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Table of Content

    05 November 2013, Volume 33 Issue 11
    Progress in research of human amniotic membrane on the skin wound healing effect
    2013, 33(11):  1367-1370. 
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    Amniotic membrane as a good biological medium has been widely applied in the research of the skin wound healing. Amniotic membrane can be used as a carrier of load cells, can release cytokines, can also be used as biological dressings, and can provide training environment to promote healing.
    Ultrastructure of Human Amniotic Mesenchymal Stem Cells
    2013, 33(11):  1371-1376. 
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    Objective To observe the ultrastructure of human amniotic mesenchymal stem cells( AMSCs) .Methods: human AMSC from full-term newborn amnion were isolated and cultured,and then subcultured,amplificated,and cell morphology was observed by microscopy.The Flow detection and identification of human AMSCs phenotype of at passage 3 were analyzed.Transmission and scanning electron microscopy were used to observe the ultrastructure of human AMSC.Results The appearance of human AMSC was spindle-shaped and polygonal.the hAMSC expressed immunophenotype CD44,CD90,CD105 and vimentin,did not express CD34,CD45.The short and thick microvilli processes were seen at the surface of human AMSC by scanning electron microscope. morpholigies of human AMSC were seen under transmissionelectron microscope,the cell was in a relatively active period in which a large and round or oval nucleus in the one cell were observed,The chromatin sparsely distributed in the nucleus,and cytoplasm was in much quantity.the organelles of mitochondria ,rough endoplasmic reticulum , golgi apparatus and small vesicle were rich.Conclusions Ultrastructure of Human Amniotic Mesenchymal Stem Cells has the biological characteristics of mesenchymal stem cells. the organelles of mitochondria ,rough endoplasmic reticulum and golgi apparatus were rich.Suggest it has a large number of secretory protein function, this is likely to be basis of cell paracrine function of amniotic mesenchymal stem cells.
    Effects of Human Adipose-derived Mesenchymal Stem Cells on Cutaneous Wound Healing
    Xiao-Yu LIU rui WANG Tao ZHANG ping shi
    2013, 33(11):  1377-1381. 
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    Objective To explore the effects of adipose derived mesenchymal stem cells(hADSCs)on cutaneous wound healing and provide a new method for clinical skin repair in the future. Methods The hADSCs were isolated by mechanical separation and subsequent enzymatic digestion. The expression profiles of CD90,CD105,CD34andCD45 were examined by immunofluorescence. The hADSCs from passage 3-5 were used for bone, osteogenic, adipogenic differentiation assays by culturing in appropriate induction medium.The mice were anesthetized and a 12-cm full-thickness excisional skin wound was made on the back of each mouse. After labeling with superparamagnetic iron oxide nanoparticles (SPIONs ), hADSCs were injected intradermally around the wound at four different injection sites. After o,7and 14d , the area of the wounds were measured and performed to evaluate the quality of wound healing compare with control. Results The morphological feature of hADSCs displayed fibroblast-like phenotype.The cells were positive for stem cells markers,including CD90 and CD105,and negative for CD34 and CD45,as shown by immunofluorescence. Osteogenic,chondrogenic, adipogenic differentiations were revealed in hADSCs when cultured in specific medium.The first 3 days of the experimental mice in each group after skin wound, wound gradually reduced, most of the wounds healed after 21d. Compared with the control group (26.7%±1.9)at 7d and(47.3%±2.3)at 14d,, the most obvious effect at 7d (74.6%±4.1)and 14d(96.5%±1.47)after transplantation(P<0.05). Conclusion hADMSCs transplantation can improve wound healing.
    Effects of covered polyethylenimine Fe3O4 superparamagnetic iron oxide on human amniotic mesenchymal cells
    2013, 33(11):  1382-1386. 
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    Objective To investigate the best method of polyethylenimine(PEI) coated superparamagnetic iron oxide(SPIO) labeled human amniotic mesenchymal stem cells(hAMSCs) and to observe its influence on cell proliferation.Methods hAMCs were incubated in DMEM/F12 containing PEI -SPIO of 6,8,10,12mg/L,using prussian blue staining method and atomic absorption spectrophotometry method analysis label efficiency. To investigate the PEI -SPIO labeled hAMSCs proliferation activity using MTT analysis.Results hAMSCs with PEI -SPIO labeled detectable amounts of iron particles.PEI -SPIO is concentration dependent; 10,12mg/L concentration significantly inhibit cell proliferation(P <0. 05).Conclusions The PEI -SPIO concentrations of 8mg/L labeled hAMSCs can obtain good labeling effect, and does not influence cell proliferation activity.
    Aquaporin 1 promote migration of human amniotic mesenchymal stem cells
    2013, 33(11):  1387-1390. 
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    Objective To investigate the effect of aquaporin 1 (AQP1) on human amniotic mesenchymal stem cells (hAMSCs) migration. Methods By immunofluorescence staining, RT-qPCR and Western blot, expression of AQP1 was detected on hAMSCs and migration ability of hAMSCs after siRNA interference was assessed with Transwell. Results The expression of AQP1 on hAMSCs was identified on membrance. After 48 hours interference, the expression of AQP1 was reduced by 60.13% for mRNA level and 40.42%for protein level. The cell numbers through the menberane of control group vs AQP1 siRNA group was signature reduction from 33.15±1.15 to 23.7±1.45, P<0.01. Conclusion AQP1 promoted migration of hAMSCs and involved in wound repair process.
    Effect Of Epidermal Growth Factor On Gene Expression Profile Of Human Amnion Mesenchymal Stem Cells And Bioinformatics Analysis
    2013, 33(11):  1391-1397. 
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    Objective To characterize the effect of epidermal growth factor on the spectrum of gene expression in the cultured human amnion mesenchymal stem cells (hAMSCs), and explore the underlying molecular mechanism. Methods Illumina RNA-Seq was used to detect the alteration in the gene expression profiles induced by 100 ng/ml epidermal growth factor in hAMSCs. Real-time PCR was employed to verify the results of selected differentially expressed genes. Results A total of 104 differential genes were found(FDR≤0.001 and |log2Ratio|≥1)in human amnion mesenchymal stem cells after treatment with epidermal growth factor for 24h, including 56 up-regulated and 48 down-regulated ones. GO enrichment of biological process analysis 26 biological processes have significant(P≤0.05);KEGG signaling pathway enrichment analysis found that significant interaction between cytokines and cytokine signaling pathway is significant(Q≤0.05).Conclusion EGF intervention hAMSCs changes in gene expression profile, Significantly differentially expressed genes and signaling pathways plays an important role in hAMSCs proliferation and migration and wound healing in the process.
    Effects of covered polyethylenimine Fe3O4 superparamagnetic iron oxide on human adipose mesenchymal stem cells
    2013, 33(11):  1398-1403. 
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    Objective To investigate the effects of superparamagnetic iron oxide covered by polyethylenimine(PEI) on human adipose mesenchymal stem cells (hADSCs).Methods The adipose tissue come from human subcutaneous,we extraction separation, culture and identification of hADSCs .The third passage of hADSCs were divided into four groups,and incubated in DMEM/F12 containing PEI -SPIO of 6,8,10,12mg/L.Detected by Prussian blue staining,Trypan blue staining,MTT test and inductions of multi-directional differentiation to evaluate the ability of SPIO on cell labeling efficiency,cell viability,proliferation and differentiation. Results 1.The labeling efficiency of SPIO (8,10,12mg/L) was 99%.However the labeling efficiency of SPIO(6mg/L)was 96%.2.In 6 ,8μg /ml group,the cell survival rate was 99%. But in10mg/L and 12mg/L group,the cell survival rate was(79.03±2.25)% and (70.23±1.36)%, Significantly lower than the control group(P<0.01).3.In 10,12 mg/L group,there were statistically significant differences in the viability,most of cells deceased in the procedure of multi-directional differentiation.Conclusions PEI-SPIO of 8mg/L can effectively label hADSCs,without influencing the cell viability,proliferation and the multiple differentiation ability.
    EGF promotes migration and expressions of MMP14 and IL-1β in human amniotic mesenchymal stem cells
    2013, 33(11):  1404-1409. 
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    Objective Research the expression regulation of epidermal growth factor (EGF) on matrix metalloproteinase 14 and interleukin-1β in the progress of human amniotic mesenchymal stem cell migration .Explore the mechanisms of EGF to promote hAMSCs migration Method Take health cesarean cultured amniotic membranes adherent mesenchymal stem cells, Using Transwell cell migration in vitro system of observation EGF on migration of hAMSCs ;By Western blotting (western blot) detection EGF action hAMSCs different time MMP14 and IL-1β protein expression changes,Using real-time quantitative PCR (Real-time PCR) detection of EGF action hAMSCs different time MMP14 and IL-1β expression levels changes of mRNA Results Primary isolated and cultured cells of human amniotic cells Identified by flow cytometry express surface marker of mesenchymal stem cells CD44 do not express CD45,Transwell chamber detect different concentration of EGF acts on hAMSCs 24h reveal that 100μg/LEGF is the best:2.0,100μg/L EGF acts on hAMSCs12h、24h and 48h,migration index namely1.6、2.0 and 1.7 ;The protein level of MMP14 and IL-1β were markedly augmentmented by 100ng/mi EGF 12h results of MMP14 are(0.81±0.20)significantly higher than control group(0.63±0.12);the 48h results of IL-1β are(0.55±0.16)significantly higher than control group(0.30±0.05), 100μg/L EGF upregulated the express of MMP14 and IL-1β mRNA significantly higher than control group(P<0.05) ; Conclusion EGF have a significant role in promoting the migration of hAMSCs,in this progess EGF also upregulated the express of MMP14 and IL-1β. MMP14 IL-1β and EGF can synergistically activate the MAPK / ERK and P13K/AKT signaling pathway and MMP14 and IL-1β are involved in the regulation of EGF to promote hAMSCs migration.
    Store bank building methods of human amniotic mesenchymal stem cells
    2013, 33(11):  1410-1417. 
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    Objective To build human amniotic mesenchymal stem cell bank for the application of MSCs provide sufficient source of cells. methods amniotic membrane were isolated from clean placenta,the human amniotic mesenchymal stem cell underwent primary culture and subculture from amniotic membrane. Cell growth pattern was detected by MTT assay. Flow cytometry was applied to analyze cell phenotype and stem cell markers. The different differential medium was used to detect its multi-directional differential ability. Such as Cell cycle, used identification of their biological characteristics. ofTransplantation of mesenchymal stem cells of magnetic nanoparticles markers under human amniotic membrane in mice of skin wound model to evaluate wound repair. results analysis shows that these amniotic -derived cells are adherent cells with typical fibroblast morphology. Cells extend not found in the process of bacteria, fungi, mold, mycoplasma, endotoxin pollution, etc. Flow cytometric analysis reveals that human amniotic mesenchymal stem cells are consistently positive for MSCs labeled with CD90 and CD105, and negative for the hematopoietic markers CD45, CD14, CD34 and HLA-DR. The cells can differentiate into osteoblasts and chondrogenic and adipogenesis under different conditioned mediumcultured conditions. Cell cycle analysis showed that most cells in G1 phase, only a few cells in the proliferation period G2. In vitro experiments show that human amniotic membrane mesenchymal stem cells can promote wound healing in mice, compared with the control group a very significant difference(p<0.05)Conclusion These results proved that these cells are human amniotic -derived MSCs. amniotic mesenchymal stem cells compared with bone marrow mesenchymal stem cells have a similar biological characteristics. Preliminary caracterizeed inventory cells biological characteristics and the established corresponding testing standards of hAMSCs in the quality control system.
    Primary culture and purification of type 2 diabetic human keratinocytes in serum-free medium
    2013, 33(11):  1418-1421. 
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    Objective To improve the methods for primary culture and purification of type 2 diabetic human keratinocytes in serum-free medium in vitro. Methods Type 2 diabetic human keratinocytes was isolated by 2 step digestion of neutral protease and trypsin, cultured in serum-free medium and purified by two-step subculture, the keratinocytes were observed using inverted phase contrast microscope, the growth curve was obtained by the WST-8 assay and phenotype and purity were analyzed by flow cytometry. Results Type 2 diabetic human keratinocytes in serum-free medium showed typical epithelial morphology, the growth curve of the keratinocyte at passage 3 is S-shaped, the average doubling time is 36.9 hours during logarithmic growth phase and the keratin-positive rate was 98.9%. Conclusion The methods for culture of high-purity type 2 diabetic human keratinocytes in serum-free media have been established.
    Probucol inhibits NF-κB activation and ICAM-1 expression in human dermal fibroblasts induced by advanced glycation end products
    Dian-Bao ZHANG ping SHI Xiao-Yu LIU
    2013, 33(11):  1422-1425. 
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    Objective To investigate the influence of Probucol on the activation of NF –κB and expression of ICAM-1 in dermal fibroblasts induced by advanced glycation end products (AGEs). Methods Fibroblasts were isolated and cultured in vitro, and then divided into 4 groups: control group, AGE-BSA group, low Probucol group(50μmol /L Probucol+100 mg /L AGE-BSA)and high Probucol group(100μmol /L Probucol+100 mg /L AGE-BSA).The content of malondialdehyde (MDA), the activities of superoxide dismutase (SOD) in Fibroblasts were detected. The Protein levels of NF – κB p65 and ICAM-1 were also measured by Western blot. Results (1) Compared with control group, the content of malondialdehyde (MDA) markedly was increased, the activities of superoxide dismutase (SOD) markedly were decreased in AGE-BSA group, the Protein levels of NF – κB P65 and ICAM-1 were markedly increased in AGE-BSA group. (2) Probucol markedly inhibited the decrease in SOD activity, the increase in the content of MDA in fibroblasts induced by AGE-BSA. In addition, the over-expression of NF–κB p65 and ICAM-1 in fibroblasts was inhibited markedly by Probucol. Conclusion Protective effect of probucol on dysfunction of fibroblasts induced by AGEs may be related with its effect on the suppressing the activation of NF–κB and subsequent expression of ICAM-1.
    The interaction of TGF-β/Smads signal transduction pathway and microRNA
    Feng Zhao ping shi
    2013, 33(11):  1426-1429. 
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    TGF-β/Smads pathway have a wide range of interactions with MicroRNAs. It has important roles in the control of gene epigenetics, transcription and post-transcriptional processing of MicroRNAs; while also from the post-transcriptional level MicroRNAs inhibit TGF-β/Smads pathway related genes expression. They also have significant relationship with a variety of human diseases, including tissue fibrosis, scar healing and tumor.
    DJ-1 relieve the mitochondrial dysfunction and cell apoptosis after rotenone exposure in MN9D cells
    2013, 33(11):  1430-1434. 
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    Objective To observe whether DJ-1 could relieve the mitochondrial dysfunctions and cell apoptosis in MN9D cellafter rotenone exposure. Methods MN9D cell lines was exposed to rotenone (100nM) for 24h after transfected with human mutant (L166P) or wild-type (WT) DJ-1.Cells were divided into Co, WT-DJ-1 (WT), L166P-DJ-1 (L166P) group. Cell viability was measured by MTT, and mitochondrial membrane potential (ΔΨm) by JC-1 and cell apoptosis by AnnexinⅤ+PI and TUNLE staining. Results Cell viability of Co group reduced to 48.2%±6.4%, and WT group was 78%±3.7% (P<0.05), there was not statistical difference between Co group and L166P group. Overexpression of WT-DJ-1 could partly rescue ΔΨm which was reduce after rotenone exposure (P<0.05), however, overexpression of L166P exacerbated the reduce of ΔΨm. And cell death in WT group was 5.4% and 9.7% after rotenone exposure for 12h or 24h respectively, accordingly 10.9% and 23.2% in Co group (P<0.05). There was not statistic difference between Co group and L166P group. TUNEL staining showed the similar trend. Conclusion Overexpression WT-DJ-1 could relieve the cell injury after rotenone exposure and dull the mitochondrial dysfunction. However, L166P had not the protective role after rotenone exposure.
    Immunization response of enterovirus 71 whole virus inactivation antigen combined with PA adjuvant by nasal mucosa in mice
    2013, 33(11):  1435-1439. 
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    Objective To study the systemic immune response intranasally immunized by type 71 whole virus inactivation antigen (EV71 Ag) combined with alpha mannan peptide (PA) adjuvant.. Methods Forty BALB/c mice that were five to six week old were randomly divided into five groups,10 mice per group. Immune groups were accepted EV71 Ag with or without adjuvants. Blood serum and bronchoalveolar lavages were collected at 0,2,3,4,5and6weeks since the first immune treatment.The level of IgG,IgA and neutralizing antibody in serum were detected by enzyme-linked immunosorbent assay(ELISA)and Neutralizing antibody tests repectively.MTT assay was involved to assess the proliferation of lymphocyte. Results The specific IgG antibody in serum could be detected in every group.There was no statistically significant different between the high-dose adjuvanted group and EV71Ag intramuscular injecting group(2.477±0.500 verse 3.040±0.120).EV71 Ag combined with alpha mannan peptide(PA)adjuvant caused lymphocyte proliferation in vitro,neutralizing antibody titer increased in all group.There were no significant difference between high-dose adjuvanted group and the EV71 Ag intramuscular injuecting group.Conclusion EV71 Ag combined with alpha mannan peptide(PA) adjuvant intranasally induced an effective systemic immune reponse.
    Construction of double interference vector targeting both Surviving and hTERT gene and its inhibitory effect on human colon cancer cell line SW480
    2013, 33(11):  1440-1445. 
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    Objective To construct the double interference vector targeting both survivin and hTERT gene and detect its silencing effects on surviving and hTERT gene in human colon carcinoma SW480 cell line. Methods Two effective targeting sequences were chosen according to hTERT and surviving cDNA sequences in Gen Bank, Then they were inserted into pGenesil-1.1 and pGenesil-1.2, pGenesil-1.1-survivin and pGenesil-1.2-hTERT were constructed. The recombinant plasmids were identified by restriction endonuclease and DNA sequencing. pGenesil-1.1-survivin and pGenesil-1.2-hTERT were digested by HindIII and BamHI separately, long fragment of pGenesil-1.1-survivin and small fragment of pGenesil-1.2-hTERT were collected. Then long fragment and small fragment were connected with T4 DNA ligase orientation, the double interference vector pGenesil1.1-survivin-hTERT was constructed. These siRNA recombinant expression vector separately were transfected into SW480 cell line, The effect of siRNA recombinant expression vector was detected by RT-PCR and MTT. Results By restriction endonuclease and DNA sequencing, specific RNAi expression vector targeting survivin and hTERT gene was constructed correctly. The growth rate of SW480 cells transfected with siRNA recombinant expression vector was significantly decreased. Conclusion The multiple siRNA recombinant expression vector targeting hTERT and Survivin gene simultaneously has been constructed successfully.This result could provide theoretical information for the further research.
    MiR-378 attenuates OGD-induced mouse N2A cell ischemic injuries by negatively regulating caspase-3 protein expression
    2013, 33(11):  1446-1451. 
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    Objective To explore the role of miR-378 in oxygen-glucose deprivation (OGD)-induced N2A cell ischemic injury and its possible molecular mechanism. Methods 3h OGD/24h reoxygenation of N2A cells was established to mimic ischemia/reperfusion in vitro, and the N2A cell survival rate was determined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The caspase-3 protein levels, and the expression levels of miR-378 and caspase-3 mRNA were determined by using the techniques of Western blot and real time RT-PCR, respectively. Luciferase reporter assay was performed to identify the direct binding of miR-378 with 3'-UTR of caspase-3 mRNA. Results The miR-378 expression levels decreased significantly (p<0.05, n=5 group) in response to reoxygenation time following 3h OGD treatment in N2A cells. Under the condition of 3h OGD/24h reoxygenation not non-OGD, up- and down-regulation of miR-378 expression level by transfection with pri-miR-378 or anti-miR-378 could enhance and reduce N2A cell survival rate (p<0.05, n=6), respectively. Similarly, miR-378 could negatively regulate caspase-3 protein expression levels, but no significant affection on caspase-3 mRNA expression levels was observed in N2A cells after 3h OGD/24h reoxygenation treatment. In addition, overexpression of miR-378 by co-transfection with pri-miR-378 could significantly decrease the luciferase activities which were expressed by plasmid containing 3'-UTR of caspase-3 mRNA in N2A cells (p<0.05, n=6). Conclusions miR-378 attenuates OGD-induced N2A cell ischemic injuries by negatively regulating caspase-3 protein expression, which may provide a potential therapeutic target for ischemic stroke in miRNAs levels.
    Endogenous PKC gamma Protein Levels Affect Cerebral Infarction Volume and Neurological Outcome of Mice with Ischemic Stroke
    2013, 33(11):  1452-1459. 
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    Objective To explore the effect of endogenous conventional protein kinase C (cPKC) γ protein expression level on cerebral infarction volume and neurological outcome of mice following ischemic stroke. Methods By using cPKCγ gene wild type (WT), heterogeneous (HET) and knockout (KO) mice to establish the 1h middle cerebral artery occlusion (MCAO)/reperfusion (R) 24h and 7d-induced ischemic stroke model, we applied the techniques of Western blot, 2,3,5-triphenyltetrazolium chloride (TTC) staining and neurological behavior tests to determine endogenous cPKCγ protein expression levels, cerebral infarction volume and neurological outcome, respectively. Results The treatments of 1h MCAO/R 24h and 7d could induce significant cerebral infarction and neurological dysfunction of WT, HET and KO mice. There were individual differences in endogenous cPKCγ protein expression levels among WT mice with two copies of cPKCγ gene, while the endogenous cPKCγ protein expression levels of HET mice with single copy of cPKCγ gene could reach about 30-100%, not simply 50% of the WT mice. The cerebral endogenous cPKCγ protein expression levels correlated negatively with the infarction size, as well as the neurological outcome could be improved with the increase of endogenous cPKCγ protein expression levels in 1h MCAO/R 24h and 7d treated mice. Conclusion These results suggested that cerebral endogenous cPKCγ protein expression levels affect the brain infarction size and the neurological outcome of mice with ischemic stroke.
    Expressions and significance of GRP78 and caspase-12 in the lungs of rats with bronchopulmonary dysplasis
    2013, 33(11):  1460-1464. 
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    Object To investigate the expression of endoplasmic reticulum stress-related protein GRP78 and caspase-12 in the lungs of bronchopulmonary dysplasis (BPD) rats. Methods Hyperoxia-induced preterm rat model of BPD was established. Forty-eight premature Sprague-Dawley rats were randomly divided into hyperoxia group and air group. The hyperoxia group was continually exposed to hyperoxia(85%) while the air group in room air. Lung tissues were obtained at 7, 14 and 21 days after exposing to either room air or hyperoxia. The changes of pulmonary histopathology were observed by hematoxylin-eosin (HE) staining and radical alveolar count (RAC). Apoptosis was detected by TdT-mediated dUTP nick end labeling (TUNEL). The expressions of GRP78 and caspase-12 mRNA and protein levels were detected by real-time quantitative RT-PCR and western blot respectively. Results The lung of hyperoxia premature rats showed simple alveolar structure, fewer and larger alveoli, which shares morphologic similarities to BPD. Compared with the air group, hyperoxia exposure resulted in lower RAC and significant apoptosis. In addition, the expression levels of GRP78 and caspase-12 mRNA and protein increased significantly (P<0.01) and had a up-regulated trend. Conclusion The increased expression of GRP78 and caspase-12 may be involved in the pathogenesis of BPD.
    L-theanine attenuates the sodium-azide-induced injury on mitochondrial in SH-SY5Y cells
    2013, 33(11):  1465-1469. 
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    Objective To investigate the effects and its mechanism of L-theanine on mitochondrial injury induced by the sodium azide in the SH-SY5Y cells. Methods The SH-SY5Y cells were exposed to different concentrations of L-theanine and the sodium azide, the cell viability was measured by the MTT method, the mitochondrial membrane potential was detected by the JC-1 method and the Scanning Confocal Microscopy. The protein levels of intracellular caspase-3, caspase-9 and cytochrome c were detected by the Western Blotting Method. Results (1) With the increase of the concentration of sodium azide, the cell activity decreases gradually. After being pretreated by L-theanine (0.25,0.50,1.00 mmol/L) for 2 hours,it can significantly reduce the cell toxicity and reverse the decline in the mitochondrial membrane potential induced by the sodium azide (0.25 mmol/L) for 24 hours(p<0.05)(2) L-theanine(0.25-1.0 mmol/L)can significantly reverse the increasing of the protein expression level of the caspase-3, caspase-9 and cytochrome c induced by the sodium azide in SH-SY5Y cell(p<0.05).Conclusion L-Theanine can stabilize mitochondrial membrane potential of the SH-SY5Y cell, reduce the mitochondrial damage.
    Decreased expression of Nneuregulin-1β in myocardium of hypertrophic rats induced by pressure-overloaded
    2013, 33(11):  1470-1474. 
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    Obective To investigate the change expression of neuregulin-1β(NRG-1β) at mRNA and protein levels in myocardium of hypertrophic rats induced by pressure-overloaded.Methods Wistar rats were randomly divided into four groups: myocardial hypertrophy groups (Model) of 4 weeks and 8 weeks, control operation groups (Control) of 4 weeks and 8 weeks. Model groups were operated with abdominal aortic constriction. Hemodynamic and echocardiogrophic characteristics were evaluated; masson staining was employed to observe the pathological lesions of myocardial tissues; radioimmunological method was used to detect angiotensin II (AngII); expression of NRG-1β mRNA and protein in myocardium were tested by Real time-PCR and western blot. Results (1) Compared with 4wC, left ventricular ejection fraction (LVEF)、left ventricular fraction shortening (LVFS)、left ventricular end systolic pressure (LVESP)、Maximal rate of increase in left ventricular pressure (±dP/dtmax) of 4wM were decreased(P <0.05),while left ventricular end-systolic diameter (LVEDD)、left ventricular end-diastolic diameter (LVESD)、left ventricular end diastolic pressure (LVEDP) 、 collagen volume fraction (CVF) and AngII of 4wM were increased(P <0.05,P <0.01),NRG-1β mRNA and protein expression of 4wM group were not exchanged(P >0.05)。 (2) Compared with 8wC, LVEF、LVFS、LVESP、±dP/dtmax of 4wM were decreased(P <0.01), LVEDD、LVESD、LVEDP、CVF and AngII were increased(P <0.05,P <0.01),NRG-1β mRNA and protein expression of 8wM group were decreased(P <0.01)。Conclusion The expression of NRG-1β in myocardium of hypertrophic rats induced by pressure-overloaded are down-regulated ,which may be involved in the myocardial hypertrophy development.
    The effect of carvedilol on TGF-beta-Smad pathways in myocardial fibrosis (MF) induced by Isoproterenol
    2013, 33(11):  1475-1479. 
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    Objective To investigate the effect of TGF-beta-Smad signaling pathway in cardiac fibrosis induced by isoproterenol(ISO)in rats and the intervention mechanism of carvedilol. Methods Thirty male Sprague-Dawley rats were randomly divided into ISO model group(M group),carvedilol treatment group (T group)and control group(C group). Cardiac fibrosis models were induced in M group and T group by subcutaneous injection of ISO(5mg/(kg?d)for 10 days). Rats of C group were injected normal saline(5ml/(kg?d)for 10days)instead, after 10 days, rats of T group were fed carvedilol(10mg/(kg?d)) for 4 weeks. Rats of M and C group were fed normal saline 10ml/(kg?d) for 4 weeks instead, after 4 weeks,all the rats were killed to measure the CWI. The pathological changes of myocardial tissue were observed by HE staining and the changes of collagen fiber hyperplasia were detected through Massion staining. The mRNA expression level of TGF-β1, Smad3 and Smad7 were determined by RT-PCR. The protein level of TGF-β1, Smad3 and Smad7 were detected by immunohistochemistry. Results Pathology changes were observed by light microscope : There were changes in M group including cardiac muscle cell hypertrophy, prolongation,fibrosis and calcification presented between the cells, in addition, myocardial collagen fibers increased significantly in masson dyeing cells in M group than that in T group and C group. T group heart muscle cells could be seen parallelism arrange. There was small amounts of areolar tissue in cellular stroma. Between the cells collagen fibers could be found, but the amount and area was decreased markedly. Compared with those of C group, all of the CWI, the expression levers of TGF-β1 mRNA, the Smad3 mRNA, TGF-β1 protein, and the Smad3 protein were significantly increased(all P<0.01), and the expression levers of Smad7 mRNA and the Smad7 protein were significantly lower in M group (P<0.01;P<0.05). However, the expression levers of TGF-β1 mRNA, the Smad3 mRNA, TGF-β1 protein, and the Smad3 protein were much lower(P<0.01;P<0.05), and the expression levers of Smad7 mRNA and the Smad7 protein were significantly higher in T group than M group(P<0.01;P<0.05). Conclusion TGF-beta-Smad signaling pathway may play an important role in myocardial fibrosis induced by ISO, and carvedilol can improve the pathological changes, which might be associated with the inhibition of TGF-beta-Smad signaling pathway.
    The application of proteomics in detecting differentially expressed proteins in serum of renal angiomyolipoma
    2013, 33(11):  1480-1483. 
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    Objective The application of WCX magnetic beads combined with MALDI-TOF MS in detecting differentially expressed proteins in serum of renal angiomyolipoma can reduce the misdiagnosis rate. Methods WCX beads combined with MALDI-TOF MS technique in detecting differentially expressed proteins in serum of renal angiomyolipoma can screen out differentially expressed proteins. And genetic algorithm was utilized to establish a diagnosis model. Results The technique screened out 142 different protein, and 13 were significantly different (P<0.05). ClinProTools2.2 software was utilized with genetic algorithm to screen out 15 differentially expressed proteins to establish a diagnosis model. The recognition rate was 100%. Conclusions The iagnosis model established by the application of WCX magnetic beads combined with MALDI-TOF MS can reduce the misdiagnosis rate of renal angiomyolipoma.
    Effect of ammonium chloride on gene expression of RHCG and AQP8 in LO2 cells
    2013, 33(11):  1484-1488. 
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    Objective: To inspect whether ammonium chloride can specifically stimulate liver cell to change the internal RHCG and AQP8 gene expression .Methods: The human liver cell line (LO2)、thyroid cancer cell line(TPC-1)、ovarian cancer cell line(SKOV-3) and esophageal cancer cell line(9706) were cultured . MTT method was used to check the proliferation ability of those 4 cell lines after cells were treated with NH4Cl(work concentration:2.5、5、10、20、40、50 mmol/L, respectively)for 24h. QT-PCR and Western blotting were used to detect the RHCG and AQP8 gene expression of LO2 cells after treated with NH4Cl(5、10、20 mmol/L)for 6、12 and 24h. The RHCG and AQP8 gene expression of TPC-1、SKOV-3 and 9706 cells was detected after treated with NH4Cl(10 mmol/L)the same method above. Results: MTT method finds the growth inhibitory rate in LO2 cells increase with ammonia concentration increasing, which is significantly greater than that in other 3 cell lines. RT-PCR finds that RHCG mRNA in LO2 cells decreased when treated with 10 mmol/L ammonium chloride for 6 hours (p<0.05). while AQP8 mRNA in LO2 cells increased when treated with 10mmol/l ammonium chloride for 12 hours (p<0.05);10 mmol/L. Western blot finds that the AQP8 protein in LO2 cells increased when treated with 10mmol/L ammonium chloride for 24 hours (p<0.05), while the RHCG protein expression has no change. No obvious change is found in the AQP8 and RHCG expression in other cells when treated with ammonia of the same concentration within the same time. Conclusion: In the environment with ammonia of high concentration, human liver cells can maintain the balance of absorbing ammonia by changing RHCG and AQP8 expression to reduce the harm of ammonia to liver cells, while other cells have no such function.
    The diagnosis and treatment of primary hyperparathyroidism in association with papillary thyroid carcinoma
    2013, 33(11):  1489-1492. 
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    Objective To describe a rare manifestation of primary hyperparathyroidism in association with papillary thyroid carcinoma and to decrease misdiagnosis and missed diagnosis.Methods We describe the clinical history, findings on physical examination, results of laboratory studies,imaging findings, and histopathologic features of three patients who were diagnosed hyperparathyroidism and papillary thyroid carcinoma in PUMCH from 1983∽2011.Results There are two women and a men,from 37 to 59 years old.They were presented to our clinic because of primary hyperparathyroidism .Serum Ca 2.78∽3.1 mmol/L,PTH 202∽241pg/mL,MIBI(+).Ultrasonography revealed solitary nodule in two people,another is no.At first, Parathyroidectomy was performed in three of them .Then ,Total thyroidectomy was performed,two of them were proposed in two step. Conclusion The concurrence of primary hyperparathyroidism and papillary thyroid carcinoma are rare. Intraoperative evaluation of the thyroid is as important as preoperative evaluation with ultrasonography in patients with thyroid and parathyroid.
    Intraoperative lymph node dissection of methylene blue calibration improves cardiac cancer survival rate
    2013, 33(11):  1493-1495. 
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    Objectives To analysis the effect of open surgery of methylene blue calibration around the lower esophageal segment cardia lymph on postoperative 3-year disease-free survival rate and 5-year overall survival rate. Methods Patients less than 70-year-old, the cardia single tumor less than 8cm, stage Ⅰ to ⅢB were randomly divided into 2 groups. Group A: D2 radical mastectomy. Group B: injected into the methylene blue around the cardia tumor and surrounding lymph nodes, so as to guide cleaning resection. Patients were followed up for 5 years.Results A postoperative 3-year disease-free survival and overall 5-year survival rates were 59% and 37%. Group B 74% and 53% respectively.Conclusions Intraoperative lymph nodes around the tumor look straight down the calibration can be more complete excision and sentinel lymph node was stained and visually difficult to find< 2mm tiny positive lymph nodes, increased after 3-year disease-free survival and overall 5-year survival rate. The technique is simple and feasible, practical, clinical applications could be extended.
    EGF regulates the expression of REST/NRSF in HaCaT cells
    2013, 33(11):  1496-1497. 
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    Progress in research of experimental animal model of multiple sclerosis
    2013, 33(11):  1500-1503. 
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    Multiple sclerosis (MS) is widely considered to be the result of an aggressive autoreactive T cell attack on myelin. Now animal models induced by myelin sheath peptide and virus are widely used in the research of pathogenesis and therapies of this disease, which are similar with human disease in the pathogenesis and symptoms of process.
    Calreticulin and Tumor Therapy
    2013, 33(11):  1504-1507. 
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    Calreticulin can express steadily on human various tumor cells surface,is a member of the heat shock protein family.It plays important roles in guiding the tumor apoptosis,improving the effect of radiotherapy and chemotherapy,and gene therapy,and so on.
    Application of Visual Slides in Experimental Exam of Histology
    2013, 33(11):  1508-1510. 
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    Objective To evaluate the application of Visual slides in experimental exam of Histology. Methods Visual slides were used in addition to the picture of traditional slides and mixed slides during the experimental exam of histology for two years. Difficulty coefficient and discrimination index were calculated. Students’ scores of different parts were compared. Results Difficulty coefficient of visual slides is 0.67, higher than traditional slides and mixed slides (p<0.01). Discrimination index of visual slide is 0.51, higher the other two. Scores of visual slides appears moderate correlation with scores of theoretical exam. Conclusion These data indicates visual slides is worthy of use in exam because of its difficulty and good discrimination.