1.Research of autophagy activity between rat bone marrow mesenchymal stem neural differentiation.
Bo LI ; Chun-tian HUANG ; Cai-fang LI ; Ping DUAN ; Xue-fei HAN ; Wen-hai YAN ; Ying XING
Chinese Journal of Applied Physiology 2015;31(1):31-34
OBJECTIVETo study the autophagy activity between rat bone marrow stem cells (BMSCs) neural differentiation in order to explore the mechanism involve in this process.
METHODSBMSCs were passed by 3 generation, then was induced with the revulsant 2% (DMSO) + 200 µmol/L (BHA), NSE expression was detected by immunocytochemical stain, the mRNA expression of autophagy associated genes L3B, Beclinl, Atg5, Atg7, Atg10 were detected by RT-PCR, the autophagy protein LC3B was examined by Western blot and flow cytometry analysis.
RESULTSBMSCs were passed by 3 generation, the purity of BMSCs could reach more than 90%, the morphology of cells were like fibroblasts, after the revulsant 2% DMSO + 200 µmol/L BRA induced, cells were extended long neurites, like nerve cells, positive rate of NSE staining was (83±5) %, RT-PCR results showed that the expression of autophagy associated genes LC3B, Beclinl, Atg5, Atg7 Atg0 were rised after BMSCs neural differentiation, Western blot analysis showed that the LC3B-II protein expression was increased after neural differentiation and the MFI of L3B was highten by flow cytometry.
CONCLUSIONAutophagy is increased after rat BMSC neural differentiation.
Animals ; Autophagy ; Cell Differentiation ; Cells, Cultured ; Flow Cytometry ; Mesenchymal Stromal Cells ; cytology ; Neurons ; cytology ; Rats
2.A QUICK AND PRECISION METHOD TO CONSTRUCT ESCHERICHIA COLI HISTIDINE AUXOTROPH
Peng WANG ; Sheng-Ling YUAN ; Ji-Ping ZHENG ; Shu-Qin LI ; Hai-Qing DUAN ; Zhao-Shan ZHANG ;
Microbiology 1992;0(02):-
Red in vivo recombination is a new kind of genetic engineering technique based on homologous recombination. In this work, plasmid pKD46 which expresses Red recombination proteins is transferred into Escherichia coli strain DH5?.The kanamycine resistant gene is generated by PCR by using primers with homology to hisDCB gene of E.coli chromosome. Thus, the hisDCB gene was replaced with kanamycine resistant gene by the plasmid recombination system, then the resistant gene was eliminated by a helper plasmid encoding the FLP recombinase. At last, a E.coli histidine auxotroph which is sensitive to kanamycine was got. The results indicate that Red in vivo recombination is a convenient method to construct auxotrophs.
3.Epidemiological investigation of an outbreak of acute gastroenteritis caused by human calicivirus in Lulong County.
Jin-Yu TANG ; Hua-Ping XIE ; Hai-Kuan HU ; Zhao-Jun DUAN ; Zhao-Yin FANG
Chinese Journal of Experimental and Clinical Virology 2007;21(3):232-234
OBJECTIVETo survey an outbreak of acute gastroenteritis in Lulong County and analyze the cause of the disease.
METHODSEpidemiological methods were applied to investigate an outbreak of acute gastroenteritis occurred in June 2000 in Lulong County. Stool specimens were collected from diarrhea patients and were tested for human calicivirus by ELISA and RT-PCR. The products of RT-PCR were cloned and sequenced, then phylogenetic analysis was carried out.
RESULTSIn total, 736 farmers were surveyed, among them 134 had acute gastroenteritis, the attack rate was 18.20%, and one elderly patient died. The age of patients was from 1 to 77 years and the incidence of the disease among young people was higher with a peak in June 25 through 30. Six stool specimens were tested for caliciviruses by ELISA and 3 were positives, one of them was confirmed by RT-PCR and belonged to norovirus genotype GI/2. No other pathogens were detected.
CONCLUSIONHuman calicivirus was confirmed to be the cause of the outbreak of acute gastroenteritis.
Acute Disease ; Caliciviridae ; genetics ; isolation & purification ; Caliciviridae Infections ; epidemiology ; China ; epidemiology ; Disease Outbreaks ; Enzyme-Linked Immunosorbent Assay ; Feces ; virology ; Gastroenteritis ; epidemiology ; virology ; Humans ; RNA, Viral ; genetics ; Reverse Transcriptase Polymerase Chain Reaction
4.Expression changes of Notch-related genes during the differentiation of human mesenchymal stem cells into neurons.
Ying XING ; Rui-Ying BAI ; Wen-Hai YAN ; Xue-Fei HAN ; Ping DUAN ; Yan XU ; Zhi-Gang FAN
Acta Physiologica Sinica 2007;59(3):267-272
The Notch signaling pathway has been implicated in the regulation of cell-fate decisions such as differentiation of embryo stem cells and neural stem cells into neurons. We cultured human mesenchymal stem cells (hMSCs) in vitro and induced hMSCs to differentiate into neural cells by beta-mercaptoethanol (beta-ME), DMSO and 3-tert-butyl-4-hydroxyanisole (BHA). Immunocytochemistry was utilized to detect neuron-specific enolase (NSE) and Nissl body, and flow cytometry was used to determine cell growth phases. The expressions of signal molecules involved in the Notch pathway such as Notch1, Jagged 1 (JAG1), presenilin 1 (PS1) and hairy and enhancer of split 1(HES1) were observed by RT-PCR and immunofluorescent techniques. The results were as follows: (1) Before induction, the percentage of hMSCs at G(0)/G(1) was 58.5%, and the percentage at S+G(2)/M was 41.5%. After induction, the percentage of hMSCs at G(0)/G(1) increased to 73.1%, 76.2% and 78.1%, respectively on days 2, 4 and 6, and the percentage at S+G(2)/M decreased to 26.8%, 24.8% and 21.9%, respectively; The percentage of NSE-positive cells reached (77+/-0.35) %; Nisslos staining was positive in cytoplasm. (2) Notch1 and JAG1 were both expressed in hMSCs before and after induction, but the mRNA expressions of both Notch1 and JAG1, detected by RT-PCR, decreased obviously after induction(P<0.05). Notch1 mRNA/beta-actin was 1.157, 0.815, 0.756 and 0.570, and JAG1 mRNA/beta-actin was 0.437, 0.350, 0.314 and 0.362, respectively, on days 0, 2, 4 and 6 after induction. The Notch pathway activation participant PS1 mRNA and Notch pathway target gene HES1 mRNA also decreased apparently after induction (P<0.05), and their mRNA/beta-actin was 0.990, 0.449, 0.441, 0.454 and 0.370, 0.256, 0.266, 0.240 on days 0, 2, 4 and 6, respectively. These observations indicate that the expressions of Notch signal molecules were suppressed when hMSCs were induced to differentiate into neural cells. Based on these findings, we propose that low level of Notch signaling activation may contribute to neural cell differentiation.
Basic Helix-Loop-Helix Transcription Factors
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genetics
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Calcium-Binding Proteins
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genetics
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Cell Cycle
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Cell Differentiation
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Flow Cytometry
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Homeodomain Proteins
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genetics
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Humans
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Intercellular Signaling Peptides and Proteins
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genetics
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Jagged-1 Protein
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Membrane Proteins
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genetics
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Mesenchymal Stromal Cells
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cytology
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Neurons
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cytology
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Receptor, Notch1
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genetics
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Receptors, Notch
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genetics
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Reverse Transcriptase Polymerase Chain Reaction
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Serrate-Jagged Proteins
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Signal Transduction
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Transcription Factor HES-1
5.Effect of Bawei Xilei powder on CD3, CD4, CD8 T-lymphocytes of rats with ulcerative colitis.
Duan-Yong LIU ; Hai-Mei ZHAO ; Ning ZHAO ; Cheng LU ; Ai-Ping LU
China Journal of Chinese Materia Medica 2008;33(11):1301-1304
OBJECTIVETo observe the effect of Bawei Xilei powder on CD3, CD4, CD8 T-lymphocytes in peripheral blood and colonic mucosa of rat with ulcerative colitis.
METHODSixty SD rats were randomly divided into 6 groups, normal group, model group, low, middle and high dosage Bawei Xilei powder group, Sulfasalazine group. Ulcerative colitis was induced by immunization with rabbit 's colonic mucous emulsified with completely Freund's adjuvant in all rats. Rats in low, middle and high dosage Bawei Xilei powder group were administered with 0.05, 0.1, 0.2 mg Bawei Xilei powder for 18 days by enema respectively. While rats in Sulfasalazine group were enema administered with 100 mg Sulfasalazine, and the rats in other group were administered with equal volume of saline enema as control. We analyzed expression of CD3, CD4, CD8 T-lymphocytes in peripheral blood by flow cytometry and in colonic mucous by immunohistochemistry.
RESULTIn peripheral blood, compared with normal group, in model group, the increased of CD4 T-lymphocytes and CD4 /CD8 ratio, the reduced of CD8 T-lymphocytes, these results were significant discrepancy (P < 0.01). Compared with model group, after treatment with Bawei Xilei powder, CD8 T-lymphocytes increased, but only high dosage Bawei Xilei powder group had discrepancy (P < 0.05). But low dosage Bawei Xilei powder group, other treatment groups' rats showed CD4/CD8 ratio were reduced significantly (P < 0.05). In colonic mucous, compared with normal group, in model group, Rats showed that expression of CD3, CD4 T-lymphocytes reduced and CD8 T-lymphocytes increased obviously (P < 0.05, P < 0.01). Compared with model group, expression of CD8 T-lymphocytes reduced significantly in all treatment groups (P < 0.05, P < 0.01).
CONCLUSIONBawei Xilei powder may regulate their balance between T-lymphocytes subgroup, consequently relieve inflammatory injury in favor of ulcer reparation and tissue regeneration.
Animals ; CD3 Complex ; metabolism ; CD4 Antigens ; metabolism ; CD8 Antigens ; metabolism ; Colitis, Ulcerative ; immunology ; metabolism ; Colon ; drug effects ; metabolism ; pathology ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; pharmacology ; Gene Expression Regulation ; drug effects ; Powders ; Rats ; T-Lymphocytes ; drug effects ; metabolism
6.Inhibition of glycogen synthase kinase 3b activity regulates Toll-like receptor 4-mediated liver inflammation.
Feng REN ; Hai-yan ZHANG ; Zheng-fu PIAO ; Su-jun ZHENG ; Yu CHEN ; De-xi CHEN ; Zhong-ping DUAN
Chinese Journal of Hepatology 2012;20(9):693-697
OBJECTIVETo determine the mechanism underlying the therapeutic activities of glycogen synthase kinase 3b (GSK3b) against hepatic ischemia-reperfusion (H-IR) injury by investigating the inhibitive effects of GSK3b on inflammation mediated by Toll-like receptor 4 (TLR4).
METHODSC57BL/6 male mice were subjected to 90 min of warm liver cephalad lobe ischemia, followed by reperfusion for various lengths of time. The mice were divided into three groups: the H-IR untreated model (control group), and the H-IR inflammation-induced models that received an intraperitoneal injection of purified lipopolysaccharide (LPS) endotoxin alone (inflammation group) or with pretreatment of the SB216763 GSK3b-specific inhibitor (intervention group). To create a parallel isolated cell system for detailed investigations of macrophages, marrow-derived stem cells were isolated from femurs of the H-IR control group of mice and used to derive primary macrophages. The cells were then divided into the same three groups as the whole mouse system: control, LPS-induced inflammation model, and inflammation model with SB216763 intervention. Differential expressions of inflammation-related proteins and genes were detected by Western blotting and real-time quantitative PCR, respectively.
RESULTSThe phosphorylation levels of ERK, JNK and p38 MAPK were induced in liver at 1 h after reperfusion, but then steadily decreased and returned to baseline levels by 4 h after reperfusion. In addition, the phosphorylation levels of ERK and JNK were induced in macrophages at 15 min after LPS stimulation, while the phosphorylation level of p38 MAPK was induced at 1 h; SB216763 pretreatment suppressed the LPS-stimulated ERK, JNK and p38 phosphorylation in macrophages. In the mouse model, GSK3b activity was found to promote the gene expression of anti-inflammatory cytokine IL-10 (control: 0.21 ± 0.08, inflammation: 0.83 ± 0.21, intervention: 1.76 ± 0.67; F = 3.16, P = 0.027) but to significantly inhibit the gene expression of pro-inflammatory cytokines IL-12 (control: 0.11 ± 0.05, inflammation: 0.85 ± 0.11, intervention: 0.43 ± 0.10; F = 2.67, P = 0.038), TNF-a, (control: 0.052 ± 0.012, inflammation: 8.11 ± 0.98, intervention: 3.9 ± 0.82; F = 4.13, P = 0.016), IL-6 (control: 0.22 ± 0.08, inflammation: 6.37 ± 0.81, intervention: 2.11 ± 0.63; F = 3.21, P = 0.024), and IL-1b (control: 0.12 ± 0.07, inflammation: 2.51 ± 0.62, and intervention: 1.28 ± 0.33; F = 2.22, P = 0.030).
CONCLUSIONInhibition of GSK3b selectively regulates the expression of anti-inflammatory and pro-inflammatory cytokines in liver Kupffer cells (liver macrophages). This process may be one of the mechanisms underlying the ability of GSK3b to ameliorate hepatic ischemia-reperfusion injury, possibly because inhibition of pro-inflammatory cytokines may indirectly mediate liver cell apoptosis.
Animals ; Cells, Cultured ; Cytokines ; metabolism ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Inflammation ; metabolism ; pathology ; Lipopolysaccharides ; adverse effects ; Liver ; pathology ; Macrophages ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Reperfusion Injury ; Toll-Like Receptor 4 ; metabolism
7.Expression of matrix metalloproteinase-9 and intercellular adhesion molecule-1 in the perihematomal tissues in human hypertensive intracerebral hemorrhage and its significance
Shu-Rong DUAN ; Song-Bin FU ; Chun-Yan WANG ; Xiao-Ran WANG ; De-Sheng WANG ; Ji-Ping QI ; Hai-Tao WANG
Chinese Journal of Neurology 1999;0(06):-
Objective To investigate the relationship between the expression of matrix metalloproteinase-9 (MMP-9) and intercellular adhesion molecule-1 (ICAM-1) in the perihematomal tissues in human hypertensive,intracerebral hemorrhage (ICH) and brain edema formation following ICH.Methods Paraffin-embedded brain tissues of 39 human fatal cases of ICH from the perihematomal tissues,1—3 cm away from the margin of the hemorrhagic lesion,as well as tissues from the corresponding area at the opposite side as controls,were stained with HE and immunohistochemistry staining.The expressions of MMP-9 and ICAM-1 in the pefihematomal tissues were analyzed with the SPSS 11.5 system.Results ①With MMP-9 immunohistochemical staining positive capillaries in the perihematomal tissues were identified at 2 h ((1.2? 0.8)/HP).The number of MMP-9 positive capillaries began to rise at 5—10 h ((4.1?0.8)/HP) reaching the peak at 45—48 h ((10.6?1.4)/HP,P
8.Apparatus for the measurement of the oxygen uptake of rats subjected to hypobaric hypoxia.
Rui-Feng DUAN ; Wen-Kao NAN ; Yi-Ping XING ; Huai-Xin WANG ; Wen-Yu CUI ; Hai WANG
Chinese Journal of Applied Physiology 2011;27(4):507-509
OBJECTIVETo construct an apparatus for the oxygen uptake measurement of rats exposed to hypobaric hypoxia at different simulated altitude.
METHODSThe capacity of this apparatus was about 0.01 m3. It included animal experimental cabin, reference cabin, altimeter, altitude vertical velocity indicator, pressure difference inductor and oxygen compensator, low scale manometer, soda lime and calcium chloride, small fan, thermometer, circulating water system and vacuum pump. The oxygen uptake of the rats at 6 000 m, 4 000 m and 1 000 m simulated altitude was measured using this apparatus.
RESULTSThe oxygen uptake of the rats at 50 m, 4 000 m and 6 000 m simulated altitude was (24.4 +/- 2.1), (10.8 +/- 2.0) and (8.8 +/- 1.6) ml O2/(kg x min) respectively (average +/- s, n = 10). The oxygen uptake decreased as altitude increased.
CONCLUSIONThis apparatus can be used to measure the oxygen uptake of the rats at different simulated altitude.
Altitude ; Altitude Sickness ; physiopathology ; Animals ; Computer Simulation ; Equipment and Supplies ; Hypoxia ; physiopathology ; Male ; Oxygen ; metabolism ; Oxygen Consumption ; physiology ; Rats ; Rats, Sprague-Dawley
9.The role of glycogen synthase kinase-3 beta in the pathogenesis of liver ischemia reperfusion injury.
Feng REN ; Hai-yan ZHANG ; Zheng-fu PIAO ; Su-jun ZHENG ; Yu CHEN ; Zhi-ming WU ; Zhong-ping DUAN
Chinese Journal of Hepatology 2011;19(7):547-551
OBJECTIVETo investigate the role of the key intracellular signaling molecule glycogen synthase kinase-3 beta in the mechanism of liver ischemia reperfusion (IR).
METHODSC57BL/6 mice were subjected to 90 min warm liver cephalad lobe ischemia, followed by various length of reperfusion. Experiment groups included sham control group, liver IRI model group and glycogen synthase kinase-3 beta inhibitor-treated group (SB216763 in DMSO, 25 g/kg, i.p, 2 hour prior to the onset of liver ischemia). The expression of glycogen synthase kinase-3 beta protein was analysed by Western blotting. The serum ALT levels were determined to reflect the function of liver. The affected liver lobes were harvested for histology analysis. The inflammatory gene expression was detected by Quantitative PCR.
RESULTSBy western blot analysis, we found that ischemia itself activated glycogen synthase kinase-3 beta by a significant decrease of its phosphorylation. Glycogen synthase kinase-3 beta inhibitor SB216763-pretreatment ameliorated the liver damages significantly as compared to the controls (sALT: 2046+/-513 U/L vs 5809+/-1689 U/L, P = 0.0153), and suppressed the gene expressions of IL-12, TNFa, IL-1b and IL-6.
CONCLUSIONSThis study demonstrated that the ischemia process modulated liver innate immune activation via a GSK-3-dependent mechanism which favored the development of a pro-inflammation response and lead to liver tissue damages. GSK-3b may be a new therapeutic target to ameliorate liver IRI in transplant patients.
Animals ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Inflammation ; metabolism ; Liver ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred C57BL ; Reperfusion Injury ; metabolism ; pathology
10.Heritability of serum uric acid in adult twins
Hai-Ping DUAN ; Zeng-Chang PANG ; Dong-Feng ZHANG ; Shao-Jie WANG ; Yao-Min ZHAI ; Dong ZHANG ; Qi-Hua TAN
Chinese Journal of Epidemiology 2010;31(4):384-388
Objective To assess the heritability of serum uric acid in adult,using the classic twin design.Methods Adult Twins were recruited from the Qingdao Twin Registry.Uric acid,height,weight were measured.Zygosity in all the same-sex twin pairs was determined by 16 polymorphic markers.Heritability was assessed by structural equation models,with age,gender and body mass index(BMI) included as covariates.Results In total,687 twin pairs were available for data analyses,including 420 pairs of monozygotic and 267 pairs of dizygotic twins.After logarithm transformed,uric acid in males ( 17.47±1.91 ) was significantly higher than in females ( 15.22±1.70,P<0.0001 ).After adjustment on age,sex and BMI,intraclass correlations for uric acid were 0.70 for monozygotic twins and 0.40 for dizygotic twins.The sex-limitation AE model,combining additive genetic and unique environmental factors,could produce the best fit for the data.Heritability estimate for uric acid was 70.5% (95% CI:65.9-74.6),with the proportion of unique environmental effects as 29.5%(95%CI:25.4-34.2).Conclusion Additive genetic effects appeared to be the major contributor to the variation of uric acid in this twins sample being studied.