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 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 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

Genotoxicity research takes an important place in traditional Chinese medicine safety evaluation. Genotoxicity test on traditional Chinese medicine has been paid great attention since 1970s. Currently, the most developed genotoxicity test methods included: bacterial reverse mutation test and mouse lymphoma assay which are used to detect relevant genetic changes, micronucleus test and chromosomal analysis which are used to measure chromosomal aberration, and single cell electrophoresis assay which is used to test DNA damage. This article reviews research progress on genotoxicity of traditional Chinese medicine, evaluation methods of genotoxicity, the problems and solutions on genotoxicity evaluation of traditional Chinese medicine, and new technique used in genotoxicity test.
Animals ; Biomedical Research ; Humans ; Medicine, Chinese Traditional ; adverse effects ; Mutagenicity Tests ; methods

Drug allergy and pseudoallergic reactions are main adverse drug reactions. Allergy is mainly induced by the immunogenicity of drug, drug metabolic products or drug additive. Pseudoallergic reactions may result from the irritation or activation of inflammatory material release. Pre-clinical evaluation of drug allergy and pseudoallergic reactions is included in immunotoxicity evaluation. Now there is no in vivo or in vitro method that could predict all kinds of allergy or pseudoallergic reactions due to the different mechanisms. In the past few years, FDA, SFDA OECD, ICH and WHO have published several guidelines on per-clinical immunotoxicity evaluation, however, no agreement has been reached on allergy and pseudoallergic reactions evaluation. This article reviews the requirements of allergy and pseudoallergic reactions in pre-clinical evaluation.
Drug Hypersensitivity ; diagnosis ; Humans ; Immune System ; drug effects ; Practice Guidelines as Topic