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Objective:To construct a lentiviral vector overexpression of micrRNA-29b and investigate the biological characteristics in mouse neuronal cell lines GT1-7.Methods:We chemically synthesized two oligonucleotide single-stranded,complete the comple-mentary by bridging extension into DNA double-stranded to form miR-29b precursor structure.The restriction enzyme digested vector plasmid FUGW was ligated to the precursor structure ofmiR-29b by homologous recombination to construct the corresponding lentiviral vector of microRNA-29b overexpression,and the stable cells were obtained in the mouse neuronal cell line GT1-7 by bleomycin drag screening.RT-PCR was used to detect the expression level of related genes at mRNA transcription level,Results:The recombinant lentiviral expression plasmid f-F-miR-29b was successfully constructed,and the expression level was about 30 times higher than that of the control group.The expressions of DCX,Vdac1 and pten were inhibited,have no changes in sex developmental related genes LH-β,kiss-l,Inshulin,IGF-I,GPR54,GnRH and leptin-R.Conclusion:Using the method of lentivirus screening,the microRNA-29b overexpressing stably transformed cells was successfully obtained in mouse neuron GT1-7 cells,which laid a foundation for the study of biological characteristics ofmicroRNA-29b.
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Objective Deletion detection and annotation of 18 lines from the population of specific chromosome 1 substitution strains ( PCSSs) derived from Chinese wild mice based on whole genome re-sequencing data. Methods Whole genome re?sequencing of the 18 lines were performed on the Illumina Hiseq platform. SpeedSeq software was used to detect the deletion after read alignment. Further annotation was obtained using SnpEff software. Results 13803 dele?tions were identified among the 18 lines, the length of deletion was ranged from 51bp to 70 kb, among them nearly 50%were less than 500 bp. Through functional annotation,we found most of the variants were located in intronic (50. 361%) and intergenic (28. 745%) regions. However, we also identified 31 protein coding genes harboring loss?of?function dele?tions. Among them, 3 genes were associated with human diseases, 7 genes were participated in 11 KEGG pathways. Conclusion The chromosome 1 of PCSSs harbors abundant deletion mutations which can be used as genetic markers in genetic studies.
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Objective To explore the potential changes of connexin Cx36 in hippocampus and cortical neurons of rats with hyperthermia -induced convulsion.Methods Rats were divided into 2 groups according to the random number table method:normal control group and experimental group.Febrile convulsion model was elicited through im-mersion in warm water.The experimental group was generated following febrile convulsion model:hyperthermia group and febrile convulsion group.Among normal control group,hyperthermia group and febrile convulsion group,western blot analysis and immunofluorescence labeling techniques were used to examine the expression of Cx36 protein in the hippo-campus and cortex area.One -Way ANOVA was used to compare the mean of multiple sample,the LSD test was used to compare the two means.Tamhane′s test was used when variance were uneven.Results The incubation period,seizure duration and temperature were (4.39 ±0.08)min,(5.38 ±0.07)min,(41 .87 ±0.06)℃ after hyperthermia-in-duced convulsion,respectively.Western blot analysis showed that the expression of Cx36 protein in the hippocampus and cortex area decreased gradually after 1 0 times of seizure in normal control group,hyperthermia group and febrile convulsion group,and the febrile convulsion group decreased most obviously.Compared with normal control group and hyperthermia group,respectively,in febrile convulsion group Cx36 expression obviously decreased in the hippocampus and cortex in rats with 1 ,5,1 0 seizure times induced by hyperthermia,and with the increase of number of induced con-vulsion,the expression of Cx36 was significantly decreased in the cortex (0.1 04 ± 0.01 2)and CA1 (0.091 ± 0.01 1 ),CA3 (0.090 ±0.01 1 )and DG (0.092 ±0.01 2)areas of hippocampal neurons compared with the normal control group (0.21 2 ±0.01 7,0.1 67 ±0.01 3,0.1 59 ±0.01 4,0.1 71 ±0.01 3)and the hyperthermia group (0.1 89 ± 0.006,0.1 44 ±0.008,0.1 29 ±0.005,0.1 65 ±0.01 1 )(all P <0.05).Furthermore,the extent of reduction in Cx36 expression seemed to correlate with the number of seizures.Conclusion With the increase of thermal seizure frequen-cy,Cx36 expression of rats was decreased obviously which may lower convulsion threshold and lead to recurrent seizures.
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Objective To establish a rapid SNP( single-nucleotide polymorphism) genetic identification method for the frozen samples, such as frozen embryos and sperm of inbred mice.Methods In this study, the frozen embryos and sperm of inbred mice were provided by Shanghai Lab.Animal Research Center.Whole genome amplification and PCR-LDR genotyping system were used to get the rich DNA sample.Forty-five SNP were genotyped by multiple polymerase chain re-action and ligase detection reaction( PCR-LDR) .Results The electrophoresis results showed that the whole genome am-plification technique could highly increase the total DNA of frozen embryos.PCR-LDR typing method was suitable for the mouse genome typing of 45 SNPs.Ten strains of inbred frozen embryos and sperms of C57BL/6, BALB/c, FVB/NJ mice were genotyping identified, and their SNP loci data obtained by PCR-LDR were as the same as those of database.The num-ber of frozen mouse embryos was proportional to the number of SNPs detected, and when the embryo number reached more than 12, the detection rate of SNP was 100%.Conclusions This method can be used to the genetic quality identification, and rapidly identify the inbreed frozen mouse embryos and sperms.
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Objective To analyze the growth phenotype and blood biochemical parameters of chromosome 1 substi-tution mouse strain(CSS1), and investigate their potential of QTL mapping .Methods Body weight, body length, tail length, organ weight of the CCS1 mice were measured at different days to create a growth curve while blood biochemical in -dexes were measured at about the 80th day.Results The CCS1 mice were different from C57BL/6 mice in several inde-xes.Compared with the C57BL/6 mice during different developmental stages , six strains including B6-Chr1KM mice were significantly different in body weight .There were five strains including B6-Chr1CM mice significantly different with C57BL/6 mice in body length, and all of the CSS1 mice were significantly different from C57BL/6 mice in tail length.Part of CCS1 mice were significantly different from C57BL/6 mice in the weight of liver, spleen, kidney and brain.The ALT of female B6-Chr1CM mice was significantly higher than that in the C 57BL/6 mice.The ALP of female B6-Chr1HZ mice was signifi-cantly higher than that in the male C57BL/6 and B6-Chr1KM mice, and was significantly lower than that in the C57BL/6 mice.The TB of male B6-Chr1CM, B6-Chr1SMX and B6-Chr1HZ mice was significantly higher than that of the C 57BL/6 mice.The TG of male B6-Chr1SMX mice and male B6-Chr1TW mice was significantly higher than that in the C 57BL/6 mice. Conclusions The phenotype of Chr1 CSS mice is quite different from commonly used inbred strain C 57BL/6 mice.CCS1 mice show great potential in QTL mapping for their characteristic growth phenotype and blood biochemical indexes .
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Objective To establish a high throughput general multiple competitive polymerase chain reaction ( cPCR) detecting method of copy number variations ( CNVs) for the population of chromosome 1 substitution strains from wild mice.Method The selected 14 loci, including 11 CNVs on chromosome 1 and internal control loci on other three chromosmes (Chr 7, Chr 19 and Chr X), were detected based on the universal fluorescent primer multiple competitive pol-ymerase chain reaction.All specific cloned plasmids were constructed as competitors.Results Altogether 11 CNVs were designed in one panel, and the copy of Chr X accurately reflects the gender.Conclusions A rapid and high-throughput fluorescent multiplex cPCR assay is established which can be used for detection of copy number variations on chromosome 1 in mice.
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Purpose Polymorphisms of candidate gene Agouti was analyzed in order to reveal the molecular mech -anisms of coat color difference in chromosome engineering mice .Methods Firstly, differences of mouse coat color was detected by color measurement spectrophotometer .Then, candidate gene Agouti was found by whole genome scanning based on DNA chip.Finally, cDNA and amino acid sequence polymorphisms were analyzed , as well as the influence of protein properties and function after mutation was predicted by bioinformatics software .Results There are five SNPs in the Agou-ti cDNA sequences , resulting in three missense mutations in the amino acid sequence of Agouti signaling protein .Bioinfor-matics analysis revealed that one βsheet deletion in the secondary structure of the mutant protein , as well as tertiary struc-ture changed , leading to decrease of binding ability .Conclusion A novel missense mutation is found in candidate Agouti gene.It plays critical role in receptor binding activity , and may reflect on mice coat color changing from light gray to dark gray eventually .
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Objective To examine the expression of follistatin-like protein 1 (FSTL1) in the peripheral blood and intestinal mucosa tissues of ulcerative colitis (UC) patients and analyze the correlation between its expression and the activity of UC.Methods From October 2010 to June 2012,sixty patients with UC were collected.From April 2012 to October 2012,thirty individuals without any obvious mucosa lesion under colonoscope and confirmed by pathological examination were set as control group.The serum expression level of FSTL1 of both UC group and control group were determined by enzyme linked immunosorbent assay (ELISA).t-test was performed for comparison between groups.The expression of FSTL1 in the intestinal mucosa of UC group and control group was detected by immunohistochemistry.Chi-square test was used for comparison between groups.The patients with UC were scored with ulcerative colitis disease activity index (UCDAI).Its correlation with plasma FSTL1 was analyzed by Pearson correlation coefficient.Results The serum expression level of FSTL1 of UC group ((14.37-±-1.80) μg/L) was higher than that of control group ((5.80±0.72) μg/L)and the difference was statistically significant (t=25.01,P＜ 0.05).The serum expression level of FSTL1 of UC group was positively correlated with UCDAI (r=0.814,P＜0.05).The positive expression rate of FSTL1 in the intestinal mucosa tissues of UC group (86.7％,52/60)was higher than that of control group (46.7％,14/30) and the difference was statistically significant (x2 =52.334,P＜0.05).Conclusions The expression of FSTL1 of UC patients increases and is positively correlated with disease activity.FSTL1 may play a role in the development of UC.
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Objective To investigate the effect of nitrate on acute experimental colitis in mice.Methods A total of 40 BALB/c mice were evenly divided into model group and treatment group.Model group were fed with 4％ dextran sulfate sodium (DSS) solution and treatment group were given 4％ DSS solution and nitrate (1.5 g/L) for seven days.The disease activity index (DAI) of mice was scored.The colon tissue of mice was taken for hematoxylin-eosin staining and myeloperoxidase (MPO)immunohistochemical staining observation.The MPO and activity of nitric oxide in colon tissue were measured by MPO and nitric oxide detecting kit.The data were analyzed by t test.Results At the 6th day and 7th day,the difference of DAI between treatment group and model group was statistically significant (t=5.12 and 6.72,P=0.012 and 0.008).At the 7th day,the tissue score of model group (2.5±0.5) was higher than that of treatment group (1.9±0.4) and the difference was statistically significant (t=3.82,P＜ 0.01).Compared with model group,the histopathological injury of colon tissue in treatment group mice significantly reduced and neutrophil infiltration also decreased.At the 7th day,the concentration of MPO,NO2-and NO3-of model group was (2.8±0.6) U/g,(10.4±4.3) mmol/g and (100.3±50.1) mmol/g respectively,treatment group was (1.5±0.3) U/g,(17.5±7.0) mmol/g and (190.7 ±85.3) mmol/g respectively.The differences were statistically significant (t=11.23,3.81 and 4.50,all P＜0.01).Conclusion Nitrate can reduce DSS-induced acute experimental colitis in mice.
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Aim To investigate the inhibitory effects and mechanism of action of isoliensinine (IL) on the proliferation of porcine coronary arterial smooth muscle cells (CASMCs) induced by phenylephrine (Phen) and its mechanisms of action. Methods MTT assay, immunohistochemical method and Western blotting were adopted. Results IL (0.03-3 μmol·L-1) could inhibit the CASMCs proliferation induced by Phen (0.1 μmol·L-1) in a concentration-dependent manner. IL (0.1 μmol·L-1) antagonized Phen-induced overexpression of PDGF-β and bFGF from 0.545±0.026 and 0.47±0.03 to 0.458±0.019 and 0.376±0.017 (P<0.01, P<0.01). IL (0.1 μmol·L-1) also decreased c-fos, c-myc and hsp70 overexpression induced by Phen from 0.57±0.04, 0.44±0.04 and (173±36)% to 0.46±0.05, 0.372±0.021 and (115±35)% respectively (P<0.01, P<0.01, P<0.01). Conclusion IL exerted antiproliferative effect on CASMCs induced by phenylephrine, and its mechanisms were related to decrease the overexpression of growth factors (PDGF-β, bFGF), protooncogene (c-fos, c-myc) and hsp70.
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AIM To study the effects of A 3 site (components), extracted from Angelica naphtha (total oil),on physiological properties of isolated rat atrium and action potential of isolated guinea pig ventricular papillary muscles. METHODS Spontaneous beats, contractile force and functional refractory period(FRP) of isolated atrial muscle were recorded according to the regular methods. Standard microelectrode intracellular recording technique was used to record action potential(AP) of ventricular papillary muscles. RESULTS A 3 site(10～160 mg?L -1 ) dramatically inhibited spontaneous beats of isolated rat right atrium and made beats stop with 160 mg?L -1 . A 3 site concentration dependently reduced contractile force of isolated rat left atrium induced by electrostimulating, with the IC 50 value of 52 3 mg?L -1 . A 3 site(40～100 mg?L -1 ) obviously prolonged FRP. The FRP was prolonged from 106 ms (as control) to 130 ms at final concentration of 100 mg?L -1 . A 3 site decreased action potential amplitude (APA), shortened 20%APD(APD 20 ) and 90%APD(APD 90 ), but had no significant effect on RP of AP. CONCLUSION The mechanism of these effects of A 3 site on myocardial physiological properties and action potential was probably associated with blocking the influx of Ca 2+ and Na +, promoting K + outflux.
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<p><b>OBJECTIVE</b>To investigate the association of two single nucleotide polymorphisms (SNPs) of beta 2-adrenoceptor (beta 2-AR) gene with hypertension in elderly patients.</p><p><b>METHODS</b>The study samples were collected from unrelated Chinese Han population of Dabie Mountain in Anhui province. Eighty-six elderly patients with hypertension and 43 controls were selected. Genotypes of +1053 and +1239 SNPs were typed by polymerase chain reaction-restriction fragment length polymorphism.</p><p><b>RESULTS</b>The frequencies of the two SNPs complied well with the Hardy-Weinberg equilibrium in normal group. The distribution of genotypes AA, GA,GG of the SNP at locus +1239 in moderate and severe hypertension group was significantly different from that in normal group (chi square=8.67, P<0.05). There were evident differences in the frequencies of alleles of the two groups (chi square=4.02, P<0.05). No significant difference was observed in the distribution of genotypes of the SNP at locus +1053 between the two groups.</p><p><b>CONCLUSION</b>These data indicate that the SNP at locus +1239 of beta 2-AR gene is associated with hypertension in elderly patients.</p>
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Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Alleles , DNA , Genetics , Gene Frequency , Genotype , Hypertension , Genetics , Polymorphism, Single Nucleotide , Genetics , Receptors, Adrenergic, beta-2 , GeneticsABSTRACT
0 05). The frequency of genotype AA (26 67 %) of the SNPs at position -1 023 bp in severe hypertension group was significantly different from that in normal group(6 98 %, P
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Objective To study the inhibitory effects of angelica A_3 active fraction (A_3) inflammation and up-regulation of cyclooxygenase-2 (Cox-2) expression of rat uterus induced by lipopolysaccharides (LPS). Methods The anti-inflammatory effects of A_3 were investigated in rats using the carrageenin-induced paw swelling model and in mice using dimethylbenzene-induced ear edema model; RT-PCR and Western blotting were used to analyze Cox-2 mRNA and the protein expression levels. Results A_3 (1, 5, and 10 mg/kg) dose-dependently inhibited dimethylbenzene-induced ear edema in mice and paw swelling in rats by ig administration. LPS 1 ?g/mL could significantly increase the level of Cox-2 mRNA and protein expression. A_3(10—320 mg/L) could concentration-dependently inhibit Cox-2 mRNA and protein over-expression stimulated by LPS. Conclusion A_3 possesses better anti-inflammatory effects than angelica oil, which maybe relates to its inhibitory effects on Cox-2 overexpression.