS inhibitors have anti-cancer and radio-chemotherapy enhancement roles.

Peroxisome proliferation-activated receptor-γ (PPAR-γ) is a ligand-activated transcription factor belonging to the nuclear hormone receptor superfamily and participates in the regulation of various metabolic pathways as well as inflammatory responses. PPAR-γ ligands significantly improve myocardial functional recovery and prevent ischemia-reperfusion induced injury. Given the increasing understanding of the cardioprotective effects of PPAR-γ ligands, we know today that the therapeutic effects of PPAR-γ ligands reach far beyond their use as insulin-sensitizers, as many of these agents exert beneficial effects in the conditions associated with ischemia-reperfusion and inflammation.

Peroxisome proliferation-activated receptor-? (PPAR-?) is a ligand-activated transcription factor belonging to the nuclear hormone receptor superfamily and participates in the regulation of various metabolic pathways as well as inflammatory responses. PPAR-? ligands significantly improve myocardial functional recovery and prevent ischemia-reperfusion induced injury. Given the increasing understanding of the cardioprotective effects of PPAR-? ligands,we know today that the therapeutic effects of PPAR-? ligands reach far beyond their use as insulin-sensitizers,as many of these agents exert beneficial effects in the conditions associated with ischemia-reperfusion and inflammation.

Pericytes are a very important cellular constituent of the blood-brain barrier.They play a regulatory role in brain angiogenesis,endothelial cell tight junction formation,blood-brain barrier differentiation,microvascular dynamic motion and structural stability.Pericytes exhibit unique functional characteristics in some diseases,such as cerebrovascular disease,neurodegenerative disease,neuroimmune disease and traumatic brain injury.This article reviews the roles of pericytes in the blood-brain barrier.

Objective To isolate CESs by dynabeads coated with the specific antibody of CD146 from peripheral blood and its origin was identified.Methods One mL blood with or without admixed HUVECs was diluted(1∶2) in PBS-0.1% BSA.Anti-CD146-coated Dynabeads were added and incubated for 30mins at(4 ℃).Cells bound to anti-CD146-coupled beads were separated from blood in Dynal MPC and then washed and resuspended in(4 mL) buffer.After 4 additional washes with PBS-0.1% BSA using the magnet,the rosetted cells were flushed from the tube wall with (100 ?L) of PBS-BSA with acridine orange or Giemsa,and counted in a hemocytometer in a inverse phase contrast fluorescence microscope.Results The amount of CECs in healthy adult was 10.5(6～16.5)/mL(n=42).The recovery rate was 91%.Isolated CECs were confirmed by positive expression of vWF and CD31.Conclusion Isolation of CECs from blood by immunomagnetic beads coated with antibody of CD146 is characterized by its accuracy,time-saving,high recovery rate,less contaimination of blood and less damage to CECs.It can be used to quantify CECs and to analyze the functional performance of CECs from patients with vascular injury diseases.

Objective To investigate the functions of Monocyte chemotactic protein-induced protein 1 (MCPIP1) in human breast cancer cell line MDA-MB-231.Methods MDA-MB-231 cells were transfected with GFP-tagged MCPIP1 by Tet-on inducing expression system.Endogenous MCPIP1 was knocked down by stable expressing shRNA.MTT assay was performed to measure the growth of MDA-MB-231 cells after overexpression or knockdown of MCPIP1.FACS method was used to analyze cell cycle in MDA-MB-231 cells.Real-time PCR was used to test the expression of cell cycle-related mRNAs expression and their half-lives.RNA-IP experiment was conducted to detect the mRNA directly enriched by MCPIP1.Luciferase assay was performed to determine whether the mRNA decay was mediated through 3′UTR.Results MCPIP1 overexpression significantly inhibited cell proliferation(P<0.05), while knockdown MCPIP1 promoted cell proliferation with statistical significances (P<0.05).MCPIP1 induced cell cycle arrest in MDA-MB-231 with statistical significance (P<0.01).MCPIP1 overexpression reduced the half-lives of cell cycle mRNAs (CDK2,CDK6,cyclin D1,cyclin E1,respectively) with significance (P<0.01).In addition, cell cycle-related mRNAs were able to be pulled down by GFP-MCPIP1 but not isotype IgG(P<0.05).Compared with control vector, MCPIP1 significant suppressed luciferase activities of all four 3′UTR reporters (P<0.05).Conclusions MCPIP1 functions as a tumor suppressor in human breast cancer cell line MDA-MB-231 through inducing G1 cell cycle arrest.

Objective To explore the method of primary isolation, cultivation and identification of rat brain microvessel pericytes. Methods The brain tissue of 10 3 week-old Wistar rats was separated sterilely. The brain microvessel fragments were separated using two-step enzyme digestion and one-step gradient centrifugation and were seeded in 35-mm dishes for primary culture. The cell morphology was observed by phase contrast microscopy; the immunofluorescence assay was used to identify the associated antigns, such as the α-smooth muscle actin (α-SMA), neuron-glial antigen 2 (NG2), von Willebrand factor (vWF), and glial fibrillary acidic protein (GFAP). Methyl thiazolyl tetrazolium was used to determine the cell growth curve. Results Pericytes climbed out from the adherent brain microvascular fragments around,showing polygonal, and the cell fusion was 95％ after 12-14 days. Immunofluorescence staining revealed that the molecular markers of the pericytes α-SMA and NG2 related antigens showed double positive, while the vWF and GFAP related antigens showed double negative and the cultured cells were confirmed as brain microvascular pericytes. The growth rate of primary cells was slower. The passage cells entered into logarithmic growth phase after 36 to 60 hours and entered into plateau phase after 72 to 108 hours. Conclusions This method may successfully isolate rat brain microvascular pericytes with higher purity.

Objective To construct pcDNA3-HIF-1? eukaryotic expression vector and to investigate its function in breast cancer MCF-7 cells.Methods RT-PCR was applied to amplify human HIF-1? cDNA from MCF-7 cells with a pair of sequence specific primers carrying a restriction enzyme site XbaⅠ or HindⅢ on each 5′end.HIF-1?(cDNA) was inserted into pMD18-T after sequencing,and then inserted into pcDNA3 vector.The recombinant vector was transfected into MCF-7 cells to observe its expression and function by Western blot and dual-luciferase reporter gene assay.Realtime-PCR was performed to detect the inducible expression of C-MET mRNA by HIF-1?,the anti-apoptotic effect of HIF-1 under hypoxia was analysed by detecting the Caspase3/7 activity.Results The sequence of pcDNA3-HIF-1? is identical with the gene bank.It enhances the expression of HRE reporter gene and C-MET mRNA,but decreases the Caspase3/7 activity in MCF-7 cells under hypoxia.Conclusion The pcDNA3-HIF-1? eukaryotic expression vector was successfully constructed,it not only has strong DNA binding and inducing activity,but also has anti-apoptotic effect in hypoxia MCF-7 cells.

Microcirculation dysfunction is involved in the onset of diabetes and its complications.The pathophysiological mechanisms behind the relationship between microcirculation and diabetes are multifactorial.Islet microcirculation dysfunction affects function of islet β cells.Impairment of microvascular vasomotion might be associated with insulin resistance.Microcirculation dysfunction of target organs mediates diabetic complications.

Objective To construct pGL3-EPO-HRE reporter gene vector and to detect its function.Methods To synthesize two single-strand DNA fragments which had overlap sequence at the 3'end,then mixed them to construct the double-strand DNA fragment which include three copies of(57 bp) human erythropoietin(EPO) HRE.There was MluⅠor BglⅡ digestive site at the end of this DNA fragment.After inserting the(87 bp) fragment into pMD18-T for sequencing,the correct sequence was inserted into pGL3-promoter to construct pGL3-EPO-HRE.The recombinant vector was co-transfected into MCF-7 cells with pcDNA3-HIF-1?or pcDNA3 respectively,(12 h) later cells were treated with(100 ?mol/L) cobalt chloride(CoCl_(2)) for(24 h), then the expression of pGL3-EPO-HRE was observed by dual-luciferase reporter gene assay method.Results The sequence of pGL3-EPO-HRE was identical with human EPO HRE sequence.pGL3-EPO-HRE luciferase activity increased about 10 fold(P