Objective To investigate the effects of a microRNA family member , let-7e, on mono-cytic cell line THP-1 with regard to cell apoptosis and cytokine secretion and to analyze the possible mecha -nism.Methods THP-1 cells were transfected with mimic negative control (cy3) and observed with immu-nofluorescence microscopy for the evaluation of transfection rate .The expression of let-7e in THP-1 cells re-spectively transfected with let-7e mimic, mimic negative control, let-7e inhibitor and inhibitor negative con-trol were detected by qRT-PCR.MTT assay and flow cytometry analysis were used to detect the activities and apoptosis of transfected THP-1 cells.Western blot assay was performed to measure the expression of the genes encoding interferon alpha-inducible protein 6( IFI6 ) , enhancer of zeste homolog 2 (EZH 2 ) and caspase -3 that were target genes of let-7e predicted by bioinformatics analysis .THP-1 cells were transfected with let-7e mimic and mimic negative control for 48 h and then stimulated with LPS for 2 h for further detec-tion.The supernatants of cell culture were collected for the detection of secreted cytokines by Human Cyto -kine Array.Results The monocytic THP-1 cells were transfected with mimic negative control with a trans-fection efficiency of about 75%.There were 8.551±0.365, 83.893±15.941, 38.858±2.743 and 0.594± 0.174, 2.427±1.229, 3.053±0.207 fold increases in let-7e expression after the transient transfection of THP-1 cells with let-7e mimic and let-7e inhibitor for 12 h, 24 h and 48 h, respectively.The transfection of let-7e mimic into THP-1 cells enhanced the cell activities and inhibited the apoptosis of the transfected cells . Bioinformatics analysis showed that let-7e bound to the genes encoding EZH 2, IFI6 and caspase-3 with the mirSVR scores of -0.1608,-0.5693 and-0.9423, suggesting them as the predicted target genes of let-7e. The expressions of IFI6, EZH2 and caspase-3 in let-7e mimic transfected THP-1 cells were decreased as in-dicated by Western blot assay .The results of Human Cytokine Array showed that the expression of LPS-in-duced cytokines including CD154, G-CSF, CD54, IL-13, IL-1RA and IL-23 were inhibited in let-7e mimic transfected THP-1 cells. Con clusion Let-7e had an anti-apoptosis effect on monocytic THP-1 cells and in-fluenced the secretion of LPS-induced cytokines in THP-1 cells.Let-7e might regulate the biological function of THP-1 cells through inhibiting the expression of target genes encoding caspase -3, IFI6 and EZH2.

Objective To study the effects of a miRNA family member,let-7e,on the LPS-induced expression of TNF-α in primary monocytes and the possible mechanism.Methods Peripheral blood mononuclear cells (PBMCs) were isolated form human blood sample by using density gradient centrifugation for further isolation of primary monocytes.Flow cytometry analysis was used to measure the purity of isolated primary monocytes.The efficiencies of transfection were evaluated by qRT-PCR and immunofluorescence assay after transfecting the primary monocytes with let-7e mimic or miRNA mimic negative control (NC) for 24 h,36 h and 48 h.To screen out the optimal stimulation time,ELISA was performed to detect the concentrations of TNF-α in the supernatants of cell culture after stimulating the primary monocytes with 1 mg/L of LPS for 0 h,1 h,3 h,6 h and 12 h,respectively.ELISA and qRT-PCR were used to measure the expression of TNF-α in the transfected cells with the interference of LPS.Western blot assay was used to detect the level of enhancer of zeste homolog 2 (EZH2) in let-7e mimic-transfected primary monocytes and the levels of NF-κB p65,ADP-ribosylation factor GTPase-activating protein 1 (ARFGAP1) and Arfaptin2 in the siEZH2-transfected monocytes.Results More than 70％ of the isolated cells were CD14+ cells.The miRNA mimics could transfect the primary monocytes effectively and the transfection rate was about 70％.High levels of let7e were detected in let-7e mimic-transfected primary monocytes 24 hours after the transfection.High levels of TNF-α were observed in the primary monocytes after stimulated with LPS for 12 h,which was considered as the optimal LPS stimulation time.Results of the ELISA indicated that let-7e mimic significantly inhibited the LPS-induced expression of TNF-α in primary monocytes at both mRNA and protein levels.Western blot assay showed that the levels of EZH2 in the let-7e mimic transfected primary monocytes were significantly lower than that in mimic NC transfected primary monocytes.Silenced expression of EZH2 significantly inhibited the expression of NF-κB p65 in nucleus as well as the expression of ARFGAP1 and Arfaptin2.Conclusion let-7e mimic significantly inhibited the LPS-induced expression of TNF-α in primary monocytes.It is possible that Let-7e regulates the expression of NF-κB p65,ARFGAP1 and Arfaptin2 by targeting EZH2 directly to inhibit the expression of TNF-α.

Objective To investigate the effects of a miRNA family member, let-7e, and a combi-nation of miR-106b and miR-20a on the expression of cytokines by THP-1 cells with Luminex xMAP technol-ogy.Methods The efficiency of transfection was evaluated by immunofluorescence assay after transfecting THP-1 cells with micrONTM mimic negative control (Cy3) for 24 h, 36 h and 48 h.The three miRNA mim-ics (let-7e, miR-106b and miR-20a) were respectively used to transfect the THP-1 cells for 24 h, 36 h and 48 h and the expression of each miRNA was analyzed by qRT-PCR analysis for screening out the optimal transfection time.The transfected THP-1 cells were stimulated with1 mg/L of LPS for 1 h.The Luminex xMAP technology was used to detect the expression of IL-8, interferon-inducible protein-10 (IP-10), mono-cyte chemotactic protein 1 (MCP-1), IL-1α, IL-6, IL-10, TNF-α, IFN-αand IFN-βin the supernatants of cell culture.A statistical analysis was performed to analyze the data obtained by using SPSS16.0 software. Results More than 90% of the transfected THP-1 cells were labeled with red fluorescence.The optimal transfection times for let-7e mimic and miR-106b/miR-20a mimics were 48 h and 24 h, respectively.Com-pared with the corresponding negative control (NC), the expression of IL-8, IP-10 and MCP-1 by THP-1 cells were enhanced after the transfection with let-7e mimic, but were inhibited after the co-transfection with miR-106b and miR-20a mimics.Conclusion The expression of IL-8, IP-10 and MCP-1 were enhanced in let-7e transfected THP-1 cells, but were inhibited in miR-106b and miR-20a co-transfected THP-1 cells.

Objective To explore the role of plasminogen activator inhibitor-1(PAI-1)in development of progressive fibrosis via the inhibition of extracellular matrix degradation,and to reveal the contributive role of PAI-1 in muscular dystrophy(MD).Methods Expression and cellular localization of PAI-1 protein were examined in frozen muscle specimens obtained via biopsy from 5 patients with duchenne muscular dystrophy(DMD),3 patients with becker muscular dystrophy(BMD),9 patients with congenital muscular dystrophy(CMD) and 4 cases with normal muscle by immunohistochemistry,double immunofluorescence and Western-blot analysis.Results PAI-1 was positive only in vascular endothelial cells of normal muscle.Both immunohistochemistry and Western-blot analysis showed that PAI-1 expression distinctly increased in most dystrophic muscles of MD than that in normal muscles.Double immunolabeling revealed that PAI-1 strongly expressed in cytoplasm and nuclei of regenerating muscle fibers,macrophages,macrophage infiltrating necrotic fibers.Some activated fibroblasts in endomysium and perimysium of DMD and CMD muscles were positive for PAI-1.Conclusions The functional consequence of overexpression of PAI-1 in dystrophic muscles is unknown but the elevated local expression of PAI-1 in diseased muscles of MD and their distinct distribution pattern provide evidence that PAI-1 participate in pathogenesis of MD.

Objective To explore the high risk factors, pathogenesis and methods of early diagnosis of periventricular leukomalacia(PVL) in premature infants.Methods The history of intrauterine hypoxia-ischemia was investigated in premature infants;TORCH-IgM antibodies in cord blood of premature infants were measured by ELISA; Nitric oxide (NO) levels in their cord blood were determined by nitric acid reducing enzyme means. Results Thirty-nine of 52 premature infants in PVL group had a history of intrauterine hypoxia-ischemia; TORCH-IgM antibody positive rate in cord blood of premature infants in PVL group was significantly higher than that in control group(P

Objective To analyze the characteristics and some related influences of interictal epileptiform discharges (IEDs) for symptomatic temporal lobe epilepsy(TLE) and extratemporal lobe epilepsy(ETLE) patients in whom with focal lesion on structural imaging .Methods The electrophysiological and clinical data of 257 epilepsy patients were retrospectively analyzed , all of whom had a focal lesion revealed by structural imaging .Patients were divided into 2 groups according to the locaton of the lesion:TLE group and ETLE group , patients were also divided into 3 subgroups according to the relationship between the location of IEDs and the lesion :TLE-1/ETLE-1 subgroup with a norm interictal EEG; TLE-2/ETLE-2 subgroup with IEDs absolutely located in brain lobes in which lesion located;TLE-3/ETLE-3 subgroup with IEDs exceed or absolutely not located in the lobes in which lesion located . Results The proportion of TLE-1 was significantly lower than ETLE-1 ( P<0.01 ) , while the proportion of TLE-2 was significantly higher than ETLE-2(P<0.01).The proportion of the TLE-3 subgroup increased along with a longer duration, and the proportion of ETLE-3 subgroup decreased along with a lower seizure frequency , and also the older the age at onset .Conclusions The positive rate of IEDs and its positioning accuracy are significantly higher in symptomatic TLE than that in ETLE patients .The distribution of IEDs is more likely to be affected by epilepsy duration in TLE , while it is more easily to be affected by seizure frequency and age at onset in ETLE .

OBJECTIVETo compare the differences of expressions of adenylyl cyclase (AC) and phosphodiesterase (PDE) in ejaculated spermatozoa between healthy volunteers and the patients with asthenospermia.

METHODSEjaculated spermatozoa were collected from healthy volunteers and the patients with asthenospermia. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect mRNA expression of AC and PDE subtypes in human spermatozoa. The concentrations of cAMP and cGMP in the samples were detected by enzyme-linked immunosorbent assay (ELISA).

RESULTSCompared with healthy volunteers, expression of sAC mRNA and concentration of cAMP were significantly decreased in the patients with asthenospermia (P < 0.01) , while the expression of PDE4C mRNA was significantly increased at the same time (P <0.01). There were no marked differences in the expression of ACIII mRNA and concentration of cGMP between the two groups.

CONCLUSIONThe sAC down-regulation and PDE4C up-regulation are possible reasons for asthenospermia.

Adenylyl Cyclases ; biosynthesis ; Asthenozoospermia ; metabolism ; Cyclic AMP ; metabolism ; Humans ; Male ; Phosphoric Diester Hydrolases ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction ; Spermatozoa ; metabolism

OBJECTIVETo analyze the penetrance of Leber hereditary optic neuropathy (LHON) individuals with mitochondrial DNA 11778 mutation in Shanxi.

METHODSAllele-specific PCR was used to detect mtDNA 11778 mutation in LHON patients and their families.

RESULTSIn 17 families of the 30 families that harbored mtDNA 11778 mutation, only the probands were LHON patients. In the other 13 families, besides the probands, 72 maternal relatives carried mtDNA 11778 mutation.

CONCLUSIONThe penetrance of LHON individuals with mtDNA 11778 mutation in the Shanxi area is 55.6%.

Adolescent ; Adult ; Child ; DNA, Mitochondrial ; genetics ; Female ; Humans ; Male ; Middle Aged ; Mutation ; Optic Atrophy, Hereditary, Leber ; genetics ; Penetrance

OBJECTIVETo investigate the expression of occludin, ZO-1, MMP-2, and MMP-9 in cerebral microvasculature following Pulse Electromagnetic Field (PEMF) induced BBB permeability change.

METHODSSprague-Dawley rats were randomized into PEMF and sham exposed groups (n = 8). After exposure to PEMF at 0.5, 1, 3, 6, and 12 h, BBB permeability was measured by Evans-Blue extravasation. The expression of occludin, ZO-1, MMP-2, and MMP-9 were detected by real-time quantitative reverse transcriptase PCR and western blotting. MMP-2 and MMP-9 activity were detected by EnzChek gelatinase assay.

RESULTSCompared with the sham group, PEMF exposure led to increased permeability of the BBB to EB, which was prolonged after exposure. BBB permeability became progressively more severe, and recovered at 6 h. The gene and protein expression of occludin and ZO-1 were significantly decreased, while MMP-2 and MMP-9 expression were significantly increased after exposure to PEMF. All levels of expression recovered 12 h following PEMF.

CONCLUSIONChanges to BBB permeability were related to the alteration expression of tight junction proteins and matrix metalloproteinase after exposure to PEMF.

Animals ; Blood-Brain Barrier ; Electromagnetic Fields ; Male ; Matrix Metalloproteinases ; metabolism ; Proteins ; metabolism ; Rats ; Rats, Sprague-Dawley ; Tight Junctions ; metabolism

BACKGROUNDCholecystokinin (CCK) is one of the richest neuropeptides in the mammalian brain, which is mainly distributed in the cerebral cortex, hippocampus, thalamus and caudate-putamen. CCK is implicated in a variety of behavioral functions such as food intake, learning, memory, anxiety, pain and neuroprotection. The current research results for CCK are obtained mainly through injection of CCK peptide into the body. The key issues of whether CCK can regulate diet by a central pathway and whether there are long-term regulation effects on diet are still unresolved. In this study, the effects of CCK on food intake in transgenic mice were investigated.

METHODSTransgenic mice were created by microinjection of the PDGF-CCK construct into male pronucleus of the zygotes. The genomic phonetype of transgenic mice were identified by PCR. The expression of PDGF-CCK was analyzed by Western blotting. Body weight, plasma glucose, cholesterol and triglycerides were assayed and analyzed.

RESULTSTwo PDGF-CCK transgenic independent lines were established and exhibited a high-levels brain-specific transgene expression compared with that of nontransgenic littermate controls. The food intake of male CCK transgenic mice was decreased by 5% - 10% with the same levels of water consumed compared with wild type mice. The food intake in female mice was not obviously changed. In the transgenic mice the bodyweight was lower and plasma glucose was higher compared with the nontransgenic littermate controls.

CONCLUSIONSThe high expression of the CCK gene in the brain can decrease body weight and increase plasma glucose. The differences in food intake between the males and females require further study.

Animals ; Blood Glucose ; genetics ; physiology ; Blotting, Western ; Body Weight ; genetics ; physiology ; Brain ; metabolism ; Cholecystokinin ; genetics ; metabolism ; Cholesterol ; blood ; Eating ; genetics ; Female ; Lipase ; blood ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic